Baked and partially baked products with heat-stable AMG variants from the Penicillium genus

Abstract

This invention relates to a method for producing baked or partially baked products, the method comprising: a first step of providing dough comprising a mature, heat-stable variant of a parental glucosylamylase having at least 70% identity with SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; and a second step of baking or partially baking the dough to produce baked or partially baked products, as well as baking compositions comprising said variants and uses of said variants.

Description

[0001] References to sequence lists

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

[0003] This invention relates to a method for producing baked or partially baked products, the method comprising: a first step of providing dough comprising a mature, heat-stable variant of a parental glucosylamylase having at least 70% identity with SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; and a second step of baking or partially baking the dough to produce baked or partially baked products, as well as baking compositions comprising said variants and uses of said variants. Background Technology

[0004] Sugar-containing baked goods (bread, cookies, etc.) are one of the most popular product segments worldwide. The sugar content in recipes typically ranges from 1% to 25% of the total flour weight.

[0005] However, due to rising sugar prices, sugar shortages in some parts of the world, and health concerns, there is a need for methods to produce baked goods with reduced added sugar without sacrificing or even improving their quality.

[0006] WO 2019 / 238423 (Novozymes A / S, Denmark) discloses methods for producing dough with reduced added sugars, which include adding raw starch-degrading α-amylase and glucosylase to the dough components. Summary of the Invention

[0007] The inventors discovered that certain thermostable variants of glucosylamylase exhibit significantly improved performance in preserving or preventing aging of baked or partially baked products. Another improvement of these thermostable variants is their ability to increase the sweetness or flavor of products, thereby reducing the amount of added sugar in traditional recipes.

[0008] Therefore, in a first aspect, the present invention relates to a method for producing baked or partially baked products, the method comprising:

[0009] a) Providing dough comprising a mature, thermostable variant of a parental glucosylamylase having at least 70% identity with SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8; and

[0010] b) Bake or partially bake the dough to produce baked or partially baked products.

[0011] A second aspect of the invention relates to baking compositions comprising a mature, thermostable variant of a parental glucosylamylase as defined in the first aspect.

[0012] Other aspects of the invention relate to the use of the baking compositions of the second aspect for: replacing sugar in methods of producing baked or partially baked products, increasing the sweetness of baked or partially baked products, reducing the amount of sugar in dough in methods of producing baked or partially baked products, and / or extending the shelf life of baked or partially baked products in methods of producing baked or partially baked products, and their use in methods as defined in the first aspect, whereby, compared to a control made without the addition of any glucosylamylase, the final fully 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 reduced firmness and / or higher elasticity after 1, 7, or 14 days.

[0013] Preferably, the mature thermostable variant of the parental glucosyl amylase of the present invention has at least 71% identity with SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, and has, for example, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. Attached Figure Description

[0014] Figure 1 Multiple alignments of the amino acid sequences of the following mature proteins are shown:

[0015] - The wild-type AMG (PoAMG) from *Penicillium oxalicum*, SEQ ID NO:1

[0016] -PoAMG variant represented as "AMG NL" of SEQ ID NO:2

[0017] -PoAMG variant represented by SEQ ID NO:3 as "AMG anPAV498"

[0018] -PoAMG variant represented as "AMG JPO001" of SEQ ID NO:4

[0019] -PoAMG variant represented as "AMG JPO124" with SEQ ID NO:5

[0020] -PoAMG variant represented as "AMG JPO172" with SEQ ID NO:6

[0021] -Wild-type AMG (PoAMG) from *Penicillium miczynskii*, SEQ ID NO:7

[0022] - The wild-type AMG (PoAMG) from *Penicillium russellii*, SEQ ID NO:8

[0023] - The wild-type AMG (PoAMG) from *Penicillium glabrum*, SEQ ID NO:9 Detailed Implementation

[0024] definition

[0025] Sequence identity: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0026] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48:443-453) was used to determine sequence identity between two amino acid sequences. This algorithm was implemented using the Needle program within the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16:276-277, preferably version 5.0.0 or later). The parameters used were a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output labeled "longest identity" by Needle (obtained using the -no brief option) was used as the identity percentage and calculated as follows:

[0027] (identical residues x 100) / (alignment length - total number of vacancies in the alignment)

[0028] Variants: The term "variant" refers to a polypeptide containing alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding one or more amino acids adjacent to and immediately following an amino acid occupying a position. Amino acid alterations can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1–30 amino acids; small N-terminal or C-terminal extensions, such as methionine residues at the N-terminus; small linker peptides of up to 20–25 residues; or small extensions that facilitate purification by altering net charge or another function (such as a polyhistidine fragment, an antigenic epitope, or a binding domain). Examples of conserved substitutions are found in the following group: 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 reactivity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in *The Proteins*, Academic Press, New York. Common substitutes 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.

[0029] Increased strength: The term “increased strength of dough” is defined herein as the following characteristics of dough that generally exhibit greater elasticity and / or require more work input for molding and shaping compared to a control.

[0030] Increased elasticity: The term “increased elasticity of dough” is defined in this paper as the following property of dough that, compared with a control, has a greater tendency to recover its original shape after being subjected to a certain physical stress.

[0031] Increased stability of dough: The term “increased stability of dough” is defined in this paper as the characteristic of dough that is less susceptible to mechanical damage than a control, and thus better retains its shape and volume, and is evaluated by the height:width ratio of the bread cross-section after normal and / or extended proofing.

[0032] Reduced stickiness of dough: The term “reduced stickiness of dough” is defined herein as the following characteristic of dough that, for example, has a lower surface area of ​​adhesion compared to a control in a dough production machine, and is assessed by a skilled test baker based on experience or measured using a texture analyzer known in the art (e.g., TAXT2).

[0033] Improved stretchability: The term “improved stretchability of dough” is defined herein as the characteristic of dough that can withstand increased stress or stretching without breaking, compared to a control.

[0034] Improved mechanical properties: The term “improved mechanical properties of dough” is defined herein as the following properties of dough that are generally less sticky and / or more compact and / or more elastic compared to a control.

[0035] Increased volume of baked goods: The term "increased volume of baked goods" refers to the volume of a given breadstick measured as a comparison with a control. Volume can be determined using methods known in the art.

[0036] Improved crumb structure of baked goods: The term “improved crumb structure of baked goods” is defined herein as the following characteristics of baked goods having finer pores and / or thinner pore walls in the crumb compared to a control, and / or a more uniform / uniform pore distribution in the crumb, and which are typically assessed by a skilled baker visually or by digital image analysis known in the art (e.g., C-cell, Calibre Control International Ltd, Appleton, Warrington, UK).

[0037] Improved softness of baked goods: The term “improved softness of baked goods” is the opposite of “firmness” and is defined herein as the characteristic of baked goods that are more easily compressed compared to a control and are measured by a skilled test baker relying on experience or, for example, using a texture analyzer known in the art (e.g., the TAXT2 or TA-XT Plus from Stable Micro Systems Ltd, Surrey, UK).

[0038] Sensory properties of baked goods: Sensory properties can be evaluated using procedures well-established in the baking industry and may include, for example, the use of a group of trained taste testers.

[0039] Improvement in thermal stability: Improvement in thermal stability (Td), expressed in OC units, is a measure of how much the variant has improved in thermal stability relative to its parent glucosylamylase under the same conditions, as illustrated in this article.

[0040] A first aspect of the present invention relates to a method for producing baked or partially baked products, the method comprising:

[0041] a) Providing dough comprising a mature, thermostable variant of a parental glucosylamylase having at least 70% identity with SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8; and

[0042] b) Bake or partially bake the dough to produce baked or partially baked products.

[0043] A second aspect of the invention relates to baking compositions comprising a mature, thermostable variant of a parental glucosylamylase as defined in the first aspect.

[0044] Other aspects of the invention relate to the use of the baking compositions of the second aspect for: replacing sugar in a method of producing a baked or partially baked product, increasing the sweetness of a baked or partially baked product, reducing the amount of sugar in dough in a method of producing a baked or partially baked product, and / or extending the shelf life of a baked or partially baked product in a method of producing a baked or partially baked product, and their use in methods as defined in the first aspect, whereby, compared to a control made without the addition of any glucosylamylase, the final fully 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 reduced firmness and / or higher elasticity after 1, 7, or 14 days.

[0045] Preferably, the mature thermostable variant of the parental glucosyl amylase of the present invention has at least 71% identity with SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, and has, for example, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0046] dough

[0047] As used in this article, "dough" refers to the dough used to prepare baked goods (especially bread).

[0048] According to the present invention, the dough used to prepare baked goods can be made from any suitable dough ingredients containing flour.

[0049] The flour can be derived from any baking grain 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 (e.g., a combination of wheat flour with one of the other flour sources; or a combination of rice flour with one of the other flour sources).

[0050] In a preferred embodiment, the flour is wheat flour.

[0051] 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... The total flour content is at least 55% or more wheat flour, for example, at least 60% or more wheat flour, for example, at least 65% or more wheat flour, for example, at least 70% or more wheat flour, for example, at least 75% or more wheat flour, for example, at least 80% or more wheat flour, for example, at least 85% or more wheat flour, for example, at least 90% or more wheat flour, for example, at least 95% or more wheat flour, for example, at least 100% wheat flour.

[0052] The dough of the present invention is typically fermented dough or dough to be fermented. The dough can be fermented in various ways, such as by adding dough ingredients like chemical leavening agents (e.g., sodium bicarbonate) or by adding a leavening agent (fermented dough), but preferably by adding a suitable yeast culture such as a culture of Saccharomyces cescerevisiae (bread yeast) (e.g., a commercially available Saccharomyces cescerevisiae strain).

[0053] 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 a partial reduction in the amount of sugar can be achieved.

[0054] In one embodiment, the amount of added sugar is reduced by at least 10% (w / w) compared to the amount of sugar added to the dough in the original recipe. For example, the amount of added sugar is reduced by at least 20% (w / w) compared to the amount of sugar added to the dough in the original recipe. For example, the amount of added sugar is reduced by at least 30% (w / w) compared to the amount of sugar added to the dough in the original recipe. For example, the amount of added sugar is reduced by at least 40% (w / w) compared to the amount of sugar added to the dough in the original recipe. For example, the amount of added sugar is reduced by at least 50% (w / w) compared to the amount of sugar added to the dough in the original recipe. For example, the amount of added sugar is reduced by at least 60% (w / w) compared to the amount of sugar added to the dough in the original recipe, for example, the amount of added sugar is reduced by at least 70% (w / w) compared to the amount of sugar added to the dough in the original recipe, for example, the amount of added sugar is reduced by at least 80% (w / w) compared to the amount of sugar added to the dough in the original recipe, for example, the amount of added sugar is reduced by at least 90% (w / w) compared to the amount of sugar added to the dough in the original recipe, for example, the amount of added sugar is reduced by 100% (w / w) compared to the amount of sugar added to the dough in the original recipe.

[0055] Dough can also contain other common dough ingredients, such as proteins like milk powder, gluten, and soy; eggs (whole eggs, yolks, or whites); oxidizing agents like ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA), or ammonium persulfate; amino acids like L-cysteine; salts like sodium chloride, calcium acetate, sodium sulfate, and calcium sulfate; diluents (such as silica); and starches from various sources. Other commonly used ingredients include hydrocolloids such as CMC, guar gum, xanthan gum, and sophora japonica gum.

[0056] Dough ingredients typically include fats (triglycerides) and / or oils and / or shortenings, especially oils such as sunflower oil or rapeseed oil.

[0057] The dough can be made using any conventional mixing process, such as continuous mixing, straight-dough, or sponge and dough.

[0058] The present invention is particularly useful for preparing dough and baked goods in industrial processes, wherein the dough used to prepare the baked goods is prepared mechanically using automated or semi-automated equipment.

[0059] The process of preparing bread typically involves the following sequential steps: making dough, sheeting or dividing the dough, shaping or rolling, and proofing, which are well known in the art.

[0060] As used herein, "baked products" means any type of baked product, including various types of bread such as pan bread, toast, open bread, covered and uncovered pan bread, rolls, Fino bread, Hammam bread, Samoli bread, baguettes, brioche, hamburger buns, rolls, rye bread, whole wheat bread, rich bread, bran bread, flatbread, tortillas, biscuits, and any variety thereof. According to the invention, baked products can also be cakes or any pastry products known in the art.

