Method for baking pulse protein fortified bread using thermally stable amyloglucosidase variants (EC 3.2. 1.3)

Abstract

Disclosed are methods of producing a baked or partially baked product, the methods comprising providing a dough comprising added legume and / or pod protein and 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; and baking or partially baking the dough to produce the baked or partially baked product.

Description

[0001] Reference to the Sequence Listing

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

[0003] The present invention relates to methods for producing baked or par-baked products, which methods comprise providing a dough comprising added legume and / or pod protein and a mature thermostable variant of a parental glucoamylase having at least 70% identity to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10; and baking or par-baking the dough to produce the baked or par-baked product. Background Art

[0004] Due to the increasing global interest in healthy foods, there is a growing commercial interest in foods with a high protein and / or fiber content, and high-protein baked products are no exception. However, simply adding flour from high-protein crops, such as legume flour, i.e., flour made from the edible seeds of leguminous plants, to the dough has a significant and detrimental effect on the volume of the resulting baked product, as shown herein. There is a great need for a technical solution to overcome the negative impact on volume.

[0005] Par-baking is a technique in which bread or another dough product is baked partially (i.e., "par") and then typically cooled or frozen for storage. When a final baked product is needed, the cooled or frozen par-baked product is baked at normal baking temperature typically for 5 to 15 minutes; the resulting type of baked product is commonly referred to as "bake-off".

[0006] WO 2021 / 239267 discloses methods for producing baked or par-baked edible products from a dough comprising added legume and / or pod protein and at least one added lipase, wherein at least 2% (w / w) of the total flour content is added legume and / or pod protein. Summary of the Invention

[0007] The inventors have found that certain thermostable variants of glucoamylase exhibit improved performance in baking with added legume and / or pod protein. One improved performance of these thermostable variants is that they increase the sweetness or sweetness of the product, which allows for a reduction in the amount of sugar added in traditional formulations and also, to some extent, can mask the additional bitterness from the added legume and / or pod protein.

[0008] Accordingly, in a first aspect, the present invention relates to methods for producing baked or partially baked products, which methods comprise providing a dough that contains added legume and / or pod protein and a mature thermostable variant of a parental glucoamylase having at least 70% identity to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10; and baking or partially baking the dough to produce the baked or partially baked product.

[0009] Preferably, the mature thermostable variant of the parental glucoamylase of the present invention has at least 71% identity to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 shows a multiple alignment of the amino acid sequences of the following mature proteins:

[0011] - wild-type AMG (PoAMG) from Penicillium oxalicum of SEQ ID NO:1

[0012] - PoAMG variant designated "AMG NL" of SEQ ID NO:2

[0013] - PoAMG variant designated "AMG anPAV498" of SEQ ID NO:3

[0014] - PoAMG variant designated "AMG JPO001" of SEQ ID NO:4

[0015] - PoAMG variant designated "AMG JPO124" of SEQ ID NO:5

[0016] - The PoAMG variant designated as "AMG JPO172" of SEQ ID NO:6

[0017] - The wild-type AMG (PoAMG) from Penicillium miczynskii of SEQ ID NO:7

[0018] - The wild-type AMG (PoAMG) from Penicillium russellii of SEQ ID NO:8

[0019] - The wild-type AMG (PoAMG) from Penicillium glabrum of SEQ ID NO:9 Detailed implementation manners

[0020] Definitions

[0021] Pod: A pod is a plant of the Fabaceae family (or Leguminosae), or the fruit or seed of such a plant (also known as pulses, especially under mature, dry conditions). Well-known pods include alfalfa, clover, kidney bean, pea, chickpea, lentil, lupin, mesquite, carob, soybean, peanut, and tamarind. Pods produce a botanically distinct type of fruit - a simple dry fruit that develops from a simple carpel and usually splits on two sides (opens along the seam).

[0022] Pulses: The Food and Agriculture Organization of the United Nations (FAO) recognizes 11 types of pulses: dry common bean, dry broad bean, dry pea, chickpea, cowpea, pigeon pea, lentil, bambara groundnut, winged bean, lupin, and pulses NES (i.e., small pulses, including: lentil, hyacinth bean (Lablab purpureus), jack bean (Canavalia ensiformis), sword bean (Canavalia gladiata), winged bean (Psophocarpus tetragonolobus), velvet bean, cowitch (Mucuna pruriens var. utilis), yam bean (Pachyrhizus erosus)).

[0023] Bean and / or pod protein: The term "bean and / or pod protein" means bean protein and / or pod protein, the desired component of bean flour and / or pod flour; the term also includes processed and / or deodorized bean and / or pod flour, wherein the processed flour has a higher protein content than the unprocessed flour. Processed or deodorized bean and / or pod flour may also be referred to as bean and / or pod protein concentrate and / or isolate, respectively.

[0024] Deodorized bean and / or pod flour or protein: In the context of the present invention, the term "deodorized" means that the flour or protein component has been processed to reduce off-flavors, such as bitterness.

[0025] Lipase activity: Triacylglycerol lipase activity (EC 3.1.1.3), i.e., the hydrolytic activity towards the carboxylic ester bond in triacylglycerols (e.g., tributyrin).

[0026] Phospholipase activity: Phospholipase activity (A1 or A2, EC 3.1.1.32 or 3.1.1.4), i.e., the hydrolytic activity towards one or two carboxylic ester bonds in phospholipids (such as lecithin).

[0027] Galactolipase activity: Galactolipase activity (EC 3.1.1.26), i.e., the hydrolytic activity towards the carboxylic ester bond in galactolipids (such as DGDG (digalactosyldiacylglycerol)).

[0028] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form after translation and any post-translational modifications (such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.).

