CN120344150A

CN120344150A - Baking at low pH with thermostable glucoamylase variants

Info

Publication number: CN120344150A
Application number: CN202280102119.2A
Authority: CN
Inventors: K·哈里斯; H·伦德奎斯特
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2022-11-30
Filing date: 2022-11-30
Publication date: 2025-07-18
Also published as: JP2026502814A; MX2025006103A; AU2022489922A1; WO2024118096A1; EP4626238A1

Abstract

本发明涉及生产烘焙或部分烘焙产品的方法，所述方法包括：a)提供面团，该面团包含与SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8或SEQ ID NO:10具有至少70％同一性的亲本葡糖淀粉酶的成熟热稳定变体，该变体以0.01‑12.40mg酶蛋白(mgEP)/kg面粉的量添加，其中所述面团具有在3.0‑6.5范围内的pH值；以及b)烘焙或部分烘焙该面团以生产烘焙或部分烘焙产品。The present invention relates to a method for producing a baked or part-baked product, the method comprising: a) providing a dough comprising a mature thermostable variant of a parent glucoamylase having at least 70% identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 10, which is added in an amount of 0.01-12.40 mg enzyme protein (mg EP)/kg flour, wherein the dough has a pH value in the range of 3.0-6.5; and b) baking or part baking the dough to produce a baked or part-baked product.

Description

Baking at low pH with thermostable glucoamylase variants

Reference to sequence Listing

The present application comprises a sequence listing in computer readable form, which is incorporated herein by reference.

Technical Field

The present invention relates to a method of producing a baked or partially baked product, the method comprising:

a) Providing a dough comprising a mature thermostable variant of a parent glucoamylase (AMG) having at least 70% identity to SEQ ID NO. 1, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 10, added in an amount of 0.01-12.40mg enzyme protein (mgEP) per kg flour, wherein the dough has a pH in the range of 3.0-6.5, and

B) Baking or partially baking the dough to produce a baked or partially baked product.

Background

Sugar-containing baked products worldwide (bread, biscuits, etc.) are one of the most popular product categories. The amount of sugar in the formulation is typically 1% to 25% of the total weight of the flour.

However, because of the rising market price of sugar, inadequate sugar supply in certain parts of the world, and health problems, there is a need for methods of producing baked products with reduced amounts of added sugar without sacrificing the quality of the baked product or even potentially improving the quality of the baked product.

WO 2019/238423 (Novozymes a/S), denmark discloses methods of producing dough with reduced amounts of added sugar, which methods comprise adding a raw starch degrading alpha-amylase and a glucoamylase to the dough ingredients.

WO 2022/090562 (Novozymes A/S), denmark discloses a method for producing baked or partially baked products from mature thermostable variants of a parent glucoamylase.

Disclosure of Invention

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

Thus, in a first aspect, the present invention relates to a method of producing a baked or partially baked product, the method comprising:

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

Preferably, the mature thermostable variant of a parent glucoamylase of the invention has at least 71% sequence identity with SEQ ID No.1, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 or SEQ ID No. 10, e.g. at least 72%, e.g. at least 73%, e.g. at least 74%, e.g. at least 75%, e.g. at least 76%, e.g. at least 77%, e.g. at least 78%, e.g. at least 79%, e.g. at least 80%, e.g. at least 81%, e.g. at least 82%, e.g. at least 83%, e.g. at least 84%, e.g. at least 85%, e.g. at least 86%, e.g. at least 87%, e.g. at least 88%, e.g. at least 89%, e.g. at least 90%, e.g. at least 91%, e.g. at least 92%, e.g. at least 93%, e.g. at least 94%, e.g. at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99% sequence identity with SEQ ID No.1, SEQ ID No. 7.

Drawings

FIG. 1 shows multiple alignments of the amino acid sequences of the following mature proteins:

Wild-type AMG (PoAMG) from Penicillium oxalicum (Penicillium oxalicum) according to SEQ ID NO. 1

PoAMG variant of SEQ ID NO.2 denoted "AMG NL

PoAMG variant of SEQ ID NO.3 denoted "AMG anPAV498

PoAMG variant of SEQ ID NO. 4 denoted "AMG JPO001

PoAMG variant of SEQ ID NO. 5 denoted "AMG JPO124

PoAMG variant of SEQ ID NO. 6 denoted "AMG JPO172

Wild-type AMG (PoAMG) from Mikrill (Penicillium miczynskii) according to SEQ ID NO. 7

Wild-type AMG (PoAMG) from Penicillium rochanterium (Penicillium russellii) according to SEQ ID NO. 8

Wild-type AMG (PoAMG) from Penicillium light (Penicillium glabrum) of SEQ ID NO 9

FIG. 2 shows the pH-activity curve of a thermostable glucoamylase variant denoted JPO-172. The pH-activity curve was determined at 40 ℃. Each data point represents the average of four measurements and the error bars represent the standard deviation. The pH 5 was set to 10%. In this curve, it can be seen that the optimum pH is around pH 5, and about 80% activity is observed from about pH 4 to 6.