[0061] α-amylase that degrades raw starch

[0062] As used in this article, "raw starch degrading α-amylase" refers to an enzyme that can directly degrade raw starch granules below the gelatinization temperature of starch.

[0063] Examples of α-amylases that degrade raw starch 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 enzymes disclosed in Tables 1-5 of U.S. Patent No. 7,326,548 and in Table 3 on page 15 of U.S. Patent Publication No. 2005 / 0054071, as well as enzymes disclosed in WO 2004 / 020499, WO 2006 / 06929, and WO 2006 / 066579.

[0064] In one embodiment, the α-amylase that degrades raw starch is GH13_1 amylase.

[0065] In one embodiment, the raw starch-degrading α-amylase has at least 70%, for example, at least 71%, 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% identity with the raw starch-degrading α-amylase shown in European Patent No. 2981170 (Novison).

[0066] In one embodiment, the raw starch-degrading α-amylase according to the present invention may be added to flour or dough at an amount of 0.01-10 mg enzyme protein / kg flour, for example, 0.1-5 mg enzyme protein / kg flour.

[0067] Glucoamylase

[0068] Glucoamylase, also known as amyloglucosidase and glucan 1,4-α-glucosidase (EC 3.2.1.3), is more commonly referred to as AMG.

[0069] According to the present invention, different types of amylases can be used as parents for producing thermostable amylase variants. For example, the amylase can be a polypeptide encoding a DNA sequence found in fungal strains of the genera *Aspergillus*, *Rhizopus*, *Talaromyces*, or *Penicillium*; preferably a DNA sequence found in fungal strains of the genus *Penicillium*, and even more preferably a DNA sequence found in fungal strains of *Penicillium oxysporum*, *Penicillium oxalate*, *Penicillium migrans*, *Penicillium rouelaceum*, or *Penicillium scintillans*. Preferably, the parental glucosylase is derived from a species of the genus *Penicillium*, and more preferably from *Penicillium oxalate*, *Penicillium migrans*, *Penicillium rouelaceum*, or *Penicillium scintillans*.

[0070] Other examples of suitable fungi include Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Rhizopus delemar, Rhizopus niveus, Rhizopus oryzae, and Talaromyces emersonii.

[0071] The following shows Figure 1 The percentage of identity between the AMG amino acid sequences compared in the sequence listing is also provided:

[0072]

[0073]

[0074] In one embodiment, the glucosyl amylase according to the invention can be added to flour or dough at 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.

[0075] Thermostable variants of PoAMG have been produced (see Table 2 below). In a preferred embodiment, the mature thermostable glucosyl amylase variant of the present invention comprises one or more or all combinations of the amino acid substitutions listed in Table 2 below.

[0076] In a preferred embodiment, the mature variant of the present invention comprises at least one amino acid modification at one 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 substitution at one or all of the positions corresponding to positions 1, 2, 4, 11, 65, 79, and 327 in SEQ ID NO:1; or preferably, the at least one amino acid modification comprises substitution at one or all of the 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 substitution at one or all of the positions corresponding to SEQ ID NO:1. In SEQ ID NO:1, positions 1, 6, 7, 31, 34, 79, 103, 132, 445, 447, 481, 566, 568, 594, and 595 contain substitutions. Preferably, the at least one amino acid modification contains substitutions at one or more positions corresponding to R1A, G6S, G7T, R31F, K34Y, K79V, S103N, A132P, D445N, V447S, S481P, D566T, T568V, Q594R, and F595S. Or preferably, the at least one amino acid modification contains substitutions at one or more positions corresponding to SEQ ID NO:1. In SEQ ID NO:1, positions 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 481, 484, 501, 539, 566, 568, 594, and 595 contain substitutions. Preferably, the at least one amino acid modification contains substitutions at one or more 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.

[0077] The thermal stability improvement (Td) of the variants in Table 2 is listed in Table 3, where the Td of the PoAMG variant denoted as "anPAV498" (parent) is set to zero. In a preferred embodiment, the mature thermally stable variants of the present invention have a thermal stability improvement (Td) relative to their parents of at least 3°C, preferably at least 4°C, 5°C, 6°C, 7°C, or 8°C, preferably as exemplified herein.

[0078] In another preferred embodiment, the mature, thermally stable variant of the present 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 OC compared to its parent.

[0079] Preferably, the mature, heat-stable variant of glucosyl amylase is included in the dough at an amount of 0.01-1,000 mg enzyme protein (mgEP) / kg flour, more preferably 0.01-500 mg enzyme protein (mgEP) / kg flour, and even more preferably 0.1-100 mg enzyme protein (mgEP) / kg flour.

[0080] amylase

[0081] α-Amylase (α-1,4-glucan-4-glucan hydrolase, 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.

[0082] Many α-amylases are called Terminyl TM and "Termamyl" TM "α-amylase", and can be found in, for example, WO90 / 11352, WO95 / 10603, WO95 / 26397, WO96 / 23873 and WO96 / 23874.

[0083] Another group of α-amylases is called Fungammyl TM and "Fungamyl" TM "-like α-amylases", which are α-amylases related to the α-amylases derived from Aspergillus oryzae disclosed in WO 01 / 34784.

[0084] Suitable commercially available α-amylase compositions according to the present invention include, for example, BAKEZYME P 300 (available from DSM) and FUNGAMYL 2500SG, FUNGAMYL 4000BG, FUNGAMYL 4000SG, FUNGAMYL 800L, FUNGAMYL ULTRA BG and FUNGAMYL ULTRA SG (available from Novozymes).

[0085] In one embodiment, the α-amylase according to the invention can be added to flour or dough at 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.

[0086] Other enzymes

[0087] Optionally, one or more other enzymes (such as α-amylase, maltodextrinase, β-amylase, aminopeptidase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, 1,4-α-maltotetrasaccharide hydrolase, glucanase, galactanase, α-galactosidase, β-galactosidase, glucose oxidase, α-glucosidase, β-glucosidase, halogenated peroxidase, hemicellulose hydrolase, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinase, peptidase, peptidase, peroxidase, phospholipase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, and xylanase) may be used with the enzyme composition according to the invention.

[0088] One or more additional enzymes can be from any source, including mammalian, plant, and microbial (bacteria, yeast, or fungi) sources.

[0089] Maltose-α-amylase (EC 3.2.1.133) can originate from the genus *Bacillus*. The maltose-α-amylase from *Bacillus stearothermophilus* strain NCIB 11837 is sourced from Novozymes under the trademark name... Available for commercial purchase.

[0090] Maltose-producing α-amylases can also be variants of maltose-producing α-amylases from *Bacillus stearothermophilus*, as disclosed in, for example, WO 1999 / 043794, WO 2006 / 032281 or WO 2008 / 148845. 3D.

[0091] The anti-aging amylase used in this invention can also be an amylase (glucan-1,4-α-maltotetrasaccharide hydrolase (EC 3.2.1.60)) or a variant thereof from Pseudomonassaccharophilia, such as any amylase disclosed in WO 1999 / 050399, WO 2004 / 111217 or WO 2005 / 003339.

[0092] Glucose oxidase can be fungal glucose oxidase, especially Aspergillus niger glucose oxidase (e.g. Available from Novozymes.

[0093] Xylanases can be of microbial origin, such as strains derived from bacteria or fungi such as Aspergillus (especially Aspergillus aculeatus, Aspergillus niger, Aspergillus foetida, or Aspergillus tubigensis), strains derived from Trichoderma (e.g., Trichoderma reesei), or strains derived from Humicola (e.g., Humicola insolens).

[0094] Suitable commercially available xylanase formulations for use in this 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).

[0095] Proteases can be derived from Bacillus species, such as *Bacillus amyloliquefaciens*. Suitable proteases are available from Novozymes.

[0096] Phospholipases may possess phospholipase A1, A2, B, C, D, or lysophospholipase activity; they may or may not have lipase activity. They can be of animal origin, such as from the pancreas, snake venom, or bee venom, or they can be of microbial origin, such as from filamentous fungi, yeasts, or bacteria, such as *Aspergillus* or *Fusarium*, for example, *Aspergillus niger*, *Aspergillus oryzae*, or *F. oxysporum*. Preferred lipases / phospholipases from *F. oxysporum* are disclosed in WO 98 / 26057. Similarly, variants described in WO 00 / 32758 may be used.

[0097] Suitable phospholipase combinations are LIPOPAN F, LIPOPAN XTRA and LIPOPAN MAX (available from Novozymes) or PANAMORE GOLDEN and PANAMORE SPRING (available from DSM).

[0098] Preferably, one or more additional enzymes are added at an amount of 0.01-1,000 mg enzyme protein (mgEP) / kg flour, more preferably 0.01-500 mg enzyme protein (mgEP) / kg flour, and even more preferably 0.1-100 mg enzyme protein (mgEP) / kg flour.

[0099] Enzyme composition

[0100] The mature, heat-stable variant of glucosylamylase of the present invention, as well as any one or more other enzymes, can be added to flour or dough in any suitable form, such as, for example, in liquid (especially stabilized liquid), or can be added to flour or dough as substantially dry powder or granules.

[0101] For example, granules can be produced 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.

[0102] One or more enzymes can be added to bread dough ingredients in any suitable manner, such as as separate components (enzymes added individually or sequentially), or as a combination of enzymes added in one step or in a composition.

[0103] Baking Composition

[0104] The present invention further relates to baking compositions comprising a mature, thermostable variant of a parental glucosylamylase as defined in the first aspect of the invention.

[0105] The baking composition may contain other dough improvers and / or bread improvers, such as any of the additives mentioned above, including enzymes.

[0106] The baking composition may be, for example, a dough composition, a flour composition, a flour premix, or a bread improver.

[0107] Preferably, the baking composition of the present invention further comprises one or more additional enzymes selected from the group consisting of: α-amylase, maltodextrinase, β-amylase, aminopeptidase, carboxypeptidase, catalase, cellulase, chitinase, keratinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, 1,4-α-maltotetrasaccharide hydrolase, glucanase, galactanase, α-galactosidase, β-galactosidase, glucose oxidase, α-glucosidase, β-glucosidase, halogenated peroxidase, hemicellulose hydrolase, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinase, peptidase, peptidase, peroxidase, phospholipase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, and xylanase.

[0108] Preferably, the baking composition of the present invention further comprises flour, sugar, yeast, salt and / or fat.

[0109] It is generally advantageous to provide a mixture of the enzymes used in the processing of the present invention with other ingredients for improving the properties of baked goods. These baking compositions are generally referred to in the art as “premixes” and typically contain flour.

[0110] Therefore, in another aspect, the present invention relates to a bread premix that improves dough quality by reducing the amount of added sugar, the premix containing the enzyme combination of the present invention.

[0111] In one embodiment, the invention further relates to a bread premix comprising the enzyme combination of the invention and flour, such as flour derived from grains, such as wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, or sorghum flour, and combinations thereof.

[0112] In another embodiment, the present invention relates to bread premixes comprising the enzyme combination of the present invention and flour, such as flour derived from grains, such as wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, sorghum, soybean flour, and combinations thereof, as well as one or more additional enzymes, as previously described.

[0113] The premix can be in the form of granules or agglomerated powder, for example, where typically 95% (by weight) of the granules or agglomerated powder has a particle size between 25 and 500 μm.

[0114] Granular and agglomerated powders can be prepared by conventional methods, such as spraying these enzymes onto a carrier in a fluidized bed granulator. The carrier can consist of microparticles with a suitable particle size. The carrier can be soluble or insoluble, such as salts (e.g., NaCl or sodium sulfate), sugars (e.g., sucrose or lactose), sugar alcohols (e.g., sorbitol), starch, rice, corn grits, or soybeans.

[0115] Bread characteristics

[0116] The sensory quality or sensory properties of bread can be measured as is known in the art. The characteristics of bread herein may be referred to as sensory properties, which include anti-staling (crust hardness), crumb characteristics and mouthfeel, or more precisely, such as the properties of bread detected in the mouth during eating (e.g., bread softness / resistance to the first bite, crumb moisture, crumb chewiness and stickiness, and crumb smoothness and melting characteristics).

[0117] In one embodiment, the sensory property obtained by using the enzyme solution according to the invention in a baked product is increased sweetness.

[0118] In one embodiment, the sensory property obtained by using the enzyme solution according to the invention in the baked product is increased sweetness of the pulp.

[0119] In a preferred embodiment of the invention, compared with a control made without the addition of any glucosylamylase, the final fully baked or partially baked products, when cooled to room temperature, packaged in a sealed container, and stored at room temperature until analysis, have reduced initial firmness and / or increased initial elasticity, and / or reduced firmness and / or higher elasticity after 1, 7, or 14 days.

