Use of glucosyltransferase in dough preparation

Abstract

The present disclosure relates, in part, to addition of glucosyltransferase type enzymes for producing and / or modifying alpha-glucan type structures in dough such as bread dough. Such enzymes encompass various glucosyltransferases where these enzymes can transfer glucose from sucrose for production and / or modification of alpha-glucan type polymers in bread dough. Further disclosed are methods of preparing a baked food product by baking a dough, comprising incorporating into the dough one or more of the foregoing enzymes, wherein the enzyme can transfer a glucose-based moiety from sucrose to the non-reducing end of a saccharide acceptor, thereby improving texture, specific volume, dough stability, dough viscosity, anti-staling attributes and / or moisture content, for example.

Description

[0001] TITLE

[0002] USE OF GLUCOSYLTRANSFERASE IN DOUGH PREPARATION

[0003] This application claims the benefit of U.S. Provisional Appl. No. 63 / 724,431 (filed November 25, 2024), which is incorporated herein by reference in its entirety.

[0004] FIELD

[0005] The present disclosure is in the field of food products. For example, the disclosure pertains to food products and food dough comprising flour and alpha-glucan, or alphadextran and methods of producing these food products. In situ production of alpha-glucan and alpha-dextran products by glycosyl-transferase in dough.

[0006] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0007] The official copy of the sequence listing is submitted electronically via EFS-Web as a file named IFF 101 lOlWOPCT SequenceListing.xml created on November 20, 2025 and having a size of about 39 kilobytes, and is filed concurrently with the specification. The sequence listing contained in this file is part of the specification and is incorporated herein by reference in its entirety.

[0008] BACKGROUND

[0009] Several additives may be used in bread baking to improve the texture, specific volume, dough stability, dough viscosity, anti-staling attributes, and / or moisture content of bakery products. These include chemicals, sugars, enzymes, or combinations of these. Well- known additives include: milk powder, gluten, emulsifiers (mono- or di-glycerides, sugar esters, lecithin, etc.), granulated fat, oxidants (ascorbic acid or potassium bromate), cysteine, sugars and salts.

[0010] Advances in biotechnology now offer additional enzymes to the baking industry. Since enzymes generally can be produced using natural ingredients, these catalysts will find greater acceptance by consumers because of demand for products without chemicals. Some families of enzymes have been shown to act as dough and / or bread improvers in situ, by being able to modify one or more major dough components. For instance, enzymes active on starch (e.g., alpha-amylase, branching and debranching enzyme, maltogenic amylase, betaamylase, amyloglucosidase) have been shown to act as anti-staling agents. Despite this progress, additional enzyme solutions are sought after for improving dough-based food products.

[0011] Addressing this need, glucosyltransferase enzymes are disclosed herein to be useful for improving bread volume and dough stability by effecting in situ production and / or modification of alpha-glucan during dough preparation. SUMMARY

[0012] In one embodiment, the present disclosure concerns a method (process) of producing a food dough, wherein the method comprises: mixing at least (a) flour and / or meal, (b) water or aqueous composition, (c) sucrose, and (d) at least one glucosyltransferase enzyme, thereby producing a food dough, wherein the at least one glucosyltransferase enzyme is selected from: (i) a glucosyltransferase enzyme that is capable of synthesizing (or that synthesizes) alpha- 1,6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1, 6- glucan are alpha- 1,6 linkages, and / or (ii) a glucosyltransferase enzyme that is capable of synthesizing (or that synthesizes) alpha- 1,3 -glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,3 -glucan are alpha-1,3 linkages, typically wherein at least one alpha-glucan is produced in the food dough, optionally wherein: (I) the food dough has a shock stability that is increased as compared to the shock stability of a control food dough that lacks the at least one glucosyltransferase enzyme, (II) the food dough has a waterbinding capacity that is increased as compared to the water-binding capacity of a control food dough that lacks the at least one glucosyltransferase enzyme, (III) the food dough has a viscosity that is increased as compared to the viscosity of a control food dough that lacks the at least one glucosyltransferase enzyme, and / or (IV) the food dough has a volume that is increased as compared to the volume of a control food dough that lacks the at least one glucosyltransferase enzyme.

[0013] In another embodiment, the present disclosure concerns a food dough or food product produced by a method herein.

[0014] In another embodiment, the present disclosure concerns a food dough comprising (a) flour and / or meal, (b) water or aqueous composition, (c) sucrose, and (d) at least one glucosyltransferase enzyme, selected from: (i) a glucosyltransferase enzyme that is capable of synthesizing (or that synthesizes) alpha- 1,6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,6-glucan are alpha-1,6 linkages, and / or (ii) a glucosyltransferase enzyme that is capable of synthesizing (or that synthesizes) alpha-1, 3- glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,3 -glucan are alpha-1,3 linkages, typically wherein the food dough comprises at least one alpha-glucan product of the at least one glucosyltransferase enzyme, optionally wherein (I) the food dough has a shock stability that is increased as compared to the shock stability of a control food dough that lacks the at least one glucosyltransferase enzyme, (II) the food dough has a waterbinding capacity that is increased as compared to the water-binding capacity of a control food dough that lacks the at least one glucosyltransferase enzyme, (III) the food dough has a viscosity that is increased as compared to the viscosity of a control food dough that lacks the at least one glucosyltransferase enzyme, and / or (IV) the food dough has a volume that is increased as compared to the volume of a control food dough that lacks the at least one glucosyltransferase enzyme.

[0015] In another embodiment, the present disclosure concerns a food product comprising (a) flour and / or meal, (b) water or aqueous composition, (c) optionally sucrose, and (d) at least one glucosyltransferase enzyme, selected from: (i) a glucosyltransferase enzyme that is capable of synthesizing (or that synthesizes) alpha- 1,6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,6-glucan are alpha-1,6 linkages, and / or (ii) a glucosyltransferase enzyme that is capable of synthesizing (or that synthesizes) alpha-1, 3- glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,3 -glucan are alpha-1,3 linkages, typically wherein the food product comprises at least one alpha-glucan product of the at least one glucosyltransferase enzyme, optionally wherein (I) the food product has a water-binding capacity that is increased (e.g., the food product is more moist) as compared to the water-binding capacity of a control food product that lacks the at least one glucosyltransferase enzyme, (II) the food product has a volume (e.g., specific volume) that is increased as compared to the volume of a control food product that lacks the at least one glucosyltransferase enzyme, (III) the food product has a crumb that is increased as compared to the crumb of a control food product that lacks the at least one glucosyltransferase enzyme, (IV) the food product has a softness that is increased as compared to the softness of a control food product that lacks the at least one glucosyltransferase enzyme, and / or (IV) the food product has a resilience that is increased as compared to the resilience of a control food product that lacks the at least one glucosyltransferase enzyme.

[0016] BRIEF DESCRIPTION OF THE SEQUENCES

[0017] Table A, Summary of Protein SEP ID Numbers

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 displays a Brabender farinograph signal (50 min) on dough development analyzed on dough made with in situ activity of glucosyltransferase enzyme or addition of GUAR 250 (reference). Refer to Example 3. FIG. 2 displays a Brabender farinograph signal (50 min) on dough development analyzed on dough made with in situ activity of glucosyltransferase enzyme or addition of GUAR 250 (reference). Refer to Example 3.

[0020] FIG. 3 shows an informal sensory dough evaluation (0-10 score) from bakers on dough body, dough development, stickiness after mixing, stickiness after resting, and extensibility. Refer to Example 6.

[0021] FIG. 4 shows a photograph of brioche bread made with in situ activity of one or two glucosyltransferase enzymes as indicated. GUAR 250 was used as a reference, and no enzyme or ingredient was used as a negative control. Refer to Example 6.

[0022] FIG. 5 shows specific volumes (cubic cm / g) of brioche breads as determined by the volume and weight of final baked bread made with in situ glucosyltransferase(s) enzyme activity. GUAR 250 was used as reference and no enzyme or ingredient in the negative control. Refer to Example 6.

[0023] FIG. 6 shows the relative % amount of sucrose according to total carbohydrate as determined according to HPLC analysis. Refer to Example 6.

[0024] FIG. 7 shows HPLC sugar analysis (bars left to right: OS [oligosaccharides, DP3+], sucrose, glucose, leucrose, fructose) in baked goods. Refer to Example 6.

[0025] FIG. 8 shows softness / hardness analysis (texture profile analysis [TP A]) of the baked brioche bread on days 1 and 7 post-baking. Refer to Example 7.

[0026] FIG. 9 shows TPA resilience analysis of the baked brioche bread on days 1 and 7 post-baking. Refer to Example 7.

[0027] FIG. 10 shows an overlay of chromatograms from samples with different time points of sucrose conversion by GTF 0768 enzyme. Black solid line: reference sample containing only sucrose. Gray solid line, black dashed line, and black dotted line: reaction mixes after 15, 60, or 120 minutes, respectively, of incubation at 40 °C with GTF 0768 enzyme. Refer to Example 8.

[0028] FIG. 11 shows the specific bread volume (mL / g) of 300-g mini -bake breads, unshocked (left bar in each pair) or shocked (right bar in each pair), according to dough weight (A) or bread weight (B). TSE-2356 addition (without added GTF enzyme) was used as reference. Refer to Example 5.

[0029] FIG. 12 shows the specific bread volume (mL / g) of 300-g mini-bake breads, unshocked (left bar in each pair) or shocked (right bar in each pair), according to dough weight (A) or bread weight (B). Refer to Example 5. FIG. 13 shows informal sensory evaluation by bakers on bread slices on day 7 postbaking for softness, moistness, and resilience. Refer to Example 7.

[0030] FIG. 14 shows the number of quantified crumb cells by C-cell analysis and crumb cell volume of brioche bread slices on day 1 post-baking. Refer to Example 6.

[0031] FIG. 15 shows bread slice brightness by C-cell analysis and cell contrast of brioche bread slices on day 1 post-baking. Refer to Example 6.

[0032] FIG. 16 shows moisture analysis of the baked brioche bread on day 1 post-baking. Refer to Example 6.

[0033] FIG. 17 shows the specific bread volume (mL / g) of punched wheat mini -bake breads, unshocked (left bar in each pair) or shocked (right bar in each pair), according to dough weight (A) or bread weight (B). Refer to Example 10.

[0034] FIG. 18 shows the specific bread volume (cm3 / g) of punched mini -bake wheat breads unshocked (left bar in each pair) or shocked (right bar in each pair), according to bread weight, made using an autolyze dough making process. Refer to Example 11.

[0035] FIG. 19 shows the specific bread volume (cm3 / g) of punched mini -bake wheat breads unshocked (left bar in each pair) or shocked (right bar in each pair), according to bread weight, made using different yeast varieties. Refer to Example 12.

[0036] FIG. 20 displays Brabender farinograph signals over 360 seconds of investigation of torque over mixing time in a simulation of kneading doughs prepared with an autolyze dough-making process. Refer to Example 13.

[0037] FIG. 21 shows the specific bread volume (cm3 / g) of punched mini -bake wheat breads, unshocked (left bar in each pair) or shocked (right bar in each pair), according to bread weight. Refer to Example 14.

[0038] FIG. 22 shows the specific bread volume (cm3 / g) of white toast bread, unshocked (left bar in each pair) or shocked (right bar in each pair). Refer to Example 15.

[0039] FIG. 23 shows overlaid chromatograms of bread crumb extracts from breads made using an autolyze process. Solid line: reference bread made without the use of a GTF enzyme ingredient in the autolyze process. Dotted line: extract composition from bread crumb made with 188 U of GTF 0768 in the autolyze process. Dashed line: extract composition from bread crumb made with 378 U of GTF 0768 in the autolyze process. Refer to Example 16.

[0040] FIG. 24 shows a spectral overlay ofJH NMR spectra of the ethanol-extracted precipitates from water-soluble fractions of brioche samples. Dark-line spectra: brioche made with GTF enzyme ingredient (as labeled). Grey-line spectra: brioche made without GTF enzyme ingredient (as labeled). Refer to Example 17.

[0041] FIG. 25 shows a spectral overlay of1H NMR spectra of the ethanol-extracted precipitates from water-soluble fractions of pound cake samples. Dark-line spectra: pound cake made with GTF enzyme ingredient (as labeled). Grey-line spectra: pound cake made without GTF enzyme ingredient (as labeled). Refer to Example 18.

[0042] DETAILED DESCRIPTION

[0043] The disclosures of all cited patent and non-patent literature are incorporated herein by reference in their entirety.

[0044] Unless otherwise disclosed, the terms “a”, “an” and “the” as used herein are intended to encompass one or more (i.e., at least one) of a referenced feature.

[0045] Where present, all ranges are inclusive and combinable, except as otherwise noted. For example, when a range of “1 to 5” (i.e., 1-5) is recited, the recited range should be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, and the like. The numerical values of the various ranges in the present disclosure, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both proceeded by the word “about”. In this manner, slight variations above and below the stated ranges can typically be used to achieve substantially the same results as values within the ranges. Also, the disclosure of these ranges is intended as a continuous range including each and every value between the minimum and maximum values.

[0046] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0047] It is to be appreciated that certain features of the present disclosure, which are, for clarity, described above and below in the context of aspects / embodiments, may also be provided in combination in a single element. Conversely, various features of the disclosure that are, for brevity, described in the context of a single aspect / embodiment, can also be provided separately or in any sub-combination. The terms “alpha-glucan”, “alpha-glucan polymer” and the like are used interchangeably herein. An alpha-glucan is a polymer comprising glucose monomeric units linked together by alpha-glycosidic linkages. In typical aspects, an alpha-glucan herein comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% alpha-glycosidic linkages. Examples of alpha-glucan polymers herein include graft copolymers as presently disclosed, as well as alpha- 1,3 -glucan and alpha- 1,6-glucan.

[0048] The terms “alpha-1, 3-glucan”, “poly alpha- 1,3 -glucan”, “alpha-1, 3-glucan polymer” and the like are used interchangeably herein. Alpha- 1,3 -glucan is a polymer comprising glucose monomeric units linked together by glycosidic linkages, wherein at least about 50% of the glycosidic linkages are alpha-1,3. Alpha- 1,3 -glucan in certain aspects comprises at least about 90% or 95% alpha- 1,3 glycosidic linkages. Most or all of the other linkages, if present, in alpha- 1,3 -glucan herein typically are alpha-1,6, though some linkages may also be alpha-1,2 and / or alpha-1,4. Alpha- 1,3 -glucan as presently disclosed can characterize an alpha- 1,3 -glucan side chain herein. In some aspects, alpha- 1,3 -glucan can characterize an alpha- 1,3 -glucan “homopolymer”, which is alpha- 1,3 -glucan that is not part of a dextranalpha- 1,3 -glucan copolymer.

[0049] The terms “dextran”, “dextran polymer”, “dextran molecule”, “alpha- 1,6-glucan” and the like herein refer to a water-soluble alpha-glucan comprising at least 50%, 60%, 70%, 80%, or 90% alpha- 1,6 glycosidic linkages (with the balance of the linkages typically being alpha-1,3). Enzymes capable of synthesizing dextran from sucrose may be described as “dextransucrases” (EC 2.4.1.5). A “substantially linear” (“mostly linear”, and like terms) dextran has 5% or less branches, before being modified herein to have with alpha- 1,3 -glucan side chains. A “linear” dextran has no branches, before being modified herein to have alpha- 1, 3-glucan side chains. Branches, if present prior to modification of dextran with alpha-1, 3- glucan side chains, can be short, being one (pendant) to three glucose monomers in length. Yet, in some aspects, dextran can be “dendritic”, which is a branched structure emanating from a core in which there are chains (containing mostly or all alpha- 1,6-linkages) that iteratively branch from each other (e.g., a chain can be a branch from another chain, which in turn is a branch from another chain, and so on). Yet, in still some aspects, dextran is not dendritic, but has a branch-on-branch structure that does not emanate from a core. Dextran as used in a glucosyltransferase reaction herein for alpha-1, 3-glucan synthesis (to produce a dextran-alpha- 1,3 -glucan copolymer) can optionally be characterized as a “primer” or “acceptor”. In some aspects, dextran can characterize a dextran “homopolymer”, which is dextran that is not part of a dextran-alpha- 1,3 -glucan copolymer.

[0050] The term “copolymer” herein refers to a polymer comprising at least two different types of alpha-glucan, such as dextran and alpha-1, 3-glucan.

[0051] The terms “graft copolymer”, “branched copolymer” and the like herein generally refer to a copolymer comprising a “backbone” (or “main chain”) and one or more side chains branching from the backbone. The side chains are structurally distinct from the backbone.

[0052] Examples of graft copolymers herein are “dextran-alpha- 1, 3-glucan graft copolymers” (and like terms) that comprise a backbone comprising dextran, and one or more side chains of alpha-1, 3-glucan. A backbone in some aspects can itself be a branched dextran as disclosed herein; the addition of alpha-1, 3-glucan side chains to such a backbone (thereby forming a graft copolymer herein) can be, for example, via enzymatic extension from non-reducing ends presented by short branches (alpha-1,2, -1,3, or -1,4 branch, each typically comprised of a single glucose monomer; i.e., pendant glucose). Short branches (that can be enzymatically extended into an alpha- 1,3 -glucan side chain) can be present on an otherwise linear or mostly linear dextran, or can be present on a branching dextran. In some aspects, alpha- 1,3 -glucan can also be synthesized from non-reducing ends of dextran main chains, such as in embodiments in which the dextran backbone is linear or mostly linear, or embodiments in which the dextran backbone is branching (e.g., dendritic, or not dendritic [branches do not emanate from a core] but has branch-on-branch structure); such alpha- 1,3 -glucan is not, technically-speaking, a side chain to the dextran, but rather an extension from the dextran main chain(s).

[0053] The percent branching in an alpha-glucan herein refers to that percentage of all the linkages in the alpha-glucan that represent branch points. For example, the percent of alpha- 1,3 branching in an alpha-glucan herein refers to that percentage of all the linkages in the glucan that represent alpha- 1,3 branch points. Except as otherwise noted, linkage percentages disclosed herein are based on the total linkages of a glucan, or the portion of a glucan for which a disclosure specifically regards.

[0054] The terms “linkage”, “glycosidic linkage”, “glycosidic bond” and the like refer to the covalent bonds connecting the sugar monomers within a saccharide compound (oligosaccharides and / or polysaccharides). Examples of glycosidic linkages include 1,6- alpha-D-glycosidic linkages (herein also referred to as “alpha-1,6” linkages), 1,3-alpha-D- glycosidic linkages (herein also referred to as “alpha- 1,3” linkages), 1,4-alpha-D-glycosidic linkages (herein also referred to as “alpha- 1,4” linkages), and 1,2-alpha-D-glycosidic linkages (herein also referred to as “alpha- 1,2” linkages). The glycosidic linkages of a glucan polymer herein can also be referred to as “glucosidic linkages”. Herein, “alpha-D-glucose” is referred to as “glucose”.

[0055] The glycosidic linkage profile of an alpha-glucan herein can be determined using any method known in the art. For example, a linkage profile can be determined using methods using nuclear magnetic resonance (NMR) spectroscopy (e.g.,13C NMR or1H NMR). These and other methods that can be used are disclosed in, for example, Food Carbohydrates:

[0056] Chemistry, Physical Properties, and (S. W. Cui, Ed., Chapter 3, S. W. Cui,

[0057] Structural Analysis of Polysaccharides, Taylor & Francis Group LLC, Boca Raton, FL, 2005), which is incorporated herein by reference.

[0058] The “molecular weight” of an alpha-glucan herein can be represented as weightaverage molecular weight (Mw) or number-average molecular weight (Mn), the units of which are in Daltons (Da) or grams / mole. In some aspects, molecular weight can be represented as DPw (weight average degree of polymerization) or DPn (number average degree of polymerization). DPw and DPn are calculated from the corresponding Mw or Mn, respectively, by dividing by the molar mass of one monomer unit Mi. In the case of glucan polymer, Mi = 162.14. In some aspects, molecular weight can sometimes be provided as “DP” (degree of polymerization), which simply refers to the number of glucoses comprised within the alpha-glucan on an individual molecule basis. Various means are known in the art for calculating these various molecular weight measurements such as with high-pressure liquid chromatography (HPLC), size exclusion chromatography (SEC), or gel permeation chromatography (GPC).

[0059] The term “sucrose” herein refers to a non-reducing disaccharide composed of an alpha-D-glucose molecule and a beta-D-fructose molecule linked by an alpha- 1,2-glycosi die bond. Sucrose is known commonly as table sugar. Sucrose can alternatively be referred to as “alpha-D-glucopyranosyl-( l ^2)-beta-D-fructofuranoside”. “Alpha-D-glucopyranosyl” and “glucosyl” are used interchangeably herein.