[0029] Sequence identity: The degree of relatedness between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0030] For the purposes of the present invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) is used to determine the sequence identity between two amino acid sequences, which is implemented as in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277, preferably version 5.0.0 or later). The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix. The output of "longest identity" marked by Needle (obtained using the non-abbreviated (-no brief) option) is taken as the percentage of identity and calculated as follows:

[0031] (Identical residues × 100) / (Alignment length - Total number of gaps in the alignment)

[0032] Variant: The term "variant" means a polypeptide that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions. Substitution means replacing the amino acid occupying a position with a different amino acid; deletion means removing the amino acid occupying a position; and insertion means adding one or more amino acids adjacent to and immediately following the amino acid occupying a position. The amino acid alterations can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1 - 30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as a methionine residue at the amino terminus; small linker peptides of up to 20 - 25 residues; or small extensions that facilitate purification by altering the net charge or another function (such as a polyhistidine segment, an epitope, or a binding domain). Examples of conservative substitutions are within the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0033] Improved thermal stability: The improvement in thermal stability (Td) in °C is a measure of how much the variant has improved in thermal stability relative to its parental glucoamylase under the same conditions, as determined as exemplified herein.

[0034] Improved crumb firmness of baked products: The term "improved crumb firmness" is defined herein as the property of a baked product that is more easily compressible compared to a baked product in which the enzyme solution of the present invention is not added to the dough.

[0035] Crumb firmness is evaluated empirically by a skilled test baker / sensory panel or measured using a texture analyzer known in the art (e.g., TAXT2 or TA-XT Plus from Stable Micro Systems, Surrey, UK).

[0036] Improved flavor of baked products: The term "improved flavor of baked products" is evaluated by a trained test panel and / or chemical analysis (e.g., headspace GC-MS analysis). The improved flavor of baked products includes reducing one or more off-flavors of the baked products.

[0037] Improved anti-aging of baked products: The term "improved anti-aging of baked products" is defined herein as a property of a baked product that has a reduced rate of deterioration of quality parameters (e.g., softness and / or elasticity) during storage.

[0038] Volume of baked products: The term "volume of baked products" is defined herein as a measure of the volume of a given loaf of bread. The volume can be determined by the rapeseed displacement method.

[0039] Bread color: The color or whiteness of a baked or partially baked product is measured as the "color L*" value in a C-Hole Chamber (Caliber Instruments Ltd, Warrington, UK) using standard methods for collecting images and standard C-Hole Chamber software for data analysis.

[0040] Dough according to the present invention

[0041] The present invention relates to a dough for a baked or partially baked product, the dough comprising added legume and / or pod protein.

[0042] The term "added" is defined herein as adding the protein and / or enzyme according to the present invention to the dough, to any ingredient to be made into dough, and / or to any mixture of dough ingredients to be made into dough.

[0043] In other words, these proteins and / or enzymes can be added at any step of dough making and can be added in one, two, or more steps. They can be added to dough ingredients, and the dough can be kneaded and processed as is known in the art for baked and / or partially baked products.

[0044] The term "effective amount" is defined herein as an amount of an enzyme composition according to the present invention that is sufficient to provide a measurable effect on at least one property of interest of the dough and / or the baked product.

[0045] The term "dough" is defined herein as a mixture of flour and other baking ingredients that is firm enough to be kneaded or rolled. In the context of the present invention, batter is encompassed within the term "dough"; preferably, the dough of the present invention comprises wheat flour.

[0046] In a preferred embodiment, the dough composition comprises wheat flour; preferably, 2% (w / w) or more of the total flour content is wheat flour; preferably, 4% (w / w) or more of the total flour content is wheat flour, preferably at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or preferably at least 95% (w / w) of the flour is wheat flour.

[0047] The dough of the present invention may comprise flours derived from any grain or other source, including wheat, emmer, spelt, einkorn, barley, rye, oats, corn, sorghum, rice, millet, amaranth, quinoa, cassava, and any combination thereof.

[0048] In a preferred embodiment of the present invention, legume and / or pod protein is added to the dough in the form of legume and / or pod flour, processed legume and / or pod flour, deodorized legume and / or pod flour, or a protein concentrate and / or isolate made essentially from legume and / or pod flour; preferably, the added legume and / or pod protein comprises lentil protein, chickpea protein, pea protein, and / or fava bean protein, or a protein concentrate and / or isolate thereof.

[0049] A preferred embodiment relates to a dough according to the first aspect, wherein at least 4% (w / w) of the total flour content is added legume and / or pod protein, preferably at least 6% (w / w) of the total flour content is added legume and / or pod protein, more preferably at least 8% (w / w) of the total flour content is added legume and / or pod protein, even more preferably at least 10% (w / w) of the total flour content is added legume and / or pod protein, and most preferably, at least 12% (w / w) of the total flour content is added legume and / or pod protein.

[0050] Preferably, the dough of the present invention further comprises gluten.

[0051] The dough may further comprise other conventional dough ingredients, such as proteins, such as milk powder, gluten, sources of dietary fiber (such as wheat, oat bran, β-glucan, and / or inulin), and eggs (whole eggs, egg yolks, or egg whites); oxidants, such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA), or ammonium persulfate; amino acids, such as L-cysteine; sugars; salts, such as sodium chloride, calcium acetate, sodium sulfate, or calcium sulfate, and / or emulsifiers.

[0052] In a preferred embodiment of the present invention, the dough of the present invention further comprises gluten.

[0053] The dough may contain a fat (triglyceride), such as particulate fat or oil.

[0054] The dough of the present invention is generally a fermented dough or a dough to be subjected to fermentation.

[0055] The dough can be fermented in various ways, such as by adding a chemical leavening agent (e.g., baking powder, sodium bicarbonate) or by adding a leavening agent (fermented dough), but preferably by adding a suitable yeast culture such as a culture of Saccharomyces cerevisiae (baker's yeast) (e.g., a commercially available strain of Saccharomyces cerevisiae) to ferment the dough.