[0120] In another preferred embodiment, the final fully baked or partially baked product has at least the same sweetness or flavor as the control product made with twice the amount of mature glucosylamylase, the amino acid sequence of which is shown in SEQ ID NO:10, preferably as exemplified herein; preferably, the final fully baked or partially baked product has a higher sweetness or flavor than the control product made with twice the amount of mature glucosylamylase, the amino acid sequence of which is shown in SEQ ID NO:10, preferably as exemplified herein.

[0121] The inventions described and claimed herein are not limited to 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.

[0122] This document cites several references, the disclosures of which are incorporated herein by reference in their entirety. The invention is further described through the following examples, which should not be construed as limiting the scope of the invention.

[0123] Example

[0124] Example 1: Building the PoAMG Library

[0125] The PoAMG library is constructed as follows:

[0126] Design forward or reverse primers with NNK or one or more desired mutations at one or more target sites, with these target sites overlapping each other by 15 bp. Perform reverse PCR using appropriate template plasmid DNA (e.g., plasmid DNA containing the JPO-0001 gene) under the following conditions, i.e., amplify the entire plasmid DNA sequence using reverse directional primers. Purify the obtained PCR fragment using the QIAquick gel extraction kit [QIAGEN] and then introduce it into E. coli ECOS competent E. coli DH5α [NIPPON GENE CO.,LTD.]. Extract plasmid DNA from E. coli transformants using the MagExtractor plasmid extraction kit [TOYOBO] and then introduce it into Aspergillus niger competent cells.

[0127] PCR reaction mixture:

[0128] PrimeSTAR Max DNA Polymerase [Takara Bio Inc. (TaKaRa)]

[0129] Total 25μl

[0130] 1.0 μl template DNA (1 ng / μl)

[0131] 9.5 μl H2O

[0132] 12.5 μl 2x PrimeSTAR Max premix

[0133] 1.0 μl forward primer (5 μM)

[0134] 1.0 μl reverse primer (5 μM)

[0135] PCR procedure:

[0136] 98℃ / 2min

[0137] 25x(98℃ / 10sec, 60℃ / 15sec, 72℃ / 2min)

[0138] 10℃ / Keep

[0139] Example 2: Screening for better thermal stability

[0140] The Bacillus subtilis library constructed as in Example 1 was fermented for 3 days at 32°C in 96-well or 24-well MTP culture media 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 salt, 10 ml / L 1M acetamide). Then, the AMG activity in the culture supernatant was measured at several temperatures using a pNPG assay as described below.

[0141] pNPG thermal stability determination:

[0142] Mix the culture supernatant containing the desired enzyme with an equal volume of pH 5.0 200 mM NaOAc buffer. Dispense 20 μL of this mixture into 96-well plates or 8-tube PCR systems and incubate at various temperatures for 30 min using a thermal cycler. Mix these samples with 10 μL of substrate solution containing 0.1% (w / v) pNPG [Wako Co., Ltd.] in pH 5.0 200 mM NaOAc buffer and incubate at 70 °C for 20 min to initiate the enzyme reaction. After the reaction, add 60 μL of 0.1 M borax buffer to stop the reaction. Collect 80 μL of the reaction supernatant and read its OD405 value using a photometer to assess enzyme activity.

[0143] Table 1a. List of relative activities of PoAMG variants compared to their parents anPAV498 or JPO-0001 (anPAV498 with a lead peptide / propeptide).

[0144]

[0145]

[0146] name Relative activity (%) at 80℃ / 75℃ AnPav498 13％ JPO-009 16％ JPO-011 15％ JPO-012 15％ JPO-013 17％ JPO-020 20％

[0147] name Relative activity (%) at 80℃ / 70℃ JPO-001 10％ JPO-004 29％ JPO-009 13％ JPO-014 21％ JPO-020 16％ JPO-021 30％ JPO-052 33％

[0148] 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％

[0149] 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％

[0150] name Relative activity (%) at 79℃ / 77℃ JPO-001 36％ JPO-029 51％ JPO-047 45％ JPO-048 81％ JPO-049 53％ JPO-050 58％ JPO-064 65％

[0151]

[0152]

[0153] Table 1b. List of relative activities of PoAMG variants compared to their parent JPO-022

[0154] name Relative activity (%) at 77℃ / 70℃ JPO-022 60％ JPO-027 67％ JPO-042 8％ JPO-044 86％ JPO-045 67％ JPO-046 48％

[0155] 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％

[0156]

[0157]

[0158] Table 1c. List of relative activities of PoAMG variants compared to their parent JPO-063

[0159] name Relative activity (%) at 79℃ / 77℃ JPO-063 91％ JPO-066 96％ JPO-071 89％ JPO-072 84％ JPO-074 103％ JPO-075 86％ JPO-076 92％ JPO-077 95％ JPO-078 88％ JPO-079 100％

[0160]

[0161]

[0162] name Relative activity (%) at 82℃ / 70℃ JPO-063 21％ JPO-093 43％ JPO-081 25％ JPO-088 39％ JPO-094 38％ JPO-096 38％ JPO-106 53％

[0163] name Relative activity (%) at 83℃ / 80℃ JPO-063 46％ JPO-051 44％ JPO-096 64％ JPO-106 88％ JPO-110 81％ JPO-111 100％ JPO-112 86％ JPO-113 83％ JPO-114 47％ JPO-115 90％

[0164] Table 1d. List of relative activities of PoAMG variants compared to their parent JPO-096

[0165]

[0166]

[0167] name Relative activity (%) at 83℃ / 80℃ JPO-051 20％ JPO-096 43％ JPO-109 51％ JPO-126 33％ JPO-129 48％ JPO-130 18％ JPO-131 51％ JPO-132 34％

[0168] Table 1e. List of relative activities of PoAMG variants compared to their parent JPO-129

[0169] name Relative activity (%) at 84℃ / 80℃ JPO-129 62％ JPO-156 51％ JPO-160 34％ JPO-161 41％ JPO-162 49％ JPO-163 21％ JPO-164 57％ JPO-165 77％ Table 1f. List of relative activities of PoAMG variants compared to their parent JPO-166

[0170] name Relative activity (%) at 84℃ / 75℃ JPO-166 19％ JPO-167 66％ JPO-168 58％ JPO-169 53％ JPO-171 47％ JPO-172 98％

[0171] Table 2. Amino acid substitutions in variants of the mature PoAMG sequence

[0172]

[0173]

[0174]

[0175]

[0176]

[0177] Example 3: Fermentation of Aspergillus niger

[0178] The Aspergillus niger strain was fermented on a rotary shaker in a 500 ml baffled flask containing 100 ml MU1 and 4 ml 50% urea at 220 rpm and 30 °C. The culture was centrifuged (10,000 x g, 20 min) and the supernatant was carefully decanted from the precipitate.

[0179] Example 4: Purification of the PoAMG (JPO-001) variant

[0180] The PoAMG variant was purified by cation exchange chromatography. The individual peak fractions were combined separately and dialyzed against 20 mM sodium acetate buffer (pH 5.0), followed by concentration using a centrifugal filter (Vivaspin Turbo 15, Sartorius). Enzyme concentration was determined by A280 value.

[0181] Example 5: Thermal Stability Measurement (TSA)

[0182] 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 was transferred to a LightCycler 480 multi-well plate (Roche Diagnostics) and the plate was sealed. TSA equipment parameters:

[0183] Instrument: LightCycler 480 real-time PCR system (Roche Applied Science)

[0184] Scan rate: 0.02℃ / sec

[0185] Scanning range: 37℃-96℃

[0186] Integrating time: 1.0 sec

[0187] Excitation wavelength 465nm

[0188] Emission wavelength 580nm

[0189] The obtained fluorescence signals were normalized to the range of 0 and 1. Td was defined as the temperature at which the signal intensity was 0.5. The improvements in thermal stability are listed in Table 3, where Td for the PoAMG variant is represented as 0 for anPAV498.

[0190] Example 6: PoAMG Activity Assay

[0191] Determination of maltodextrin (DE11) by the GOD-POD method

[0192] substrate solution

[0193] 30g maltodextrin (pindex #2 from Matsutani Chemical Industry Co., Ltd.)

[0194] 100 ml 120 mM sodium acetate buffer, pH 5.0

[0195] Glucose CII test kit (Wako Pure Chemical Industries, Ltd., Japan)

[0196] Mix 20 μl of enzyme sample with 100 μl of substrate solution and incubate at the set temperature for 2 h. Cool the sample on an aluminum block for 3 min, then mix 10 μl of reaction solution with 590 μl of 1M Tris-HCl (pH 8.0) to stop the reaction. Mix 10 μl of the solution with 200 μl of the working solution of the test kit and let stand at room temperature for 15 min. Read the absorbance at A505. The activities are listed in Table 3 as relative activities of the PoAMG variant denoted as anPAV498.

[0197] Table 3

[0198]

[0199]

[0200]

[0201]

[0202] Example 7: The Preservative Effect of AMG on Bread (Part 1)

[0203] Bake bread using the direct fermentation process according to the recipe in Table 4. Bake the bread in a covered baking pan to ensure all loaves are of uniform volume. Combine the ingredients in a spiral mixer at 17 rpm and 35 rpm for 3+7 minutes to form a dough. Divide the dough into 450g portions, round them, flatten them, and place them in a baking pan. Proof the dough in the baking pan at 32°C and 86% relative humidity for 55 minutes. Bake the proofed dough in a box oven at 230°C for 35 minutes.

[0204] Table 4.

[0205]

[0206] Table 5. Seven dough treatments prepared using different enzyme additions according to Table 4; AMG 3300BG It is commercially available AMG (Novozymes, Denmark) for baking; AMG NL and AMG anPAV498 are artificial variants of PoAMG (see Table 2).

[0207]

[0208] The dough was baked, and the resulting bread was packaged in a sealed plastic bag 2 hours after baking and stored at room temperature until analysis.

[0209] The texture of each loaf was assessed using a texture analyzer (TA-XT plus, Stable Microsystems, Godalmine, UK). Bread 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 performed using a 40mm diameter cylindrical probe. When a 25mm thick slice of bread is pressed down with the cylindrical probe at a deformation rate of 1mm / s to 40% stress, the force on the cylindrical probe is recorded. The probe is then held in this position for 30 seconds while the force is recorded, and then the probe is returned to its initial position.

[0210] Toughness (in grams) is defined as the force required to compress the probe to 25% stress (corresponding to compression of 6.25 mm into a slice of bread crumb with a thickness of 25 mm).

[0211] Elasticity (in %) is defined as the force recorded after compression for 30 seconds under 40% stress (corresponding to the force of a 25mm thick bread slice at time = 40s) divided by the force required to press the probe into the crumb by 10mm (corresponding to the force of a 25mm thick bread slice at time = 10s) multiplied by 100.

[0212] The results of the texture analysis are shown in Table 6 (Durability) and Table 7 (Elasticity).

[0213] Fresh bread without enzymes (control) has low firmness and high elasticity; as the bread is stored, firmness decreases over time while elasticity decreases. Conventional AMGs used in baking applications (such as Goldcrust) do not affect firmness or elasticity.

[0214] AMG anPAV498 added at 25 or 50 mg EP / kg and AMG NL added at 50 mg EP / kg flour improve (reduce) initial firmness and reduce the increase in firmness over time. AMG anPAV498 added at 25 or 50 mg EP / kg and AMG NL added at 50 mg EP / kg flour improve (increase) initial elasticity and prevent loss of elasticity over time.

[0215] Table 6. Clarity (g) of bread treated with enzymes according to Table 5 on days 1, 3, and 7.

[0216]

[0217] Table 7. Elasticity (g) of bread treated with enzymes according to Table 5 on days 1, 3, and 7.

[0218]

[0219] Sugars were extracted from bread crumbs using 0.1 M phosphate buffer (pH 8.0) in 70% EtOH. Bread crumbs (180 mg) were added to the extraction buffer (1.8 mL) and incubated at 70 °C for 20 min during mixing. The bread crumbs were centrifuged at 12,000 rpm for 5 min, and 500 μL of the supernatant was collected and diluted 200-fold with 20 mM phosphate buffer (pH 8.0) + 10 mg / L cellobiose as an internal standard. Sugars (glucose, fructose, maltose, and maltotriose) were extracted and quantified using an ICS-5000 HPLC system with a CarboPac PA1 column. Theoretical sweetness was calculated using sweetness intensity factors based on the levels of glucose, fructose, and maltose. The sweetness factors in Table 8 are based on determinations in Portmann MO, Birch GJ Sci Food Agric [Food and Agricultural Science Journal] 69(3):275-81, 1995.