Detailed Description

Definition of the definition

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

For the purposes of the present invention, sequence identity between two amino acid sequences is determined using the Needleman-Wellman application algorithm (Needleman-Wunsch algorism) (Needleman and Wunsch,1970, J.mol. Biol. [ J. Mol. Biol. ] 48:443-453) as implemented by the Needle program of the EMBOSS software package (EMBOSS: the European Molecular Biology Open Software Suite [ European molecular biology open software suite ], rice et al, 2000,Trends Genet. [ genetics trend ]16:276-277, preferably version 5.0.0 or newer). The parameters used are gap opening penalty of 10, gap extension penalty of 0.5, and EBLOSUM62 (the emoss version of BLOSUM 62) substitution matrix. The output of the "longest identity" of the Needle label (obtained using the non-simplified (-no brief) option) was used as a percentage of identity and calculated as follows:

(identical residue. Times.100)/(alignment Length-total number of gaps in the alignment)

Variant the term "variant" means a polypeptide comprising 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 changes may have minor properties, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein, small deletions, typically of 1-30 amino acids, small amino-terminal or carboxy-terminal extensions, such as amino-terminal methionine residues, small linker peptides of up to 20-25 residues, or small extensions that facilitate purification by changing the net charge or another function (such as a polyhistidine segment, epitope, or binding domain). Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H.Neurath and R.L.Hill,1979,The Proteins [ proteins ], ACADEMIC PRESS [ academic Press ], new York. Common substitutions are Ala/Ser、Val/Ile、Asp/Glu、Thr/Ser、Ala/Gly、Ala/Thr、Ser/Asn、Ala/Val、Ser/Gly、Tyr/Phe、Ala/Pro、Lys/Arg、Asp/Asn、Leu/Ile、Leu/Val、Ala/Glu、 and Asp/Gly.

Increased strength the term "increased strength of a dough" is defined herein as the characteristic of a dough that generally has greater elastic properties and/or requires more work input to mold and shape than a control.

Increased elasticity the term "increased elasticity of the dough" is defined herein as the property of the dough that has a higher tendency to recover its original shape after being subjected to a certain physical stress than a control.

Increased stability of dough the term "increased stability of dough" is defined herein as the property of dough that is less susceptible to mechanical damage than controls, thus better retaining its shape and volume, and is assessed by the ratio of the height to width of the cross section of the bread after normal and/or prolonged proofing.

Reduced stickiness of dough the term "reduced stickiness of dough" is defined herein as a characteristic of dough that has a lower tendency to adhere to a surface, e.g., in a dough production machine, than a control, and is evaluated empirically by a skilled test baker or measured using a texture analyzer (e.g., TAXT 2) known in the art.

Improved stretchability the term "improved stretchability of the dough" is defined herein as the property of the dough that can withstand increased stress or stretching without breaking as compared to a control.

Improved mechanical ability the term "improved mechanical ability of a dough" is defined herein as the characteristic of a dough that is generally less viscous and/or more compact and/or more elastic than a control.

The term "increased volume of baked product" is measured for the volume of a given bread stick as compared to a control. The volume may be determined using methods known in the art.

Improved crumb structure of baked products the term "improved crumb (crumb) structure of baked products" is defined herein as the property of baked products that have finer cells and/or thinner cell walls in the crumb and/or a more uniform/homogenous cell distribution in the crumb compared to a control and that are typically assessed visually or by digital image analysis known in the art (e.g., C-cell, international fine control company (Calibre Control International Ltd), aprton (applyton), warriongton (Warrington), uk) by a skilled baker.

Improved softness of a baked product the term "improved softness of a baked product" is contrary to "firmness" and is defined herein as the characteristic of a baked product that is more easily compressed than a control and is evaluated empirically by a skilled test baker or measured for example using a texture analyzer known in the art, such as, for example, TAXT2 or TA-XTplus from Stable microsystems company (Stable Micro SYSTEMS LTD) in Sari, england.

Sensory attributes of baked products sensory attributes may be evaluated using well established procedures in the baking industry and may include, for example, the use of a trained panel of taste testers.

Thermostability improvement in units of °c (Td) is a measure of how much a variant improves in thermostability relative to its parent glucoamylase under the same conditions, as measured as exemplified herein.

A first aspect of the invention relates to a method of producing a baked or partially baked product, the method comprising:

a) Providing a dough comprising a mature thermostable variant of a parent glucoamylase having at least 70% identity with SEQ ID NO. 1, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 10, and

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

Dough

As used herein, "dough" means dough used to prepare baked products (particularly bread).

The dough used to prepare the baked product according to the present invention may be made from any suitable dough ingredient comprising flour.

The flour may be from any baked cereal known in the art, such as wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, sorghum flour, potato flour, soybean flour, and any combination thereof (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).

In a preferred embodiment, the flour is wheat flour.