[0056] The dough of the present invention may contain at least one added lipase, preferably a lipase and / or phospholipase, preferably a mature lipase and / or mature phospholipase. Preferably, the at least one added lipase comprises a mature lipase having an amino acid sequence that has at least 70% identity with one or more of the sequences shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21; preferably having at least 75% identity, at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or preferably at least 99% identity with one or more of the sequences shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21.

[0057] Preferably, the at least one added lipase is added in an amount in the range of 0 to 100 mg of enzyme protein / kg of flour; preferably in the range of 0 to 50 mg of enzyme protein / kg of flour; more preferably in the range of 0 to 25 mg of enzyme protein / kg of flour; even more preferably in the range of 0 to 10 mg of enzyme protein / kg of flour; still more preferably in the range of 0 to 5 mg of enzyme protein / kg of flour; and most preferably in the range of 0 to 2.5 mg of enzyme protein / kg of flour.

[0058] Preferably, the dough of the first aspect further comprises at least one additionally added enzyme, preferably at least one mature α-amylase, more preferably a mature maltogenic α-amylase; preferably a mature maltogenic α-amylase from Bacillus stearothermophilus; more preferably having an amino acid sequence with at least 70% identity to the sequence of SEQ ID NO:6, preferably at least 75%, at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or preferably at least 99% identity to the sequence of SEQ ID NO:11. Preferably, the mature maltogenic α-amylase is added in an amount in the range of 0 to 10,000 MANU / kg of flour; preferably in the range of 0 to 7,500 MANU / kg of flour; preferably in the range of 0 to 5,000 MANU / kg of flour.

[0059] Preferably, the at least one additionally added enzyme comprises a mature α-amylase; preferably a mature fungal α-amylase; more preferably a mature α-amylase from Aspergillus oryzae; preferably, the additional mature α-amylase is added in an amount in the range of 0 to 1,000 FAU / kg of flour; preferably in the range of 0 to 500 FAU / kg of flour; more preferably in the range of 0 to 100 FAU / kg of flour; even more preferably in the range of 0 to 50 FAU / kg of flour; and most preferably in the range of 0 to 25 FAU / kg of flour.

[0060] Preferably, the dough of the first aspect further comprises at least one additionally added enzyme, and the at least one additionally added enzyme comprises at least one mature xylanase, preferably a GH5, GH8 and / or GH11 xylanase.

[0061] The present invention is particularly useful for preparing yeast-fermented dough, baked or partially baked products in an industrial process, wherein the dough used to prepare the baked or partially baked products is mechanically prepared using automated or semi-automated equipment.

[0062] The process for preparing bread generally involves the following sequential steps: making the dough (with an optional proofing step), sheeting or dividing, shaping or rolling the dough, and proofing, which steps are well known in the art. If an optional proofing step is used, preferably more flour is added and an alkali can be added to neutralize the acid produced or to be produced during the second proofing step. In the industrial baking production process according to the present invention, the following automated or semi-automated equipment is used to perform one or more of these steps, for example:

[0063] Horizontal mixer: A roller mixer equipped with rotating arms, which had two speed settings in older models, typically slow mixing at 35 rpm and fast mixing at 70 rpm, while newer models more often have variable speed settings in the range of 15 - 120 rpm.

[0064] Vertical mixer: A spiral mixer is typically a mixer with a rotating bowl and a spiral that counteracts the rotation. Some spiral mixers can be bidirectional to provide better ingredient distribution.

[0065] The purpose of mixing is the uniform mixing and hydration of dry materials, kneading the dough to form a gluten network and introducing air into the dough. Both types of mixers typically mix at two speeds: slow to gather the dough without pushing it to the sides of the bowl, and fast to help form the gluten network.

[0066] In a preferred embodiment, the dough is mixed as follows:

[0067] a) At a slow mixing speed, preferably in the range of 5 - 50 rpm, more preferably in the range of 10 - 40 rpm, for at least 5 minutes; more preferably for at least 10 minutes at a slow mixing speed, even more preferably for at least 15 minutes at a slow mixing speed; and optionally

[0068] b) Subsequently mix the dough at a faster speed.

[0069] Partially baked product

[0070] Partial baking is a technique of partially baking bread or dough products and then typically rapidly cooling / freezing for storage.

[0071] The raw dough is baked normally but stopped at about 80% of the normal cooking time and then rapidly cooled.

[0072] Partially baked dough product bread can be easily transported and stored until needed. The partially baked dough products are kept in a sealed container to prevent moisture loss. They can be stored at room temperature; or in the refrigerator, or in the freezer.

[0073] The freezing step can cause ice crystal formation and subsequently damage to starch granules and amylose leakage. Therefore, the amount of leaked amylose and unbound water may be higher in bread baked without a freezing step before the second full baking. These are two parameters known to increase crumb firmness.

[0074] When the final dough product is required, the partially baked product is "finished" by baking it for a further period at normal temperature (typically 5 to 15 minutes). The exact time must be determined by testing as it varies according to the product.

[0075] Thus, the partially baked product is manufactured by the following steps:

[0076] a) shaping the dough into a product,

[0077] b) baking the product,

[0078] c) storing the product, and

[0079] d) rebaking the product into a partially baked product.

[0080] The product can be stored at ambient temperature / room temperature or the product can be stored at low temperature, which means it will typically be stored at a temperature below 5 degrees Celsius. In one embodiment, the product will be stored in a freezer.

[0081] The process of the present invention can be used for any kind of partially baked product prepared from dough, especially soft, whether of the white, light or dark type.

[0082] Examples are bread (especially white, wholemeal or rye bread), typically in the form of loaves or rolls, bread, flatbreads, pitta breads, tortillas, cakes, pancakes, biscuits, wafers, cookies, pie crusts, pizza, etc.