[0220] Table 8.

[0221]

[0222] The amounts of simple sugars (glucose, fructose, maltose, and maltotriose) and the theoretical sweetness calculated based on the amount of each sugar are shown in Table 9. All three AMGs have increased amounts of simple sugars. In comparison, both AMG NL and AMGanPAV498 are more efficient at producing glucose, resulting in a higher theoretical sweetness.

[0223] Table 9. Amount of sugar extracted from dough treated with the enzymes according to Table 5 (g / kg bread crumb)

[0224]

[0225] Example 8. The Preservation Effect of AMG (Part 2)

[0226] Bake bread using the direct fermentation process according to the recipe in Table 10. Bake the bread in a covered baking pan to ensure all loaves are of uniform volume. Combine the ingredients in a spiral mixer at 17 rpm and 35 rpm for 3+7 minutes to form a dough. Divide the dough into 450g portions, round them, flatten them, and place them in a baking pan. Proof the dough in the baking pan at 32°C and 86% relative humidity for 55 minutes. Bake the proofed dough in a box oven at 230°C for 35 minutes.

[0227] Table 10

[0228]

[0229]

[0230] Table 11. Preparation of seven dough treatments using different enzyme additions

[0231]

[0232] The dough was baked, and the resulting bread was packaged in a sealed plastic bag 2 hours after baking and stored at room temperature until analysis.

[0233] The texture of each loaf was assessed using a texture analyzer (TA-XT plus, Stable Microsystems, Godellmin, UK). Bread crumb texture characteristics are characterized by the firmness (the same as "hardness" and the opposite of "softness") and elasticity of the baked product.

[0234] The standard method for measuring firmness and elasticity is based on force-deformation of baked goods. Force-deformation of baked goods can be performed using a 40mm diameter cylindrical probe. When the cylindrical probe is applied to a 25mm thick slice of bread at a deformation rate of 1mm / s with 40% stress, the force on the probe is recorded. The probe is then held in this position for 30 seconds while the force is recorded, and then the probe is returned to its initial position.

[0235] Toughness (in grams) is defined as the force required to compress the probe to 25% stress (corresponding to compression of 6.25 mm into a slice of bread crumb with a thickness of 25 mm).

[0236] Elasticity (in %) is defined as the force recorded after compression for 30 seconds under 40% stress (corresponding to the force of a 25mm thick bread slice at time = 40s) divided by the force required to press the probe into the crumb by 10mm (corresponding to the force of a 25mm thick bread slice at time = 10s) multiplied by 100.

[0237] The results of the texture analysis are shown in Table 12 (Durability) and Table 13 (Elasticity).

[0238] Fresh bread without enzymes (control) has low firmness and high elasticity; as the bread is stored, firmness decreases over time while elasticity decreases. Conventional AMGs used in baking applications (such as Goldcrust) do not affect firmness or elasticity (Example 7).

[0239] All three AMGs (AMG anPAV498, JPO124, and JPO172) added at 25 or 50 mg EP / kg improve (reduce) initial firmness and reduce the increase in firmness over time. All three AMGs (AMG anPAV498, JPO124, and JPO172) added at 25 or 50 mg EP / kg improve (increase) initial elasticity and prevent loss of elasticity over time.

[0240] Table 12. Crust (g) of bread with enzymes treated according to Table 11 on days 1, 3 and 7.

[0241]

[0242] Table 13. Elasticity (%) of bread with enzyme treatment according to Table 11 on days 1, 3 and 7.

[0243]

[0244] Sugars were extracted from bread crumbs using 0.1 M phosphate buffer (pH 8.0) in 70% EtOH. Bread crumbs (180 mg) were added to the extraction buffer (1.8 mL) and incubated at 70 °C for 20 min during mixing. The bread crumbs were centrifuged at 12,000 rpm for 5 min, and 500 μL of the supernatant was collected and diluted 200-fold with 20 mM phosphate buffer (pH 8.0) + 10 mg / L cellobiose as an internal standard. Sugars (glucose, fructose, maltose, and maltotriose) were extracted quantitatively using an ICS-5000 HPLC system with a CarboPac PA1 column. Theoretical sweetness was calculated using sweetness intensity factors based on the levels of glucose, fructose, and maltose. Sweetness factors are listed in Table 13.

[0245] Determination based on Portmann MO, Birch GJ Sci Food Agric [Food and Agricultural Science Journal] 69(3):275-81, 1995.

[0246] Table 14.

[0247]

[0248] The amounts of simple sugars (glucose, fructose, maltose, and maltotriose) and the theoretical sweetness calculated based on the amount of each sugar are shown in Table 1. All three AMGs increased the amount of simple sugars and increased the calculated sweetness. The higher the dosage of the AMG, the more glucose was produced, and the higher the theoretical sweetness. JPO0172 and JPO124 were more effective than AMG anPAV498 in increasing glucose and theoretical sweetness.

[0249] Table 15. Simple sugars extracted from bread treated with the enzymes according to Table 11 (g / kg bread crumb).

[0250]

[0251]

[0252] The changes in sugar levels in bread crumbs as a function of bread storage time at ambient temperature are shown in Tables 16-20. Glucose levels in AMG products (Table 16) remained stable over bread storage time. The same pattern was observed for other confectionery sugars extracted from bread crumbs (Table 17), maltose (Table 18), maltotriose (Table 19), and maltotetraose (Table 20). Table 16. Changes in glucose levels (g / kg bread crumbs) in bread crumbs over time as a function of enzyme treatment.

[0253]

[0254] Table 17. Maltose levels in bread crumbs (g / kg bread crumbs) over time as a function of enzyme treatment.

[0255]

[0256]

[0257] Table 18. Changes in fructose levels (g / kg bread crumb) over time as a function of enzyme treatment.

[0258]

[0259] Table 19. Changes in maltotriose levels (g / kg bread crumb) in bread crumb as a function of enzyme treatment over time.

[0260]

[0261]

[0262] Table 20. Changes in maltotetraose levels (g / kg bread crumb) in bread crumb as a function of enzyme treatment over time.

[0263]

[0264] Example 9. The preservation effect of combining AMG and Novamyl

[0265] Bake bread using the direct fermentation process according to the recipe in Table 21. Bake the bread in a covered baking pan to ensure all loaves are of uniform volume. Combine the ingredients in a spiral mixer at 17 rpm and 35 rpm for 3+7 minutes to form a dough. Divide the dough into 450g portions, round them, flatten them, and place them in a baking pan. Proof the dough in the baking pan at 32°C and 86% relative humidity for 55 minutes. Bake the proofed dough in a box oven at 230°C for 35 minutes.

[0266] Table 21.

[0267]

[0268] Table 22. Preparation of seven treatments using different enzyme additions

[0269]

[0270] The bread was packaged in a sealed plastic bag 2 hours after baking and stored at room temperature until analysis.

[0271] The texture of each loaf of bread was assessed using a texture analyzer (TA-XT plus, Stable Microsystems, Godellmin, UK). Bread crumb texture characteristics are characterized by the firmness (the same as "hardness" and opposite to "softness") and elasticity of the baked goods. The standard method for measuring firmness and elasticity is based on the force-deformation of the baked goods. The force-deformation of the baked goods can be performed using a cylindrical probe with a diameter of 40 mm. When the cylindrical probe is applied to a 25 mm thick slice of bread at a deformation rate of 1 mm / s with 40% stress, the force on the cylindrical probe is recorded. The probe is then held in this position for 30 seconds while the force is recorded, and then the probe is returned to its initial position.

[0272] Toughness (in grams) is defined as the force required to compress the probe to 25% stress (corresponding to compression of 6.25 mm into a slice of bread crumb with a thickness of 25 mm).

[0273] Elasticity (in %) is defined as the force recorded after compression for 30 seconds under 40% stress (corresponding to the force of a 25mm thick bread slice at time = 40s) divided by the force required to press the probe into the crumb by 10mm (corresponding to the force of a 25mm thick bread slice at time = 10s) multiplied by 100.

[0274] The results of the texture analysis are shown in Tables 23 (firmness) and 24 (elasticity). Fresh bread without enzymes (control) had low firmness and high elasticity; as the bread was stored, firmness decreased over time while elasticity decreased.

[0275] AMG anPAV498 improves initial firmness and elasticity, and reduces changes in firmness and elasticity over time.

[0276] 3D does not affect initial firmness or elasticity. However, 3D reduces changes in firmness and elasticity over time.

[0277] With no enzyme or used alone Compared to 3D, the AMG anPAV498 and The 3D combination improves initial firmness and elasticity, as well as how firmness and elasticity change over time. This combination allows bread to maintain optimal firmness and elasticity after 7 days of storage.

[0278] Table 23. Crust (g) of bread with enzymes treated according to Table 22 on days 1, 3 and 7.

[0279]

[0280]

[0281] Table 24. Elasticity (%) of bread with enzyme treatment according to Table 22 on days 1, 3 and 7.

[0282]

[0283] Sugars were extracted from bread crumbs using 0.1 M phosphate buffer (pH 8.0) in 70% EtOH. Bread crumbs (180 mg) were added to the extraction buffer (1.8 mL) and incubated at 70 °C for 20 min during mixing. The bread crumbs were centrifuged at 12,000 rpm for 5 min, and 500 μL of the supernatant was collected and diluted 200-fold with 20 mM phosphate buffer (pH 8.0) + 10 mg / L cellobiose as an internal standard. Extracted sugars (glucose, fructose, maltose, and maltotriose) were quantified using an ICS-5000 HPLC system with a CarboPac PA1 column. Theoretical sweetness was calculated using sweetness intensity factors based on the levels of glucose, fructose, and maltose. The sweetness factors in Table 25 are based on determinations in Portmann MO, Birch GJ Sci Food Agric [Food and Agricultural Science Journal] 69(3):275-81, 1995.

[0284] Table 25.

[0285]

[0286]

[0287] The amounts of different sugars extracted from bread and the theoretically calculated sweetness based on the sugar content are shown in Table 26. The higher the dose of AMGanPAV498, the more glucose is present in the bread. The higher the dosage of 3D, the more maltose and maltotriose are in the dough. AMG anPAV498 and The 3D combination increases glucose, maltose, and maltotriose. The main factor in calculating sweetness is the dosage of AMG anPAV498, because glucose has a greater impact on sweetness than maltose and maltotriose.

[0288] Table 26. Simple sugars extracted from bread treated with enzymes according to Table 22 (g / kg bread crumb).

[0289]

[0290] Example 10. Dose-response of AMG NL (partial sugar substitution)

[0291] Bake bread using the direct fermentation process according to the recipe in Table 27. Mix the ingredients in a spiral mixer at 17 rpm and 35 rpm for 3+7 minutes to form a dough. Divide the dough into 450g portions, round them, flatten them, and place them in a baking pan. Proof the dough in the pan at 32°C and 86% relative humidity for 55 minutes. Bake the proofed dough in a box oven at 230°C for 25 minutes.

[0292] Table 27. 4000SG is a commercially available fungal amylase for baking (Novozymes, Denmark). BG is a commercially available bacterial xylanase for baking (Novozymes, Denmark).

[0293]

[0294] Table 28. Preparation of eight treatments using different enzyme additions, AMG AMG (Novozymes, Denmark) is a commercially available product for baking, while JA126 is a starch-degrading amylase (Novozymes, Denmark).

[0295]

[0296] Dough characteristics were assessed by trained bakers, and bread volume was measured using a Volscan profiler (Stable Microsystems, Godellmin, UK). The assessment results are shown in Table 29. Doughs containing 44.6 and 53.6 mg EP / kg flour (doughs 5 and 6) of AMG NL had similar characteristics to those containing 112.5 mg EP / kg flour of AMG NL. The dough has a similar volume, the same stretchability and elasticity, but the dough is slightly less sticky and soft.

[0297] Table 29.