[0060] The terms “sugar” or “sugars”, unless used to specifically refer to sucrose only, refer to any monosaccharide (e.g., fructose, glucose, and / or galactose) and / or disaccharide (e.g., sucrose, leucrose, and / or lactose; and / or optionally DP2 gluco-oligosaccharide), and / or optionally any oligosaccharide (e.g., ranging from DP3 to DP4, DP5, DP6, DP7, DP8, DP9, DP10, DP12, DP14, DP15, DP16, DP18, or DP20; typically gluco-oligosaccharide) such as those disclosed herein. Sugars herein typically are water-soluble. The terms “glucosyltransferase”, “glucosyltransferase enzyme”, “GTF”, “glucansucrase” and the like are used interchangeably herein. The activity of a glucosyltransferase herein catalyzes the reaction of the substrate sucrose to make the products alpha-glucan and fructose. Other products (by-products) of a GTF reaction can include glucose, various soluble gluco-oligosaccharides, and leucrose. Wild type forms of glucosyltransferase enzymes generally contain (in the N-terminal to C-terminal direction) a signal peptide (which is typically removed by cleavage processes), a variable domain, a catalytic domain, and a glucan-binding domain. A glucosyltransferase herein is classified under the glycoside hydrolase family 70 (GH70) according to the CAZy (Carbohydrate- Active EnZymes) database (Cantarel et al., Nucleic Acids Res. 37:D233-238, 2009). The term “dextransucrase” (and like terms) can optionally be used to characterize a glucosyltransferase enzyme that produces dextran.

[0061] The term “glucosyltransferase catalytic domain” herein refers to the domain of a glucosyltransferase enzyme that provides alpha-glucan-synthesizing activity to a glucosyltransferase enzyme. A glucosyltransferase catalytic domain typically does not require the presence of any other domains to have this activity.

[0062] The terms “enzymatic reaction”, “glucosyltransferase reaction”, “glucan synthesis reaction”, “reaction composition”, “reaction formulation” and the like are used interchangeably herein and generally refer to a reaction that initially comprises water, sucrose, at least one active glucosyltransferase enzyme, and optionally other components. Components that can be further present in a glucosyltransferase reaction typically after it has commenced include fructose, glucose, leucrose, soluble gluco-oligosaccharides (e.g., DP2- DP7) (such may be considered as products or by-products, depending on the glucosyltransferase used), and / or insoluble alpha-glucan product(s) of DP8 or higher. It would be understood that certain glucan products, such as alpha- 1,3 -glucan with a degree of polymerization (DP) of at least 8 or 9, typically are water-insoluble and thus not dissolved in a glucan synthesis reaction. The term “under suitable reaction conditions” as used herein refers to reaction conditions that support conversion of sucrose to alpha-glucan product(s) via glucosyltransferase enzyme activity. It is during such a reaction that glucosyl groups originally derived from the input sucrose are enzymatically transferred and used in alphaglucan polymer synthesis; glucosyl groups as involved in this process can thus optionally be referred to as the glucosyl component or moiety (or like terms) of a glucosyltransferase reaction. The term “zw situ” as used herein typically characterizes a glucosyltransferase reach on(s) that occurs inside a food dough or precursor thereof (e.g., incompletely mixed dough) and thereby produces alpha-glucan within the food dough itself (or precursor). Such produced alpha-glucan (e.g., graft copolymer, alpha- 1,3 -glucan, and / or alpha- 1,6-glucan) can be soluble or insoluble. While an alpha- 1,3 -glucan product is typically insoluble and an alpha- 1,6-glucan product is typically soluble, a graft copolymer product can either be soluble or insoluble, in a food dough / precursor or food product herein. In situ production of alphaglucan in a food product / precursor typically substitutes for adding alpha-glucan herein as an ingredient in food, though such addition can be performed if desired (e.g., to supplement the alpha-glucan produced in situ). Typically, a glucosyltransferase enzyme(s) used for an in situ reaction herein is added to the food dough or precursor thereof as an exogenous ingredient, for example as a purified or otherwise isolated enzyme. Enzymatic product(s) produced in situ in a food dough or precursor thereof typically persist through downstream food dough / precursor processing (e.g., baking), and thus can exist in a food product made using the enzymatically treated food dough / precursor.

[0063] The terms “percent by volume”, “volume percent”, “vol %”, “v / v %” and the like are used interchangeably herein. The percent by volume of a solute in a solution can be determined using the formula: [(volume of solute) / (volume of solution)] x 100%.

[0064] The terms “percent by weight”, “weight percentage (wt%)”, “weight-weight percentage (% w / w)” and the like are used interchangeably herein. Percent by weight refers to the percentage of a material on a mass basis as it is comprised in a composition, mixture, or solution.

[0065] The terms “weight / volume percent”, “w / v%” and the like are used interchangeably herein. Weight / volume percent can be calculated as: ((mass [g] of material) / (total volume [mL] of the material plus the liquid in which the material is placed)) x 100%. The material can be insoluble in the liquid (i.e., be a solid phase in a liquid phase, such as with a dispersion), or soluble in the liquid (i.e., be a solute dissolved in the liquid).

[0066] The terms “ingestible product” and “ingestible composition” are used interchangeably herein, and refer to any substance that, either alone or together with another substance, may be taken orally (i.e., by mouth), whether intended for consumption or not. Thus, an ingestible product includes food / beverage products. “Food / beverage products” refer to any edible product intended for consumption (e.g., for nutritional purposes) by humans or animals, including solids, semi-solids, or liquids. A “food” herein can optionally be referred to as a “foodstuff’, “food product”, or other like term, for example. Herein, unless otherwise disclosed, a beverage or other ingestible liquid is an example of a food product. While the present disclosure generally regards food and food precursors that are by definition intended for ingestion or eventual ingestion (food precursor first made into food before being eaten), the disclosure likewise regards other ingestible products (e.g., supplement, nutraceutical, pharmaceutical product) comprising in .s / / / / -produced alpha-glucan. A food precursor herein can be (i) a food as it exists before one or more processing steps (e.g., fermentation, aging, cooling / freezing, heating, baking, mixing) that render it to be a food product intended for direct consumption, and / or (ii) an ingredient for use in preparing a food product, for example. In some aspects, a food precursor can characterize a food product or ingredient as it exists before treatment with one or more GTF enzymes in a method herein.

[0067] “Flour” and like terms herein refer to powder made by grinding (milling) grains / cereals, roots / tubers, beans / legumes, or nuts / seeds, for example. Typically, the material that is ground into flour is entered into the grinding process in raw, dried form. A flour herein that is made from grain can optionally be referred to as a “grain flour”. “Meal” and other like terms herein refer to a substance that is similar to flour, but with a grain or particle size that is larger / coarser. A meal is not ground / milled as finely as flour. A meal herein that is made from grain can optionally be referred to as a “grain meal”. Flour and meal are generally used as ingredients in various food products. Flour and meal produced from a grain can optionally be characterized as grain derivatives herein.

[0068] “Dough”, “food dough” and like terms herein typically refer to a mixture comprising at least (i) flour and / or meal and (ii) a water-comprising liquid (e.g., water and / or milk), and typically is in a suitable form (stiff / firm) for kneading or rolling. A dough can optionally be referred to with reference to the grain, grain derivative, or other material from which it was derived (e.g., wheat dough, wheat flour dough, com flour dough, cornmeal dough). Since dough typically is not eaten as a food prior to further processing (e.g., baking), dough can optionally be characterized as a “food precursor”.

[0069] A “baked food” (and like terms) herein refers to a food that has been baked during its preparation. Baking herein typically refers to a process of applying dry heat to a food / food precursor (e.g., dough) for a period of time during preparation of the food. In general, baking is conducted in an enclosed (typically confined) space such as within an oven. Bread is an example of a food for which its preparation process typically comprises baking.

[0070] An “extruded food” (and like terms) herein refers to a food that has been extruded during its preparation. Food extrusion is a process by which a mix of ingredients (e.g., dough) is forced through an opening in a perforated device (e.g., plate or die), which typically is specifically designed for the food being extruded. After this step, an extruded food typically is then cut to a particular size.

[0071] The terms “dietary fiber”, “glucan fiber” and the like herein refer to an alpha-glucan that is indigestible and / or that does not increase blood-glucose levels when enterally administered to a mammal. In general, a dietary fiber herein is not significantly hydrolyzed by endogenous enzymes in the upper gastrointestinal tract of mammals such as humans.

[0072] “Gluten” herein refers to a group of proteins, termed prolamins and glutelins, which occur with starch in the endosperm of cereal grains. Gluten has been previously described by Wieser (2007, Food Microbiol. 2007 24: 115-119), for example, which is incorporated herein by reference.

[0073] “Fermentation” and like terms herein as applied to food product / precursor refer to the conversion of carbohydrates in a food product / precursor into alcohol(s) and / or acid(s) through the action of one or more microorganisms (e.g., bacteria, yeast).

[0074] A composition herein that is “dry” or “dried” typically has less than 5, 4, 3, 2, 1, 0.5, or 0.1 wt% water comprised therein.

[0075] The terms “aqueous liquid”, “aqueous fluid”, “aqueous conditions”, “aqueous setting”, “aqueous system” and the like as used herein can refer to water or an aqueous solution. An “aqueous solution” herein can comprise one or more dissolved salts, where the maximal total salt concentration can be about 3.5 wt% in some embodiments. Although aqueous liquids herein typically comprise water as the only solvent in the liquid, an aqueous liquid can optionally comprise one or more other solvents (e.g., polar organic solvent) that are miscible in water. Thus, an aqueous solution can comprise a solvent having at least about 10 wt% water.

[0076] An “aqueous composition” herein has a liquid component that comprises about, or at least about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100 wt% water, for example. Examples of aqueous compositions include mixtures, solutions, dispersions (e.g., suspensions, colloidal dispersions) and emulsions, for example.

[0077] An alpha-glucan herein that is “insoluble”, “aqueous-insoluble”, “water-insoluble” (and like terms) herein does not dissolve (or does not appreciably dissolve) in water or other aqueous conditions, optionally where the aqueous conditions are at a pH of 4-9 (e.g., pH 6-8) and / or a temperature of about 1 to 130 °C (e.g., 20-25 °C). In some aspects, less than 1.0 gram (e.g., no detectable amount) of an aqueous-insoluble alpha-glucan dissolves in 1000 milliliters of such aqueous conditions (e.g., water at 23 °C). In contrast, an alpha-glucan that is “soluble”, “aqueous-soluble”, “water-soluble” and the like appreciably dissolves under the above aqueous conditions.

[0078] Alpha-glucan in some aspects of the present disclosure can provide stability to a dispersion or emulsion of a food product / precursor. The “stability” (or the quality of being “stable”) of a dispersion or emulsion herein is, for example, the ability of dispersed particles of a dispersion, or liquid droplets dispersed in another liquid (emulsion), to remain dispersed (e.g., about, or at least about, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100 wt% of the particles of the dispersion or liquid droplets of the emulsion are in a dispersed state) for a period of about, or at least about, 2, 4, 6, 9, 12, 18, 24, 30, or 36 months following initial preparation of the dispersion or emulsion. A stable dispersion or emulsion can resist total creaming, sedimentation, flocculation, and / or coalescence of dispersed / emulsified material, for example.

[0079] The term “viscosity” as used herein refers to the resistance of a food product / precursor to deformation at a given rate. Viscosity may also be defined as a measure of the extent to which a fluid (aqueous or non-aqueous) resists a force tending to cause it to flow. Furthermore, viscosity can be defined as the shear stress resulting from an applied shear rate. Both dynamic and kinematic viscosity are meant by the term viscosity, as both parameters are directly correlated through the density of a food product / precursor. Various units of viscosity that can be used herein include centipoise (cP, cps) and Pascal-second (Pa s), for example. A centipoise is one one-hundredth of a poise; one poise is equal to 0.100 kg-m’^s’1.

[0080] As used herein, the term “polypeptide” is defined as a chain of amino acid residues, usually having a defined sequence. As used herein the term polypeptide is interchangeable with the terms “peptides” and “proteins”. Typical amino acids contained in polypeptides herein include (respective three- and one-letter codes shown parenthetically): alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine (Gin, Q), glycine (Gly, G), histidine (His, H), isoleucine (He, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), valine (Vai, V).

[0081] The terms “sequence identity”, “identity” and the like as used herein with respect to polynucleotide or polypeptide sequences refer to the nucleic acid residues or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window. Thus, “percentage of sequence identity”, “percent identity” and the like refer to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. It would be understood that, when calculating sequence identity between a DNA sequence and an RNA sequence, T residues of the DNA sequence align with, and can be considered “identical” with, U residues of the RNA sequence. For purposes of determining “percent complementarity” of first and second polynucleotides, one can obtain this by determining (i) the percent identity between the first polynucleotide and the complement sequence of the second polynucleotide (or vice versa), for example, and / or (ii) the percentage of bases between the first and second polynucleotides that would create canonical Watson and Crick base pairs.

[0082] Percent identity can be readily determined by any known method, including but not limited to those described in: 1) Computational Molecular Biology (Lesk, A.M., Ed.) Oxford University: NY (1988); 2) Biocomputing: Informatics and Genome Projects (Smith, D.W., Ed.) Academic: NY (1993); 3) Computer Analysis of Sequence Data, Part I (Griffin, A.M., and Griffin, H.G., Eds.) Humana: NJ (1994); 4) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991), all of which are incorporated herein by reference.

[0083] Preferred methods for determining percent identity are designed to give the best match between the sequences tested. Methods of determining identity and similarity are codified in publicly available computer programs, for example. Sequence alignments and percent identity calculations can be performed using the MEGALIGN program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI), for example. Multiple alignment of sequences can be performed, for example, using the Clustal method of alignment which encompasses several varieties of the algorithm including the Clustal V method of alignment (described by Higgins and Sharp, CABIOS. 5: 151-153 (1989); Higgins, D.G. et al., Comput. AppL Biosci., 8: 189-191 (1992)) and found in the MEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values can correspond to GAP PENAL TY= 10 and GAP LENGTH PENAL TY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method can be KTUPLE=1, GAP PENAL TY=3, WIND0W=5 and DIAGONALS SAVED=5. For nucleic acids, these parameters can be KTUPLE=2, GAP PENAL TY=5, WIND0W=4 and DIAGONALS SAVED=4. Additionally, the Clustal W method of alignment can be used (described by Higgins and Sharp, CABIOS. 5: 151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci. 8: 189-191(1992); Thompson, J.D. et al, Nucleic Acids Research, 22 (22): 4673-4680, 1994) and found in the MEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (protein / nucleic acid) can be: GAP PENALTY=10 / 15, GAP LENGTH PENALTY=0.2 / 6.66, Delay Divergent Seqs(%)=30 / 30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB.

[0084] Various polypeptide amino acid sequences and polynucleotide sequences are disclosed herein as features of certain embodiments. Variants of these sequences that are at least about 70-85%, 85-90%, or 90%-95% identical to the sequences disclosed herein can be used or referenced. Alternatively, a variant amino acid sequence or polynucleotide sequence can have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with a sequence disclosed herein. A variant amino acid sequence or polynucleotide sequence herein has the same function / activity of the disclosed sequence, or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% of the function / activity of the disclosed sequence. Any polypeptide amino acid sequence disclosed herein not beginning with a methionine or valine can typically further comprise at least a start-methionine or start-valine at the N- terminus of the amino acid sequence. In contrast, any polypeptide amino acid sequence disclosed herein beginning with a methionine or valine can optionally lack such a methionine or valine residue. In some aspects, any polypeptide amino acid sequence disclosed herein beginning with a methionine or valine can instead have, respectively, a valine or methionine as the first amino acid residue.

[0085] The term “isolated” means a substance (or process) in a form or environment that does not occur in nature. A non-limiting example of an isolated substance herein includes any non-naturally occurring substance such as a food product (e.g., baked dough product) or food precursor (e.g., food dough or precursor thereof) (as well as enzymatic reactions / processes used to prepare these materials). It is believed that the embodiments disclosed herein are synthetic / man-made (could not have been made except for human intervention / involvement), and / or have properties that are not naturally occurring.

[0086] The term “increased” as used herein can refer to a quantity or activity that is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 50%, 100%, or 200% more than the quantity or activity for which the increased quantity or activity is being compared. The terms “increased”, “elevated”, “enhanced”, “greater than”, “improved” and the like are used interchangeably herein.

[0087] Some aspects of the present disclosure concern a method / process of producing a food dough (flour dough or flour slurry) (a flour slurry is an example of a dough precursor or dough ingredient herein). Such a method can comprise mixing (combining, stirring, and / or kneading) at least (a) flour and / or meal, (b) water or aqueous composition, (c) sucrose, and (d) one or more glucosyltransferase (GTF) enzymes, thereby producing a food dough. At least one of the GTF enzymes can be selected from:

[0088] (i) a GTF enzyme that is capable of synthesizing alpha- 1,6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,6-glucan are alpha- 1,6 linkages, and / or

[0089] (ii) a GTF enzyme that is capable of synthesizing alpha- 1,3 -glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,3 -glucan are alpha-1,3 linkages. Typically, at least one alpha-glucan is produced in the food dough by virtue of the activity of the one or more GTF enzymes present in the food dough. Optionally, a food dough produced by this method can be characterized by one or more of the following features:

[0090] (I) the food dough has a shock stability that is increased as compared to the shock stability of a control food dough that lacks the at least one GTF enzyme,

[0091] (II) the food dough has a water-binding capacity that is increased as compared to the water-binding capacity of a control food dough that lacks the at least one GTF enzyme,

[0092] (III) the food dough has a viscosity that is increased as compared to the viscosity of a control food dough that lacks the at least one GTF enzyme, and / or

[0093] (IV) the food dough has a volume that is increased as compared to the volume of a control food dough that lacks the at least one GTF enzyme.

[0094] A “control food dough” (or “corresponding food dough” and like terms) herein can be, for example, a food dough that is produced the same way, or substantially the same way, as the first food dough (food dough of the present disclosure can optionally also be referred to as “first food dough”, which can be instructive for comparison purposes), with the exception that the GTF enzyme(s) is / are not included / added during production of the control food dough. A “control food product” (and like terms) is similarly distinguished from a food product made from a first food dough.

[0095] The foregoing method can optionally be characterized herein as a dough production method or process. A dough production method herein comprises addition of one or more GTF enzymes, which processing can optionally lead to one or more enhancements of the dough product, such as increased dough strength (e.g., increased dough shock stability and / or increased dough viscosity) and / or water-binding capacity. Increased dough strength can be taken advantage of by, for example, (i) reducing the amount of gluten and / or related proteins that otherwise would be necessary as a dough component / ingredient to achieve a level of dough strength, and / or (ii) achieving greater volumes of baked products made using dough herein.

[0096] A food dough production method as presently disclosed comprises provision of flour and / or meal. In some aspects, only flour or only meal is the flour / meal ingredient (one or more other ingredients that are not flour or meal can optionally also be present), while in other aspects there can be a mix of flour and meal. For example, flour and meal ingredients can comprise at least about 50, 60, 70, 80, 90, 95, 98, or 99 wt% flour, and less than about 50, 40, 30, 20, 10, 5, 2, or 1 wt% meal, or vice versa. There can be one, two, three, or more different types of flour and / or meal, for example, in the flour / meal component of food dough herein. Flour and / or meal herein can be that of a grain / cereal, root / tuber, bean / legume, or nut / seed in some aspects. Meal can be bolted or unbolted in some aspects.

[0097] Suitable examples of a grain / cereal for making a flour and / or meal herein include grain of wheat (e.g., Triticum aeslivum. T. compactum, T. sphaerococcum), Hordeum vulgare (barley), Avena sativa (oat), Secale cereale (rye), Triticosecale spp. (triticale), Zea mays (maize / com), sorghum, millet (e.g., Digitaria, Echinochloa, Eleusine, Panicum, Setaria, Pennisetum), Phalaris canariensis (canary seed), rice (e.g., Oryza species such as 0. sativa, Zizania), Eragrostis abyssinica (teff), Coix lacryma-jobi (Job's tears), amaranth, buckwheat (e.g., Fagopyrum, Eriogonum) and quinoa. A grain can be dehulled or hulled. Examples of wheat varieties herein include winter and spring wheat, hard and soft wheat, and red wheat and white wheat; some particular wheat varieties are winter wheat (e.g., hard red winter, soft red winter), spring wheat (e.g., hard red spring), durum (e.g., for making semolina), hard white wheat, and soft white wheat. Soft wheat flour herein can contain about 5-9 wt% gluten (or total protein), whereas hard wheat flour can contain about 11-15 wt% gluten (or total protein), for example. Examples of com varieties herein include dent com, flour corn, sweet com, flint corn, heirloom corn, and waxy com. Examples of rice varieties herein include long grain rice, medium grain rice, short grain rice, sticky rice, basmati rice, jasmine rice, wild rice, Manmibyeo, Jinsumi, Seolgaeng, Hanareumbyeo, Chenmaai, and Goamibyeo. Examples of barley varieties herein include malting barley, Lacey, and Taylor. Flour made with grain can be whole grain flour in some aspects, which includes the germ and bran of the grain.