[0083] Glucoamylase

[0084] Glucoamylase is also known as amyloglucosidase and dextran 1,4-α-glucosidase (EC 3.2.1.3), and more commonly they are referred to as AMG.

[0085] According to the present invention, different types of glucoamylases can be used as parents for generating thermostable glucoamylase variants. For example, the glucoamylase can be a polypeptide encoded by a DNA sequence found in a fungal strain of the genus Aspergillus, Rhizopus, Talaromyces (Rasamsonia) or Penicillium; preferably a DNA sequence found in a fungal strain of the genus Penicillium, even more preferably a DNA sequence found in a fungal strain of Penicillium oxysporum, Penicillium oxalicum, Penicillium meleagrinum, Penicillium roqueforti or Penicillium glabrum. Preferably, the parental glucoamylase is from a species of the genus Penicillium, preferably from Penicillium oxalicum, Penicillium meleagrinum, Penicillium roqueforti or Penicillium glabrum.

[0086] Examples of other suitable fungi include Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Rhizopus delemar, Rhizopus niveus, Rhizopus oryzae, and Talaromyces emersonii (Rasamsonia emersonii).

[0087] The following shows Figure 1 the % identity between the AMG amino acid sequences aligned in

[0088]

[0089]

[0090] Thermostable variants of PoAMG have been generated (see Table 2 below). In a preferred embodiment, the mature thermostable glucoamylase variant of the invention comprises one or more or all combinations of the amino acid substitutions listed in Table 2 below.

[0091] In a preferred embodiment, the mature variant of the present invention comprises at least one amino acid modification at one or more or all positions corresponding to positions 1, 2, 4, 6, 7, 11, 31, 34, 50, 65, 79, 103, 132, 327, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595 in SEQ ID NO:1; preferably, the at least one amino acid modification comprises substitutions 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 substitutions at one or more or all positions corresponding to R1A, P2N, P4S, P11F, T65A, K79V and Q327F in SEQ ID NO:1; or preferably, the at least one amino acid modification comprises substitutions 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 substitutions at one or more or all positions corresponding to R1A, G6S, G7T, R31F, K34Y, K79V, S103N, A132P, D445N, V447S, S481P, D566T, T568V, Q594R and F595S in SEQ ID NO:1; or preferably, the at least one amino acid modification comprises substitutions 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 substitutions at one or more or all 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;Alternatively, preferably, the at least one amino acid modification comprises substitutions 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 substitutions at one or more or all positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, K79V, S103N, A132P, D445N, V447S, S481P, T484P, E501A, N539P, D566T, T568V, Q594R, and F595S in SEQ ID NO:1; or preferably, the at least one amino acid modification comprises substitutions 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 substitutions at one or more or all 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.;

[0092] The improvement in thermal stability (Td) of the variants in Table 2 is listed in Table 3, where the Td of the PoAMG variant designated as "anPAV498" (parent) is set to zero. In a preferred embodiment, the mature thermostable variant of the invention has an improvement in thermal stability (Td) of at least 5°C, preferably at least 6°C, 7°C, or 8°C relative to its parent, preferably as determined as exemplified herein.

[0093] In another preferred embodiment, the mature thermostable variant of the invention has a relative activity of at least 150, preferably at least 200, more preferably at least 250, and most preferably at least 300 at 91°C compared to its parent.

[0094] Source of phospholipase

[0095] The phospholipase can be prokaryotic, particularly bacterial, or eukaryotic, such as from fungal or animal sources.

[0096] Phospholipases can be derived from, for example, the following genera or species: Thermomyces, Thermomyces lanuginosus (also known as Humicola lanuginosa); Humicola, Humicola insolens; Fusarium, Fusarium oxysporum, Fusarium solani, Fusarium heterosporum; Aspergillus, Aspergillus tubigensis, Aspergillus niger, Aspergillus oryzae; Rhizomucor; Candida, Candida antarctica, Candida rugosa; Penicillium, Penicillium camembertii; Rhizopus, Rhizopus oryzae; Absidia; Dictyostelium; Mucor; Neurospora; Rhizopus, Rhizopus arrhizus, Rhizopus japonicus; Sclerotinia; Trichophyton, Whetzelinia; Bacillus; Citrobacter; Enterobacter; Edwardsiella; Erwinia; Escherichia, Escherichia coli; Klebsiella; Proteus, Providencia; Salmonella, Serratia, Shigella; Streptomyces; Yersinia; Pseudomonas, or Pseudomonas cepacia.

[0097] Phospholipases can be produced in suitable host cells known in the art.

[0098] Phospholipases can also be obtained from bee or snake venom or from mammalian pancreas such as porcine pancreas.

[0099] WO 98 / 26057 discloses lipases / phospholipases from Fusarium oxysporum and their use in baking.

[0100] WO 2004 / 099400 discloses various phospholipases and their use in baking for reducing dough stickiness.

[0101] Suitable commercial phospholipase preparations are Lipopan F TM 、Lipopan Xtra TM and Lipopan Prime TM (available from Novozymes A / S).

[0102] Other available phospholipases are, for example, Panamore available from DSM TM 。

[0103] Commercial lipase preparations are, for example, Lipopan F available from Novozymes A / S TM and Lipopan 50BG TM 。

[0104] α-Amylase

[0105] α-Amylase (α-1,4-glucan-4-glucanohydrolase, EC.3.2.1.1) constitutes a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glycosidic oligosaccharides and polysaccharides.

[0106] A number of α-amylases are known as Termamyl TM 、 SC and "Termamyl TM -like α-amylase", and can be learned from, for example, WO 90 / 11352, WO 95 / 10603, WO 95 / 26397, WO 96 / 23873 and WO 96 / 23874.

[0107] Another group of α-amylases are known as Fungamyl TM and "Fungamyl TM -like α-amylase", which are α-amylases related to the α-amylase derived from Aspergillus oryzae disclosed in WO 01 / 34784.