In preferred embodiments, at least 10% (w/w) or more of the total flour content is wheat flour, e.g., at least 15% or more of the total flour content is wheat flour, e.g., at least 20% or more of the total flour content is wheat flour, e.g., at least 25% or more of the total flour content is wheat flour, e.g., at least 30% or more of the total flour content is wheat flour, e.g., at least 35% or more of the total flour content is wheat flour, e.g., at least 40% or more of the total flour content is wheat flour, e.g., at least 45% or more of the total flour content is wheat flour, e.g., at least 50% or more of the total flour content is wheat flour, e.g., at least 55% or more of the total flour content is wheat flour, e.g., at least 60% or more of the total flour content is wheat flour, e.g., at least 65% or more of the total flour content is wheat flour, e.g., at least 70% or more of the total flour content is wheat flour, e.g., at least 75% or more of the total flour content is wheat flour, e.g., at least 80% or more of the total flour is at least 100% or more of the total flour.

The dough of the present invention is typically a leavened dough or a dough to be subjected to leavening. The dough may be leavened in various ways, such as by the addition of dough ingredients such as chemical leavening agents (e.g., sodium bicarbonate) or by the addition of leavening agents (leavening dough), but preferably by the addition of a suitable yeast culture such as a culture of Saccharomyces cerevisiae (Saccharomyces cerevisiae) (baker's yeast) (e.g., a commercially available strain of Saccharomyces cerevisiae).

The dough of the present invention may typically contain some added sugar because, while the method according to the present invention is capable of reducing the amount of added sugar, it is normally only partially possible to reduce the amount of sugar.

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 formulation, e.g., by at least 20% (w/w) compared to the amount of sugar added to the dough in the original formulation, e.g., by at least 30% (w/w) compared to the amount of sugar added to the dough in the original formulation, e.g., by at least 40% (w/w) compared to the amount of sugar added to the dough in the original formulation, e.g., by at least 50% (w/w) compared to the amount of sugar added to the dough in the original formulation, e.g., by at least 60% (w/w) compared to the amount of sugar added to the dough in the original formulation, e.g., by at least 70% (w/w) compared to the amount of sugar added to the dough in the original formulation, e.g., by at least 80% (w/w) compared to the amount of sugar added to the dough in the original formulation, e.g., by at least 90% (w/w) compared to the amount of sugar added to the original formulation.

The dough may also contain other conventional dough ingredients such as proteins like milk powder, gluten and soy, eggs (whole egg, egg yolk or egg white), oxidants such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammonium persulfate, amino acids such as L-cysteine, salts such as sodium chloride, calcium acetate, sodium sulfate, calcium sulfate, diluents such as silica and starches of different origin. Other commonly used ingredients also include hydrocolloids such as CMC, guar gum, xanthan gum, locust bean gum, and the like.

The dough ingredients typically may comprise fats (triglycerides) and/or oils and/or shortening, in particular oils such as sunflower oil or rapeseed oil.

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

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

The dough may be prepared using any conventional mixing process, such as a continuous mixing process, a direct-fermentation (dough) process, or a medium-fermentation (sponge and dough) process.

The invention is particularly useful for preparing dough and baked products in an industrial process wherein the dough used to prepare the baked product is mechanically prepared using automated or semi-automated equipment.

The process of making bread generally involves the sequential steps of making a dough, sheeting (sheeting) or dividing, shaping or rolling (rolling), and proofing (proofing) the dough, which are well known in the art.

As used herein, "baked product" means any type of baked product, including a variety of bread types, such as molded bread (pan bread), toast bread, open bread (open bread), molded bread with and without lids, buns, feno bread (Fino bread), ha Mam bread (Hammam bread), samoli bread (Samoli bread), croissants, british bread (brioche), hamburger buns, rolls (roll), black bread, whole wheat bread, high oil sugar bread (rich bread), bran bread, flat bread, mexico tortilla (tortilla), biscuits and any variety thereof. The baked product according to the invention may also be a cake or any pastry product known in the art.

Raw starch degrading alpha-amylase

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

Examples of raw starch degrading alpha-amylase include those disclosed in WO 2005/003311, U.S. patent publication No. 2005/0054071 and U.S. patent No. 7,326,548. Examples also include those disclosed in tables 1 to 5 of the examples in U.S. patent No. 7,326,548 and U.S. patent publication No. 2005/0054071 (page table 3 of 15), as well as the enzymes disclosed in WO 2004/020499 and WO 2006/06929 and WO 2006/066579.

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

In one embodiment, the raw starch degrading alpha-amylase has at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity with a raw starch degrading alpha-amylase as shown in european patent No. 2981170 (novelia).

In one embodiment, the raw starch degrading alpha-amylase according to the invention may be added to flour or dough in an amount of 0.01-10mg enzyme protein per kg flour, for example in an amount of 0.1-5mg enzyme protein per kg flour.

Glucoamylase enzyme

Glucoamylases are also known as amyloglucosidase and glucan 1, 4-alpha-glucosidase (EC 3.2.1.3), more commonly they are known as AMG.