[0298] dough 1 2 3 4 5 6 7 8 viscosity 5 4 4 4 4 4 4 4 Softness 5 4 4 4 4 4 4 4 Stretchability 5 3 3 3 5 5 5 5 elasticity 5 6 6 6 5 5 5 5 Specific volume (ml / g) 4.4 4.3 4.2 4.2 4.3 4.2 4.1 4.1

[0299] Sugars were extracted from bread crumbs using 0.1 M phosphate buffer (pH 8.0) in 70% EtOH. Bread crumbs (180 mg) were added to the extraction buffer (1.8 mL) and incubated at 70 °C for 20 min during mixing. The bread crumbs were centrifuged at 12,000 rpm for 5 min, and 500 μL of the supernatant was collected and diluted 200-fold with 20 mM phosphate buffer (pH 8.0) + 10 mg / L cellobiose as an internal standard. Extracted sugars (glucose, fructose, maltose, and maltotriose) were quantified using an ICS-5000 HPLC system with a CarboPac PA1 column. Theoretical sweetness was calculated using sweetness intensity factors based on the levels of glucose, fructose, and maltose. The sweetness factors in Table 30 are based on determinations in Portmann MO, Birch GJ Sci Food Agric [Food and Agricultural Science Journal] 69(3):275-81, 1995.

[0300] Table 30.

[0301]

[0302] The sugar content (g / kg bread crumb) and theoretical sweetness are shown in Table 31. Based on these sugar levels, it can be calculated that the dough containing 44.6 mg EP / kg flour of AMG NL is superior to that containing 112.5 mg EP / kg flour of AMG NL. The dough has a higher theoretical sweetness and contains 53.6 mg EP / kg flour. The dough of AMG NL contains 112.5 mg EP / kg flour. The dough produces more glucose.

[0303] Table 31.

[0304]

[0305] Example 11. Dose-response relationship of AMG anPAV498 (partial sugar substitution)

[0306] Bake bread using the direct fermentation process according to the recipe in Table 32. Mix the ingredients in a spiral mixer at 17 rpm and 35 rpm for 3+7 minutes to form a dough. Divide the dough into 450g portions, round them, flatten them, and place them in a baking pan. Proof the dough in the pan at 32°C and 86% relative humidity for 55 minutes. Bake the proofed dough in a box oven at 230°C for 25 minutes.

[0307] Table 32.

[0308]

[0309]

[0310] Table 33. Preparation of eight treatments using different enzyme additions

[0311]

[0312] The dough properties were evaluated by trained bakers, and the volume of the bread was measured using a food volume analyzer (Stable Microsystems, Goldrmyn, UK). The evaluation results are shown in Table 34. All doughs exhibited similar dough properties and produced breads with similar volumes.

[0313] Table 34.

[0314] dough 1 2 3 4 5 6 7 8 viscosity 5 5 5 5 5 5 5 5 Softness 5 5 5 5 6 6 6 6 Stretchability 5 5 5 6 6 6 6 6 elasticity 5 6 6 5 5 5 5 5 Average specific volume (ml / g) 4.3 4.4 4.3 4.3 4.3 4.2 4.2 4.2 Specific volume index % 100 101 100 99 100 97 98 96

[0315] Sugars were extracted from bread crumbs using 0.1 M phosphate buffer (pH 8.0) in 70% EtOH. Bread crumbs (180 mg) were added to the extraction buffer (1.8 mL) and incubated at 70 °C for 20 min during mixing. The bread crumbs were centrifuged at 12,000 rpm for 5 min, and 500 μL of the supernatant was collected and diluted 200-fold with 20 mM phosphate buffer (pH 8.0) + 10 mg / L cellobiose as an internal standard. Extracted sugars (glucose, fructose, maltose, and maltotriose) were quantified using an ICS-5000 HPLC system with a CarboPac PA1 column. Theoretical sweetness was calculated using sweetness intensity factors based on the levels of glucose, fructose, and maltose. The sweetness factors in Table 35 are based on determinations in Portmann MO, Birch GJ Sci Food Agric [Food and Agricultural Science Journal] 69(3):275-81, 1995.

[0316] Table 35.

[0317]

[0318] The sugar content (g / kg bread crumb) and theoretical sweetness are shown in Table 36. Based on these sugar levels, it can be calculated that dough containing AMG anPAV498 with 24.1 mg EP / kg flour is superior to dough containing 112.5 mg EP / kg flour. The dough produced a higher theoretical sweetness, and the dough containing 27.1 mg EP / kg flour (AMG anPAV498) was superior to the dough containing 112.5 mg EP / kg flour. The dough produces more glucose.

[0319] Table 36.

[0320] Example 12. Sensory comparison of sweetness with AMG NL and AMG anPAV498 (partial sugar substitution)

[0321] Bake bread using the direct fermentation process according to the recipe in Table 37. Mix the ingredients in a spiral mixer at 17 rpm and 35 rpm for 3+8 minutes to form a dough. Divide the dough into 350g pieces, round them, flatten them, and place them in a baking pan. Proof the dough in the pan at 35°C and 85% relative humidity for 85 and 115 minutes, respectively. Bake the proofed dough in a box oven at 230°C for 25 minutes.

[0322] Table 37.

[0323] Baked goods % flour 100 water 57 fresh yeast 3 Salt 1 sugar 5 ascorbic acid 0.06 Fungamyl 4000SG 7ppm Panzea BG 25ppm

[0324] Table 38. Preparation of three treatments using different enzyme additions

[0325]

[0326] Table 39. Dough Properties

[0327] dough 1 2 3 Average specific volume (ml / g) 7.1 7.0 7.2 Specific volume index % 100 99 102

[0328] Sugars were extracted from bread crumbs using 0.1 M phosphate buffer (pH 8.0) in 70% EtOH. Bread crumbs (180 mg) were added to the extraction buffer (1.8 mL) and incubated at 70 °C for 20 min during mixing. The bread crumbs were centrifuged at 12,000 rpm for 5 min, and 500 μL of the supernatant was collected and diluted 200-fold with 20 mM phosphate buffer (pH 8.0) + 10 mg / L cellobiose as an internal standard. Extracted sugars (glucose, fructose, maltose, and maltotriose) were quantified using an ICS-5000 HPLC system with a CarboPac PA1 column. Theoretical sweetness was calculated using sweetness intensity factors based on the levels of glucose, fructose, and maltose. The sweetness factors in Table 40 are based on determinations in Portmann MO, Birch GJ Sci Food Agric [Food and Agricultural Science Journal] 69(3):275-81, 1995.

[0329] Table 40.

[0330]

[0331] The amount of sugar (mg / g bread crumb) and its theoretical sweetness can be found in Tables 41 and 316. (AMG) They produce more glucose, while AMG NL and AMG anPAV498 have higher maltose levels. However, the calculated sweetness of AMG NL at 52.2 mg EP / kg flour and AMG anPAV498 at 23.6 mg EP / kg flour is actually much higher than that of a dose of 124.3 mg EP / kg flour. The sweetness is similar (Table 32).

[0332] Table 41.

[0333] dough 1 2 3 Glucose, mg / g 18.9 17.7 15.9 Fructose, mg / g 8.8 8.3 7.9 Maltose, mg / g 4.8 8.6 11.5 Maltotriose, mg / g M M M Sweetness calculation* 19.3 18.9 18.2

[0334] Sensory evaluation methods

[0335] Two slices of each bread type were provided to each sensory evaluator (Day 1). Samples were provided blinded, coded with 3 digits, and in a randomized order. Seven evaluators participated in the evaluation. The intensity of the sweetness of the bread crumb was assessed on an intensity scale of 1-9, from weak to very strong.

[0336] There were no significant differences in sweetness between the samples, nor were any other significant differences found between the samples.

[0337] Table 42.

[0338] dough 1 2 3 sweet 5.6 6.0 5.6

[0339] Example 13. Sensory evaluation of full sugar substitution

[0340] Toast bread (molded bread, open-faced) - no added sucrose in the dough

[0341] Table 43. Formulation, % (w / w):

[0342]

[0343] *) Enzyme solution:

[0344] Control (= without starch-degrading enzymes and glucoamylase)

[0345] Enzyme solution A: 0.35 mg raw starch-degrading α-amylase (JA126) protein / kg flour and 112.5 mg Glucoamylase protein / kg flour

[0346] Enzyme solution B: 0.35 mg raw starch-degrading α-amylase (JA126) protein / kg flour, and 53.6 mg AMG NL glucosylamylase protein / kg flour.

[0347] Enzyme solution C: 0.35 mg raw starch-degrading α-amylase (JA126) protein / kg flour, and 21.1 mg AMGanPAV498 glucoamylase protein / kg flour.

[0348] Table 44. Baking Procedure

[0349] program Time, min Low / high speed (17rpm / 35rpm) mix 3 / 7 Temperature after mixing, ℃ 25.3-26.2 Floor time 20 Weigh 320g into a baking pan. 10 Bench time (time spent sitting on the workbench) 15 Fermentation time at 32℃, min 80 Baking at 230℃ 25

[0350] Sensory evaluation methods

[0351] Two slices of each bread type were provided to each evaluator (Day 1). Samples were provided blinded, with 3-digit codes, and in a randomized order. Moisture and softness were assessed manually, and sweetness was assessed by tasting the crumb. Sensory attributes were evaluated on an intensity scale of 1-9, from weak to very strong. Four trained evaluators participated in the evaluation. Two replicate sensory assessments were conducted.

[0352] result:

[0353] The doughs have the same stickiness and softness. AMG-NL provides doughs with better stretch and less elasticity (Table 45).

[0354] Table 45. Dough Parameters

[0355] Comparison A B C viscosity 5 6 6 6 Softness 5 5 5 5 Stretchability 5 5 6 5 elasticity 5 5 4 5

[0356] The data shown in Table 45 indicate that solution C provided the moistest and softest bread, while there was no significant difference in sweetness. No other differences were found between the samples. There were no significant differences in bread specific volume (Table 46).

[0357] Table 46. Specific volume index, %

[0358] Comparison A B C 100 101 102 103

[0359] Table 47. Average sensory scores of enzyme bread 1 day after baking.

[0360] Sensory attributes A B C p-value Humidity 5.1AB 5.0B 5.8B 0.0338 Softness 5.1B 5.1B 6.6A 0.002 sweet 4.0 3.6 4.0 0.7998

[0361] Tukey HSD: Mean values ​​(followed by different letters) within sensory attributes showed significant differences between samples (P<0.05).

[0362] Sugars were extracted from bread crumbs using 0.1 M phosphate buffer (pH 8.0) in 70% EtOH. Bread crumbs (180 mg) were added to the extraction buffer (1.8 mL) and incubated at 70 °C for 20 min during mixing. The bread crumbs were centrifuged at 12,000 rpm for 5 min, and 500 μL of the supernatant was collected and diluted 200-fold with 20 mM phosphate buffer (pH 8.0) + 10 mg / L cellobiose as an internal standard. Sugars (glucose, fructose, maltose, and maltotriose) were quantified using an ICS-5000 HPLC system equipped with a CarboPac PA1 column.

[0363] The glucose levels in breads using B and C were slightly higher than those using A (Table 48), meaning that B and C were sweeter than control A. Breads using B and C also had higher maltose levels than those using A.

[0364] Table 48. Sugar levels in bread (mg / g bread crumb)

[0365]

[0366]

[0367] Example 14. The preservation effect of AMG in American sponge fermentation formula

[0368] Bake bread using the sponge fermentation process according to the recipe in Table 49. Bake the bread in a covered baking pan to ensure all loaves are of equal volume. Combine the sponge ingredients in a needle mixer at 50 rpm and 150 rpm for 2+1 minutes to form a dough. Proof the sponge at 27°C and 75% RH for 2 hours. Combine the sponge with the remaining dough ingredients in a needle mixer and combine at 50 rpm and 150 rpm for 1+3 minutes to form a dough.

[0369] Divide the dough into 400g pieces, round them, flatten them, and place them in a covered baking pan. Let the pan containing the dough rise for 60 minutes at 43°C and 80% relative humidity. Bake the risen dough in a revolving oven at 215°C for 20 minutes.

[0370] Table 49. Formula

[0371]

[0372]

[0373] Table 50. Preparation of seven treatments using different enzyme additions

[0374]

[0375] Two hours after baking, the bread was packaged in sealed plastic bags and stored at room temperature until analysis. The texture of the bread was assessed using a texture analyzer (TA-XT plus, Stable Microsystems, Godellmin, UK). Bread 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 performed using a 40 mm diameter cylindrical probe. When the cylindrical probe is applied to a 25 mm thick slice of bread at a deformation rate of 1 mm / s with 40% stress, the force on the cylindrical probe is recorded. The probe is then held in this position for 30 seconds while the force is recorded, and then the probe is returned to its initial position.