[0108] A preferred group of α - amylases are called maltogenic α - amylases (EC 3.2.1.133), typically derived from Bacillus stearothermophilus. Preferred maltogenic α - amylases have an amino acid sequence with at least 70% identity to SEQ ID NO:11 herein, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO:11 herein.

[0109] Also preferred is a group of sugar - tolerant maltogenic α - amylase variants, such as those disclosed in WO 2006 / 032281 (Novozymes A / S), where a number of sugar - tolerant variants are provided, each of which contains an amino acid alteration that is a substitution or deletion or insertion adjacent to I15, R18, K44, N86, T87, G88, Y89, H90, Y92, W93, F188, T189, O190, P191, A192, F194, L196, O329, N371, O372, P373, N375 or R376. Herein, we refer to one such preferred variant as sugar - tolerant maltogenic α - amylase 1 (ST - MAA1), and it has the following three substitutions: F188L, D261G and T288P. WO 2008 / 148845 also discloses a number of preferred sugar - tolerant maltogenic α - amylase variants, each of which contains the two substitutions D261G and T288P and at least one additional amino acid alteration that is a substitution or deletion or insertion adjacent to Y89, W93, P191, F194, Y360 or N375. We refer to one such preferred variant as sugar - tolerant maltogenic α - amylase 2 (ST - MAA2), and it has the following four substitutions: F194Y, D261G, T288P and N375S.

[0110] Yet another group of preferred mature α - amylases are mature non - maltose - producing maltotetraose - hydrolyzing amylases. WO2004 / 111217 (Danisco A / S) discloses a number of preferred thermostable variants of Pseudomonas saccharophilia amylases or maltotetraose - hydrolyzing amylases having the amino acid sequence shown in SEQ ID NO:12 that do not produce maltose, each of these variants containing one or more of the following substitutions: G69P, A141P, G223A, A268P, G313P, S399P, and G400P.

[0111] WO 2007 / 148224 (Danisco A / S) discloses more preferred mature non - maltose - producing anti - aging variants of Pseudomonas saccharophilia amylases having the amino acid sequence shown in SEQ ID NO:12, each of these variants containing an amino acid substitution of lysine (K) or arginine (R) at position 307.

[0112] WO 2010 / 133644 discloses other preferred mature non - maltose - producing variants of Pseudomonas saccharophilia amylases having the amino acid sequence shown in SEQ ID NO:12, each of these variants containing one or more substitutions at positions including 42, 88, 205, 223, 235, 240, 311, 392, and 409. One such preferred variant of Pseudomonas saccharophilia amylase is disclosed in SEQ ID NO:31 of WO 2010 / 133644, the amino acid sequence of which is also shown in SEQ ID NO:13 herein, and we designate it as: HPL G+.

[0113] Another preferred variant of Pseudomonas saccharophilia amylase is disclosed in SEQ ID NO:21 of WO 2007 / 148224, the amino acid sequence of which is also shown in SEQ ID NO:14 herein, and we designate it as: HPL G4.

[0114] Other preferred non - maltose - producing α - amylases are disclosed in WO 2005003339 (Danisco A / S), WO 2005007818 (Danisco A / S), and WO 2022 / 216801 (DuPont Nutrition Biosciences ApS).

[0115] Preferred mature maltose-non-producing α-amylases have an amino acid sequence that has at least 70% identity with SEQ ID NO:12, 13, or 14 herein, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity with SEQ ID NO:12, 13, or 14 herein; in addition, preferred mature maltose-non-producing α-amylases have one or more of the substitutions listed in the above paragraph.

[0116] Yet another group of preferred mature α-amylases are raw starch-degrading α-amylases. As used herein, "raw starch-degrading α-amylase" refers to an enzyme that can directly degrade raw starch granules below the starch gelatinization temperature.

[0117] Examples of raw starch-degrading α-amylases include those disclosed in WO 2005 / 003311, US Patent Publication No. 2005 / 0054071, and US Patent No. 7,326,548. Examples also include those enzymes disclosed in Tables 1 to 5 of the examples in US Patent No. 7,326,548, in US Patent Publication No. 2005 / 0054071 (Table 3 on page 15), together with the enzymes disclosed in WO2004 / 020499, WO 2006 / 06929, and WO 2006 / 066579, and those enzymes disclosed in the sequence listing and specification of WO 2006 / 069290 (Novozymes A / S) or in WO 2013 / 006756 (Novozymes A / S), both of which are incorporated herein by reference in their entirety.

[0118] In one embodiment, the raw starch-degrading α-amylase is a GH13_1 amylase.

[0119] In one embodiment, the raw starch degrading α-amylase has the following amino acid sequence, which has at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity with the raw starch degrading α-amylase shown in European Patent No. 2981170 (Novozymes A / S) or SEQ ID NO: 15 or 16 herein.

[0120] Additional enzyme

[0121] In a preferred embodiment of the first aspect, one or more additional enzymes are added to the dough, and the additional enzymes may be selected from the group consisting of: α-amylase, maltogenic amylase, raw starch degrading α-amylase, β-amylase, aminopeptidase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, dextran 1,4-α-maltotetraohydrolase, dextranase, β-glucanase, galactanase, α-galactosidase, β-galactosidase, glucose oxidase, ɑ-glucosidase, β-glucosidase, haloperoxidase, hemicellulase, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinase, peptidylglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, protease, pullulanase, raw starch degrading α-amylase, ribonuclease, transglutaminase, and xylanase.

[0122] Enzyme composition

[0123] The mature thermostable variant glucoamylase of the present invention and any one or more additional enzymes can be added in any suitable form, such as in liquid (especially stabilized liquid) form, or can be added as a substantially dry powder or granule.

[0124] For example, the granules can be produced as disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452. The liquid enzyme preparation can 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.