According to the invention, different types of amyloglucosidase may be used as parents for producing thermostable amyloglucosidase variants, e.g. amyloglucosidase may be a polypeptide encoded by a DNA sequence found in a fungal strain of Aspergillus (Aspergillus), rhizopus (Rhizopus), penicillium (Talaromyces) or Penicillium (Penicillium), preferably a DNA sequence found in a fungal strain of Penicillium, even more preferably a DNA sequence found in a fungal strain of Penicillium, penicillium aculeatum (Penicillium oxysporum), penicillium oxalate, penicillium michaelii, penicillium roseum or Penicillium photospora. Preferably, the parent glucoamylase is derived from a species of the genus penicillium, preferably from penicillium oxalicum, penicillium michaeli, penicillium rochanterium or penicillium photospora.

Examples of other suitable fungi include Aspergillus niger (Aspergillus niger), aspergillus awamori (Aspergillus awamori), aspergillus oryzae (Aspergillus oryzae), rhizopus delemar (Rhizopus delemar), rhizopus niveus (Rhizopus niveus), rhizopus oryzae (Rhizopus oryzae), and Emerson basket (Talaromyces emersonii).

The identity between the aligned AMG amino acid sequences in fig. 1 is shown below and is also provided in the sequence listing:

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

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

In preferred embodiments, the mature variants of the invention comprise at least one amino acid modification at one or more or all 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 a substitution at one or more or all positions corresponding to positions 1,2, 4, 11, 65, 79 and 327 in SEQ ID NO. 1, preferably, the at least one amino acid modification comprises a substitution at one or more or all positions corresponding to R1 24 11, 65V and Q327F in SEQ ID NO. 1; or preferably the at least one amino acid modification comprises a substitution at one or more or all positions corresponding to positions 1, 6, 7, 31, 34, 79, 103, 132, 445, 447, 481, 566, 568, 594 and 595 in SEQ ID NO:1, preferably the at least one amino acid modification comprises a substitution at one or more or all positions corresponding to R1, 7, 31, 132 445 447 566 568R and F595S in SEQ ID NO:1, or preferably the at least one amino acid modification comprises a substitution at one or more or all positions corresponding to positions 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595 in SEQ ID NO:1, preferably the at least one amino acid modification comprises a substitution at one or more or all positions corresponding to R1, 6, 7, 445, 31, 103, 484, 566V 566, 539 and 539 in SEQ ID NO:1 Substitution at one or more or all positions of Q594R and F595S.

The thermal stability improvements (Td) of the variants in table 2 are listed in table 3, wherein Td of the PoAMG variant, denoted "anPAV498" (parent), is set to zero. In a preferred embodiment, the mature thermostable variants of the present invention have a thermostability improvement (Td) of at least 3 ℃, preferably at least 4 ℃,5 ℃,6 ℃, 7 ℃ or 8 ℃ relative to their parent, preferably as determined as exemplified herein.

In another preferred embodiment, the mature thermostable variants of the invention have a relative activity at 91 ℃ as compared to their parent of at least 150, preferably at least 200, more preferably at least 250, most preferably at least 300.

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

Amylase enzyme

Alpha-amylases (alpha-1, 4-glucan-4-glucan hydrolase, EC.3.2.1.1) constitute a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1, 4-glycosidic oligosaccharides and polysaccharides.

A variety of alpha-amylases are known as Termamyl ^TM and "Termamyl ^TM -like alpha-amylases" and are known, for example, from WO 90/11352, WO 95/10603, WO 95/26397, WO 96/23873 and WO 96/23874.

Another group of alpha-amylases are known as Fungamyl ^TM and "Fungamyl ^TM -like alpha-amylases", which are alpha-amylases associated with the alpha-amylases derived from Aspergillus oryzae disclosed in WO 01/34784.

Suitable commercially available alpha-amylase compositions according to the invention include, for example, BAKEZYME P (available from Di Siman Co., ltd.)) and FUNGAMYL 2500SG, FUNGAMYL 4000BG, FUNGAMYL 4000SG, FUNGAMYL 800L, FUNGAMYL ULTRA BG and FUNGAMYL ULTRA SG (available from NoveXin Co.).

In one embodiment, the alpha-amylase according to the invention may be added to flour or dough in an amount of 0.01-1,000mg enzyme protein (mgEP) per kg of flour, preferably in an amount of 0.01-500mg enzyme protein (mgEP) per kg of flour, even more preferably in an amount of 0.1-100mg enzyme protein (mgEP) per kg of flour.

Additional enzymes

Optionally, one or more additional enzymes (e.g., alpha-amylase, maltogenic amylase, beta-amylase, aminopeptidase, carboxypeptidase, catalase, cellulolytic enzyme, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1, 4-alpha-maltotetrahydrolase, glucanase, galactanase, alpha-galactosidase, beta-galactosidase, glucose oxidase, alpha-glucosidase, beta-glucosidase, haloperoxidase, hemicellulolytic enzyme, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinolytic enzyme, peptide glutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, and xylanase) may be used with the enzyme composition according to the invention.

The one or more additional enzymes may be of any origin, including mammalian origin, plant origin, and microbial (bacterial, yeast, or fungal) origin.

Maltogenic alpha-amylase (EC 3.2.1.133) may be from Bacillus. Maltogenic alpha-amylase from Bacillus stearothermophilus strain NCIB 11837 was obtained from Norwesternum under the trade nameCommercially available below.