[0098] Suitable examples of roots and tubers for making a flour and / or meal herein include arrowroot, cassava / yucca / manioc / tapioca (e.g., sweet or bitter), potatoes, sweet potatoes, yams, taro root, carrot, beetroot, parsnip, ginger, lotus root and turmeric. Potatoes can be starchy potatoes (e.g., Russet, Idaho, Yukon gold) or waxy potatoes (e.g., red, blue, fingerling), for example. Other vegetables that are fruit (e.g., squash, pumpkins, tomatoes) or leaves (e.g., kale, spinach) of plants can be used to make flour or meal in some aspects.

[0099] Suitable examples of beans and legumes for making a flour and / or meal herein include chickpeas, soybeans, mung beans, peas (yellow or green), black gram (urad dal), fava beans and lentils, while suitable examples of nuts and seeds include almonds, coconuts, Brazil nuts, cashews, pistachios, macadamias, peanuts, pecans, walnuts, hazelnuts, pine nuts, flaxseeds, sunflower seeds, chia seeds, pumpkin seeds, hemp seeds, and tiger nuts.

[0100] Flour made from wheat in some aspects herein can be in the form of whole wheat flour (includes the germ and bran), graham flour, cake flour, pastry flour, all-purpose (plain) flour, atta flour, or maida flour. Corn flour herein typically is made of finely ground cornmeal. Cornmeal in some aspects can be unprocessed or further processed (e.g., leached with lye to make corn masa).

[0101] In some aspects, the gluten content of a flour (e.g., wheat flour) is about, or less than about, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0% (or otherwise undetectable), 0.001-0.01%, 0.001-0.1%, 4-6%, 4-8%, 4-9%, 5-9%, 6-8%, or 6- 10% by weight.

[0102] Typically, a flour or dough herein comprises alpha- 1,4-glucan such as in the form of starch, amongst other species of alpha- 1,4-glucan (e.g., dextrin, malto-oligosaccharides). Such starch-containing flour or dough can thus optionally be characterized as being farinaceous.

[0103] One or more glucosyltransferase (GTF) enzymes used in a method as presently disclosed can comprise, for example: (i) a GTF enzyme that is capable of synthesizing alpha- 1,6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,6-glucan are alpha- 1,6 linkages (or, a GTF enzyme with alpha- 1,6-glucan / dextran synthesis activity), and / or

[0104] (ii) a GTF enzyme that is capable of synthesizing alpha- 1,3 -glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,3 -glucan are alpha-1,3 linkages (or, a GTF enzyme with alpha- 1,3 -glucan synthesis activity).

[0105] In some aspects, a GTF enzyme (dextransucrase) that is capable of synthesizing alpha- 1,6-glucan herein can comprise an amino acid sequence that is about 100% identical to, or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, SEQ ID NO: 1, 2, 11, or 12 (GTF 0768), or 14 or 15 (GTF 6831), and have GTF activity. Yet, in some aspects, a GTF enzyme that is capable of synthesizing alpha- 1,6-glucan can be as disclosed in any of U.S. Patent Appl. Publ. Nos. 2017 / 0218093, 2018 / 0282385, 2018 / 0291311, or 2016 / 0122445, which are each incorporated herein by reference. For example, the GTF identified as GTF 8117 (SEQ ID NO:30), GTF 6831 (SEQ ID NO:32), or GTF 5604 (SEQ ID NO:33) in US2018 / 0282385 can be used, or the GTF identified as GTF 2919 (SEQ ID NO: 5), GTF 2918 (SEQ ID NO: 9), GTF 2920 (SEQ ID NO: 13), or GTF 2921 (SEQ ID NO: 17) in US2016 / 0122445 can be used, or a GTF comprising an amino acid sequence that is about 100% identical to, or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, the amino acid sequence of any of these GTF enzymes (and having GTF activity) can be used.

[0106] A dextransucrase herein is capable of producing dextran comprising about, or at least about, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% alpha-1,6 glycosidic linkages, for example. Such a percent alpha- 1,6 linkage profile takes into account the total of all linkages in the dextran (main chains of alpha-1,6 glucan and, if present, branch portions therefrom). Dextran as disclosed elsewhere herein such as in a homopolymer or graft-copolymer can have any of the foregoing linkage profiles, for example.

[0107] A dextransucrase herein is capable of producing dextran having a weight-average molecular weight (Mw) of about, at least about, or less than about, 1000, 2500, 5000, 7500, 10000, 25000, 50000, 75000, 100000, 150000, 200000, 250000, 500000, 750000, 1000000, 1000-10000, 1000-100000, 1000-1000000, 10000-100000, 10000-1000000, or 100000- 1000000 Daltons, for example. In some aspects, the Mw is about, at least about, or less than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 10-50, 10-70, 10-80, 10-100, 10-120, 10-130, 10-150, 10-200, 25- 50, 25-70, 25-80, 25-100, 25-120, 25-130, 25-150, 25-200, 50-70, 50-80, 50-100, 50-120, 50- 130, 50-150, 50-200, 70-80, 70-100, 70-120, 70-130, 70-150, 70-200, 80-100, 80-120, 80- 130, 80-150, 80-200, 100-120, 100-130, 100-150, 100-200, 120-130, 120-150, 120-200, 130- 150, or 130-200 million Daltons, for example. Dextran as disclosed elsewhere herein, such as in a homopolymer or graft-copolymer, can have any of the foregoing molecular weight profiles, for example.

[0108] In some aspects, a GTF enzyme that is capable of synthesizing alpha- 1,3 -glucan herein can comprise an amino acid sequence that is about 100% identical to, or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 26, 28, 30, 34, or 59, or amino acid residues 55-960 of SEQ ID NO:4, residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20, and have GTF activity; these amino acid sequences are as disclosed in U.S. Patent Appl. Publ. No. 2019 / 0078063, which is incorporated herein by reference. It is noted that such a GTF enzyme comprising SEQ ID NO:2, 4, 8, 10, 14, 20, 26, 28, 30, 34, or amino acid residues 55-960 of SEQ ID NO:4, residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20, can synthesize alpha-glucan comprising at least about 90% (-100%) alpha-1,3 linkages. A GTF enzyme that is capable of synthesizing alpha- 1,3 -glucan in some aspects can be that identified as GTF 0974 (SEQ ID NO: 13 herein, SEQ ID NO: 110 in US2018 / 0291311), or a GTF comprising an amino acid sequence that is about 100% identical to, or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, the foregoing amino acid sequence of GTF 0974 (and having GTF activity). Any of the foregoing GTF enzyme amino acid sequences can be modified as described herein to increase product yield, modify product molecular weight, and / or enhance GTF performance and / or stability.

[0109] A GTF enzyme for producing alpha- 1,3 -glucan herein can, in some aspects, synthesize alpha- 1,3 -glucan at a yield of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%. Yield in some aspects can be measured based on the glucosyl component of the reaction, and / or as measured using HPLC or NIR spectroscopy. Yield can be achieved in a reaction conducted for about 16-24 hours (e.g., ~20 hours), for example. Examples of such a GTF enzyme are those having an amino acid sequence modified such that the enzyme produces more products (alpha- 1,3- glucan and fructose), and less by-products (e.g., glucose, oligosaccharides such as leucrose), from a given amount of sucrose substrate. For example, one, two, three, four, or more amino acid residues of the catalytic domain of an alpha-l,3-glucan-producing GTF herein can be modified / substituted to obtain a GTF enzyme that produces more products. Examples of a suitable modified GTF enzyme are disclosed in Tables 3-7 of U.S. Patent Appl. Publ. No. 2019 / 0078063, which is incorporated herein by reference. A modified GTF enzyme, for example, can comprise one or more amino acid substitutions corresponding with those in Tables 3-7 (ibid.) that is / are associated with an alpha- 1,3 -glucan yield of at least 40% (the position numbering of such at least one substitution corresponds with the position numbering of SEQ ID NO:62 as disclosed in U.S. Patent Appl. Publ. No. 2019 / 0078063). A set of amino acid modifications as listed in Tables 6 or 7 ibidd) can be used, for example.

[0110] The amino acid sequence of a GTF enzyme for alpha- 1,3 -glucan synthesis in some aspects has been modified such that the enzyme produces alpha- 1,3 -glucan with a molecular weight (DPw) that is lower than the molecular weight of alpha- 1,3 -glucan produced by its corresponding parent GTF. Examples of a suitable modified GTF enzyme are disclosed in Tables 3 and 4 of U.S. Patent Appl. Publ. No. 2019 / 0276806, which is incorporated herein by reference. A modified GTF enzyme, for example, can comprise one or more amino acid substitutions corresponding with those in Tables 3 and / or 4 (ibid.) that is / are associated with an alpha- 1,3 -glucan product molecular weight that is at least 5% less than the molecular weight of alpha- 1,3 -glucan produced by parent enzyme (the position numbering of such at least one substitution corresponds with the position numbering of SEQ ID NO:62 as disclosed in U.S. Patent Appl. Publ. No. 2019 / 0276806). A set of amino acid modifications as listed in Table 4 (ibid.) can be used, for example.

[0111] The amino acid sequence of a GTF enzyme for alpha- 1,3 -glucan synthesis in some aspects has been modified such that the enzyme produces alpha- 1,3 -glucan with a molecular weight (DPw) that is higher than the molecular weight of alpha- 1,3 -glucan produced by its corresponding parent GTF. Examples of a suitable modified GTF enzyme are disclosed in Tables 3, 4 and 5 of U.S. Patent Appl. Publ. No. 2019 / 0078062, which is incorporated herein by reference. A modified GTF enzyme, for example, can comprise one or more amino acid substitutions corresponding with those in Tables 3, 4 and / or 5 (ibid.) that is / are associated with an alpha- 1,3 -glucan product molecular weight that is at least 5% higher than the molecular weight of alpha- 1,3 -glucan produced by parent enzyme (the position numbering of such at least one substitution corresponds with the position numbering of SEQ ID NO:62 as disclosed in U.S. Patent Appl. Publ. No. 2019 / 0078062). A set of amino acid modifications as listed in Table 5 (ibid.) can be used, for example.

[0112] In some aspects, a modified GTF for alpha- 1,3 -glucan synthesis (i) comprises at least one amino acid substitution or a set of amino acid substitutions (as described above regarding yield or molecular weight), and (ii) comprises or consists of a GTF catalytic domain that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to amino acid residues 55-960 of SEQ ID NO:4, amino acid residues 54-957 of SEQ ID NO:65, amino acid residues 55-960 of SEQ ID NO:30, amino acid residues 55-960 of SEQ ID NO:28, or amino acid residues 55-960 of SEQ ID NO:20 (each of these sequences as disclosed in U.S. Patent Appl. Publ. No. 2019 / 0078063, which is incorporated herein by reference). Each of these subsequences are the approximate catalytic domains of each respective reference sequence, and produce alpha- 1,3 -glucan comprising at least about 50% (e.g., >90% or >95%) alpha-1,3 linkages. In some aspects, a modified GTF (i) comprises at least one amino acid substitution or a set of amino acid substitutions (as described above), and (ii) comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:62 or a subsequence thereof such as SEQ ID NO:4 (without start methionine thereof) or positions 55-960 of SEQ ID NO:4 (approximate catalytic domain) (each of these sequences as disclosed in U.S. Patent Appl. Publ. No. 2019 / 0078063).

[0113] In the present disclosure, SEQ ID NOs:5, 6, 7, 8, 9 and 10 (Table A) are the same amino acid sequences as, respectively, SEQ ID NOs:4, 65, 30, 28, 20 and 62 as disclosed in U.S. Patent Appl. Publ. No. 2019 / 0078063. Thus, each of presently disclosed SEQ ID NOs:5, 6, 7, 8, 9 and 10 can be used in any of the disclosed aspects, as appropriate. For example, a GTF enzyme that is capable of synthesizing alpha- 1,3 -glucan herein can comprise an amino acid sequence that is about 100% identical to, or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, SEQ ID NO:5, 6, 7, 8, 9, or 10, or amino acid residues 55-960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55-960 of SEQ ID NO:8, or residues 55-960 of SEQ ID NO:9. Any of these sequences can be modified as described herein to affect alpha- 1,3 -glucan yield and / or molecular weight and / or stability, for example.

[0114] In some aspects, a GTF enzyme for alpha- 1,3 -glucan synthesis has been modified such that the enzyme has enhanced performance and / or stability benefit(s). Such a GTF enzyme can be as disclosed, for example, in Int. Patent Appl. Publ. No. W02023 / 055902, which is incorporated herein by reference. Modification of such a GTF can be, for example, by having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid substitutions as compared to a corresponding parent GTF enzyme (e.g., a wild type mature GTF or active subsequence thereof such as a catalytic domain). Exemplary performance and / or stability benefits herein include one or more of increased thermal stability, increased storage stability, increased solubility, better pH profile, increased specific activity, modified substrate specificity, modified substrate binding, modified pH-dependent activity, modified pH-dependent stability, increased oxidative stability, increased expression, and / or increased glucan product yield (and / or decreased byproduct [e.g., leucrose] yield). In some aspects, a performance benefit is realized at a relatively low temperature (e.g., <5 °C) or at a relatively high temperature (e.g., >40 °C). An increase in any of the foregoing features can be by about, or at least about, 5%, 10%, 15%, 20%, 25%, or 30%, for example, as compared to the respective activity of a parent GTF enzyme that has not been modified.

[0115] Some examples of modified alpha-l,3-glucan-producing GTF enzymes herein having enhanced performance and / or stability benefit(s) comprise or consist of SEQ ID NO:3 (vGTFJ) or 4. It is noted that SEQ ID NOs:3 and 4 are both derivable from SEQ ID NO:5 (GTF 6855), for example (e.g., SEQ ID NO:5 can be a backbone for making substitutions to render SEQ ID NOs:3 and 4).

[0116] In some aspects of the present disclosure, a modified GTF enzyme can comprise or consist of an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:3, and have one or more of (or all of) the following amino acid residues: 8-Asn, 9- Ala, 336-Tyr, 411-Leu, 430-Tyr, 448-Ala, 564-Ser, 1254-Gln, and / or 1273-Phe. The valine at position 1 of SEQ ID NO:3 in any of the foregoing aspects can optionally instead be a methionine, or can be deleted.

[0117] In some aspects of the present disclosure, a modified GTF enzyme can comprise or consist of an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:4, and have one or more of (or all of) the following amino acid residues: 336-Tyr, 430-Tyr, 431-Val, 448-Ala, 564- Ser, 1011-Glu, 1150-His, 1155-Ala, 1241-Lys, 1242-Glu, 1243-Gly, 1244-Ser, 1247-Leu, and / or 1248-Val. The valine at position 1 of SEQ ID NO:4 in any of the foregoing aspects can optionally instead be a methionine, or can be deleted.

[0118] Although it is believed that a modified alpha-l,3-glucan-producing GTF enzyme in some aspects need only have a catalytic domain, the modified GTF can be comprised within a larger amino acid sequence. For example, a catalytic domain may be linked at its C-terminus to a glucan-binding domain, and / or linked at its N-terminus to a variable domain and / or signal peptide.

[0119] Although amino acid substitutions in a modified alpha-l,3-glucan-producing GTF enzyme are generally disclosed in some aspects with respect to corresponding positions in SEQ ID NO: 10, such substitutions can alternatively be stated simply with respect to its / their position number in the amino acid sequence used to produce the modified GTF itself (e.g., SEQ ID NO:5 [optionally without start methionine thereof] or positions 55-960 of SEQ ID NO:5 [approximate catalytic domain]), as convenience may dictate. Such can be done simply by aligning the amino acid sequence with SEQ ID NO: 10 and identifying the position number(s) of interest in the amino acid sequence based on its / their direct alignment with the corresponding position(s) in SEQ ID NO: 10.

[0120] An alpha-l,3-glucan-producing GTF herein is capable of producing alpha- 1,3 -glucan comprising about, or at least about, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% alpha-l,3-glycosidic linkages, for example. Alpha- 1,3 -glucan as disclosed elsewhere herein such as in a homopolymer or graftcopolymer (e.g., as a side chain) can have any of the foregoing linkage profiles, for example.

[0121] An alpha-l,3-glucan-producing GTF herein is capable of producing alpha- 1,3 -glucan with a DPw, DPn, or DP of about, less than about, or at least about, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, or 1650, for example. DPw, DPn, or DP can optionally be expressed as a range between any two of these values. Merely as examples, the DPw, DPn, or DP can be about 100-1650, 200-1650, 300-1650, 400-1650, 500-1650, 600- 1650, 700-1650, 100-1250, 200-1250, 300-1250, 400-1250, 500-1250, 600-1250, 700-1250, 100-1000, 200-1000, 300-1000, 400-1000, 500-1000, 600-1000, 700-1000, 100-900, 200- 900, 300-900, 400-900, 500-900, 600-900, 700-900, 11-25, 12-25, 11-22, 12-22, 11-20, 12- 20, 20-300, 20-200, 20-150, 20-100, 20-75, 30-300, 30-200, 30-150, 30-100, 30-75, 50-300, 50-200, 50-150, 50-100, 50-75, 75-300, 75-200, 75-150, 75-100, 100-300, 100-200, 100-150, 150-300, 150-200, or 200-300. Alpha- 1,3 -glucan as disclosed elsewhere herein such as in a homopolymer or graft-copolymer (e.g., as a side chain) can have any of the foregoing molecular weight profiles, for example. In some aspects, a GTF enzyme can be any as disclosed herein and include 1-300 (or any integer there between [e.g., 10, 15, 20, 25, 30, 35, 40, 45, or 50]) residues on the N- terminus and / or C-terminus. Such additional residues can be from a corresponding wild type sequence from which the GTF enzyme is derivable, or can be a heterologous sequence such as an epitope tag (at either N- or C-terminus) or a heterologous signal peptide (at N- terminus), for example. A GTF enzyme herein typically lacks an N-terminal signal peptide; such an enzyme can optionally be characterized as being mature if its signal peptide was removed during a secretion process.

[0122] A GTF enzyme herein can typically be derived from bacteria. Examples of bacterial GTF enzymes are those derived from a Streptococcus species, Leuconostoc species, or Lactobacillus species. Examples of Streptococcus species include S. salivarius, S. sobrinus. S. denlirouselli. S. downei. S. mulans. S. oralis, S. gallolyticus and S. sanguinis. Examples of Leuconostoc species include L. mesenleroides, L. amelibiosum, L. argenlinum, L. carnosum, L. cilreum, L. cremoris, L. dextranicum and L. fructosum. Examples of Lactobacillus species include L. acidophilus, L. delbrueckii, L. helveticus, L. salivarius, L. casei, L. curvatus, L. plantarum, L. sakei, L. brevis, L. buchneri, L. fermentum and L. reuteri.

[0123] A GTF enzyme herein can be prepared by fermentation of an appropriately engineered microbial strain, for example. Recombinant enzyme production by fermentation can be done, for example, using microbial species such as E. coli, Bacillus strains (e.g., B. subtilis), Ralstonia eutropha, Pseudomonas jluorescens, Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, and species of Aspergillus (e.g., A. awamori) and Trichoderma (e.g., T. reesei) (e.g., see Adrio and Demain, Biomolecules 4: 117-139, 2014, which is incorporated herein by reference). A nucleotide sequence encoding a GTF amino acid sequence is typically linked to a heterologous promoter sequence to create an expression cassette for the enzyme, and / or is codon-optimized accordingly. Such an expression cassette can be incorporated in a suitable plasmid or integrated into the microbial host chromosome. The expression cassette can include a transcriptional terminator nucleotide sequence following the amino acid coding sequence. The expression cassette can also include, between the promoter sequence and GTF amino acid coding sequence, a nucleotide sequence encoding a signal peptide (e.g., heterologous signal peptide) that is designed for direct secretion of the GTF enzyme. At the end of fermentation, cells can be ruptured accordingly (generally when a signal peptide for secretion is not employed) and the GTF enzyme can be isolated using methods such as precipitation, filtration, and / or concentration. Alternatively, a lysate or extract comprising a GTF can be used without further isolation. If the GTF was secreted (i.e., it is present in the fermentation broth), it can optionally be used as isolated from, or as comprised in, the fermentation broth. The activity of a GTF enzyme can be confirmed by biochemical assay, such as measuring its conversion of sucrose to glucan polymer.