[0125] One or more enzymes can be added in any suitable manner, such as in separate components (adding the enzymes separately or sequentially), or adding these enzymes together in one step or in one composition.

[0126] Granules and agglomerated powders can be prepared by conventional methods, for example, by spraying enzymes onto a carrier in a fluidized bed granulator. The carrier can consist of fine particle nuclei with suitable particle sizes. 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.

[0127] Examples

[0128] Example 1: Construction of the PoAMG library

[0129] The PoAMG library was constructed as follows:

[0130] Forward or reverse primers designed to have NNK or one or more desired mutations at one or more target sites, with 15 bp overlaps between these target sites. Reverse PCR was performed using an appropriate template plasmid DNA (e.g., plasmid DNA containing the JPO - 0001 gene) under the following conditions, i.e., amplifying the entire plasmid DNA sequence with reverse - oriented primers. The resulting PCR fragments were purified using a QIAquick Gel Extraction Kit [QIAGEN], and then introduced into competent Escherichia coli DH5α [NIPPON GENE CO., LTD.] of Escherichia coli ECOS. Plasmid DNA was extracted from the Escherichia coli transformants using a MagExtractor Plasmid Extraction Kit [TOYOBO], and then introduced into Aspergillus niger competent cells.

[0131] PCR reaction mixture:

[0132] PrimeSTAR Max DNA polymerase [TaKaRa]

[0133] Total 25 μl

[0134] 1.0 μl template DNA (1 ng / μl)

[0135] 9.5 μl H 2 O

[0136] 12.5 μl 2x PrimeSTAR Max Premix

[0137] 1.0 μl forward primer (5 μM)

[0138] 1.0 μl reverse primer (5 μM)

[0139] PCR procedure:

[0140] 98°C / 2 min

[0141] 25 x (98°C / 10 sec, 60°C / 15 sec, 72°C / 2 min)

[0142] 10°C / hold

[0143] Example 2: Screening for better thermal stability

[0144] The Bacillus subtilis library constructed as in Example 1 was fermented in 96-well or 24-well MTPs containing COVE liquid medium (2.0 g / L sucrose, 2.0 g / L isomaltose, 2.0 g / L maltose, 4.9 mg / L, 0.2 ml / L 5N NaOH, 10 ml / L COVE salts, 10 ml / L 1M acetamide) at 32°C for 3 days. Then, the AMG activity in the culture supernatant was measured by the pNPG assay described below at several temperatures.

[0145] pNPG thermal stability assay:

[0146] The culture supernatant containing the desired enzyme was mixed with an equal volume of 200 mM NaOAc buffer at pH 5.0. Twenty microliters of this mixture was dispensed into 96-well plates or 8-strip PCR tubes and then heated in a thermal cycler at various temperatures for 30 min. Those samples were mixed with 10 μl of a substrate solution containing 0.1% (w / v) pNPG [Wako Pure Chemical Industries, Ltd.] in 200 mM NaOAc buffer at pH 5.0 and incubated at 70°C for 20 min for the enzyme reaction. After the reaction, 60 μl of 0.1M Borax buffer was added to stop the reaction. Eighty microliters of the reaction supernatant was taken out and its OD 405 value was read by a photometer to evaluate the enzyme activity.

[0147] Table 1a. List of relative activities of PoAMG variants compared to their parental anPAV498 or JPO-0001 (anPAV498 with leader peptide / prepropeptide)

[0148] Name Relative activity (%) at 80°C / 75°C anPAV498 17％ JPO-004 32％ JPO-005 15％ JPO-006 16％ JPO-007 3％

[0149]

[0150]

[0151] Name Relative activity (%) at 80°C / 70°C JPO-001 10％ JPO-004 29％ JPO-009 13％ JPO-014 21％ JPO-020 16％ JPO-021 30％ JPO-052 33％

[0152] Name Relative activity (%) at 79°C / 70°C JPO-001 23％ JPO-021 46％ JPO-022 39％ JPO-023 44％ JPO-025 51％ JPO-027 49％ JPO-029 37％

[0153]

[0154]

[0155] Name Relative activity (%) at 79°C / 77°C JPO-001 36％ JPO-029 51％ JPO-047 45％ JPO-048 81％ JPO-049 53％ JPO-050 58％ JPO-064 65％

[0156] Name Relative activity (%) at 79°C / 77°C JPO-001 41％ JPO-021 60％ JPO-022 48％ JPO-023 57％ JPO-025 56％ JPO-027 64％ JPO-029 66％ JPO-047 50％ JPO-048 72％ JPO-051 82％ JPO-058 73％ JPO-062 72％ JPO-063 85％ JPO-064 83％

[0157] Table 1b. List of relative activities of PoAMG variants compared to their parent JPO-022

[0158] Name Relative activity (%) at 77°C / 70°C JPO-022 60％ JPO-027 67％ JPO-042 8％ JPO-044 86％ JPO-045 67％ JPO-046 48％

[0159] Name Relative activity (%) at 77°C / 70°C JPO-022 76％ JPO-023 75％ JPO-025 80％ JPO-027 84％ JPO-058 92％ JPO-059 88％ JPO-060 86％ JPO-061 83％ JPO-062 87％

[0160]

[0161]

[0162] Table 1c. List of relative activities of PoAMG variants compared to their parent JPO-063 at different temperatures

[0163] Name Relative activity (%) at 79°C / 77°C JPO-063 91％ JPO-066 96％ JPO-071 89％ JPO-072 84％ JPO-074 103％ JPO-075 86％ JPO-076 92％ JPO-077 95％ JPO-078 88％ JPO-079 100％

[0164] Name Relative activity (%) at 84°C / 80°C JPO-063 16％ JPO-065 26％ JPO-067 21％ JPO-070 12％ JPO-071 13％ JPO-074 32％ JPO-081 17％ JPO-082 24％ JPO-083 46％ JPO-084 26％ JPO-044 37％