The maltogenic alpha-amylase may also be a variant of a maltogenic alpha-amylase from Bacillus stearothermophilus, e.g. as disclosed in WO 99/43794, WO 2006/032581, or WO 2008/148845, e.g.3D。

The anti-aging amylase used in the present invention may also be an amylase from Pseudomonas saccharophila (Pseudomonas saccharophilia) (glucan 1, 4-alpha-maltotetraohydrolase (EC 3.2.1.60)) or a variant thereof, such as any of the amylases disclosed in WO 99/50399, WO 2004/111217 or WO 2005/003339.

The glucose oxidase may be a fungal glucose oxidase, particularly Aspergillus niger glucose oxidase (e.g.)Available from novelin).

The xylanase may be of microbial origin, for example a strain derived from a bacterium or fungus such as aspergillus (in particular aspergillus aculeatus, aspergillus niger, aspergillus awamori or aspergillus tubingensis (a. Tubingensis)), a strain derived from Trichoderma (e.g. Trichoderma reesei (t. Reesei)), or a strain derived from Humicola (Humicola) (e.g. Humicola insolens).

Suitable commercially available xylanase preparations for use in the present invention include PANZEA BG, PENTOPAN MONO BG and PENTOPAN BG (available from novelica), GRINDAMYL POWERBAKE (available from Danisco), and BAKEZYME BXP 5000 and BAKEZYME BXP 5001 (available from diesman).

The protease may be from the genus Bacillus, for example Bacillus amyloliquefaciens. Suitable proteases may be available from Novelis

The phospholipase may have phospholipase A1, A2, B, C, D or lysophospholipase activity, and it may or may not have lipase activity. It may be of animal origin, for example from pancreas, snake venom or bee venom, or it may be of microbial origin, for example from a filamentous fungus, yeast or bacteria, such as Aspergillus or Fusarium (Fusarium), for example Aspergillus niger, aspergillus oryzae or Fusarium oxysporum. Preferred lipases/phospholipases from Fusarium oxysporum are disclosed in WO 98/26057. Also, variants described in WO 00/32758 may be used.

Suitable phospholipase compositions are LIPOPAN F, LIPOPAN XTRA and LIPOPAN MAX (available from NoveXin) or PANAMORE GOLDEN and PANAMORE SPRING (available from Dissman).

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

Enzyme composition

The mature thermostable variant glucoamylase of the invention and any one or more additional enzymes may be added to the flour or dough in any suitable form, such as, for example, in liquid (particularly stabilized liquid) form, or it may be added to the flour or dough as a substantially dry powder or granules.

The particles may be produced, for example, as disclosed in U.S. Pat. nos. 4,106,991 and 4,661,452. The liquid enzyme preparation may be stabilized, for example, by adding sugar or sugar alcohol or lactic acid according to established procedures. Other enzyme stabilizers are well known in the art.

The enzyme(s) may be added to the bread dough ingredients in any suitable manner, such as in separate components (separate or sequential addition of enzymes), or by adding the enzymes together in one step or in one composition.

Bread characteristics

The organoleptic quality or attribute of the bread may be measured as known in the art. The characteristics of bread may be referred to herein as organoleptic properties, which include anti-aging (crumb firmness), crumb characteristics and mouthfeel, or more precisely, properties of bread as detected in the mouth during eating (e.g., softness of bread/resistance to first bite, crumb moisture, crumb chewiness and gumminess, and crumb smoothness and thawing characteristics).

In one embodiment, the organoleptic attribute obtained by using the enzyme solution according to the invention for the baked product is increased sweetness.

In one embodiment, the organoleptic attribute obtained by using the enzyme solution according to the invention for the baked product is increased crumb sweetness.

In preferred embodiments of the invention, the final fully baked or partially baked product has reduced initial firmness and/or increased initial elasticity when cooled to room temperature, packaged in a sealed container and stored at room temperature until analyzed, and/or has reduced firmness increase and/or higher elasticity after 1, 7 or 14 days, as compared to a control made without any glucoamylase added.

In another preferred embodiment, the final fully baked or partially baked product has at least the same sweetness or sweetness as a control product made with twice the amount of mature glucoamylase having an amino acid sequence shown in SEQ ID NO:10, preferably as determined as exemplified herein, and preferably the final fully baked or partially baked product has a higher sweetness or higher sweetness than a control product made with twice the amount of mature glucoamylase having an amino acid sequence shown in SEQ ID NO:10, preferably as determined as exemplified herein.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, as these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments, as well as combinations of one or more of the embodiments, are intended to be included within the scope of the present invention.

Various references are cited herein, the disclosure of which is incorporated by reference in its entirety. The invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

Examples

Example 1 construction of PoAMG library

The PoAMG library was constructed as follows:

forward or reverse primers were designed with NNK or one or more desired mutations at one or more target sites that had 15bp overlap with each other. Inverse PCR is performed using the appropriate template plasmid DNA (e.g., plasmid DNA containing the JPO-0001 gene) by reverse-targeting primers to amplify the entire plasmid DNA sequence. The resulting PCR fragment was purified by QIAquick gel extraction kit [ QIAquick company ], and then introduced into escherichia coli ECOS competent escherichia coli dh5α [ lithon GENE co., ltd.) ]. Plasmid DNA was extracted from E.coli transformants by the MagExactor plasmid extraction kit [ TOYOBO ], and then introduced into Aspergillus niger competent cells.