[0124] Alpha-glucan produced in a food dough / precursor or product thereof in some aspects can comprise a graft copolymer comprising:

[0125] (i) an alpha- 1,6-glucan (dextran) backbone, wherein at least about 50% of the glycosidic linkages of the alpha- 1,6-glucan (dextran) backbone are alpha-1,6 linkages, and

[0126] (ii) at least one alpha- 1,3 -glucan side chain, wherein at least about 50% of the glycosidic linkages of the alpha- 1,3 -glucan chain are alpha-1,3 linkages.

[0127] Such a graft copolymer can be aqueous-soluble or aqueous-insoluble. Dextran backbone of an alpha-glucan graft copolymer herein can be dextran as presently disclosed, for example, or can be as disclosed (e.g., molecular weight, linkage / branching profile, production method) in U.S. Patent Appl. Publ. Nos. 2016 / 0122445, 2017 / 0218093, 2018 / 0282385, 2020 / 0165360, or 2019 / 0185893, which are each incorporated herein by reference. In some aspects, a dextran backbone (before being integrated into a graft copolymer) has been alpha-1,2- and / or alpha- 1,3 -branched; the percent alpha-1,2 and / or alpha-1,3 branching of a backbone of a graft copolymer herein can be about, at least about, or less than about, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 2-25%, 2-20%, 2-15%, 2-10%, 5-25%, 5-20%, 5-15%, 5-10%, 7-13%, 8-12%, 9-11%, 10-25%, 10-20%, or 10-15%, for example. Alpha- 1,3-glucan side chain(s) of an alpha-glucan graft copolymer herein can be alpha- 1,3 -glucan as presently disclosed, for example, or can be as disclosed (e.g., molecular weight, linkage profile), in U.S. Patent Nos. 7000000, 8871474, 10301604, or 10260053, or U.S. Patent Appl. Publ. Nos. 2019 / 0112456, 2019 / 0078062, 2019 / 0078063, 2018 / 0340199, 2018 / 0021238, 2018 / 0273731, 2017 / 0002335, 2015 / 0232819, 2015 / 0064748, 2020 / 0165360, 2020 / 0131281, or 2019 / 0185893, which are each incorporated herein by reference.

[0128] One, two, three, or more different GTF enzymes that synthesize alpha- 1,6-glucan herein can be used, for example, in a method as presently disclosed. In some aspects, only an alpha-1, 6-glucan-producing GTF(s) is used (i.e., an alpha-l,3-glucan-producing GTF is not used). One, two, three, or more different GTF enzymes that synthesize alpha- 1,3 -glucan herein can be used, for example, in a method as presently disclosed. In some aspects, only an alpha-1, 3-glucan-producing GTF(s) is used (i.e., an alpha- 1,6-glucan-producing GTF is not used). In some aspects, an alpha- 1,6-glucan-producing GTF(s) can be added to (made to contact) a food dough / precursor before adding an alpha- 1, 3-glucan-producing GTF(s), while in some aspects both these types of GTF enzymes can be added at about the same time (simultaneously). Still, in some aspects, an alpha- 1, 3-glucan-producing GTF(s) can be added to a food dough / precursor before adding an alpha- 1,6-glucan-producing GTF(s). Still, in some aspects, a dextran as disclosed herein, but produced exogenously to the food dough / precursor, can be added as an ingredient to a food dough / precursor to which an alpha- 1,3 -glucan-producing GTF has already been added or will be added. While not being held to any particular theory, it is believed that addition of at least one alpha- 1,6-glucan-producing GTF (and / or exogenously produced dextran) and at least one alpha- 1,3 -glucan-producing GTF in a method of producing a food dough / precursor allows for production of a dextranalpha- 1,3 -glucan graft copolymer as presently disclosed, possibly along with production of dextran and / or alpha- 1,3 -glucan homopolymer(s) (i.e., alpha- 1,6-glucan and / or alpha-1, 3- glucan produced independent from the production of graft copolymer). However, it is believed possible that, in some aspects, only dextran and / or alpha- 1,3 -glucan homopolymer(s) is / are produced with little (e.g., < 5 wt% of all glucan products) or no production of graft copolymer. Still, in some aspects, it is believed possible that alpha-1, 6- glucan and / or alpha- 1,3 -glucan as presently disclosed is / are not produced when using an alpha-1, 6-glucan-producing GTF(s) and / or an alpha- 1,3 -glucan-producing GTF(s) in a method herein.

[0129] The molecular weight and / or linkage profile of alpha-glucan produced by a GTF enzyme (dextransucrase or alpha- 1,3 -glucan-producing GTF) as generally disclosed above can be as observed, for example, in an isolated reaction consisting of, or essentially of, water, sucrose, GTF enzyme and optionally one or more salts and / or buffer. In some aspects, the molecular weight and / or linkage profile of alpha-glucan as produced by one or both of these types of GTF enzyme in a food dough / precursor herein may be different from what is produced in the foregoing isolated reaction.

[0130] The content of at least one of an alpha- 1,6-glucan-producing GTF and / or alpha-1, 3- glucan-producing GTF in a food dough / precursor herein can be about, or at least about, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75., 2, 2.5, 0.1-1, 0.1-0.75, 0.1-0.5, 0.1-0.3, 0.2-1, 0.2-0.75, 0.2-0.5, 0.2-0.3, 0.5-2.5, 0.5-2, 0.5-1.5, 0.5-1, 1-2.5, 1-2, or 1-1.5 wt%, for example. In some aspects, a single GTF is used, whereas two, three, or more GTF enzyme(s) can be used in other aspects. The foregoing enzyme contents can be with respect to one GTF enzyme, or a combination of GTF enzymes. The foregoing GTF enzyme contents are typically with respect to active enzyme(s), but in some aspects can be with respect to total protein of isolated / purified enzyme(s). In some aspects, the amount of a GTF enzyme(s) used can be as disclosed in the below Examples, or within 5 to 10% or 5 to 20% of the disclosed amount.

[0131] In some aspects, the ratio of a GTF enzyme that is capable of synthesizing alpha- 1,6- glucan to a GTF enzyme that is capable of synthesizing alpha- 1,3 -glucan in a method herein is about 95:5 to about 5:95. Yet, in some aspects, an alpha- 1,6-glucan-producing GTF to alpha-1, 3-glucan-producing GTF ratio can be about 97.5:2.5, 95:5, 92.5:7.5, 91 :9, 90: 10, 89: 11, 87.5: 12.5, 85: 15, 82.5: 17.5, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 5:95, or 2.5:97.5, or range between any two of these ratios (e.g., about 95:5 to about 50:50, about 95:5 to about 75:25, or about 80:20 to 60:40). The amount of each enzyme (active enzyme) for purposes of determining a ratio thereof herein can be on a molar, weight, or GTF activity basis, for example. The activity of a GTF enzyme for preparing a ratio herein can optionally be determined as disclosed in U.S. Patent Appl. Publ. No. 2014 / 0087431 or Int. Patent Appl. Publ. No. W02023 / 055902, which are incorporated herein by reference, or as disclosed in Example 2 herein. In some aspects, a full (e.g., “100%”) complement of a GTF enzyme for setting up a ratio herein can be that amount of enzyme that can convert most of (e.g., >95%, >98%, >99%), or all of, sucrose in a GTF reaction comprising or consisting of water, sucrose (e.g., 50 or 100 g / L), the GTF, and optionally buffer / salt in a given amount of time (e.g., 6, 12, 18, 24, 30, or 36 hours); such a measured amount can optionally be characterized as a normalized amount of GTF.

[0132] A GTF enzyme (or any other enzyme as presently disclosed) for use in a method herein is typically in purified (isolated) form. A purified enzyme can be essentially free from insoluble and / or soluble components of an organism / cell used to produce the enzyme, and / or any medium that was used for cellular fermentation of the enzyme. In some aspects, a purified enzyme denotes an enzyme preparation that contains less than 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% by weight of other material (e.g., polypeptide material) with which the enzyme is natively or recombinantly associated. In some aspects, a GTF and / or any other enzyme herein is not comprised in or otherwise associated with (e.g., expressed by) a microbial (e.g., bacterial, yeast, fungal, algal) cell that might be present (e.g., endogenously or purposely added) in a food product / precursor herein; however, in some aspects a GTF and / or any other enzyme herein is comprised in or otherwise associated with (e.g., expressed by) a microbial (e.g., bacterial, yeast, fungal, algal) cell such as one that heterologously expresses the enzyme(s) (i.e., recombinant cells). Contacting a food product / precursor with a GTF enzyme(s) herein typically is not performed in an oral cavity or other environment in which unpurified / non-isolated GTF enzymes can possibly be present.

[0133] A GTF enzyme (or any other enzyme as presently disclosed) for use in a method herein can be comprised in a sterile-filtered preparation, for example. In some aspects, an enzyme can be sterile-filtered inline while applying the enzyme to a food product / precursor during a contacting step. In some aspects, an enzyme can be added to a food product / precursor that has been pasteurized (after pasteurization), or alternatively an enzyme can be added before pasteurizing the food product / precursor. In some aspects, an enzyme can be added to a food product / precursor that has been fermented (after fermentation), or alternatively an enzyme can be added during or before fermenting the food product / precursor. A GTF enzyme (or any other enzyme as presently disclosed) in some aspects for use in a method herein can be comprised in a preparation that is substantially free of (e.g., <0.5, <0.1, <0.05 wt%) any other enzyme(s) such as a lipase, protease, amylase, mannanase, pectinase, cellulase, and / or p-nitrobenzylesterase; such a preparation typically has little or no detectable activity(ies) of such other enzyme(s).

[0134] An aqueous composition used in a food dough production method herein typically is water, an aqueous solution, and / or an aqueous mixture. An example of an aqueous mixture herein is a dispersion such as a homogenized product (e.g., milk such as skim milk, low-fat milk, whole milk, or other dispersed dairy product). Further examples of an aqueous composition herein include whole eggs (comprise -70-80 wt% water), egg whites (comprise -85-90 wt% water), egg yolks (comprise -45-50 wt% water), buttermilk, yogurt, sour cream, cottage cheese, soft cheese (e.g., ricotta), coconut milk, juice (e.g., fruit or vegetable juice), beer (e.g., ale, lager), cider, wine, liquor, or soda. One, two, three, four, or more of the foregoing aqueous ingredients can be in the aqueous composition component of a dough recipe herein.

[0135] The content of flour and / or meal (or the total solids content) in a flour dough or flour slurry (or other dough precursor / ingredient) herein can be about, or at least about, 30, 35, 40, 45, 50, 55, 60, 30-60, 30-50, 30-40, 35-60, 35-50, 35-40, 40-60, 40-50, or 50-60 wt%, for example. Typically, the balance of the dough or slurry (in weight) can be of water. Optionally, the balance can be of water plus some amount of other food-suitable liquid where the water typically is over 80%, 90%, or 95% by weight of the total liquid, for example. Examples of a food-suitable liquid include oil (e.g., coconut oil, palm kernel oil, palm oil, cottonseed oil, wheat germ oil, soybean oil, olive oil, corn oil, sunflower oil, safflower oil, hemp oil, canola / rapeseed oil, vegetable oil, lecithin oil) and any other suitable liquid as presently disclosed.

[0136] A food (e.g., bread), food dough (e.g., bread dough), or precursor in some aspects of the present disclosure can comprise sucrose that is endogenous to the food, dough, or precursor (e.g., its sucrose is native), and / or can comprise sucrose that has been added to the food, dough, or precursor (either during or after its preparation as an ingredient) (e.g., can be characterized as being “sweetened”). The sucrose content of a food, dough, or precursor provided and / or prepared in a method herein, regardless of the original source of the sucrose (exogenous ingredient and / or endogenous), can be about, at least about, or less than about, 1, 3, 5, 7, 10, 15, 20, 25, 30, 3-15, 3-10, 5-15, 5-10, 7-15, or 7-10 wt%, for example. In some aspects, sucrose can be provided as white refined sucrose, or in an unrefined form such as disclosed in U.S. Patent No. 9719121, for example, which is incorporated herein by reference. The foregoing sucrose content can be, for example, with respect to the initial sucrose content (i.e., pre-GTF-reacted basis), or the final sucrose content (i.e., post-GTF- reacted basis), of a food, dough, or precursor herein.

[0137] A food (e.g., bread), food dough (e.g., bread dough), or precursor herein can optionally comprise other ingredients in addition to its water (for dough and food products comprising water), flour / meal, sucrose (sometimes optional, such as in a food product herein), GTF enzyme(s), and alpha-glucan product (typically as produced following GTF activity) components. For example, at least one component selected from lipids, emulsifiers, enzymes, salt, sugar (e.g., other sugar beside sucrose, such as fructose and / or dextrose), added protein source (e.g., whey protein, egg), added starch, added gluten, leavening agent (e.g., yeast, baking powder, baking soda), natural and / or artificial flavors / colors, aqueous liquid (e.g., water, milk), and / or artificial sweetener can be included. A yeast in some aspects is a standard baking yeast, while in some aspects it is a yeast that has reduced (e.g., less than about 200, 100, 50, 25, 10, 5, 2.5, 1, 0.1, or 0.01 U / g invertase enzyme activity), non- detectable, or no invertase activity; the latter type of yeast (reduced or no invertase activity; e.g., a “brown yeast”) can be as produced or described in U.S. Pat. Nos. 4693898 or 5801049, for example, which are each incorporated herein by reference. Examples of lipid ingredients include triglycerides and phospholipids, such as soybean oil, soybean lecithin, butter, lard, margarine, corn oil, peanut oil, canola oil, or olive oil. Examples of emulsifiers include monoglycerides, diglycerides and esters of these glycerides and acids such as monoglyceride lactate or monoglyceride diacetyltartrate. Examples of enzymes (in addition to a GTF herein) include proteolytic enzymes, amylolytic enzymes and hemicellulolytic enzymes (e.g., alphaamylase, beta-amylase, endo-xylanase, protease). Additional examples of enzymes include lipase, alpha-amylase, xylanase, non-maltogenic exoamylase, glucoamylase, glucose oxidase, and hexose oxidase. Examples of other agents that can be included in a food, food dough, or precursor herein include oxidizing agents, reducing agents, and water-binding components such as hydrocolloids (e.g., pectin, gelatin, carboxymethyl cellulose, carrageenan, guar, locust bean gum, exogenously added alpha- 1,3 -glucan, exogenously added dextran). Bread, bread dough, or dough precursor in some aspects can comprise one or more ingredients as disclosed in any of U.S. Pat. Appl. Publ. Nos. 2005 / 0013900, 2009 / 0297663, or 2018 / 030310, which are incorporated herein by reference. In addition to these references, a food, food dough, or precursor herein such as a bread, bread dough, or dough slurry can optionally comprise one or more ingredients according to any of U.S. Patent Nos. 3889003, 3930055, 3987206, 4367241, 4645673, 4687673, 4849230, 5403610, 5409717, 7947319, 7815952, 9883679, or 8486469, all of which are incorporated herein by reference.

[0138] In some aspects, a food (e.g., bread), food dough (e.g., bread dough), or precursor herein further comprises at least one disaccharide in addition to sucrose, and / or at least one oligosaccharide. An oligosaccharide can have 3-15 or 3-20 monomeric units (i.e., DP3-DP15 or DP3-DP20), for example (e.g., DP3-DP5, DP3-DP6); thus, in some aspects, a polysaccharide herein has more than 15 or 20 monomeric units. A disaccharide and / or oligosaccharide herein can comprise only glucose monomeric units, for example, and / or one or more other types of monosaccharides (e.g., galactose, fructose, mannose) as monomeric units. Examples of disaccharides herein (in addition to sucrose) include maltose, isomaltose, lactose, lactosucrose, nigerose, leucrose, trehalulose, maltulose, isomaltulose, and turanose. Examples of oligosaccharides herein include gluco-oligosaccharides (gluco-oligomers) such as malto-oligosaccharides (MOS) and isomalto-oligosaccharides (IMO), and galactooligosaccharides (GOS).

[0139] In some aspects, a food (e.g., bread), food dough (e.g., bread dough), or precursor herein further comprises one or more enzymes in addition to one or more GTF enzymes. Examples of other enzymes include lipases, alpha-amylases, xylanases, non-maltogenic exoamylases, glucoamylases, glucose oxidases, and / or hexose oxidases. Such one or more other enzymes can be added to a flour dough or flour slurry production method in the same manner as one or more GTF enzymes herein are added, for example.

[0140] A food dough production method herein can comprise a step of mixing / blending / stirring at least (a) flour and / or meal, (b) water or aqueous composition, (c) sucrose and (d) one or more GTF enzymes herein, to produce a food dough. Yet, in some aspects, a food dough production method can simply be characterized to comprise providing a food dough comprising at least (a) flour and / or meal, (b) water or aqueous composition, (c) sucrose and (d) one or more GTF enzymes herein. Mixing herein typically produces a flour dough or slurry.

[0141] The mixing step of a food dough production method herein can comprise, for example, addition of all of at least the recipe ingredients of (a) flour and / or meal, (b) water or aqueous composition, (c) sucrose and (d) one or more GTF enzymes herein together into a mixing vessel, followed by mixing, or mixing two or three of the ingredients first followed by admixture of the remaining ingredient(s) (e.g., [a], [b], [c], and then [d]; [b], [c], [d], and then [a]; [a], [b], [d], and then [c]; [a], [c], [d], and then [b]). One or more other ingredients (e.g., any as disclosed herein such as a leavening agent and / or salt) can optionally be included in any of these mixing schemes, accordingly. Mixing herein can be done manually or via automation. Mixing can optionally also be characterized herein as stirring, blending, or any other like term.

[0142] In some aspects, a food dough or slurry produced herein is allowed to incubate for a time (e.g., ~0.5 hour to about, or at least about, 1, 2, 4, 6, 8, 10, or 12 hours) and / or temperature (e.g., -5-50 °C, -20-40 °C, -30-40 °C, -20-30 °C, -20-25 °C, -20 °C, -25 °C, -30 °C, -35 °C, -37 °C, or -40 °C) allowing one or more of the ingredient GTF enzyme(s) to produce alpha-glucan (and / or exert other effects such as compound glycosylation) in the dough or slurry. It is contemplated that such in situ GTF activity can also occur while initially mixing and / or kneading a dough having all of at least ingredients (a), (b), (c) and (d) (above).

[0143] Yet, in some aspects, incubation of one or more GTF enzymes (and / or any other enzyme as presently disclosed) in a flour dough or slurry can be for about, or at least about, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 24, 30, 36, 42, 48, 72, 96, 0.5-3, 0.5-2.5, 0.5-2, 0.5-1.5, 1-3, 1-2.5, 1-2, 1-1.5, 1.5-3, or 1.5-2 hours, or for about, or at least about, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 days (or a range between any two of these hours and / or days), for example. The temperature for incubating one or more enzymes in a dough or slurry herein can be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 2-5, 2-10, 2-15, 2-20, 2-25, 2-30, 2-35, 2-40, 2-45, 2-50, 3-5, 3-10, 3-15, 3-20, 3-25, 3-30, 3-35, 3-40, 3-45, 3-50, 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 15-20, 15-25, 15-30, 15-35, 15-40, 15-45, 15-50, 20-25, 20-30, 20-35, 20- 40, 20-45, 20-50, 25-30, 25-35, 25-40, 25-45, 25-50, 30-35, 30-40, 30-45, or 30-50 °C, for example.

[0144] In some aspects, one or more GTF enzymes (and / or any other enzyme as presently disclosed) can be added during a food dough production method in dry form (e.g., powder, flakes, lyophilized enzyme preparation) or wet form, either of which enzyme form typically is added to a wet preparation. In some aspects, a flour can be combined with GTF enzyme(s) under dry conditions (resulting combination is dry) (optionally also including sucrose), after which time water or an aqueous composition (optionally including sucrose) is added. The water content of a food dough or slurry (or food product) herein can be about, or at least about, 10, 20, 30, 40, 50, 60, 70, 80, or 90 wt%, for example. The pH of a food dough or slurry herein, which is typically the pH for incubating one or more enzymes in the food dough or slurry, can be about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 5.0-9.0, 5.0-8.0, 5.5-9.0, 5.5-8.0, 6.0-9.0, or 6.0-8.0, for example.