[0376] Toughness (in grams) is defined as the force required to compress the probe to 25% stress (corresponding to compression of 6.25 mm into a slice of bread crumb with a thickness of 25 mm).

[0377] Elasticity (in percentage) was defined as the force recorded after compression for 30 seconds under 40% stress (corresponding to the force on a 25 mm thick bread slice at time = 40 s) divided by the force required to press the probe into the crumb by 10 mm (corresponding to the force on a 25 mm thick bread slice at time = 10 s) multiplied by 100. The results of the texture assessment are shown in Tables 46 and 47, respectively.

[0378] The firmness of the control bread increased with storage time (Table 51) and it lost its elasticity (Table 52), also known as bread staling.

[0379] Surprisingly, all three types of AMG tested in the study showed anti-aging effects, with less increase in firmness over storage time – Table 46. AMG also had a positive effect on elasticity, starting at a higher level, and after 14 days of storage, AMG bread had higher elasticity than the control – Table 47.

[0380] Table 51. Firmness over storage time

[0381]

[0382] Table 52. Elasticity with Storage Time

[0383]

[0384] Example 15. Applications of heat-stabilized AMG in cakes

[0385] Use a commercially available cake mix (Tegral Satin Creame Cake Neutral SG, Puratos, UK) to bake muffins using the recipe on the bag.

[0386] Table 53. Pancake Recipes.

[0387]

[0388]

[0389] Table 54. Preparation of nine muffin treatments using different enzyme additions:

[0390] 1. Blank

[0391] 2. JPO124 100mgEP / kg cake mixture

[0392] 3. JPO124 900mgEP / kg cake mixture

[0393] 4. JPO172 100mgEP / kg cake mixture

[0394] 5. JPO172 900mgEP / kg cake mixture

[0395] 6. OC50 1250 MANU / kg cake mix (6.25 mg EP / kg cake mix)

[0396] 7. OC50 2500 MANU / kg cake mix (12.5 mg EP / kg cake mix)

[0397] 8. OC50 3750 MANU / kg cake mix (18.75 mg EP / kg cake mix)

[0398] 9. OC50 5000 MANU / kg cake mix (25 mg EP / kg cake mix) Table 55. Muffin making process:

[0399] 1. Add the eggs, oil, and water according to Table 48 to the mixing bowl.

[0400] 2. Add the individual processing ingredients according to Table 49 to each dough ball.

[0401] 3. Add the cake mixture to the mixing bowl and mix with a hand mixer at speed 1 for 1 minute to form a cake batter.

[0402] 4. Use a piping bag to place the cake batter into muffin tins (50g batter per tin).

[0403] 5. Bake the muffins in a box oven for 28 minutes at 200°C top and 180°C bottom, with the tray upside down on the bottom of the oven.

[0404] 6. Allow the muffin to cool for 1 hour, then place it in a sealed plastic bag with modified atmosphere packaging and store at room temperature until analysis.

[0405] The texture properties of the muffins were analyzed using a texture analyzer for texture analysis (TPA). For the analysis, the top of the muffin was cut off at the same height as the muffin tin, leaving a 3 cm portion. The muffin was placed on the texture analyzer, and a 25 mm diameter cylindrical probe was pressed down twice at a constant upward and downward speed of 1 mm / s to a depth of 7 mm, with a 5-second interval between the two compressions. Force (g) was recorded as a function of time (seconds) and distance (mm).

[0406] • The peak force of the first compression corresponds to the hardness (grams) of the muffin.

[0407] • The area under the force-distance curve of the second compression divided by the area under the force-distance curve of the first compression corresponds to cohesiveness, expressed as a percentage.

[0408] • The force-distance under the curve during the first upward movement divided by the area under the curve during the first downward movement corresponds to the resilience, expressed as a percentage.

[0409] Table 56 below illustrates the benefits of using JPO172 and JPO124 in muffins. Muffins treated with JPO124 and JPO172 exhibit surprisingly improved (higher) resilience and cohesiveness; even better than other known solutions for improving cake freshness.

[0410] Table 56. Texture characteristics of muffins treated with different enzymes.

[0411]

[0412]

[0413] Example 16. AMG AMG NL, AMG AnPAV498, JP172 and Novamyl Sensory comparison of freshness

[0414] Bake bread using the direct fermentation process according to the recipe in Table 57. Mix the ingredients in a spiral mixer at 17 rpm and 35 rpm for 3+6 minutes to form a dough. Divide the dough into 450g portions, round them, flatten them, and place them in a baking pan. Proof the dough in the pan at 32°C and 86% relative humidity for 55 minutes. Bake the proofed dough in a box oven at 230°C for 35 minutes.

[0415] Table 57.

[0416] Baked goods % flour 100 water 57 fresh yeast 4.5 Salt 1.5 sugar 1.5 Calcium propionate 0.25 ascorbic acid 0.04 Fungamyl 4000SG 7ppm Panzea BG 25ppm

[0417] Table 58. Preparation of treatments using different enzyme additions

[0418]

[0419]

[0420] *Note: In this experiment, AMG AnPAV498 was mistakenly added in excess by ten times; it should have been 500 mg EP / kg, but it was 500 mg EP / kg.

[0421] Sensory evaluation methods

[0422] Sensory assessments were conducted on Day 1 and Day 8. A training session was held prior to the assessments to define the relevant attributes and procedures (Table 59). Texture was assessed manually. Four to five trained assessors participated in the assessments. Each assessor was provided with two slices of each bread type (without crust). Samples were provided blinded, with three-digit codes, and in a randomized order. The intensity of the sensory attributes was assessed on a scale of 1–9, from weak to very strong. Two repeated sensory assessments were conducted on each assessment day.

[0423] Table 59. Description of sensory attributes, procedures, and assessments

[0424]

[0425] Sensory results

[0426] For all freshness attributes (moisture, softness, and foldability) assessed on day 1 and day 8, JPO172 and AMG AnPAV498 received the highest scores. There was no difference compared to the control group.

[0427] Table 60. Average sensory scores of bread on day 1.

[0428]

[0429] Table 61. Average sensory scores of bread on day 8.

[0430] Example 17. AMG's preservation effect in the first 24 hours

[0431] Bake the bread using the direct fermentation mini-baking process according to the recipe in Table 62. Bake the bread in a covered baking pan to ensure all loaves are of equal volume. Combine the ingredients in a spiral mixer at 90 rpm for 4 minutes to form a dough. Divide the dough into 20g portions, round them, and place them in a baking pan. Proof the dough-filled pans on a conveyor belt at 36°C and 80% relative humidity for 55 minutes. Bake the proofed dough in a small tunnel oven at 210°C for 12 minutes.

[0432] Table 62.

[0433]

[0434] Table 63. Preparation of ten dough treatments using different enzyme additions

[0435] 1. Compare (blank)

[0436] 2. Datem 0.5%

[0437] 3. JPO172 50mgEP / kg flour

[0438] 4. Opticake 50BG 200MANU / kg

[0439] 5. JPO124 50mgEP / kg flour

[0440] 6. Novmayl 3D 440 MANU / kg flour

[0441] 7. SSL 0.5%

[0442] 8. 0.5% of distilled monoglycerides

[0443] 9. Novamyl 10,000 BG 750 MANU / kg

[0444] 10. Lipopan Extra 200 LU / kg

[0445] The dough was baked, and the resulting bread was packaged in a sealed plastic bag 0.5 hours after baking and stored at room temperature until analysis.

[0446] The texture of each loaf was assessed using a texture analyzer (TA-XT plus, Stable Microsystems, Godellmin, UK). Bread crumb texture characteristics are characterized by the firmness (the same as "hardness" and the opposite of "softness") and elasticity of the baked product.

[0447] The standard method for measuring firmness and elasticity is based on force-deformation of baked goods. Force-deformation of baked goods can be performed using a 20mm diameter spherical probe. When the probe is applied to a 25mm thick slice of bread at a deformation rate of 1mm / s with 40% stress, the force on the probe is recorded. The probe is then held in this position for 30 seconds while the force is recorded, and then the probe is returned to its initial position.

[0448] Toughness (in grams) is defined as the force required to compress the probe to 25% stress (corresponding to compression of 6.25 mm into a slice of bread crumb with a thickness of 25 mm).

[0449] Elasticity (in %) is defined as the force recorded after compression for 30 seconds under 40% stress (corresponding to the force of a 25mm thick bread slice at time = 40s) divided by the force required to press the probe into the crumb by 10mm (corresponding to the force of a 25mm thick bread slice at time = 10s) multiplied by 100.

[0450] The results of the texture analysis can be found in Table 64 (Durability) and Table 65 (Elasticity).

[0451] Fresh bread without enzymes (control) has low firmness and high elasticity; as the bread is stored, firmness decreases over time while elasticity decreases. Traditional AMG for baking applications (e.g., AMG) It does not affect firmness or elasticity (see Example 7).

[0452] The two AMGs (JPO124 and JPO172) added at 50 mg EP / kg improved (reduced) the firmness over time. Both AMGs (JPO124 and JPO172) added at 50 mg EP / kg improved (increased) initial elasticity and prevented loss of elasticity over time.

[0453] Table 64. Clarity (g) of bread with enzymes treated according to Table 63 at 2, 5 and 24 hours after baking.

[0454]

[0455] Table 65. Elasticity (g) of bread with enzymes treated according to Table 63 at 2, 5 and 24 hours after baking.

[0456] Example 18. Preservative effect of high-dose AMG

[0457] Bake the bread using the direct fermentation mini-baking process according to the recipe in Table 66. Bake the bread in a covered baking pan to ensure all loaves are of equal volume. Combine the ingredients in a spiral mixer at 90 rpm for 4 minutes to form a dough. Divide the dough into 20g portions, round them, and place them in a baking pan. Proof the dough-filled pans on a conveyor belt at 36°C and 80% relative humidity for 55 minutes. Bake the proofed dough in a small tunnel oven at 210°C for 12 minutes.

[0458] Table 66.

[0459]

[0460] Table 67. Preparation of ten treatments using different enzyme additions

[0461] 1. Comparison

[0462] 2. Opticake 50BG 200MANU / kg

[0463] 3. JPO124 50mgEP / kg flour

[0464] 4. JPO124 100mgEP / kg flour

[0465] 5. JPO124 300mgEP / kg flour

[0466] 6. JPO124 500mgEP / kg flour

[0467] 7. JPO172 50mgEP / kg flour

[0468] 8. JPO172 100mgEP / kg flour

[0469] 9. JPO172 300mgEP / kg flour

[0470] 10. JPO172 500mgEP / kg flour

[0471] The dough was baked, and the resulting bread was packaged in a sealed plastic bag 0.5 hours after baking and stored at room temperature until analysis.

[0472] The texture of each loaf was assessed using a texture analyzer (TA-XT plus, Stable Microsystems, Godellmin, UK). Bread crumb texture characteristics are characterized by the firmness (the same as "hardness" and the opposite of "softness") and elasticity of the baked product.

[0473] The standard method for measuring firmness and elasticity is based on force-deformation of baked goods. Force-deformation of baked goods can be performed using a 20mm diameter spherical probe. When the probe is applied to a 25mm thick slice of bread at a deformation rate of 1mm / s with 40% stress, the force on the probe is recorded. The probe is then held in this position for 30 seconds while the force is recorded, and then the probe is returned to its initial position.

[0474] Toughness (in grams) is defined as the force required to compress the probe to 25% stress (corresponding to compression of 6.25 mm into a slice of bread crumb with a thickness of 25 mm).

[0475] Elasticity (in %) is defined as the force recorded after compression for 30 seconds under 40% stress (corresponding to the force of a 25mm thick bread slice at time = 40s) divided by the force required to press the probe into the crumb by 10mm (corresponding to the force of a 25mm thick bread slice at time = 10s) multiplied by 100.

[0476] The results of the texture analysis can be found in Table 68 (Durability) and Table 69 (Elasticity).

[0477] Fresh bread without enzymes (control) has low firmness and high elasticity; as the bread is stored, firmness decreases over time while elasticity decreases. Traditional AMG for baking applications (e.g., AMG) It does not affect firmness or elasticity (see Example 7).

[0478] Two new AMGs (JPO124 and JPO172) improve (reduce) initial firmness and firmness over time. Higher doses result in lower firmness over time. Both AMGs (JPO124 and JPO172) improve (reduce) initial elasticity and prevent loss of elasticity over time. Higher AMG doses result in higher initial elasticity and lower loss of elasticity over time.