PCR reaction mixture:

PRIMESTAR MAX DNA polymerase [ TaKaRa ] of Takara Shuzo Co., ltd

PCR procedure:

98°C/2min

25x(98°C/10sec,60°C/15sec,72°C/2min)

10 ℃ per hold

Example 2 screening for better thermal stability

The bacillus subtilis library constructed as in example 1 was fermented in 96-well or 24-well MTP containing COVE liquid medium (2.0 g/L sucrose, 2.0g/L isomaltose, 2.0g/L maltose, 4.9mg/L, 0.2ml/L5N NaOH, 10ml/L COVE salt, 10 ml/L1M acetamide) at 32 ℃ for 3 days. AMG activity in the culture supernatant was then measured by the pNPG assay described below at several temperatures.

Thermal stability determination of pNPG:

culture supernatants containing the desired enzyme were mixed with the same volume of pH5.0 200mM NaOAc buffer. Twenty microliters of this mixture was dispensed into 96-well plates or 8-way PCR tubes and then heated by a thermal cycler for 30min at various temperatures. Those samples were mixed with 10. Mu.l of a substrate solution containing 0.1% (w/v) pNPG [ and light Co., ltd. (wako) ] in NaOAc buffer pH5.0 and incubated at 70℃for 20min to perform an enzyme reaction. After the reaction, 60. Mu.l of 0.1M Borax buffer was added to stop the reaction. 80 microliters of the reaction supernatant was removed and its OD ₄₀₅ value was read by photometer to assess enzyme activity.

TABLE 1A list of relative Activity of PoAMG variants compared to their parents anPAV498 or JPO-0001 (anPAV 498 with leader peptide/propeptide)

Name of the name	Relative Activity at 80 ℃/75 (%)
		anPAV498	17%
JPO-004	32%
		JPO-005	15%
JPO-006	16%
		JPO-007	3%

Name of the 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%

Name of the 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%

Name of the 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%

Name of the name	Relative Activity at 79 ℃/77 (%)
		JPO-001	41%
JPO-021	60%
		JPO-022	48%
JPO-023	57%
		JPO-025	56%
JPO-027	64%
		JPO-029	66%
JPO-047	50%
		JPO-048	72%
JPO-051	82%
		JPO-058	73%
JPO-062	72%
		JPO-063	85%
JPO-064	83%

TABLE 1b list of relative Activity of PoAMG variants compared to their parent JPO-022

Name of the 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%

Name of the name	Relative Activity at 79 ℃/77 (%)
		JPO-022	49%
JPO-023	51%
		JPO-025	52%
JPO-027	58%
		JPO-058	69%
JPO-059	36%
		JPO-060	41%
JPO-061	44%
		JPO-062	57%

TABLE 1c list of relative Activity of PoAMG variants compared to their parent JPO-063

Name of the 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%

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

Name of the 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%

TABLE 1 list of relative Activity of PoAMG variants compared to their parent JPO-096

Name of the name	Relative Activity at 83 ℃/70 (%)
		JPO-082	53%
JPO-088	70%
		JPO-091	69%
JPO-092	65%
		JPO-093	62%
JPO-094	74%
		JPO-095	69%
JPO-096	67%
		JPO-097	65%
JPO-098	65%

Name of the 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%

TABLE 1 list of relative Activity of PoAMG variants compared to their parent JPO-129

Name of the 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 1 list of relative Activity of PoAMG variants compared to their parent JPO-166

Name of the name	Relative Activity at 84 ℃/75 (%)
		JPO-166	19%
JPO-167	66%
		JPO-168	58%
JPO-169	53%
		JPO-171	47%
JPO-172	98%

TABLE 2 amino acid substitutions in variants of the PoAMG mature sequence

Example 3 fermentation of Aspergillus niger

Aspergillus niger strains were fermented at 220rpm, 30℃in 500ml baffled flasks containing 100ml MU1 and 4ml 50% urea on a rotary shaker. The culture broth was centrifuged (10,000Xg, 20 min) and the supernatant carefully decanted from the precipitate.

EXAMPLE 4 purification of PoAMG (JPO-001) variants

The PoAMG variant was purified by cation exchange chromatography. The respective peak fractions were pooled individually and dialyzed against 20mM sodium acetate buffer (pH 5.0), and then the samples were concentrated using a centrifugal filtration device (Vivaspin Turbo 15, sartolius Co.). The enzyme concentration was determined by the a280 value.