[0145] A GTF enzyme (and / or any other enzyme as presently disclosed) herein can optionally be provided by introducing a recombinantly engineered cell (e.g., a microbial cell such as a bacterial or fungal / yeast cell) during food dough / slurry production, wherein the cell recombinantly (heterologously) expresses and secretes the enzyme(s) in the dough or slurry. Such a cell can be that of a microbe that is amenable to recombinant engineering and useful in food processing (e.g., fermentation), such as a microbial cell disclosed herein (as applicable). In some aspects, a recombinantly engineered cell that is provided in a flour dough or slurry herein can be inactive and / or non-viable in some manner, such as by having been killed (but preferably in a manner that otherwise retains cellular shape / structure). For example, a cell can be rendered inactive and / or non-viable by being irradiated or being treated with a sterilizing agent / chemical (e.g., ethylene oxide). Typically, the means for cell inactivation and / or killing preserves at least some of the three-dimensional shape / structure of the cell, and / or ensures that enzyme(s) that had been expressed by the cell remains active and typically remains associated with the inactive / non-viable cell (e.g., such as by being associated with a cellular membrane via an optional transmembrane domain or membranebinding domain of the enzyme(s) [e.g., fused to the enzyme]). An inactive / non-viable cell typically is porous, and optionally can be immobilized on a support (e.g., an inert, waterinsoluble material, such as of a particle or surface).

[0146] In some aspects, flour and / or meal (optionally further with added water and / or sucrose) can be brought into contact with one or more GTF enzymes by virtue of adding the flour and / or meal to an aqueous composition (a preprepared aqueous composition) comprising at least sucrose and the one or more GTF enzymes. Such a GTF / sucrose- containing aqueous composition can optionally be referred to herein as a “GTF / sucrose starter composition”. Optionally, one or more food precursors as presently disclosed (e.g., ingredients such as a liquid food product / precursor herein, fruit / vegetable puree, syrup, or juice or juice concentrate) can be added to a GTF / sucrose starter composition. The initial sucrose concentration of a GTF / sucrose starter composition can be, for example, about 1-5%, 1-10%, 2-5%, 2-10%, 5-60%, 5-50%, 5-40%, 10-60%, 10-50%, 10-40%, 20-60%, 20-50%, 20-40%, 30-60%, 30-50%, 30-40%, 40-60%, or 40-50% by weight. In some aspects, a GTF / sucrose starter composition has few (e.g., less than about 1, 0.5, 0.1, 0.05, or 0.01 wt%) or no saccharide compounds (e.g., one or more monosaccharides, disaccharides, oligosaccharides and / or polysaccharides, such as presently disclosed) aside from sucrose. A GTF / sucrose starter composition can comprise at least one alpha- 1,6-glucan-producing GTF and / or an alpha-l,3-glucan-producing GTF as presently disclosed, for example. The temperature, pH and / or any other condition / parameter of this methodology (before and / or after adding at least flour and / or meal to a GTF / sucrose starter composition) can be as disclosed herein, for example. In some aspects, a GTF / sucrose starter composition can be incubated for about, at least about, or up to about, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 120, 180, 240, 300, 360, 420, 480, 540, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15- 45, 15-40, 15-35, 15-30, 15-25, 15-20, 20-45, 20-40, 20-35, 20-30, 20-25, 25-45, 25-40, 25- 35, or 25-30 minutes, before adding at least flour and / or meal. Any of these foregoing time periods can also apply to the period of time allowed to proceed after adding the flour and / or meal to the GTF / sucrose starter composition, until optionally terminating the GTF activity (e.g., heat-inactivation at 90-100 °C or 70-110 °C) of the thus prepared flour dough or flour slurry. In some aspects, the activity of GTF enzyme(s) in a GTF / sucrose starter composition can be terminated before combining the starter composition with flour and / or meal (and other dough ingredients). Optionally, in such aspects, no active GTF enzyme is added to the combination (i.e., the only source of GTF-produced alpha-glucan in such aspects is from the GTF / sucrose starter composition made before GTF activity termination). However, in some aspects, active GTF enzyme(s) (typically a GTF different from the now-terminated GTF) can be added and incubated in the combination in any manner as presently disclosed.

[0147] A dough production method herein can further include, for example, a step of processing the food dough into a food product. Such aspects can therefore be characterized as a food production method, if desired. In some aspects, dough processing for food production can comprise at least baking, frying, boiling, steaming, drying, leavening, chilling, extruding, and / or flattening / rolling the food dough. Any of these processing steps can be performed as disclosed in any of U.S. Pat. Appl. Publ. Nos. 2005 / 0013900, 2009 / 0297663, or 2018 / 030310, or U.S. Patent Nos. 3889003, 3930055, 3987206, 4367241, 4645673, 4687673, 4849230, 5403610, 5409717, 7947319, 7815952, 9883679, or 8486469 (all of which are incorporated herein by reference), for example. Example conditions for making a bread product herein include letting a bread dough rise at a temperature of about 20 to 40 °C (e.g., for about 20 to 45 minutes), followed by baking at a temperature of about 350 to 425 °F (e.g., for a time of about 25 to 45 minutes). In some aspects, parameters for conducting dough rising and / or baking (as well as other steps such as mixing), and can be as disclosed in any of U.S. Patent Nos. 4957040, 4538509, 5615605, 6035763, or 6113966, for example, which are all incorporated herein by reference.

[0148] A food as presently disclosed, such as one made using a food dough herein, can be a baked food and / or extruded food, for instance. In some aspects, and optionally in addition to being baked and / or extruded, a food can have been fried, boiled, steamed, dried, leavened, chilled, and / or flattened / rolled during its production.

[0149] Examples of a baked food herein include bread (e.g., buns, sourdough, rye, whole wheat, pita, flatbread, tortilla, combread, brioche, white, baguette, bagels, banana, ciabatta, brown, challah, focaccia, multigrain, bread sticks, soda bread, pumpernickel, potato bread, biscuits, English muffins, whole grain, matzo, lavash, croutons, pizza crust), cakes (e.g., carrot cake, red velvet, angle food, pound cake, chocolate, white, black forest, tiramisu, coffee cake, cheesecake, devil’s food, upside-down cake, Boston cream pie, Swiss roll, lemon cake, short cake, chiffon cake, butter cake, spice cake, rum cake, sponge cake, marble cake, coconut cake, pandan cake), muffins, brownies, scones, cookies, bars, custards, pies, crackers (e.g., saltines, oyster crackers, cream crackers, water biscuits, cheese crackers, graham crackers, digestive biscuits, RITZ style crackers), pretzels, pastries, pudding and tarts. A food herein such as bread can be leavened or unleavened.

[0150] Examples of an extruded food herein include pasta (e.g., spaghetti, rotini, fusilli, penne, bucatini, macaroni / maccheroni, rigatoni, fettuccine, linguine, vermicelli, ziti, farfalle, gomiti / elbow, rotelle), cereal (e.g., direct expanded cereal, filled cereal, flakes, breakfast cereal), some bread products (e.g., croutons, bread sticks, flat breads), pre-made cookiedough, dry and semi-moist pet food (e.g., kibbles), and snacks (e.g., cheese curls, filled pillow puffs, chips [e.g., corn chips, pita chips, processed potato chips, tortilla chips], snack sticks [e.g., vegetable sticks], puffed shaped products such as curls [e.g., cheese curls], balls, tubes, bananas, cups, bowls, disks, baby food puffs). Pasta herein can be extruded (e.g., see above) and / or flattened / rolled (e.g., lasagna), fresh or dried, long or short, minute / soup pasta (pastina), filled (e.g., tortellini, ravioli, agnolotti, tortelli), stretched (e.g., cencioni, corzetti, foglie d’ulivo, orecchiette), and / or egg pasta.

[0151] Examples of a fried food herein include beignets, churros, fried dough (e.g., elephant ears), doughnuts, falafel, fritters, funnel cake, hushpuppies, jin deui, fish cakes, empana, curry puffs, curry bread, croquettes, noodles (wheat, rice), frybread / popovers, haliva, kachori, luchi, ma hua, papadum, paratha, pastel, prawn cracker, puri, papri, samosa, shuangbaotai, fried bread sticks, pierogi, pancakes / hotcakes, naan, roti, chapati, waffles, and batter. A fried food herein can be prepared by cooking food in hot vegetable or animal oil / fat, and / or by heating on a pan / griddle or other vessel typically containing at least a small amount of (much less than used in a deep-frying process) vegetable or animal oil / fat on its cooking surface.

[0152] A food dough herein can be that used to prepare any of the foregoing products, for example. Semolina flour dough optionally can be used to prepare pasta herein.

[0153] A food dough, food product, or any other composition, such as any as presently disclosed, can comprise one or more GTF enzymes herein. In some aspects, a GTF enzyme(s) in any of the foregoing materials has been inactivated, such as by heat-inactivation or another inactivation means.

[0154] A food product or food dough as presently disclosed in some aspects (e.g., a bread or bread dough) can have the same or improved handling / machinability, shelf life, texture, moisture, moisture retention, shape, volume, cohesiveness, strength, viscosity, shock stability, crumb pore size, uniformity of gas bubbles, non-separation of crust and crumb, crust crispiness, oven spring, resilience, softness, staling resistance, water-binding capacity and / or dietary fiber characteristics as compared to a control food product or control food dough. If improved, such improvement of one or more of these characteristics can be by about, or at least about, 1%, 2%, 5%, 7.5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 1-30%, 1-25%, 1-20%, 1-15%, 5-30%, 5-25%, 5-20%, 5-15%, or 7.5-12.5%, for example, as compared to a control food product or control food dough. In some aspects, the shape, cohesiveness, and / or strength of a bread or bread dough herein is / are retained while keeping the benefit of reduced gluten (or gluten-free). In some aspects, an improved aspect herein can persist for about, or at least about, 1, 2, 5, 10, 15, 20, 30, 45, 60, 90, or 120 days (e.g., under standard temperature [e.g., 20-25 °C] and / or humidity conditions [e.g., 30-55% relative humidity]). In some aspects, a food product (e.g., bread product) has increased crumb pore size (e.g., by about, or at least about, 1%, 2%, 5%, 7.5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 1- 30%, 1-25%, 1-20%, 1-15%, 5-30%, 5-25%, 5-20%, 5-15%, or 7.5-12.5%, for example, as compared to a control food product). Food crumb pore size (e.g., bread crumb pore size) can be measured using any suitable means, such as disclosed in the below Examples (e.g., using a digital imaging system, such as C-Cell, according to AACC method 10-18.01, which is incorporated herein by reference) or U.S. Pat. Appl. Publ. No. 2022 / 0125060, which is incorporated herein by reference.

[0155] In some aspects, a food product (e.g., bread product) has increased moisture or moisture retention (e.g., by about, or at least about, 1%, 2%, 5%, 7.5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 1-30%, 1-25%, 1-20%, 1-15%, 5-30%, 5-25%, 5-20%, 5-15%, or 7.5- 12.5%, for example, as compared to a control food product). Food moisture / moisture retention (e.g., bread moisture / retention) can be measured using any suitable means, such as disclosed in the below Examples (e.g., using an analyzer, such as a Mettler Toledo moisture analyzer, according to AACC method 23.8310.F15, which is incorporated herein by reference) or U.S. Pat. No. 5515718, which is incorporated herein by reference.

[0156] In some aspects, a food product (e.g., bread product) has increased softness (e.g., by about, or at least about, 1%, 2%, 5%, 7.5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 1-30%, 1- 25%, 1-20%, 1-15%, 5-30%, 5-25%, 5-20%, 5-15%, or 7.5-12.5%, for example, as compared to a control food product). Food softness (e.g., bread softness) can be measured using any suitable means, such as disclosed in the below Examples (e.g., using an analyzer, such as a texture analyzer from Stable Micro Systems, and / or by AACC Method 74-09, which is incorporated herein by reference) or U.S. Pat. Appl. Publ. No. 2009 / 0297663, which is incorporated herein by reference. Optionally, food softness herein can instead be referred to in terms of reduced food hardness (e.g., reduced bread hardness).

[0157] In some aspects, a food product (e.g., bread product) has increased resilience (e.g., by about, or at least about, 1%, 2%, 5%, 7.5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 1-30%, 1- 25%, 1-20%, 1-15%, 5-30%, 5-25%, 5-20%, 5-15%, or 7.5-12.5%, for example, as compared to a control food product). Food resilience (e.g., bread resilience) can be measured using any suitable means, such as disclosed in the below Examples, or U.S. Pat. Appl. Publ. No. 2018 / 0242598, which is incorporated herein by reference.

[0158] In some aspects, a food dough has a shock stability that is increased as compared to the shock stability of a control food dough. Typically, shock stability of a dough is with regard to a dough that has risen (e.g., dough that has been leavened, such as by comprising an added yeast, and subjected to conditions suitable for allowing the dough to rise from such leavening) (e.g., a raised bread dough loaf / roll). Shock stability in some aspects can be assessed by (i) dropping a risen bread dough at least one time over a distance of about, or at least about, 1.0, 1.5, 2.0, 2.2, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 1.0-2.5, 1.5-2.5, 2.0-2.5, 5.0-7.0, or 5.5-6.5 cm (i.e., a shocking step), or (ii) spinning the risen dough at about 450, 460, 470, 480, 490, 500, 450-500, or 470-490 revolutions-per-minute (rpm) for a time of about, or at least about, 0.5, 1.0, 1.5, 2.0, or 0.5-1.5 minutes (i.e., a shocking step), and then optionally measuring the height and / or volume of the now-shocked bread dough. Such a shocking step in some aspects can simply be practiced by virtue of moving a risen bread dough from one place to another in a bread production facility (e.g., bakery, kitchen), where, during the course of such movement, there is ample vertical movement constituting dropping the raised bread dough by the foregoing distance. Dropping rate typically is about g (9.8 m / s2), for example. Shock stability in some additional or alternative aspects can be assessed by subjecting a risen bread dough to horizontal shaking movement at about 630-650 rpm (e.g., -640 rpm) for about 45-75 seconds (e.g., -60 seconds) (e.g., on a shaking table). In some aspects, height and / or volume assessments can be done with bread that has been baked using the shocked dough. In some aspects, the height or volume of a shocked risen bread dough (or bread baked therewith) is about, or at least about, 5%, 10%, 15%, 20%, 5-20%, 10-20%, 15- 20%, 5-15%, or 10-15% greater than it would be if the shocked risen bread dough (or shocked baked bread) did not comprise a GTF enzyme(s) as presently disclosed during the dough production method. Yet, in some aspects, increased volume can be with respect to unshocked risen dough (e.g., bread dough), or food product made therewith (e.g., baked bread). Volume herein can be in terms of specific volume (e.g., interchangeably provided herein in units of mL / g or cm3 / g), for example, which can optionally be measured with a procedure disclosed in the below Examples (or with parameters within 5% or 10% of those disclosed in the Examples).

[0159] In some aspects, a flour dough or slurry has a viscosity that is increased as compared to the viscosity of a control flour dough or slurry. Such a viscosity increase can optionally manifest during the mixing step of a dough production method by the dough or slurry exhibiting a torque that is about, or at least about, 2%, 3%, 4%, 5%, 6%, 2-6%, 3-6%, or 4- 6% greater than it would be if the dough / slurry did not comprise a GTF enzyme(s) as presently disclosed during the dough / slurry production method. Torque can be measured in Brabender units (BU), for example. Viscosity herein can be assessed using any suitable methodology, such as with a procedure disclosed in the below Examples (or with parameters within 5% or 10% of those disclosed in the Examples).

[0160] Non-limiting examples of compositions and methods disclosed herein include:

[0161] 1. A method (process) of producing a food dough (flour dough or flour slurry), the method comprising: mixing (combining, stirring, and / or kneading) at least (a) flour and / or meal, (b) water or aqueous composition, (c) sucrose, and (d) at least one glucosyltransferase enzyme, thereby producing a food dough, wherein the at least one glucosyltransferase enzyme is selected from: (i) a glucosyltransferase enzyme that is capable of synthesizing (or that synthesizes) alpha- 1,6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,6-glucan are alpha- 1,6 linkages, and / or (ii) a glucosyltransferase enzyme that is capable of synthesizing (or that synthesizes) alpha- 1,3 -glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,3 -glucan are alpha-1,3 linkages, typically wherein at least one alpha-glucan is produced in the food dough, optionally wherein: (I) the food dough has a shock stability that is increased as compared to the shock stability of a control food dough that lacks the at least one glucosyltransferase enzyme, (II) the food dough has a waterbinding capacity that is increased as compared to the water-binding capacity of a control food dough that lacks the at least one glucosyltransferase enzyme, (III) the food dough has a viscosity that is increased as compared to the viscosity of a control food dough that lacks the at least one glucosyltransferase enzyme, and / or (IV) the food dough has a volume that is increased as compared to the volume of a control food dough that lacks the at least one glucosyltransferase enzyme (e.g., the control food dough is produced the same way as the food dough, except that the at least one glucosyltransferase enzyme is not included in producing the control food dough).

[0162] 2. The method of embodiment 1, wherein the at least one glucosyltransferase enzyme is the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 6-glucan.

[0163] 3. The method of embodiment 1 or 2, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:1, 2, 11, 12, 14, or 15 (e.g., an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO: 14 or 15).

[0164] 4. The method of embodiment 1, 2, or 3, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical (e.g., at least 95% identical) to SEQ ID NO: 1, 2, 11, or 12 (e.g., an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO: 1 or 2). 5. The method of embodiment 1, wherein the at least one glucosyltransferase enzyme is the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan.

[0165] 6. The method of embodiment 1, 3, 4, or 5, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical (e.g., at least 95% identical) to residues 55-960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55-960 of SEQ ID NO:8, or residues 55-960 of SEQ ID NO:9.

[0166] 7. The method of embodiment 1, 3, 4, 5, or 6, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical (e.g., at least 95% identical) to residues 55-960 of SEQ ID NO:5.

[0167] 8. The method of embodiment 1, 3, 4, 5, 6, or 7, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical (e.g., at least 95% identical) to SEQ ID NO:3, 4, 5, 6, 7, 8,

[0168] 9. or 13.

[0169] 9. The method of embodiment 1, 3, 4, 5, 6, 7, or 8, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical (e.g., at least 95% identical) to SEQ ID NO:5.

[0170] 10. The method of embodiment 1, 3, 4, 6, 7, 8, or 9, wherein both of the (i) glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan and (ii) glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan are used in the mixing step.

[0171] 11. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein the alpha-glucan produced in the food dough comprises a graft copolymer comprising: (i) an alpha- 1,6-glucan backbone, wherein at least about 50% of the glycosidic linkages of the alpha- 1,6-glucan backbone are alpha- 1,6 linkages, and (ii) at least one alpha- 1,3 -glucan side chain, wherein at least about 50% of the glycosidic linkages of the alpha- 1,3 -glucan chain are alpha-1,3 linkages, wherein the graft copolymer is aqueous-soluble or aqueous-insoluble.

[0172] 12. The method of embodiment 10 or 11, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan in the mixing step is about 95:5 to about 5:95.

[0173] 13. The method of embodiment 10, 11, or 12, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan in the mixing step is about 95:5 to about 50:50 (e.g., about 95:5 to about 75:25).

[0174] 14. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, wherein the flour is used in the method, optionally wherein the flour is wheat flour.

[0175] 14a. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, wherein the mixing comprises (A) first combining (and mixing) the (b) water or aqueous composition, (c) sucrose, and (d) at least one glucosyltransferase enzyme (typically under conditions herein [e.g., temperature, time, pH, sucrose cone.] suitable for synthesis of alpha-glucan by the at least one glucosyltransferase enzyme), and then (B) combining the combination of (b), (c) and (d) with the (a) flour and / or meal (optionally, [i] the activity of the at least one glucosyltransferase can be terminated prior to the further mixing with the flour and / or meal, and / or [ii] additional glucosyltransferase enzymefs] can be added to the mixture of [a]-[d]). 14b. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, wherein the mixing comprises (A) first combining (and mixing) (a) a portion of (e.g., about 10-50, 10-40, 10-30, 20-50, 20-40, or 25-35 wt% of) the total amount of the flour and / or meal to be used in the mixing, (b) most of (e.g., at least about 90, 95, 98, or 99 wt% of) or all of the total amount of the water or aqueous composition to be used in the mixing, (c) optionally the sucrose, and (d) optionally the at least one glucosyltransferase enzyme, thereby producing a flour slurry or pre-mix (i.e., an autolyze composition, and / or a dough precursor) (and incubating / holding the flour slurry / pre-mix under suitable conditions for conducting an autolyze process [e.g., a time of about 10-60, 10-30, 20-60, or 20-30 minutes, and / or at a temperature of about 20-25 °C]), typically wherein at least one alpha-glucan is produced in the flour slurry / pre-mix if the sucrose and at least one glucosyltransferase enzyme were included in the flour slurry / pre- mix, and then (B) combining (and mixing) the flour slurry / pre-mix with the remaining amount of the flour and / or meal to be used in the mixing, and any remaining amount of water or aqueous composition, sucrose and at least one glucosyltransferase enzyme to be used in the mixing (as well as any additional ingredients such as salt, leavening agent [e.g., yeast], and / or optionally other enzymes), thereby producing the food dough.