[0479] Table 68. Crust (g) of bread with enzymes treated according to Table 67 on day 1 and day 7.

[0480]

[0481] Table 69. Elasticity (%) of bread with enzyme treatment according to Table 67 on day 1 and day 7.

[0482]

[0483] Example 19. The preservation effect of the combination of AMG and Lip182

[0484] Bake the bread using the direct fermentation mini-baking process according to the recipe in Table 10. Bake the bread in a covered baking pan to ensure all loaves are of equal volume. Combine the ingredients in a spiral mixer at 90 rpm for 4 minutes to form a dough. Divide the dough into 20g portions, round them, and place them in a baking pan. Proof the dough-filled pans on a conveyor belt at 36°C and 80% relative humidity for 55 minutes. Bake the proofed dough in a small tunnel oven at 210°C for 12 minutes.

[0485] Table 70.

[0486]

[0487] Table 71. Preparation of various treatments using different enzyme additions

[0488]

[0489]

[0490] The dough was baked, and the resulting bread was packaged in a sealed plastic bag 0.5 hours after baking and stored at room temperature until analysis.

[0491] The texture of each loaf was assessed using a texture analyzer (TA-XT plus, Stable Microsystems, Godellmin, UK). Bread crumb texture characteristics are characterized by the firmness (the same as "hardness" and the opposite of "softness") and elasticity of the baked product.

[0492] The standard method for measuring firmness and elasticity is based on force-deformation of baked goods. Force-deformation of baked goods can be performed using a 20mm diameter spherical probe. When the probe is applied to a 25mm thick slice of bread at a deformation rate of 1mm / s with 40% stress, the force on the probe is recorded. The probe is then held in this position for 30 seconds while the force is recorded, and then the probe is returned to its initial position.

[0493] Toughness (in grams) is defined as the force required to compress the probe to 25% stress (corresponding to compression of 6.25 mm into a slice of bread crumb with a thickness of 25 mm).

[0494] Elasticity (in %) is defined as the force recorded after compression for 30 seconds under 40% stress (corresponding to the force of a 25mm thick bread slice at time = 40s) divided by the force required to press the probe into the crumb by 10mm (corresponding to the force of a 25mm thick bread slice at time = 10s) multiplied by 100.

[0495] The results of the texture analysis can be found in Table 72 (Durability) and Table 73 (Elasticity).

[0496] Fresh bread without enzymes (control) has low firmness and high elasticity; as the bread is stored, firmness decreases over time while elasticity decreases. Traditional AMG for baking applications (e.g., AMG) It does not affect firmness or elasticity (see Example 7).

[0497] AMG JPO172 improved (decreased) initial firmness and firmness over time. Compared to the control bread, the use of lipase Lip182 alone had no effect on firmness. The combination of Lip182 and JPO172 produced bread with the lowest firmness on both day 1 and day 7.

[0498] AMG JPO172 improves (increases) initial elasticity and prevents loss of elasticity over time. Lipase Lip182 has similar elasticity to the control, and the combination of JPO172 and Lip182 is similar to using JPO172 alone.

[0499] Table 72. Crust (g) of bread with enzymes treated according to Table 71 on day 1 and day 7.

[0500]

[0501] Table 73. Elasticity (%) of bread with enzyme treatment according to Table 71 on day 1 and day 7.

[0502]

[0503]

[0504] Example 20. The preservation effect of combining AMG with Gluzyme Fortis

[0505] Bake the bread using the direct fermentation mini-baking process according to the recipe in Table 74. Bake the bread in a covered baking pan to ensure all loaves are of equal volume. Combine the ingredients in a spiral mixer at 90 rpm for 4 minutes to form a dough. Divide the dough into 20g portions, round them, and place them in a baking pan. Proof the dough on a conveyor belt at 36°C and 80% relative humidity for 55 minutes. Bake the proofed dough in a small tunnel oven at 210°C for 12 minutes.

[0506] Table 74.

[0507] Table 75. Preparation of various treatments with different enzyme additions. Additional water was added to obtain similar dough rheological properties.

[0508]

[0509] The dough was baked, and the resulting bread was packaged in a sealed plastic bag 0.5 hours after baking and stored at room temperature until analysis.

[0510] The texture of each loaf was assessed using a texture analyzer (TA-XT plus, Stable Microsystems, Godellmin, UK). Bread crumb texture characteristics are characterized by the firmness (the same as "hardness" and the opposite of "softness") and elasticity of the baked product.

[0511] The standard method for measuring firmness and elasticity is based on force-deformation of baked goods. Force-deformation of baked goods can be performed using a 20mm diameter spherical probe. When the probe is applied to a 25mm thick slice of bread at a deformation rate of 1mm / s with 40% stress, the force on the probe is recorded. The probe is then held in this position for 30 seconds while the force is recorded, and then the probe is returned to its initial position.

[0512] Toughness (in grams) is defined as the force required to compress the probe to 25% stress (corresponding to compression of 6.25 mm into a slice of bread crumb with a thickness of 25 mm).

[0513] Elasticity (in %) is defined as the force recorded after compression for 30 seconds under 40% stress (corresponding to the force of a 25mm thick bread slice at time = 40s) divided by the force required to press the probe into the crumb by 10mm (corresponding to the force of a 25mm thick bread slice at time = 10s) multiplied by 100.

[0514] The results of the texture analysis can be found in Table 76 (Durability) and Table 77 (Elasticity).

[0515] Fresh bread without enzymes (control) has low firmness and high elasticity; as the bread is stored, firmness decreases over time while elasticity decreases. Conventional AMGs used in baking applications (such as Goldcrust) do not affect firmness or elasticity (Example 7).

[0516] AMG JPO172 improved (reduced) initial firmness and firmness over time. Compared to the control bread, the use of glucose oxidase (Gluzyme Fortis) alone reduced firmness to some extent. The combination of glucose oxidase and JPO172 produced bread with the lowest firmness on both day 1 and day 7.

[0517] AMG JPO172 improves (increases) initial elasticity and prevents loss of elasticity over time. Glucose oxidase alone provides similar elasticity to the control, and the combination of JPO172 and glucose oxidase is similar to JPO172 alone.

[0518] Table 76. Crust (g) of bread with enzymes treated according to Table 75 on day 1 and day 7.

[0519]

[0520] Table 77. Elasticity (%) of bread with enzyme treatment according to Table 75 on day 1 and day 7.

[0521]

[0522] Example 21.AMG Sensory comparison of the preservation effects of AMG NL, AMG AnPAV498, and JPO124 in sponge fermentation formulations

[0523] Bake bread using the sponge fermentation process according to the recipe in Table 78. Bake the bread in a covered baking pan to ensure all loaves are of uniform volume. Combine the starter ingredients in a needle mixer at 50 rpm and 150 rpm for 2+1 minutes to form a dough. Proof the starter at 27°C and 75% RH for 2 hours. Combine the starter with the remaining dough ingredients in a needle mixer and combine at 50 rpm and 150 rpm for 1+3 minutes to form a dough.

[0524] Divide the dough into 400g pieces, round them, flatten them, and place them in a covered baking pan. Let the pan containing the dough rise for 60 minutes at 43°C and 80% relative humidity. Bake the risen dough in a rotisserie oven at 215°C for 20 minutes.

[0525] Table 78. Formula

[0526]

[0527] Table 79. Preparation of treatments using different enzyme additions

[0528]

[0529]

[0530] Sensory evaluation methods

[0531] Sensory assessments were conducted on days 1 and 7. Training sessions were held prior to the assessments to define relevant attributes and procedures (Table 80). Texture was assessed manually. Five trained assessors participated in the assessments. Each assessor was provided with two slices of each bread type. Samples were provided blind-coded in a 3-digit, randomized order. The intensity of sensory attributes was assessed on a scale of 1-9, from weak to very strong. Sensory assessments were repeated twice on each assessment day.

[0532] Table 80. Description of Sensory Attributes, Procedures, and Evaluation

[0533]

[0534] Sensory results

[0535] JPO124 received the highest rating for moisture retention, softness, and foldability on day 7, followed by AMG AnPAV498.

[0536] Table 81. Average sensory scores of bread on Day 1.

[0537]

[0538]

[0539] Table 82. Average sensory scores of bread on day 7.

[0540]

[0541] Example 22. Preservation effect of JPO172 in low-pH rye / wheat mixed sourdough bread

[0542] Bread was baked using the direct fermentation process according to the recipe in Table 83. Nine different treatments were performed according to Table 84. The ingredients were mixed in a spiral mixer at 17 rpm and 35 rpm for 6+4 minutes respectively to form dough. The dough was divided into 650g pieces, rounded, flattened, and placed in baking pans. The final dough pH was 4.3. The bread was baked in covered baking pans to ensure all loaves were of uniform volume. The baking pans containing the dough were proofed at 32°C and 85% relative humidity for 60 minutes. The proofed dough was then baked in a box oven at 225°C for 20 minutes.

[0543] Table 83. Formula

[0544]

[0545]

[0546] Table 84. Processing.

[0547]

[0548] After baking, allow the bread to cool for 2 hours and then place it in a sealed plastic bag. Store the bread at room temperature until analysis.

[0549] The texture of each loaf was assessed using a texture analyzer (TA-XT plus, Stable Microsystems, Godellmin, UK). The texture characteristics of bread crumbs are characterized by the firmness (the same as "hardness" and opposite to "softness") and elasticity of the baked goods. The standard method for measuring firmness and elasticity is based on the force-deformation of the baked goods. The force-deformation of the baked goods can be performed using a cylindrical probe with a diameter of 40 mm. When the cylindrical probe is pressed down at a deformation rate of 1 mm / s, the force on the probe is recorded. The probe is then held in this position for 30 seconds while the force is recorded, and then the probe is returned to its initial position.

[0550] Toughness (in grams) is defined as the force required to compress the probe to 25% stress (corresponding to compression of 6.25 mm into a slice of bread crumb with a thickness of 25 mm).

[0551] Elasticity (in %) is defined as the force recorded after compression for 30 seconds under 40% stress (corresponding to the force of a 25mm thick bread slice at time = 40s) divided by the force required to press the probe into the crumb by 10mm (corresponding to the force of a 25mm thick bread slice at time = 10s) multiplied by 100.

[0552] The results of the texture analysis can be found in Table 85 (Durability) and Table 86 (Elasticity).

[0553] Fresh bread without any treatment (control) has low firmness and high elasticity. As the bread is stored, it becomes harder and loses its elasticity. Bread with JPO172 has lower firmness and higher elasticity after baking. Compared with the control bread, the change in firmness and elasticity over time is also less, making bread with JPO172 less firm and more elastic on day 7 than the control bread on day 1.

[0554] Table 85. Effects of various treatments on firmness

[0555] deal with Day 1 Day 3 Day 7 Comparison 1316 2331 3040 12.5 mg EP / kg flour JPO172 1012 1592 2072 25mgEP / kg flour JPO172 872 1217 1668 50mgEP / kg flour JPO172 858 1129 1312 100mgEP / kg flour JPO172 879 992 1097

[0556] Table 86. Effects of various treatments on elasticity.

[0557] deal with Day 1 Day 3 Day 7 Comparison 55.4 44.9 43.6 12.5 mg EP / kg flour JPO172 61.9 54.0 48.2 25mgEP / kg flour JPO172 64.6 60.6 54.6 50mgEP / kg flour JPO172 65.4 63.1 61.1 100mgEP / kg flour JPO172 66.2 65.6 65.0

[0558] Example 23. The preservation effect of JPO172 in tortillas

[0559] Make tortillas using the recipe in Table 87, adding different enzyme solutions according to Table 88. Mix the ingredients in a needle mixer at low and high speeds for 1+6 minutes respectively. Let the dough rest for 2 minutes. Divide the dough into 30g pieces and roll them up. Bake the tortillas using a two-step method: first, bake the dough pieces at 160°C for 6 seconds in a hot air gun, then bake the tortillas for 20 seconds, flip them, and bake for another 20 seconds.

[0560] Table 87. Formula.

[0561] Element quantity,% flour 100.0 water 54 Baking powder 3.0 glycerin 4.5 Salt 2.0 sugar 2.00 Citric acid 0.40 Calcium propionate 0.50 DMG 0.50 SSL 0.25 Guar gum 0.30 Sunflower seed oil 6.00

[0562] Table 88. Preparation of various treatments using different enzyme additions.