[0165] Name Relative activity (%) at 82°C / 70°C JPO-063 21％ JPO-093 43％ JPO-081 25％ JPO-088 39％ JPO-094 38％ JPO-096 38％ JPO-106 53％

[0166] Name Relative activity (%) at 83°C / 80°C JPO-063 46％ JPO-051 44％ JPO-096 64％ JPO-106 88％ JPO-110 81％ JPO-111 100％ JPO-112 86％ JPO-113 83％ JPO-114 47％ JPO-115 90％

[0167] Table 1d. List of relative activities of PoAMG variants compared to their parent JPO-096

[0168]

[0169]

[0170] Name Relative activity (%) at 83°C / 80°C JPO-051 20％ JPO-096 43％ JPO-109 51％ JPO-126 33％ JPO-129 48％ JPO-130 18％ JPO-131 51％ JPO-132 34％

[0171] Table 1e. List of relative activities of PoAMG variants compared to their parent JPO-129

[0172] Name Relative activity (%) at 84°C / 80°C JPO-129 62％ JPO-156 51％ JPO-160 34％ JPO-161 41％ JPO-162 49％ JPO-163 21％ JPO-164 57％ JPO-165 77％

[0173] Table 1f. List of relative activities of PoAMG variants compared to their parent JPO-166

[0174]

[0175]

[0176] Table 2. Amino acid substitutions in variants of the mature sequence of PoAMG

[0177]

[0178]

[0179]

[0180]

[0181]

[0182] Example 3: Fermentation of Aspergillus niger

[0183] The Aspergillus niger strain was fermented in 500 ml baffled flasks on a rotary shaker at 220 rpm and 30 °C. These flasks contained 100 ml of MU1 and 4 ml of 50% urea. The culture broth was centrifuged (10,000 x g, 20 min) and the supernatant was carefully decanted from the pellet.

[0184] Example 4: Purification of PoAMG (JPO - 001) variant

[0185] The PoAMG variant was purified by cation exchange chromatography. The respective peak fractions were combined separately and dialyzed against 20 mM sodium acetate buffer (pH 5.0), and then the samples were concentrated using a centrifugal filtration device (Vivaspin Turbo 15, Sartorius). The enzyme concentration was determined by the A280 value.

[0186] Example 5: Thermal stability assay (TSA)

[0187] The purified enzyme was diluted to 0.5 mg / ml with 50 mM sodium acetate buffer (pH 5.0) and mixed with an equal volume of SYPRO Orange (Invitrogen) diluted with Milli - Q water. 18 μl of the mixture solution was transferred to a LightCycler 480 multi - well plate 384 (Roche Diagnostics) and the plate was sealed.

[0188] Device parameters of TSA:

[0189] Instrument: LightCycler 480 real - time PCR system (Roche Applied Science)

[0190] Scanning rate: 0.02 °C / sec

[0191] Scanning range: 37°C - 96°C

[0192] Integration time: 1.0 second

[0193] Excitation wavelength 465 nm

[0194] Emission wavelength 580 nm

[0195] The obtained fluorescence signal is normalized to the range of 0 and 1. Td is defined as the temperature at which the signal intensity is 0.5. The improvement in thermal stability is listed in Table 3, where the Td of the PoAMG variant designated as anPAV498 is 0.

[0196] Example 6: PoAMG activity assay

[0197] Determination of maltodextrin (DE11) by the GOD-POD method

[0198] Substrate solution

[0199] 30 g of maltodextrin (pindex#2 from MATSUTANI chemical industry Co., Ltd.)

[0200] 100 ml of 120 mM sodium acetate buffer, pH 5.0

[0201] Glucose CII test kit (Wako Pure Chemical Industries, Ltd.)

[0202] Mix 20 μl of the enzyme sample with 100 μl of the substrate solution and incubate at the set temperature for 2 hours. Cool the sample on an aluminum block for 3 min, then mix 10 μl of the reaction solution with 590 μl of 1 M 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 then let it stand at room temperature for 15 min. Read the absorbance at A505. The activity is listed in Table 3 as the relative activity of the PoAMG variant designated as anPAV498.

[0203] Table 3.

[0204]

[0205]

[0206]

[0207] Example 7: JPO172 in legume bread

[0208] Bake the bread using the formulation according to Table 4 in a direct fermentation baking process. Conduct different treatments according to Table 5. Bake the bread in an open pan. Mix the ingredients in a spiral mixer at 17 rpm for 3 min and 35 rpm for 7 min to form dough. Let the dough rest for 10 minutes and divide it into 320 g dough pieces. Round the dough pieces, roll them out and place them in baking trays. Let the baking trays with the dough proof at 32 °C and 86% relative humidity for 60 min. Bake the proofed dough in an oven at 180 °C for 20 min.

[0209] Package the bread in a sealed plastic bag 2 hours after baking and store it at room temperature until analysis.

[0210] Table 4 formulation

[0211]

[0212] Table 5 treatments

[0213]

[0214]

[0215] Evaluate the texture of the bread using a texture analyzer (TA-XT plus, Stable Micro Systems, Godalming, UK). The crumb texture properties are characterized by the firmness (the same as "hardness" and opposite to "softness") and elasticity of the baked product. The standard method for measuring firmness and elasticity is based on the force-deformation of the baked product. The force-deformation of the baked product can be carried out using a 40 mm diameter cylindrical probe. When the cylindrical probe is pressed down on a 25 mm thick bread slice at a deformation speed of 1 mm / s with 40% stress, record the force on the cylindrical probe. Then, hold the probe at this position for 30 s while recording the force, and then the probe returns to its initial position.

[0216] Firmness (in grams) is defined as the force required to compress the probe to 25% stress (corresponding to compressing 6.25 mm into a bread crumb slice with a thickness of 25 mm).