[0176] 15. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14a, or 14b, further comprising: processing the food dough into a food (food product).

[0177] 16. The method of embodiment 15, wherein the processing comprises at least baking, frying, boiling, drying, chilling, extruding, and / or flattening the food dough. 17. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14a, 14b, 15, or

[0178] 16, wherein the food dough comprises a leavening agent, optionally wherein the leavening agent is yeast (e.g., a standard baking yeast).

[0179] 17a. The method of embodiment 17, wherein the yeast has reduced invertase activity (e.g., less than about 200, 100, 50, 25, 10, 5, 2.5, or 1 U / g invertase enzyme activity) or no detectable invertase activity (e.g., the yeast is a “brown yeast”).

[0180] 18. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14a, 14b, 15, 16,

[0181] 17, or 17a, wherein the food dough is a bread dough.

[0182] 19. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14a, 14b, 15, 16, 17, 17a, or 18, further comprising allowing the food dough to rise, thereby providing risen food dough.

[0183] 20. The method of embodiment 19, wherein the risen food dough is risen bread dough, optionally wherein the volume of the risen bread dough (or the volume of the risen food dough) is at least 5% greater than it would be if the risen bread dough (or the risen food dough) did not comprise the at least one glucosyltransferase enzyme, optionally wherein the risen bread dough (or risen food dough) has not been shocked, or optionally the risen bread dough (or risen food dough) has been shocked.

[0184] 21. The method of embodiment 19 or 20, further comprising dropping the risen food dough at least one time over a distance of at least 1 cm, wherein, following the dropping, the volume of the risen food dough is at least 2% greater than it would be if the risen food dough did not comprise the at least one glucosyltransferase enzyme.

[0185] 22. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14a, 14b, 15, 16, 17, 17a, 18, 19, 20, or 21, wherein the mixing exhibits a torque that is at least 2% greater than it would be if the food dough did not comprise the enzyme.

[0186] 23. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14a, 14b, 15, 16, 17, 17a, 18, 19, 20, 21, or 22, wherein the flour has a gluten content that is less than about 12 wt% (e.g., gluten-free).

[0187] 24. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14a, 14b, 15, 16, 17, 17a, 18, 19, 20, 21, 22, or 23, further comprising adding at least one of a lipase, alphaamylase, xylanase, non-maltogenic exoamylase, glucoamylase, glucose oxidase, or hexose oxidase during production of the food dough.

[0188] 25. A food dough produced by the method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14a, 14b, 15, 16, 17, 17a, 18, 19, 20, 21, 22, 23, or 24. 26. A food product produced by the method of embodiment 15 or 16, and / or as produced using a food dough of embodiment 19 or 43 (e.g., a baked food such as baked bread).

[0189] 27. A food dough comprising (a) flour and / or meal, (b) water or aqueous composition, (c) sucrose, and (d) at least one glucosyltransferase enzyme, selected from: (i) a glucosyltransferase enzyme that is capable of synthesizing (or that synthesizes) alpha-1, 6- glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,6-glucan are alpha-1,6 linkages, and / or (ii) a glucosyltransferase enzyme that is capable of synthesizing (or that synthesizes) alpha- 1,3 -glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,3 -glucan are alpha-1,3 linkages, typically wherein the food dough comprises at least one alpha-glucan product of the at least one glucosyltransferase enzyme, optionally wherein (I) the food dough has a shock stability that is increased as compared to the shock stability of a control food dough that lacks the at least one glucosyltransferase enzyme, (II) the food dough has a water-binding capacity that is increased as compared to the waterbinding capacity of a control food dough that lacks the at least one glucosyltransferase enzyme, (III) the food dough has a viscosity that is increased as compared to the viscosity of a control food dough that lacks the at least one glucosyltransferase enzyme, and / or (IV) the food dough has a volume that is increased as compared to the volume of a control food dough that lacks the at least one glucosyltransferase enzyme (e.g., the control food dough is produced the same way as the food dough, except that the at least one glucosyltransferase enzyme is not included in producing the control food dough).

[0190] 28. The food dough of embodiment 27, wherein the at least one glucosyltransferase enzyme is the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 6-glucan.

[0191] 29. The food dough of embodiment 27 or 28, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:1, 2, 11, 12, 14, or 15 (e.g., an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO: 14 or 15).

[0192] 30. The food dough of embodiment 27, 28, or 29, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical (e.g., at least 95% identical) to SEQ ID NO: 1, 2, 11, or 12 (e.g., an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO: 1 or 2).

[0193] 31. The food dough of embodiment 27, wherein the at least one glucosyltransferase enzyme is the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan.

[0194] 32. The food dough of embodiment 27, 29, 30, or 31, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical (e.g., at least 95% identical) to residues 55-960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55-960 of SEQ ID NO:8, or residues 55-960 of SEQ ID NO:9.

[0195] 33. The food dough of embodiment 27, 29, 30, 31, or 32, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical (e.g., at least 95% identical) to residues 55-960 of SEQ ID NO:5.

[0196] 34. The food dough of embodiment 27, 29, 30, 31, 32, or 33, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical (e.g., at least 95% identical) to SEQ ID NO:3, 4, 5, 6, 7, 8, 9, or 13.

[0197] 35. The food dough of embodiment 27, 29, 30, 31, 32, 33, or 34, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical (e.g., at least 95% identical) to SEQ ID NO:5.

[0198] 36. The food dough of embodiment 27, 29, 30, 32, 33, 34, or 35, wherein the food dough comprises both of the (i) glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan and (ii) glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3- glucan.

[0199] 37. The food dough of embodiment 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36, wherein the food dough comprises the at least one alpha-glucan product, and the alpha-glucan product comprises a graft copolymer comprising: (i) an alpha-1, 6-glucan backbone, wherein at least about 50% of the glycosidic linkages of the alpha- 1, 6-glucan backbone are alpha- 1,6 linkages, and (ii) at least one alpha- 1,3 -glucan side chain, wherein at least about 50% of the glycosidic linkages of the alpha- 1,3 -glucan chain are alpha-1,3 linkages, wherein the graft copolymer is aqueous-soluble or aqueous-insoluble.

[0200] 38. The food dough of embodiment 36 or 37, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha- 1, 6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan is about 95:5 to about 5:95.

[0201] 39. The food dough of embodiment 36, 37, or 38, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha- 1, 6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan is about 95:5 to about 50:50 (e.g., about 95:5 to about 75:25). 40. The food dough of embodiment 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39, wherein the food dough comprises the flour, optionally wherein the flour is wheat flour.

[0202] 41. The food dough of embodiment 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or

[0203] 40, wherein the food dough comprises a leavening agent, optionally wherein the leavening agent is yeast.

[0204] 42. The food dough of embodiment 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41, wherein the food dough is a bread dough.

[0205] 43. The food dough of embodiment 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,

[0206] 41, or 42, wherein food dough is a risen food dough.

[0207] 44. A food product comprising (a) flour and / or meal, (b) water or aqueous composition, (c) optionally sucrose (in some aspects, all the sucrose has been consumed / reacted by a GTF enzymefs] and no additional sucrose has been added, and in other aspects some residual non- consumed / unreacted sucrose remains and / or sucrose was later added such as following terminating the activity of the GTF enzymefs]), and (d) at least one glucosyltransferase enzyme, selected from: (i) a glucosyltransferase enzyme that is capable of synthesizing (or that synthesizes) alpha- 1,6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,6-glucan are alpha- 1,6 linkages, and / or (ii) a glucosyltransferase enzyme that is capable of synthesizing (or that synthesizes) alpha- 1,3 -glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,3 -glucan are alpha-1,3 linkages, typically wherein the food product comprises at least one alpha-glucan product of the at least one glucosyltransferase enzyme, optionally wherein (I) the food product has a water-binding capacity that is increased (e.g., the food product is more moist) as compared to the waterbinding capacity of a control food product that lacks the at least one glucosyltransferase enzyme, (II) the food product has a volume (e.g., specific volume) that is increased as compared to the volume of a control food product that lacks the at least one glucosyltransferase enzyme, (III) the food product has a crumb that is increased as compared to the crumb of a control food product that lacks the at least one glucosyltransferase enzyme, (IV) the food product has a softness that is increased as compared to the softness of a control food product that lacks the at least one glucosyltransferase enzyme, and / or (IV) the food product has a resilience that is increased as compared to the resilience of a control food product that lacks the at least one glucosyltransferase enzyme (e.g., the control food product is produced the same way as the food product, except that the at least one glucosyltransferase enzyme is not included in producing the control food product). 45. The food product of embodiment 44, wherein the at least one glucosyltransferase enzyme is the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 6-glucan.

[0208] 46. The food product of embodiment 44 or 45, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1, 6-glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 1, 2, 11, 12, 14, or 15 (e.g., an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO: 14 or 15).

[0209] 47. The food product of embodiment 44, 45, or 46, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1, 6-glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical (e.g., at least 95% identical) to SEQ ID NO: 1, 2,

[0210] 11, or 12 (e.g., an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO: 1 or 2).

[0211] 48. The food product of embodiment 44, wherein the at least one glucosyltransferase enzyme is the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan.

[0212] 49. The food product of embodiment 44, 46, 47, or 48, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical (e.g., at least 95% identical) to residues 55-960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55-960 of SEQ ID NO:8, or residues 55-960 of SEQ ID NO:9.

[0213] 50. The food product of embodiment 44, 46, 47, 48, or 49, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical (e.g., at least 95% identical) to residues 55-960 of SEQ ID NO:5.

[0214] 51. The food product of embodiment 44, 46, 47, 48, 49, or 50, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical (e.g., at least 95% identical) to SEQ ID NO:3, 4, 5, 6, 7, 8, 9, or 13.

[0215] 52. The food product of embodiment 44, 46, 47, 48, 49, 50, or 51, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical (e.g., at least 95% identical) to SEQ ID NO:5.

[0216] 53. The food product of embodiment 44, 46, 47, 49, 50, 51, or 52, wherein the food product comprises both of the (i) glucosyltransferase enzyme that is capable of synthesizing alpha- 1, 6-glucan and (ii) glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan. 54. The food product of embodiment 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53, wherein the food product comprises the at least one alpha-glucan product, and the alpha-glucan product comprises a graft copolymer comprising: (i) an alpha-1, 6-glucan backbone, wherein at least about 50% of the glycosidic linkages of the alpha- 1, 6-glucan backbone are alpha- 1,6 linkages, and (ii) at least one alpha- 1,3 -glucan side chain, wherein at least about 50% of the glycosidic linkages of the alpha- 1,3 -glucan chain are alpha-1,3 linkages, wherein the graft copolymer is aqueous-soluble or aqueous-insoluble.

[0217] 55. The food product of embodiment 53 or 54, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha- 1, 6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan is about 95:5 to about 5:95.

[0218] 56. The food product of embodiment 53, 54, or 55, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha- 1, 6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan is about 95:5 to about 50:50 (e.g., about 95:5 to about 75:25).

[0219] 57. The food product of embodiment 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or 56, wherein the food product comprises the flour, optionally wherein the flour is wheat flour.

[0220] 58. The food product of embodiment 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, or

[0221] 57, wherein the food product is a baked food product.

[0222] 59. The food product of embodiment 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58, wherein the food product is a bread product.

[0223] EXAMPLES

[0224] The present disclosure is described in further detail in the following examples, which are not in any way intended to limit the scope of the disclosure. The attached figures are meant to be considered as integral parts of the specification and description of the disclosure. The following examples are offered to illustrate, but not to limit, the present disclosure.

[0225] Example 1. Glucosyltransferase (GTE) enzyme preparations for alpha-1, 3-glucan and dextran (alpha-1, 6-glucan) production

[0226] In this Example, the GTF enzymes were used to produce various graft copolymers having a dextran (alpha- 1, 6-glucan) backbone with various alpha- 1,3 -glucan side chains. This production was done in dough in situ using a combination of a dextransucrase and an alpha- 1,3 -glucan sucrase in the dough preparation process. This alpha-glucan production coincided with sugar reduction and texturizing effects in the dough. The sugar reduction and texturing effects of using GTF 0768 (SEQ ID NO: 1, also represented by SEQ ID NOs:2, 11 and 12) and an amino acid-substituted GTF 6855 variant (SEQ ID NO:3, “vGTFJ” herein) alone, or in combination, was investigated in dough production set-ups (various scales). Both these GTF enzymes use sucrose as a substrate to produce fructose and glucan (i.e., they are examples of glucansucrases). GTF 0768 (a dextransucrase) produces a soluble alpha-glucan with a high alpha-1,6 linkage content (i.e., a type of dextran; refer to U.S. Patent Appl. Publ. No. 20160122445, which is incorporated herein by reference), whereas vGTFJ stably produces at high yield an insoluble alpha-glucan having about 100% alpha-1,3 linkages (the variant GTF of SEQ ID NO:4 similarly can be used herein to stably produce at high yield insoluble alpha-glucan having about 100% alpha- 1,3 linkages).

[0227] Example 2. Protocol for GTF activity assay (GTF U / g)

[0228] GTF enzymes initially hydrolyze sucrose to produce glucose and fructose. Then glucose becomes the acceptor of another glucosyl unit from the enzyme-glucosyl intermediate to form DP2, then DP3, DP4 and so on forming long glucose polymer (i.e., glucan). The vGTFJ enzyme forms alpha-1,3 linkages and an insoluble polymer where GTF 0768 forms alpha-1,6 linkages and a soluble polymer. For each actual GTF catalytic event, approximately 1 molecule of fructose will be released. The rate of fructose release (indirectly indicating alpha-glucan polysaccharide formation) was measured with MEGAZYME D- Fructose and D-Glucose Assay Procedure K-FRUGL (Megazymes, Ireland). One GTF unit (GTF U) was defined as the amount of fructose changed per minute at 30 °C (mg fructose / min / g sample) at assay conditions.

[0229] The enzyme reaction was run for 5 minutes in 25 mM sodium acetate, 0.1 % CaCh and 0.1% BSA, pH 5.5, with 10% sucrose as substrate at 30 °C. GTF enzyme was inactivated by 15 minutes incubation with 8% IM NaOH. Afterwards, pH was neutralized by adding 0.77: 1 volume of IM HEPES buffer pH 7.0. The mg / mL concentration of fructose released was then determined for each sample from the linear regression of a fructose / glucose standard curve. Sample absorbance must have been within the linear range of the standard curve.

[0230] Fructose and glucose standards: Weighed 0.150 g of dried glucose and 1.00 g fructose into a 200-mL beaker. Recorded the weight of each. Added 80 mL of MILLI-Q water to the beaker and mixed. Poured into a 100-mL volumetric and QS to 100 mL. Prepared standard dilutions and measured using MEGAZYME D-Fructose and D-Glucose Assay Procedure K-FRUGL (Megazymes, Ireland). The GTFU activity of GTF 0768 (SEQ ID NO: 1) was determined according to the above assay to be 448 GTFU / g and the GTFU activity of vGTFJ (SEQ ID NO:3) was determined to be 103 GTFU / g.

[0231] Example 3. Dough development analyzed by Brabender farinograph with in situ addition of GTF enzyme

[0232] The impact of GTF enzymes on dough development was analysed using a Brabender farinograph (calibrated and operated according to Brabender ICC BIPEA 50). The procedure was similar to AACC method 51-21-01 (incorporated herein by reference) with minor modifications. 50 g of reform flour (Lantmannen Cerealia, Vejle, Denmark, 12.5% protein, 140528, lot 1000501977) and 2%b (bakers percentage) salt was dry-mixed for 1 minute in a FARINOGRAPH-E (Brabender, Duisburg, Germany) equipped with a 50-g moulding chamber (MIXER S 50N, model 820608), equilibrated at 30.0 °C and set speed of 63 rounds / minute, and then 56.5% tap water (adjusted to 30.0 °C) was added and the dough was mixed for 20 minutes (to achieve a torque of 380 BU after arrival time). The water content of the flour was assumed to be constant at 14%. Two dosages of sucrose were tested: 1 ,5%b (0.92% absolute) and 15%b (8.75% absolute). Torque (BU) was recorded throughout the mixing for up to 45 minutes. A blank consisting of the flour, salt, sucrose and water, but no added enzyme herein, had a BU (Brabender unit) of 380±5 at 12 minutes of development; see FIG. 1. As reference, GRINDSTED GUAR 250 (“GUAR 250” herein, 810006, IFF Brabrand Denmark) was used as polysaccharide derived from the seeds of the guar plant recognized for its remarkable thickening, stabilizing, and emulsifying properties in baking and dough preparation. GUAR 250 was added at a dosage of 0.5%b of the flour. Using the high concentration of 15%b sucrose, a sample of GTF 0768 was dosed at 173 and 1553 GTFU / kg flour, and a sample of vGTFJ was dosed at 107 and 966 GTFU / kg flour. The dough development (torque) as analyzed by the Brabender farinograph is shown in FIG. 1 with high (15%b) addition of sucrose. It was clear from the results that the addition of 0.5% GUAR 250 increased the torque of the dough development from 381 to 404 BU (see Brabender consistency in Table 1), and maintained this increased torque / stability throughout the mixing time of 45 min. GTF 0768 in the low dose (107 GTFU / kg) showed a minor increased torque of the dough development to 398 BU, which was slightly increased over the mixing time. GTF 0768 in the high dose (966 GTFU / kg) demonstrated surprisingly highly increased torque of the dough development to 534 BU, which dropped accordingly over mixing time. The low dose (107 GTFU / kg) of vGTFJ delivered a greater consistency of 438 BU, being higher than GTF 0768 and GUAR 250, and in the high dose (966 GTFU / kg) demonstrated very high consistency increase up to 587 BU, with an increasing torque throughout mixing, thereby indicating increased dough stability; this increased dough stability was even further enhanced with mixing time (FIG. 1). Table 1. Dough development analyzed by Brabender farinograph with 15%b sucrose

[0233] In addition, using the high concentration of 15%b sucrose, the following combinations of GTF enzymes were tested: (i) GTF 0768 high (1553 GTFU / kg) + vGTFJ low (107 GTFU / kg), and (ii) GTF 0768 low (173 GTFU / kg) + vGTFJ high (966 GTFU / kg). The results of the combinatorial enzyme additions are shown in FIG. 2. The combination of GTF 0768 high (1553 GTFU / kg) + vGTFJ low (107 GTFU / kg) showed significantly more torque throughout mixing as compared to the combination of GTF 0768 low (173 GTFU / kg) + vGTFJ high (966 GTFU / kg). The highly increased torque (584 BU) and dough stability obtained by the combination of GTF 0768 high (1553 GTFU / kg) + vGTFJ low (107 GTFU / kg) were obtained as additive performance in comparison to the results of using these two enzymes separately.

[0234] Example 4. Sugar analysis of simple dough prepared with GTF enzyme addition

[0235] The amount of GTF enzyme needed to convert sucrose in a simple dough system (no yeast, salt or butter added) was determined in 100-g scale samples. Table 2, Simple dough constituent in % of the flour weight (Bakers percentage)

[0236] Increasing enzyme volumes were added to the water. The dry and wet ingredients were each mixed separately before combining and kneading them together. 5-g samples were extracted after 150 min. and frozen in liquid nitrogen for 1 min before putting the samples in the freeze-dryer overnight. The freeze-dried material was ground to fine powder and added to 20 g 60% ethanol to solubilize sugars and oligosaccharides while keeping polysaccharides and proteins undissolved. The samples were centrifuged at 10000 rpm for 10 minutes. 1-mL supernatants were extracted and freeze-dried again overnight to remove ethanol and the dry matter was redissolved in 1 mL water for analysis of sugars and oligosaccharides.