Example 5 thermal stability measurement (TSA)

Purified enzyme was diluted to 0.5mg/ml with 50mM sodium acetate buffer (pH 5.0) and mixed with an equal volume of SYPRO Orange (Invitrogen) diluted with Milli-Q water. An 18ul of the mixture solution was transferred to a LightCycler 480 multiwell plate 384 (roses diagnostics company (Roche Diagnostics)) and the plate sealed. Device parameters of TSA:

Instrument LightCycler 480 real-time PCR System (Roche APPLIED SCIENCE, department of application of Roche)

Scanning Rate 0.02 ℃/sec

Scanning range of 37-96 DEG C

Integration time 1.0 seconds

Excitation wavelength 465nm

Emission wavelength 580nm

The obtained fluorescence signal is normalized to the range of 0 and 1. Td is defined as the temperature at which the signal strength is 0.5. The thermal stability improvements are listed in table 3, where Td of the PoAMG variant, represented as anPAV498, is 0.

Example 6 PoAMG Activity assay

Maltodextrin (DE 11) assay by GOD-POD method

Substrate solution

30G maltodextrin (pindex #2 from Song Gu chemical Co. (MATSUTANI chemical industry Co., ltd.))

100Ml 120mM sodium acetate buffer, pH 5.0

Glucose CII test kit (Japan and light pure chemical industry Co., ltd. (Wako Pure Chemical Industries, ltd.))

20Ul of enzyme sample was mixed with 100ul of substrate solution and incubated at the set temperature for 2 hours. The sample was cooled on an aluminum block for 3min, and then 10ul of the reaction solution was mixed with 590ul of 1M Tris-HCl (pH 8.0) to stop the reaction. 10ul of the solution was mixed with 200ul of the working solution of the test kit and then allowed to stand at room temperature for 15min. Absorbance was read at a 505. The activities are listed in table 3 as relative activities of PoAMG variants denoted anPAV 498.

Table 3.

Example 7 baking with reduced dose of JPO-172

Bread was baked in a direct fermentation baking process with the recipe according to table 4. The different treatments were performed according to Table 5, wherein the anti-aging commercial baked maltogenic alpha-amylase product with the best performance of JPO-172 compared to the current performance was performed3D (novelin, denmark) for comparison. The bread was baked in a covered baking pan. The ingredients were mixed in a pin mixer for 1min at a first speed to form a dough, and then mixed at a second speed until the dough was fully formed. The dough was left to stand for 5 minutes and divided into 645g dough pieces and rounded. The round dough pieces were left to stand for 10min, sheeted, and placed in a baking pan. The dough-filled bakeware was proofed to a certain height at 104-109F and 85% relative humidity. For covered pans, the dough is proofed until the dough is 3/4 "from the top. The proofed dough was baked in a roller oven at about 227 ℃ (440 ℃) for 17 minutes.

TABLE 4 formulation

TABLE 5 treatment of

The bread was packaged in sealed plastic bags 2 hours after baking and stored at room temperature until analysis.

The texture of the bread was evaluated using a texture analyzer (TA-XTplus, stability microsystems, godelming, UK). Crumb texture properties are characterized by the firmness (same as "hardness" and opposite to "softness") and elasticity of the baked product. The standard method for measuring firmness and elasticity is based on force-deflection of baked products. Force-deformation of the baked product can be performed with a 40mm diameter cylindrical probe. The force on the cylindrical probe was recorded when it was pressed down 27% stress on a 25mm thick slice of bread at a deformation rate of 1 mm/sec. The probe was then held in this position for 30 seconds while the force was recorded, and then the probe was returned to its original position.

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

Elasticity (in%) is defined as the force recorded after 30 seconds of compression at 27% stress (corresponding to the force at time=36.75 s for a slice of bread having a thickness of 25 mm) divided by the force required to press the probe 6.75mm into the flesh (corresponding to the force at time=6.75 s for a slice of bread having a thickness of 25 mm) times 100.

The results of the texture analysis can be seen in tables 6 and 7. Fresh bread is soft (low firmness) and elastic. When the bread is stored, it becomes stiffer and less elastic. The higher the dosage of JPO-172, the lower the firmness and higher the elasticity of the bread at days 7-21. However, under these conditions and at these doses, the most soft and elastic is of the type having, over a period of 1-21 daysBread of 3D 750MANU/kg flour.

Table 6. Crumb firmness (g) at different time points.

Table 7. Crumb elasticity (%) at different time points.

Example 8 baking with reduced dose of JPO-172 at lower pH

Bread was baked in a direct-ferment baking process with the recipe according to table 8. Different treatments were performed according to table 9. The bread was baked in a covered baking pan. The ingredients were mixed in a pin mixer for 1min at a first speed to form a dough, and then mixed at a second speed until the dough was fully formed. The dough was left to stand for 5 minutes and divided into 645g dough pieces and rounded. The round dough pieces were left to stand for 10min, sheeted, and placed in a baking pan. The dough-filled bakeware was proofed to a certain height at 104-109F and 85% relative humidity. For covered pans, the dough is proofed until the dough is 3/4 "from the top. The proofed dough was baked in a roller oven at about 227 ℃ (440 ℃) for 17 minutes.

TABLE 8 formulation

TABLE 9 treatment of

Texture analysis was performed as in example 7. The results of the texture analysis can be seen in tables 10 and 11. Fresh bread is soft (low firmness) and elastic. When the bread is stored, it becomes stiffer and less elastic in the absence of anti-staling enzymes.