[0563]

[0564] After baking, allow the tortillas to cool for 30 minutes, then place them in a sealed plastic bag and store at room temperature until analysis.

[0565] The textural properties of tortillas were evaluated using a texture analyzer (Stable Microsystems, Godellmin, UK) with a tortilla / pastry burst rig (HDP / TPB). In the test procedure, the sample was held between two plates, and a 1" spherical probe was driven through the center. The force and distance of stretching the sample were measured and used as indicators of "deformation resistance" and "stretchability," respectively.

[0566] Tortillas are typically used as wrappers, where they are wrapped around different types of fillings. An important parameter is the stretchability, which describes resistance to breakage. Fresh tortillas are stretchable. However, as shown in Table 90, they quickly lose this stretchability during storage. The addition of JPO172 produces tortillas with similar stretchability to freshly baked tortillas after 28 days.

[0567] Table 89. Deformation resistance of corn tortillas, g

[0568] deal with dose Day 1 Day 14 Day 28 JPO172, mgEP / kg flour 0 663 482 333 400 727 634 492 2000 868 616 498 Sensea Wrap, ppm 0 663 482 333 200 649 543 420 400 689 542 445

[0569] Table 90. Extensibility of Corn Tortillas (mm)

[0570] Example 24. The preservation effect of JPO172 in brioche bread

[0571] Bread was baked using the direct fermentation process according to the recipe in Table 91. Eight different treatments were performed according to Table 92. The ingredients were mixed in a spiral mixer at 17 rpm and 35 rpm for 4+8 minutes respectively to form dough. The dough was divided into 420g pieces, rounded, flattened, and placed in a baking pan. The dough was proofed at 30°C and 75% RH for 2.5 hours. The bread was baked at 175°C for 34 minutes.

[0572] Table 91. Formula.

[0573]

[0574] Table 92. Processing

[0575]

[0576] After baking, allow the bread to cool for 2 hours and then place it in a sealed plastic bag. Store the bread at room temperature until analysis.

[0577] The texture of each loaf of bread was assessed using a texture analyzer (TA-XT plus, Stable Microsystems, Godellmin, UK). Bread crumb texture characteristics are characterized by the firmness (the same as "hardness" and opposite to "softness") and elasticity of the baked goods. The standard method for measuring firmness and elasticity is based on the force-deformation of the baked goods. The force-deformation of the baked goods can be performed using a cylindrical probe with a diameter of 34 mm. When the cylindrical probe is pressed down on a 25 mm thick slice of bread at a deformation rate of 1 mm / s to achieve 28% stress, the force on the cylindrical probe is recorded. The probe is then held in this position for 30 seconds while the force is recorded, and then the probe is returned to its initial position.

[0578] Toughness (in grams) is defined as the force required to compress the probe to 25% stress (corresponding to compression of 6.25 mm into a slice of bread crumb with a thickness of 25 mm).

[0579] Elasticity (in %) is defined as the force recorded after compression for 30 seconds under 28% stress (corresponding to the force of a 25mm thick bread slice at time = 40s) divided by the force required to press the probe into the crumb by 10mm (corresponding to the force of a 25mm thick bread slice at time = 10s) multiplied by 100.

[0580] The results of the texture analysis can be found in Table 93 (Durability) and Table 94 (Elasticity).

[0581] Fresh bread without any treatment (control) had low firmness and high elasticity after baking, becoming harder and losing elasticity as it was stored. Compared to the control, bread with JPO172 had lower firmness and higher elasticity after baking. When storing bread with JPO172, the firmness and elasticity changed only slightly, resulting in brioche bread with JPO172 having similar firmness and better elasticity to the control on day 1 at day 60.

[0582] Table 93. Effects of various treatments on firmness.

[0583]

[0584]

[0585] Table 94. Effects of various treatments on elasticity.

[0586] deal with Day 1 Day 21 Day 39 Day 60 Comparison 52.3 42.2 40.4 40.9 50mg EP JP0172\Kg flour 58.0 51.9 51.0 50.0 75mgEP JP0172\Kg flour 59.9 55.5 54.9 53.1 100mg EP JP0172\Kg flour 60.0 56.7 55.5 54.8 150mg EP JP0172\Kg flour 60.3 57.2 56.5 55.9 200mg EP JP0172\Kg flour 60.5 56.9 56.6 55.9

[0587] Example 25. JPO124 and JPO172 in Lebanese double-layer flatbread

[0588] Lebanese double-layer flatbread was baked using a direct fermentation process with the ingredients listed in Table 95. Seven different treatments were performed according to Table 96. The ingredients were mixed in a spiral mixer at 35 rpm for 2.5 minutes to form a dough. The dough was proofed at 32°C and 82% RH for 40 minutes. The dough was rolled to a thickness of 2 mm, and 20 cm round dough pieces were cut from this sheet. The round dough pieces were proofed at room temperature for 20 minutes. The dough pieces were placed in an oven at 750°C and baked for 9 seconds.

[0589] Table 95. Formula.

[0590] Element quantity,% flour 100.0 water 51 Instant dry yeast 0.7 sucrose 4.0 Salt 0.4 Calcium propionate 0.20

[0591] Table 96. Processing.

[0592]

[0593] After baking, allow the flatbread to cool for 30 minutes, then place it in a sealed plastic bag and store at room temperature until analysis.

[0594] On day 3, the textural properties of Lebanese flatbread were evaluated using a texture analyzer (Stable Microsystems, Godellmin, UK) with a tortilla / pasta burst device (HDP / TPB). In the testing procedure, the sample was held between two plates, and a 4mm spherical probe was driven through the center. The force and distance of tension on the sample were measured and used as indicators of “deformation resistance” and “tensile strength,” respectively.

[0595] Sensory evaluation was conducted on day 3. A training course was held prior to the evaluation to define the relevant attributes and procedures (Table ZZ). Texture was evaluated manually. Four to five trained evaluators participated in the evaluation. Each evaluator was provided with two slices of each bread type (without crust). Samples were provided blind-sampled, with three-digit codes and in random order. The intensity of sensory attributes was assessed on an intensity scale of 1-9, from weak to very strong. Two repeated sensory evaluations were conducted on each evaluation day.

[0596] Table 97. Sensory evaluation.

[0597]

[0598]

[0599] The results of the sensory evaluation are shown in Table 98, and the results of the texture evaluation are shown in Table 99. For all sensory parameters, the bread without any added enzymes (control) scored lower (2-3). For all parameters, the flat bread with JPO172 and JPO124 scored higher, and the higher the dosage, the higher the score. The improvements detected in the sensory evaluation were also seen in the texture analysis, where the bread with JPO124 or JPO172 had higher stretchability compared to the flat bread without any enzymes (control).

[0600] Table 98. Sensory evaluation of Lebanese flatbread on day 3.

[0601]

[0602] Table 99. Texture assessment of Lebanese flatbread on day 3.

[0603] toughness Stretchability Comparison 271 3.8 400mgEP / kg JPO172 266 5.1 2000mgEP / kg JPO172 204 5.0 4000mgEP / kg JPO172 217 5.5 400mgEP / kg JPO124 323 4.9 4000mgEP / kg JPO124 268 6.4

Claims

1. A method for producing baked or partially baked products, the method comprising: a) Provide dough containing a mature, thermostable variant of a parental glucosylamylase that is 100% identical to SEQ ID NO: 6; as well as b) Baking or partially baking the dough to produce baked or partially baked products; This mature, thermally stable variant exhibits at least a 5°C improvement in thermal stability compared to its parent.

2. The method of claim 1, wherein the baked or partially baked product is a type of bread.

3. The method according to claim 2, wherein the bread is molded bread.

4. The method according to claim 2, wherein the bread is toast, open loaf, roll, fino bread, hamburger bread, samori bread, baguette, brioche, hamburger roll, bread roll, rye bread, whole wheat bread, high-fat and high-sugar bread, bran bread, or flatbread.

5. The method of claim 1, wherein the baked or partially baked product is a pastry.

6. The method of claim 5, wherein the pastry is a tortilla, biscuit, or cake.

7. The method according to any one of claims 1-6, wherein the parental glucosylamylase is derived from a species of the Penicillium genus.

8. The method according to claim 7, wherein the parental glucosylamylase is derived from Penicillium oxalate, Penicillium migrans, Penicillium rovolaceum, or Penicillium sclerotiorum.

9. The method according to any one of claims 1-6, wherein the mature thermally stable variant has an improvement in thermal stability relative to its parent at at least 6°C, 7°C or 8°C.

10. The method according to any one of claims 1-6, wherein the mature, thermostable variant has a relative activity of at least 150 compared to its parent at 91°C.

11. The method of claim 10, wherein the mature, thermostable variant has a relative activity of at least 200 compared to its parent at 91°C.

12. The method of claim 11, wherein the mature, thermostable variant has a relative activity of at least 250 compared to its parent at 91°C.

13. The method of claim 12, wherein the mature, thermostable variant has a relative activity of at least 300 compared to its parent at 91°C.

14. The method according to any one of claims 1-6, wherein, compared with a control made without the addition of any glucosylamylase, the final fully 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 reduced firmness and / or increased elasticity after 1, 7, or 14 days.

15. The method according to any one of claims 1-6, wherein the baked product after final full baking has at least the same sweetness as the control product made with twice the amount of mature glucosylamylase, the amino acid sequence of which is shown in SEQ ID NO:

10.

16. The method according to any one of claims 1-6, wherein the mature, heat-stable variant of glucosylamylase is contained in the dough at an amount of 0.01-1,000 mg enzyme protein / kg flour.

17. The method of claim 16, wherein the mature, heat-stable variant of glucosylamylase is contained in the dough at an amount of 0.01-500 mg enzyme protein / kg flour.

18. The method of claim 17, wherein the mature, heat-stable variant of glucosylamylase is contained in the dough at an amount of 0.1-100 mg enzyme protein / kg flour.

19. The method according to any one of claims 1-6, wherein the dough further comprises one or more additional enzymes selected from the group consisting of: α-amylase, maltodextrinase, β-amylase, aminopeptidase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, 1,4-α-maltotetrasaccharide hydrolase, glucanase, galactanase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, hemicellulase, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinase, peptidase, glutaminase, phospholipase, phytase, proteolytic enzyme, ribonuclease, transglutaminase, and xylanase.

20. The method according to claim 19, wherein the α-amylase is a starch-degrading α-amylase.

21. The method of claim 19, wherein the oxidase is a polyphenol oxidase, a peroxidase, or a glucose oxidase.

22. The method of claim 21, wherein the peroxidase is a halogenated peroxidase.

23. The method of claim 19, wherein the one or more additional enzymes are contained in an amount of 0.01-1,000 mg enzyme protein / kg flour.

24. The method of claim 23, wherein the one or more additional enzymes are contained in an amount of 0.01-500 mg enzyme protein / kg flour.

25. The method of claim 24, wherein the one or more additional enzymes are contained in an amount of 0.1-100 mg enzyme protein / kg flour.

26. A baking composition comprising a mature, thermostable variant of a parental glucosylamylase as defined in any one of claims 1-6.

27. The baking composition of claim 26, further comprising one or more additional enzymes selected from the group consisting of: α-amylase, maltodextrinase, β-amylase, aminopeptidase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, 1,4-α-maltotetrasaccharide hydrolase, glucanase, galactanase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, hemicellulose hydrolase, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinase, peptidase, glutaminase, phospholipase, phytase, proteolytic enzyme, ribonuclease, transglutaminase, and xylanase.

28. The baking composition according to claim 27, wherein the oxidase is a peroxidase, a polyphenol oxidase, or a glucose oxidase.

29. The baking composition according to claim 28, wherein the peroxidase is a halogenated peroxidase.

30. The baking composition according to claim 26 or 27, further comprising flour, sugar, yeast, salt and / or fat.

31. The baking composition according to any one of claims 26-29 is used for: replacing sugar in a method of producing a baked or partially baked product, increasing the sweetness of a baked or partially baked product, reducing the amount of sugar in dough in a method of producing a baked or partially baked product, and / or extending the shelf life of a baked or partially baked product in a method of producing a baked or partially baked product.

32. Use of the baking composition according to any one of claims 26-29 in the method according to any one of claims 1-25, wherein the baked product after final full 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 reduced firmness and / or higher elasticity after 1, 7 or 14 days, compared with a control made without the addition of any glucosylamylase.

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