[0237] HPLC sugar content analysis

[0238] Sugar composition was measured by high performance liquid chromatography (HPLC) with a Waters® 2695 Serrations module or a ThermoScientific DionexTM UltiMate 3000 HPLC, equipped with a Phenom onex Rezex™ RPM-Monosaccharide Pb2+column (300 mm x 7.8 mm), and an Rl-detector. Water was used as the mobile phase at a flow rate of 0.400 mL / min. The column temperature was 70 °C. Samples were prepared for HPLC injection by an appropriate dilution in water, optionally centrifugation (10 min at 15000 rpm), and sterile filtration. Signals from the HPLC were quantified against calibration standards of sugars eluting at the same time. Sugar reduction was calculated by subtracting the total sum of mono- and di-saccharides in the test sample from the total sum of mono- and disaccharides in the reference sample without enzymes added.

[0239] The results are shown in Table 3. It was concluded that at least 0.56 mL vGTFJ (6.3 GTFU / g sucrose) or 0.392 mL GTF 0768 (14.5 GTFU / g sucrose) was needed to deplete the sucrose. Although it seems that there was still some sucrose left with the GTF 0768 dosing, it was likely close to 0% sucrose as nigerose (a DP2) has the same retention time as sucrose and would mistakenly be labelled as sucrose. Table 3, Relative distribution (%- weight) of sugars in a simple dough with varying glucosyltransferase

[0240] Example 5. Determination of specific volumes of baked wheat dough punched rolls using GTF enzymes including dough shock test

[0241] Soft wheat flour (Valsemollen, Esbjerg Denmark, 10% protein) with an initial water content of 14% was tempered to 44 °C in a heating cabinet. 300 g tempered soft flour, l%b salt, 1.6%b sugar, 2%b dry yeast (SAF instant Yeast, S. I. Lesaffre, France), 0.33%b GRIND AMYL A1000 (A1000 diluted to 16.2 FAU / g, IFF, Brabrand, Denmark) and 0.125%bTSE-2356 sample (technical enzyme sample consisting of lipase, amylase, xylanase and oxidase to provide increased bread volume; provided by IFF, Brabrand, Denmark) were all mixed in a KITCHEN AID (Artisan 5KSM125, Kitchen Aid, MI, USA) for 1 minute, then 55.5%b water tempered to 44 °C (and enzyme) was added and mixed at low speed for 2 minutes followed by high speed for 8 minutes. The dough was then placed at 34 °C, 86% humidity in a proofing cabinet (Bago-raskeskab BMR-77, Bago-Line, Faaborg, Denmark) for 10 minutes. Eight 25-30 g dough samples were punched out with a round form (4 cm in diameter), after having used a rolling pin to form a uniform level of dough, and placed in silicone muffin molds on baking sheets with eight rolls per sheet. The weight of doughs was determined. Next, the punched doughs were proofed at 34 °C, 86% humidity for 45 minutes exactly. The rounded rolls on one of the two baking sheets (one for preparing 4 unshocked rolls, and one for 4 shocked rolls) were shocked on a shaking table (IKA KS 130 basic, Bie and Berntsen AS, Rodovre, Denmark) at 480 rpm for exactly 1 minute (fixed silicone muffin molds). The baking sheet with the shocked rounded doughs in molds was placed in an oven (Bago-mini oven BMO-77, Bago-Line, Faaborg, Denmark) together with the baking sheet with the unshocked rounded doughs and baked for 8 minutes at 220 °C. The unshocked rolls and shocked rolls were cooled at ambient temperature for 20 minutes before they were weighed, and volume determined using a dry sand displacement technique. Initially, the density of the dried fine sand was carefully determined. The volume of the rolls was then calculated based on gravimetric measurement of the rolls alone and the rolls placed in a glass jar of known volume and weight, where dry sand was filled to the rim of the jar. Volume determination by sand displacement was performed in duplicates. The specific volume of rolls was determined from volume and weight of the baked goods or weight of dough; e.g., volume of the rolls (4) divided by the weight of the rolls (4). Specific volumes of the unshocked rolls and shocked rolls were calculated individually.

[0242] Trial A GTF enzymes were added according to GTFU activity as described in Example 2: the activity of GTF 0768 (SEQ ID NO: 1) was determined according to the above assay to 448 GTFU / g and the activity of glucosyltransferase vGTFJ (SEQ ID NO:3) was determined to 103 GTFU / g; see also the trial setup in Table 4 below. Adjustment of the fungal alpha-amylase content was achieved by addition of 0.33% GRIND AMYL® A 1000 (A1000 diluted to 16.2 FAU / g, provided by IFF, Brabrand, Denmark) to obtain better dough handling and slightly increased final volume. 0.125%b TSE-2356 sample was added to obtain higher final bread volume.

[0243] Table 4, Ingredient table for wheat dough punched rolls using 300 g wheat flour with GTF

[0244] 0768

[0245] The specific volumes of the unshocked and shocked breads (4 unshocked rolls and 4 shocked rolls) were determined in duplicate. The results of Trial A are shown in FIG. 11 (A) based on dough weight, and the specific volume of the unshocked and shocked breads are given in FIG. 11 (B) based on bread weight.

[0246] With the GTF 0768 enzyme in this Example - at 0.04%, 0.08%, 0.16%, 0.32% and 0.64% - all except a single dose surprisingly improved the specific volumes of the unshocked breads (dough weight) (FIG. 11 [A]). Also, GTF 0768 treatment demonstrated increased specific volume, with increasing enzyme doses, of 3.10 mL / g, 3.53 mL / g, 3.32 mL / g, 3.65 mL / g and 3.78 mL / g, respectively, versus the blank references (TSE-2356 only, no GTF) specific volume of 3.23 mL / g (FIG. 11 [A]). The relative volume ranged from 95% to 117%, as compared to blank reference (TSE-2356 only, no GTF), which was set to 100%; thus, there was a relative increase of 17%. A somewhat similar trend was observed when evaluating the specific volume based on bread weight, which was significantly improved with the unshocked breads having GTF 0768 added (above doses), with 3.35 mL / g, 3.18 mL / g, 3.28 mL / g, 3.25 mL / g and 3.37 mL / g, respectively, versus the blank reference (TSE-2356 only, no GTF) specific volume of 3.05 mL / g. These results indicated no difference between the individual doughs with different enzymes in water evaporation during bake-off.

[0247] The specific volumes of the shocked breads, based on either dough weight or bread weight, increased with increasing addition of GTF 0768. The specific volumes of the shocked breads (dough weight) increased from 2.85 mL / g to 3.06 mL / g, a relative increase of 7%.

[0248] Trial B GTF enzymes were added according to GTFU activity as described in Example 2: The GTFU activity of glucosyltransferase vGTFJ (SEQ ID NO:3) was determined to be 103 GTFU / g and the GTFU activity of GTF 0768 (SEQ ID NO: 1) was determined to be 448 GTFU / g; see also the trial setup in Table 5 below. Table 5, Ingredient table for wheat dough punched rolls using 300 g wheat flour with GTF

[0249] 0768 and vGTFJ

[0250] The specific volume of the unshocked and shocked breads (4 unshocked rolls and 4 shocked rolls) were determined in duplicate; the results of trial B are shown in FIG. 12 (A) based on dough weight and the specific volume of the unshocked and shocked breads are given in FIG. 12(B) based on bread weight.

[0251] The GTF 0768 in this Example at the 0.16% dose showed improved specific volumes of the unshocked breads (dough weight), 3.74 mL / g, versus the blank reference (TSE-2356 and no GTF) specific volume of 3.36 mL / g (FIG. 12 [A]). Similarly, all dosages of vGTFJ (0.16%, 0.32%, 0.63% and 1.26%) showed improved specific volumes of the unshocked breads (dough weight), of 3.55 mL / g, 3.82 mL / g, 3.71 mL / g and 3.68 mL / g, respectively, versus the blank reference (TSE-2356 only, no GTF) (FIG. 12 [A]). The relative volume increases ranged from 105% to 113% as compared to blank reference, which was set to 100%; thus, there was a relative increase of about 13%. A very similar trend was observed evaluating the specific volume based on the bread weight, with significantly improved specific volumes of the unshocked breads having GTF 0768 (4.71 mL / g) and vGTFJ (4.45 mL / g, 4.83 mL / g, 4.68 mL / g, and 4.71 mL / g, respective to the above doses) versus the blank reference (TSE-2356 only, no GTF) specific volume of 4.22 mL / g (FIG. 12 [B]). The relative volume increases ranged from 105% to 114% as compared to blank reference, which was set to 100%; thus, there was a relative increase of about 14%. The fact that the results based on bread and dough weights followed the same trend indicates that there was no difference in water evaporation between different doughs.

[0252] The specific volumes of the shocked breads (based on dough weight or based on bread weight) increased with increasing addition of vGTFJ (FIG. 12 [A-B]). The specific volumes of the shocked breads (dough weight) increased to a high of 3.05 mL / g as compared to the control volume of 2.57 mL / g, a relative increase of 18%.

[0253] It was clear that both GTF 0768 and vGTFJ enzymes, which generate alpha- 1,6- glucan and alpha- 1,3 -glucan, respectively, improved the unshocked specific volume of baked goods as well as the dough stability in terms of specific volume of the shocked baked goods.

[0254] Example 6. Protocol for brioche bread baking trials using GTF enzymes

[0255] All dry ingredients and yeast (SAF instant Yeast, S. I. Lesaffre, France) were mixed (see Table 6) for 1 minute at 1stspeed (Diosna prog. 05.08 Brioche), then adding eggs, water and enzyme - mixing for 2 minutes at 1stspeed - 6 minutes 2ndspeed, and afterwards adding butter - mix 3 minutes 2ndspeed - dough temperature was 25 °C. As a reference, GRINDSTED GUAR 250 (810006, IFF Brabrand Denmark) was used. Guar is a polysaccharide derived from the seeds of the guar plant recognized for its remarkable thickening, stabilizing, and emulsifying properties in baking and dough preparation. GRIND AMYL™ Al 000 (diluted to 16.2 FAU / g) fungal alpha-amylase was added 0.47%b guided by the falling number of the flour (International Flavors & Fragrances Inc., DK-8220 Brabrand, Denmark). A sample of GTF 0768 (SEQ ID NO: 1) having 388 GTFU / g according to Example 2 was used and a sample vGTFJ (SEQ ID NO:3) having 103 GTFU / g according to Example 2 were used. The dosages of these GTF enzymes are given as GTFU / g sucrose in Table 6. Varying combinations of GTF 0768:vGTFJ were also applied.

[0256] Dough was rested for 30 minutes under cloth at ambient temperature. Mould 640 g on Bernier MS 500, following settings: Preform: -18, Drum press.: 3, Pressure board: 4.0 cm front, 3.5 cm back, Width: 370 mm front, 330 mm back, and put into tins (DK toast tins). Dough was proofed for 110 minutes at 30 °C, 85% relative humidity (RH) and then baked for 10 minutes at 210 °C + 13 minutes at 200 °C Reed Rack Oven. + 5 minutes at 180°C with damper open (Miwe prog 19). The final loaf temperature was verified to be 96- 98 °C and after baking the breads were cooled for 70 minutes at ambient temperature before weighing and measuring of volume using a rapeseed displacement method. The bread used for softness, resilience, and adhesiveness measurements were packed with vacuum.

[0257] A slice of each bread was analyzed in C-Cell at day 1 according to AACC method 10- 18.01 (incorporated herein by reference) and analyzed for moisture level. At day 1 and 7, it was analyzed on TP A, to document resilience. Samples of dough and final bread were taken after mixing, after resting, after proofing, and after baking, as appropriate, and analyzed for sugar profile using HPLC.

[0258] Table 6, Relative distribution (% weight of flour) of ingredients for brioche breads with varying GTF amounts (unit addition given in last two lines) Informal sensorial evaluation of brioche bread: An informal sensorial evaluation was made on the final breads slices at day 1 and day 7, following the instructions provided in Table 7

[0259] Informal sensorial evaluation of brioche bread (similar to Table 7), which is a high fat and high sugar type of bread, was performed according to sensory scoring listed in Table 7. Brioche bread with GTF enzymes was compared to addition of 0.3% GUAR 250 (a hydrocolloid) to see if the same or improved effect could be achieved with in situ GTF activity. Different dough parameters were measured by the baker after mixing, after resting, and after proofing the dough. The evaluation results are shown in FIG. 3. It was observed that GTF 0768:vGTFJ (90: 10) treatment matched the effects of 0.3% guar in all parameters, and therefore provided desired dough properties and machinability, while only increasing dough stickiness compared to the negative control. Also, GTF 0768:vGTFJ 95:5, 50:50 and 0: 100 treatments provided satisfactory properties; only the GTF 0768:vGTFJ 100:0 and 10:90 treatments resulted in dough that was too sticky to be easily used in a day-to-day bakery operation.

[0260] After the dough was baked, pictures of the resulting bread loaves were taken (FIG. 4). It was clear that all the GTF enzyme treatments increased volumes, especially with GTF 0768-only and the GTF 0768:vGTFJ 90: 10 blend, as compared to both the negative control and positive control (0.3% GUAR 250) breads.

[0261] The specific volumes of the breads were also measured using a rapeseed displacement method (FIG. 5). While all the GTF treatments resulted in increased specific volumes of bread, it was most striking that bread from trial 3 with GTF 0768 and trial 5 with GTF 0768:vGTFJ (90: 10) resulted in specific volume increases to 5.98 and 5.92 cm3 / g, respectively, as compared to the controls having a specific volume of about 5.10-5.11 cm3 / g. Thus, the addition of GTF enzyme(s) for in situ alpha- 1,6-glucan and / or alpha- 1,3 -glucan generation have very beneficial influence on the specific volume of the baked bread.

[0262] The dough samples after mixing, resting and proofing were collected as well as samples of the baked goods. No dough samples after proofing of trials 1, 2, 3 and 4 were obtained. Dough and bread samples were prepared as described in Example 4, and the relative amount of sucrose was quantified by HPLC. The results are shown in FIG. 6.

[0263] The relative amount of sucrose as compared to total soluble carbohydrates was quantified by HPLC analysis of samples taken after mixing, resting, proofing and baking the final breads. The sucrose amount was initially at 40% relative to total soluble carbohydrate after mixing in the negative control value. Already after mixing, all the samples with GTF enzyme(s), had half or less the sucrose. The GTF 0768:vGTFJ 100:0, 95:5 and 90: 10 enzyme treatments were the fastest to convert the sucrose. After baking the breads, all samples with GTF enzyme(s) had very little detectable sucrose level, and possibly they had 0% sucrose content as nigerose (a DP2) has the same HPLC retention time as sucrose.

[0264] HPLC sugar analysis (sucrose, glucose, fructose, leucrose and oligosaccharides (OS, DP3+) in the baked goods were analyzed essentially as described in Example 9 and the absolute amounts of sugars were quantified by HPLC are shown in FIG. 7. It was clear that GTF 0768, vGTFJ and all tested combinations thereof resulted in significant decreases in sucrose and glucose, and increases in production of OS.

[0265] Image analysis of bread slices was performed with a C-cell apparatus according to IFF departmental instruction 23.8310.F21, which is similar to AACC method 10-18.01 (incorporated herein by reference). The C-cell analysis was done on bread slices of brioche bread made according to the Table 6 trial setup. A scan was performed on day-1 slices of the eight samples, at day 1, in the C-cell machine. The machine showed differences in the number of cells in each slice (FIG. 14) and in the brightness of each slice (FIG. 15). A high number of cells is a positive attribute, as it correlates with better crumb of the bread. FIG. 14 shows that breads made with GTF 0768:vGTFJ 100:0, 95:5 and 90:10 enzyme treatments all had a higher number of cells than the negative control bread and the guar-containing bread. The differences in bread slice brightness was readily apparent with the unaided eye, as reflected in the results shown in FIG. 15 quantitating brightness. The control slices (samples 1 and 2) appeared much more yellow and dark, as compared to the bread slices made with GTF enzyme treatment (samples 3-8). The GTF 0768:vGTFJ 100:0, 95:5 and 90: 10 treated bread slices appeared very white and bright, while those made with the GTF 0768:vGTFJ 50:50 to 0: 100 enzyme treatments progressively appeared more yellow. Thus, brightness in this analysis correlated with the addition of GTF 0768 during bread production.

[0266] The moisture content in brioche bread slices was determined with a Mettler Toledo moisture analyzer according to AACC (23.8310. F15; incorporated herein by reference) (target weight 1.5 g, target temperature 130 °C). The results of moisture content (%) analyses conducted at day 1 with brioche bread slices made according to trials / samples 1-8 (Table 6) are shown in FIG. 16. Slices of samples 3, 4 and 5 (GTF 0768:vGTFJ 100:0, 95:5 and 90: 10 enzyme treatments, respectively) had the highest moisture content (significantly higher than that of control samples 1-2), thereby rendering them to be the freshest bread samples. It was clear that the increased moisture retention in these studies correlated with the addition of GTF 0768. This also correlated well with the surprisingly much higher requirement for water addition compared to when using 0.3% GUAR 250. It was therefore probable that poly- and / or oligo-saccharides formed by GTF 0768 in bread have a high water binding capacity, which allows for greater incorporation of water in the breads, and in turn having a positive effect on bread volume.

[0267] Example 7. Protocol for Evaluation of Softness and Resilience of Brioche bread products.

[0268] Texture profile analysis (TP A) of bread firmness, resilience and cohesiveness were determined by analyzing bread slices by TPA using a texture analyzer from Stable Micro Systems, UK. Calculation of softness, resilience, and adhesiveness were done according to preset standards supplied by Stable Micro Systems. The probe used was aluminum 35 mm round. Bread was sliced with the width of 11.0 mm. Measurement was performed by placing two bread slices on top of each other and then compressing them with a depth of 15 mm. Softness (expressed in grams) was determined during the first compression as the peak value. The figure is the force needed to compress the bread slice to a depth of 15 mm. The lower the softness value, the softer the bread, which is a preferred attribute.

[0269] The following settings were used: pre-test speed: 4 mm / s, test speed: 5 mm / s, post test speed: 5 mm / s, compression distance: 15 mm, trigger force: 20.0 g, time between measurements: 5.00 sec, count: 5, load cell: 5 kg, trigger type: auto - 20.0 g. The mode of compression was a modification to the one used in Standard method AACC 74-09 (incorporated herein by reference). The sample was compressed twice in the test. This assay may be referred to as the “Softness Evaluation Protocol”. Protocol for Evaluation of Resilience

[0270] Area under the curve was a measure of work applied during the test. The area under the curve in the compression part is defined as ‘Al’ and the withdrawal part is defined as ‘ A2’ during the first compression. The ratio between A2 and Al is defined as the resilience of the sample and is expressed as Resilience Units (A2 / A1). True elastic material will give a symmetric curve, as the force applied during the first part will be equal to the force in the second part. For bread and bread-like material, A2 is normally smaller than Al due to disturbance of the structure during compression. Hence, resilience is always lower than 1. This assay may be referred to as the “Resilience Evaluation Protocol”.

[0271] Protocol for Evaluation of Adhesiveness

[0272] Adhesiveness was defined as the negative area of the curve after the withdrawal of the probe. Thus, the area of curve was defined by negative force. The larger negative area that was associated with a negative compression force, the larger negative value of adhesiveness was given in units g s. For bread and bread-like material, adhesiveness is always lower than 0. This assay may be referred to as the “Adhesiveness Evaluation Protocol”.

[0273] TPA was performed on day 1 and day 7 on the brioche breads baked according to Example 6. TPA is a popular double compression test for determining the textural properties of foods. The beauty of TPA as an analytical method is that it can quantify multiple textural parameters in just one experiment. For breads, softness / hardness and resilience are important parameters, and the TPA results in these parameters are seen in FIGs. 8-9.

[0274] The lower HPa / g was, the softer and better the bread. As seen in FIG. 8, all samples with GTF enzyme(s) were softer than both the negative control and GUAR 250-containing breads at day 1 and 7. Softness was measured at TPA as how much the machine can press down in HPa on the bread per g. All GTF enzyme treatments provided an improvement, but the GTF 0768:vGTFJ 100:0, 95:5 and 90: 10 treatments provided bread that was much softer than the references on days 1 and 7.