We conclude that adding 2% vinegar, thereby lowering the pH of the dough, allows the dosage of JPO-172 to be reduced below 12mgEP/kg flour while still providing a good fit3D is equivalent or better in anti-aging effect.

Table 10. Crumb firmness (g) at different time points.

Table 11. Crumb elasticity (%) at different time points.

EXAMPLE 9 Activity of JPO-172 as a function of pH

The pH activity curve is a characteristic property of enzymes, which is of great importance for their use in different applications. The pH profile (in the range of pH 2-10) of JPO-172 was determined at 40℃and 30 min incubation.

A30 mM maltose (CAS number: 6363-53-7) substrate solution was prepared in buffer (0.1M acetic acid; 0.1M MES;0.1M HEPES;0.1M glycine) and adjusted to pH 2-10 using HCl or NaOH. The pH activity profile was prepared by adding 15. Mu.L of diluted enzyme sample (10 ppm, diluted in 20mM MES, pH 5) or buffer to 135. Mu.L of substrate solution in an Eppendorf tube. The pH of the reaction mixture was determined. The mixture was incubated at 40℃and 800rpm for 30 minutes. After 30 minutes, the reaction was stopped by adding 16. Mu.L of 0.5MNaOH and placed on ice. The reaction mixture was diluted 10-fold in a microtiter plate with 20mM MES (pH 5). mu.L of the diluted solution was mixed with 160. Mu.L of GOD-POD reaction mixture in a new microtiter plate and incubated for 30 minutes in the absence of light. After incubation, absorbance at 420nm was measured. The average of the reaction mixture was subtracted from the average of the blank sample as shown in fig. 2.

The pH-activity curve is shown in FIG. 2. All data points are averages of four measurements and give a relative pH of 5, which is set at 100%. In this curve, it can be seen that the optimum pH is around pH 5, and about 80% activity is observed from about pH 4 to 6.

Claims

1. A method for producing a baked or partially baked product, the method comprising:

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

b) baking or part baking the dough to produce a baked or part baked product.

2. The method according to claim 1, wherein the baked or part-baked product is a type of bread, preferably tinned bread, toasted bread, open-faced bread, buns, fino bread, hammam bread, samoli bread, baguette, brioche, hamburger bun, roll, brown bread, wholemeal bread, galette bread, bran bread, flatbread, tortillas, or biscuits, cakes or pastries.

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

4. The method of any one of claims 1 to 3, wherein the mature variant comprises at least one amino acid modification at one or more or all of the positions corresponding to positions 1, 2, 4, 6, 7, 11, 31, 34, 65, 79, 103, 132, 327, 445, 447, 481, 566, 568, 594 and 595 in SEQ ID NO: 1.

5. The method according to claim 4, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 2, 4, 11, 65, 79 and 327 in SEQ ID NO: 1, preferably, the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to R1A, P2N, P4S, P11F, T65A, K79V and Q327F in SEQ ID NO: 1.

6. The method of claim 4, wherein the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to positions 1, 6, 7, 31, 34, 79, 103, 132, 445, 447, 481, 566, 568, 594 and 595 in SEQ ID NO: 1, preferably, the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to R1A, G6S, G7T, R31F, K34Y, K79V, S103N, A132P, D445N, V447S, S481P, D566T, T568V, Q594R and F595S in SEQ ID NO: 1.

7. The method according to any one of claims 1 to 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595 in SEQ ID NO: 1, preferably, the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, K79V, S103N, A132P, D445N, V447S, S481P, T484P, E501A, N539P, D566T, T568V, Q594R and F595S in SEQ ID NO: 1.

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

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

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

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

12. The method according to any one of claims 1 to 11, wherein the baked or partly baked product after final full baking has at least the same sweetness as a control product made with twice the amount of mature glucoamylase, the amino acid sequence of which is shown in SEQ ID NO: 10.

13. The method of any one of claims 1-12, wherein the dough further comprises one or more additional enzymes selected from the group consisting of: alpha-amylase, maltogenic amylase, raw starch degrading alpha-amylase, beta-amylase, aminopeptidase, carboxypeptidase, catalase, cellulolytic enzyme, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1,4-alpha-maltotetraose hydrolase, glucanase, galactanase, alpha-galactosidase, beta-galactosidase, glucose oxidase, alpha-glucosidase, beta-glucosidase, haloperoxidase The one or more additional enzymes may be selected from the group consisting of enzymes, hemicellulolytic enzymes, invertases, laccases, lipases, mannanases, mannosidases, oxidases, pectinolytic enzymes, peptidylglutaminases, peroxidases, phospholipases, phytases, polyphenol oxidases, proteolytic enzymes, ribonucleases, transglutaminases and xylanases; preferably, the one or more additional enzymes are comprised in an amount of 01-1,000 mg enzyme protein (mgEP)/kg flour, preferably in an amount of 0.01-500 mg enzyme protein (mgEP)/kg flour, even more preferably in an amount of 0.1-100 mg enzyme protein (mgEP)/kg flour.