[0217] Elasticity (in %) is defined as the force recorded after compressing for 30 s at 40% stress (corresponding to the force at time = 40 s for a 25 mm thick bread slice) divided by the force required to press the probe 10 mm into the crumb (corresponding to the force at time = 10 s for a 25 mm thick bread slice) multiplied by 100.

[0218] The results of the texture analysis can be seen in Table 6 (firmness) and Table 7 (elasticity). The bread made with 33 mg EP / kg flour JPO172 and 40 ppm Lipopan Xtra is more elastic than the bread made with 100 ppm Novamyl G and 40 ppm Lipopan Xtra. At the same time, the breads have the same firmness.

[0219] When additional vital wheat gluten was added to both treatments, the bread made with 33 mg EP / kg flour, 40 ppm Lipopan Xtra, and 6% vital wheat gluten was generally more elastic and had the same firmness throughout the study period compared to the bread made with 100 ppm Novamyl G, 40 ppm Lipopan Xtra, and 6% vital wheat gluten.

[0220] Adding 33 mg EP / kg flour JPO172 alone was also able to improve elasticity. However, this bread was much harder than the bread made with the combination of 33 mg EP / kg flour JPO172 and 40 ppm Lipopan Xtra.

[0221] Conclusion: The combination of JPO172 and Lipopan Xtra can produce bread with a combination of low firmness and high elasticity throughout the study period.

[0222] Table 6 Firmness of the crumb of bread at different time points. The letters after the numbers represent the significance level using the student t-test, where the significance level is 0.05.

[0223]

[0224] Table 7 Elasticity of the crumb of bread at different time points. The letters after the numbers represent the significance level using the student t-test, where the significance level is 0.05.

[0225]

[0226]

Claims

1. A method for producing a baked or partially baked product, the method comprising: a) providing a dough comprising added legume and / or pod protein and a mature thermostable variant of a parental glucoamylase having at least 70% identity to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10; and b) baking or partially baking the dough to produce the baked or partially baked product.

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

3. The method according to any one of claims 1-2, wherein the mature variant comprises at least one amino acid modification at one or more or all of the positions corresponding to positions 1, 2, 4, 6, 7, 11, 31, 34, 50, 65, 79, 103, 132, 327, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595 in SEQ ID NO:

1.

4. The method according to claim 3, wherein the at least one amino acid modification comprises substitutions at one or more or all of the positions corresponding to positions 1, 2, 4, 11, 65, 79 and 327 in SEQ ID NO:1, preferably, the at least one amino acid modification comprises substitutions at one or more or all of the positions corresponding to R1A, P2N, P4S, P11F, T65A, K79V and Q327F in SEQ ID NO:

1.

5. The method according to claim 3, wherein the at least one amino acid modification comprises substitutions at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 79, 103, 132, 445, 447, 481, 566, 568, 594 and 595 in SEQ ID NO:1, preferably, the at least one amino acid modification comprises substitutions at one or more or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, K79V, S103N, A132P, D445N, V447S, S481P, D566T, T568V, Q594R and F595S in SEQ ID NO:

1.

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

1.

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

1.

8. The method according to any one of claims 1-3, wherein the at least one amino acid modification comprises substitutions 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 substitutions at one or more or all positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, K79V, S103N, A132P, D445N, V447S, S481P, T484P, E501A, N539P, D566T, T568V, Q594R and F595S in SEQ ID NO:

1.

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

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

11. The method according to any one of claims 1 - 10, wherein at least 2% (w / w) of the total flour content is added legume and / or pod protein, preferably at least 4% (w / w) of the total flour content is added legume and / or pod protein, preferably at least 6% (w / w) of the total flour content is added legume and / or pod protein, more preferably at least 8% (w / w) of the total flour content is added legume and / or pod protein, even more preferably at least 10% (w / w) of the total flour content is added legume and / or pod protein, most preferably at least 12% (w / w) of the total flour content is added legume and / or pod protein.

12. The method according to any one of claims 1 - 11, wherein the dough further comprises gluten.

13. The method according to any one of claims 1 - 12, wherein the dough further comprises at least one added lipase, preferably the at least one added lipase comprises lipase and / or phospholipase, preferably mature lipase and / or mature phospholipase, preferably wherein the at least one added lipase comprises mature lipase, and the mature lipase has an amino acid sequence having at least 70% identity with one or more of the sequences shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:

21.

14. The method according to claim 13, wherein the at least one added lipase is added in an amount in the range of 0 to 100 mg enzyme protein / kg flour; preferably in the range of 0 to 50 mg enzyme protein / kg flour; more preferably the amount is in the range of 0 to 25 mg enzyme protein / kg flour; even more preferably the amount is in the range of 0 to 10 mg enzyme protein / kg flour; still more preferably the amount is in the range of 0 to 5 mg enzyme protein / kg flour; and most preferably the amount is in the range of 0 to 2.5 mg enzyme protein / kg flour.

15. The method according to any one of claims 1 - 14, wherein the dough is mixed as follows: a) at a slow mixing speed, preferably in the range of 5 - 50 rpm, more preferably in the range of 10 - 40 rpm, for at least 5 minutes; more preferably for at least 10 minutes at a slow mixing speed, even more preferably for at least 15 minutes at a slow mixing speed; and optionally b) subsequently mixing the dough at a faster speed.

16. The method according to any one of claims 1-15, the method further comprising adding one or more additional enzymes selected from the group consisting of: α-amylase, amylomaltase, raw starch-degrading α-amylase, β-amylase, aminopeptidase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1,4-α-maltotetraohydrolase, glucanase, β-glucanase, galactanase, α-galactosidase, β-galactosidase, glucose oxidase, ɑ-glucosidase, β-glucosidase, haloperoxidase, hemicellulase, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinase, peptidylglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, protease, pullulanase, ribonuclease, transglutaminase, and xylanase.

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