[0275] Resilience, which can also be measured with TPA, is the ability to withstand adversity / pressure and bounce back after being pressed down; the higher the resilience value, the better the quality of bread. As shown in FIG. 9, all bread samples that had GTF enzyme(s) treatment during preparation were better as they had higher resilience than both the negative control and GUAR 250-containing breads at day 1 and 7. All GTF enzyme treatments provided an improvement, but the GTF 0768:vGTFJ 90: 10 and 0: 100 treatments provided bread that was much more resilient than the references on days 1 and 7. Taken together, GTF enzyme treatments during bread preparation clearly improved anti-staling properties of baked bread products, especially with both softness and resilience (desired shelf-life features of baked goods). Informal sensorial evaluation of the brioche bread samples in this Example were evaluated according to sensory scoring Table 7. Brioche bread made with GTF enzyme(s) was compared to bread made containing 0.3% GUAR 250 to see if the same or better effects (that guar typically provides to bread) could be achieved with GTF enzyme treatment. The evaluation results are provided on FIG. 13 on final bread samples (slices) at day 7 post-baking. The scope of this sensory analysis was to evaluate the effects of adding GTF enzymes using the standard brioche toast bread recipe with an expected shelf-life of 14 days. By hand only, an evaluation on softness, moistness, resilience and overall freshness was done by four individuals. FIG. 13 shows the average score for the breads in the four parameters. By considering softness, moistness and overall freshness levels (FIG. 13), it is apparent that the three bread samples with the highest values are those made with GTF 0768:vGTFJ 100:0, 95:5 and 90: 10 treatments. These results are consistent with what was observed in the technical analysis of softness / hardness and moisture % (above).

[0276] Example 8. HPLC-SEC for analysis of sucrose conversion by GTF enzyme

[0277] A solution of 1% sucrose was made in a 10 mM Na2HPO4 buffer, pH 6.0. An aliquot of 5 mL was transferred to a glass vial with lid and placed in an incubator at 40 °C for 10 min. To start the reaction, 1.4 U of GTF 0768 enzyme (SEQ ID NO: 1) was added and the vial was then left to incubate at 40 °C. At timepoints t = 15, 60 and 120 min, 500 pL of reaction mix was transferred to a plastic tube containing 500 pL pure water at 99 °C. The tube was left at 99 °C for 10 min to inactivate the enzyme. When all timepoints from the experiment had been collected, the tubes were centrifuged at 14000 rpm for 10 min. Supernatants were further filtered through 0.22-pm syringe filters. The filtered samples were injected on an HPLC for analysis. For reference (t=0) sample, an amount of water corresponding to the enzyme addition was added to the 1% sucrose / buffer solution and treated in the same way as the enzyme sample.

[0278] Size exclusion chromatography (SEC) was done using UHPLC equipment from Thermo Scientific (Ultimate 3000) equipped with an SEC column (SUPREMA, Linear-XL 8 x 300 mm) and an RI detector (ERC RefractoMAX 520). Chromatographic conditions: Mobile phase was 10 mM NaCl in ultrapure water. Elution was isocratic with a run time of 30 min. The flow was 0.5 mL / min. The column temperature was 50 °C. Rl-detector was set to positive mode and a data collection speed of 2 Hz. Injection volume was 25 pL. Pullulan standards were used to make a correlation between polymer MW and elution time.

[0279] Results: Overlaid chromatograms from different timepoints of the reaction between sucrose and GTF 0768 enzyme can be seen in FIG. 10. It was observed with the reference sample (black line) that sucrose comes out at a single peak at around 22 min. For the 15-min reaction time sample (gray line), it was observed that the sucrose peak was reduced to about half and fructose was formed. Formation of larger polymers are not immediately visible at the level of detail in FIG. 10. After 60 min reaction time (dark dashed line), the sucrose peak disappeared. Reaction products were fructose and a polymer of MW of -2000 kDa. After 120 min reaction time (dotted line), only minor differences compared to the 60 min reaction timepoint were observed. This indicates that the reaction had run to completion between about 15 and 60 minutes.

[0280] Example 9. Digestibility of carbohydrates in brioche bread made with GTF enzyme addition

[0281] Brioche bread samples 1 and 5 were again produced as described in Example 6 (Table 6), after which the bread samples were subjected to a digestibility test. HPLC analysis was performed according to following method: HPLC carbohydrate analysis

[0282] Soluble carbohydrate composition was measured by HPLC with a Waters® 2695 Serrations module or a ThermoSci entific DionexTM UltiMate 3000 HPLC, equipped with a Phenomonex Rezex™ RPM-Monosaccharide Pb2+column (300 mm x 7.8 mm), and an RI- detector. Water was used as the mobile phase at a flow rate of 0.400 mL / min. The column temperature was 70 °C. Samples were prepared for HPLC injection by centrifugation (10 min at 13,000 rpm) and then using appropriate dilution of the supernatant in water, and a sterile filtration. Signals from the HPLC were quantified against calibration standards of sugars eluting at the same time. Sugar reduction was calculated by subtracting the total sum of mono- and di-saccharides in the test sample from the total sum of mono- and disaccharides in the reference sample without enzymes added. Digestibility of carbohydrates analysis

[0283] Approximately 5 g of the brioche bread was cut out and freeze-dried before grinding it to a powder. Moisture in the powder was determined to be 5.8% in the negative control and 5.2% in the sample produced with GTF using a moisture analyzer.

[0284] Three aliquots of 450 pL 5% dry solids (DS) slurry in water were then prepared from each of the two powders and tempered at 37 °C. At time 0, to each slurry was added either 150 pL water (time 0 reference) or 150 pL carbohydrate digestive enzyme mix of 128 U / g PLUSWEET G (glucoamylase) and 144 U / g FOODPRO I (invertase). The samples with the carbohydrate digestive enzymes were incubated either 20 min or 120 min at 37 °C before inactivating the enzymes at 95 °C (10 min incubation). To all the samples was then added 350 pL ethanol, and then the samples were heated at 83 °C for 10 min to precipitate protein and non-digested polysaccharides. Supernatant was collected for HPLC analysis after centrifugation for 10 min at 13000 rpm to remove precipitate.

[0285] Results: Theoretically, both samples should contain the same levels of total carbohydrates relative to the dry solids as they differed only in addition of water and enzyme. As previously observed (above), it was clear that more oligosaccharides were present in the GTF enzyme-treated sample vs. the reference sample without GTF enzyme addition (compare respective time 0 samples), and that more free glucose and fructose were released in the reference sample due to yeast invertase activity (Table 8). It was also observed that less maltose and glucose were present in the GTF-treated samples, most likely due to it being glucosylated into oligosaccharides by the GTF enzymes during bread preparation.

[0286] In the digestion test, glucose release increased over time, as expected, from hydrolysis of starch, oligosaccharides and residual sucrose. It was, however, clear that the reference sample had a much higher release of glucose over time; therefore, the reference sample contained more digestible carbohydrates (polysaccharides) compared to the GTF-treated sample.

[0287] Table 8, Carbohydrate analysis of brioche bread samples after digestibility treatment Example 10. Determination of specific volumes of baked wheat dough punched rolls using ex situ GTF dextran process

[0288] The effect of increasing dosages of GTF dextran on the shocked and unshocked specific volumes of punched wheat rolls was examined. A 2.9% solution of sucrose in ultrapure water was incubated with GTF 0768 (SEQ ID NO: 1) at a concentration of 69.4 U / g sucrose for 65 minutes at room temperature to convert sucrose to dextran. The resulting GTF dextran solution was then heated to boiling point, and tempered to 34 °C. This dextran solution was then added in increasing doses to replace a 2.9% sucrose solution used to produce the doughs for punched wheat doughs. At an expected sucrose conversion rate of 100% by GTF 0768, this corresponded to a GTF 0768 dextran addition ranging from 0.32 to 1 ,60%b. The ingredients for the tests with increasing dextran amounts are listed in Table 9. Details on these ingredients, as well as the mixing, proofing, shaping, shocking, baking and volume determination process can be found in Example 5.

[0289] Table 9, Ingredients for Tests 1 to 6 with increasing ex .s / V / i-produced dextran addition

[0290] Addition of ex .s / 7 / / -produced GTF 0768 dextran, ranging from 0.32%b to 1.6%b, increased the specific volume of the unshocked breads (based on dough weight) from 3.62 mL / g (Test 1, the negative control without GTF dextran solution) to 4.11 mL / g (FIG. 17 [A]), and similarly increased the specific volume of the unshocked breads (based on bread weight) from 4.55 mL / g to 5.26 mL / g (FIG. 17 [B]). These relative volume increases corresponded to 114% and 116% (dough weight and bread weight bases, respectively) as compared to the respective blank reference (100%).

[0291] Addition of ex .sz / zz-produced GTF 0768 dextran also increased the specific volume of the shocked breads (based on dough weight) from 2.86 mL / g (Test 1, the negative control without GTF dextran solution) to 3.53 mL / g (FIG. 17 [A]), and similarly increased the specific volume of the shocked breads (based on bread weight) from 3.31 mL / g to 4.45 mL / g (FIG. 17 [B]). These relative volume increases corresponded to 123% and 134% (dough weight and bread weight bases, respectively) as compared to the respective blank reference (100%).

[0292] Thus, addition of ex .sz7 / / -produced GTF 0768 dextran significantly increased both unshocked and shocked volumes of wheat dough rolls.

[0293] Example 11. Determination of specific volumes of baked wheat dough punched rolls using GTF dextran in an autolyze dough making process

[0294] The effects were tested of using GTF 0768 (SEQ ID NO: 1) alone or a combination of GTF 0768 and vGTFJ (SEQ ID NO:3), with or without TSE-2356, on the specific volume and stability of punched wheat rolls produced using an autolyze (or “autolyse”) process. Reform flour (300 g, Lantmannen Cerealia, Vejle, Denmark, 12% protein) with an initial water content of 14% was tempered to room temperature. For the autolyze process of the controls (Tests 1 and 2, Table 10), 1.6%b sucrose was dissolved in 59.5%b room temperature water, 30.55%b of reform flour was added, and the autolyze was mixed by hand for 1 minute. The autolyze was incubated at room temperature for 20 minutes. Then, the remaining 69.45%b of flour was briefly blended by hand with l%b salt, 2%b dry yeast (SAF instant yeast, S. I. Lesaffre, France) and 0.56%b GRIND AMYL A1000 (diluted to 16.2 FAU / g, IFF, Brabrand, Denmark). For Test 1, additionally, 0.125%b TSE-2356 (technical enzyme sample consisting of lipase, amylase, xylanase and oxidase to provide increased bread volume; provided by IFF, Brabrand, Denmark) was added. This blend was then added to the autolyze, and mixing was performed in a KITCHEN AID (Artisan 5KSM125, Kitchen Aid, MI, USA) mixer at low speed for 2 minutes followed by high speed for 8 minutes. Proofing, punching out of the rolls, baking, shocking and volume determination were performed as described in Example 5.

[0295] For Tests 3-6 (Table 10), containing GTF 0768 or a combination of GTF 0768 and vGTFI, with or without the addition of 0.125%b TSE-2356, the same process as for Tests 1 and 2 was followed, but the weight of the GTF enzyme(s) was subtracted from the weight of the water added. An overview of the ingredients of Tests 1-6 are listed in Table 10. Table 10. Ingredients for Tests 1 to 6 with for testing effects of GTF enzyme(s) in an autolyze dough making process

[0296] The results of specific volume measurements of shocked and unshocked wheat rolls made using doughs of Tests 1-6 can be found in FIG. 18. In the presence of TSE-2356, addition of GTF 0768 resulted in a specific unshocked volume of 5.14 cm3 / g (Test 3), and addition of a combination of GTF 0768 and vGTFJ resulted in a specific unshocked volume of 4.80 cm3 / g (Test 5), while the negative control containing TSE-2356 (Test 1) showed an unshocked specific volume of 3.95 cm3 / g. This corresponded to unshocked volume increases to 130.1% and 121.5%, respectively, compared to the negative control (100%). Regarding shocked volumes in the presence of TSE-2356, the addition of GTF 0768 resulted in a specific volume of 3.90 cm3 / g (Test 3), and addition of a combination of GTF 0768 and vGTFJ resulted in a specific volume of 3.72 cm3 / g (Test 5), which are increases to 120.0% and 114.5%, respectively, compared to the specific shocked volume of the negative control containing TSE-2356 (Test 1) of 3.25 cm3 / g (100%). These results indicate that the addition of GTF 0768, or a combination of GTF 0768 and vGTFJ, can further increase the specific volume and stability of bread formulations that already contain enzymes aimed at increasing bread volume, such as lipases, amylases, xylanases and oxidases. Without the addition of TSE-2356, addition of GTF 0768 resulted in a specific unshocked volume of 4.44 cm3 / g (Test 4), and using of a combination of GTF 0768 and vGTFJ resulted in a specific unshocked volume of 4.14 cm3 / g (Test 6), while the negative control not containing TSE-2356 (Test 2) showed an unshocked specific volume of 2.86 cm3 / g (FIG. 18). This corresponded to unshocked specific volume increases to 155.2% and 144.8%, respectively, compared to the negative control (100%). Regarding shocked volumes without the use of TSE-2356, the addition of GTF 0768 resulted in a specific volume of 3.72 cm3 / g (Test 4), and addition of a combination of GTF 0768 and vGTFJ showed a specific volume of 3.34 cm3 / g (Test 6), which are increases to 122.8% and 110.2%, respectively, compared to the specific shocked volume of the negative control not containing TSE-2356 of 3.03 cm3 / g (Test 2). These results indicate that the addition of GTF 0768, or a combination of GTF 0768 and vGTFJ, increase the specific volume and stability of bread formulations, even without the use of additional enzymes aimed at increasing bread volume, such as lipases, amylases, xylanases and oxidases.

[0297] Example 12. Determination of specific volumes of baked wheat dough punched rolls made using GTF 0768 and different yeast varieties

[0298] The effect of GTF 0768 (SEQ ID NO: 1) on the specific volume of shocked and unshocked punched wheat rolls produced with two different types of yeast was studied. Two different yeasts were used in this Example: a yeast intended for use in recipes with low or no sugar, and a yeast intended for use in sweet dough. The ingredients used for this trial can be found in Table 11. Reform flour (300 g, Lantmannen Cerealia, Vejle, Denmark, 12% protein) with an initial water content of 14% was tempered to room temperature. l%b salt, 1.6%b sugar, 2%b dry yeast (SAF instant yeast, S. I. Lesaffre, France, for Tests 1 and 3, or Instant dry yeast - brown, FERMIPAN, hereafter referred to as Brown Yeast, for Tests 2 and 4), and 0.56%bGRIND AMYL A1000 (A1000 diluted to 16.2 FAU / g, IFF, Brabrand, Denmark) were all mixed in a KITCHEN AID mixer (Artisan 5KSM125, Kitchen Aid, MI, USA) for 1 minute, and then 59.5%b room temperature water was added. The mixing, proofing, punching out of the rolls, shocking, baking, and volume determination were performed as described in Example 5. Table 11. Ingredients for the test of the effect of GTF 0768 on the specific volume of shocked and unshocked punched wheat rolls produced with two different types of yeast

[0299] The results of measuring specific volumes of shocked and unshocked wheat rolls of Tests 1-4 can be found in FIG. 19. Using Brown Yeast, a product intended for sweet doughs, in combination with GTF 0768, did not increase the specific unshocked volume. The specific unshocked volume of the negative control with Brown Yeast (Test 2) was 3.85 cm3 / g, and the specific unshocked volume of Test 4 with GTF 0768 and Brown Yeast was 3.79 cm3 / g. However, the stability of the GTF 0768-containing doughs increased, as can be seen by the comparison of the specific volumes of the breads baked from shocked doughs. In Test 4, which was made with GTF 0768 in an autolyze process and Brown Yeast, the specific volume of the shocked breads was 3.76 cm3 / g and thus close to the unshocked specific volume of Test 4. This is an increase to 117.9% of the shocked specific volume as compared to the negative control made with Brown Yeast (Test 2) (100%), which had a specific shocked volume of 3.19 cm3 / g.

[0300] Using GTF 0768 with SAF Instant Yeast (Test 3), which is a yeast product intended for low to no sugar doughs, resulted in unshocked and shocked specific volumes of 3.64 and 3.29 cm3 / g, respectively. This is an increase to 108.7% and 108.2%, respectively, of the unshocked and shocked specific volumes as compared to the respective unshocked and shocked negative controls made with SAF Instant Yeast (Test 1).

[0301] Comparing the specific volume of the breads from Tests 3 and 4, the bread rolls produced with GTF 0768 and Brown Yeast in Test 4 resulted in overall higher unshocked and shocked specific volumes compared to the bread rolls produced with GTF 0768 and SAF Instant Yeast in Test 3. This observed higher bread volume and stability in Test 4 might be attributed to the lower invertase activity typically found in yeast varieties used for high sugar doughs, resulting in more available sucrose as a substrate for GTF 0768 during the mixing and proofing time.

[0302] Example 13. Dough development with addition of GTF enzyme in a simulated autolyze process, as analyzed by Brabender farinograph

[0303] The impact of GTF 0768 (SEQ ID NO: 1) in an autolyze process on dough development was analysed using a Brabender farinograph (calibrated and operated according to Brabender ICC_BIPEA_50).

[0304] AACC method 51-21-01 (incorporated herein by reference) was modified to simulate dough preparation with an autolyze process. For the blanks (Test nos. 1 and 3, Table 12), 15%b of the total amount of 50 g of reform flour (Lantmannen Cerealia, Vejle, Denmark, 12% protein) was mixed by hand with 1.66%b sugar and 59.5 to 61.5 %b of room temperature water. For Test nos. 2 and 4 (Table 12), 59.22 to 61.22 %b room temperature water was added, followed by addition of 1366 GTFU of GTF 0768 per kg of total flour. Table 12 provides an overview of the ingredients of each test. The autolyze was incubated at room temperature for 20 minutes.

[0305] Table 12, Ingredients for farinograph tests

[0306] The residual 85%b of reform flour and 2%b salt were dry-mixed for 1 minute in a FARINOGRAPH-E (Brabender, Duisburg, Germany) equipped with a 50-g moulding chamber (MIXER S 50N, model 820608), equilibrated at 30.0 °C, and set to a speed of 63 rounds / minute. Then, the autolyze was added and the dough was mixed for 6 minutes. Torque (FU) was recorded throughout the mixing.

[0307] The dough development (torque) of the doughs as analyzed by the Brabender farinograph, with or without GTF 0768, is shown in FIG. 20. The mean torque of the final ten seconds of mixing time, referred to as final torque, is presented in Table 13. Table 13, Mean torque plus standard deviation of tests over the final ten seconds of mixing time

[0308] Table 13 shows that the use of GTF 0768 increased the final torque by 87 FU at 59.5%b WA (water addition) (Tests 1 and 2), and by 70 FU at 61.5%b WA (Tests 3 and 4). Increased water addition by 2%b without the use GTF 0768 decreased the final torque by 32 FU (Tests 1 and 3). Using GTF 0768, a 2%b point increase in WA still resulted in a 38 FU higher final torque compared to a system with lower WA and no GTF 0768 (Test 1 and 4). Thus, these results show that addition of GTF 0768 provides optionality to increase WA while keeping dough torque constant.

[0309] Example 14. Determination of specific volumes of baked wheat dough punched rolls with increased water addition using GTF dextran in an autolyze process

[0310] The effect of GTF 0768 (SEQ ID NO: 1) on the specific volume of shocked and unshocked punched wheat rolls produced with increased water addition (WA) was studied. Tests 1 and 2 (Table 14) were performed at a WA of 59.5%b, which is the amount of water necessary to achieve the standard torque of 400 farinograph units. This value was determined prior to the experiment (data not shown). The effect of GTF 0768 on a bread roll system with an increased WA of 61 ,5%b was studied. The ingredients used for the tests in this Example can be found in Table 14. The autolyze process used to produce the bread rolls was performed as described in Example 11. The proofing, punching out of the rolls, baking, shocking and volume determination were performed as described in Example 5.

[0311] Table 14, Ingredients for a trial with and without the addition of GTF 0768 to bread rolls with a water addition of 59,5 %b or 61,5%b

[0312] As can be seen in FIG. 21, the use of a GTF increased the unshocked and shocked specific volumes from 3.38 and 2.82 cm3 / g in the negative control with a WA of 59.5%b (Test 1), to 5.16 and 3.99 cm3 / g (Test 2), respectively. Increasing the WA to 61.5%b increased the unshocked specific volume of the negative control from 3.38 (Test 1) to 3.61 cm3 / g (Test 3), while the shocked specific volume (and thus stability) for the negative controls slightly decreased from 2.82 to 2.80 cm3 / g. However, with the addition of GTF 0768 to the doughs with an increased WA of 61.5%b, the unshocked and shocked specific volumes of the breads were found to be 4.41 and 3.32 cm3 / g, respectively (Test 4), which is an increase to 122.2 and 118.6% relative to the unshocked and shocked volumes of the negative control with a WA of 61.5%b (100%). This Example therefore shows that, by addition of GTF 0768, unshocked specific volume and stability (as indicated by the shocked specific volume) can be increased, even at increased levels of WA.

[0313] Example 15. Determination of specific volumes of white toast bread using GTF dextran in an autolyze process

[0314] An autolyze process was initiated by mixing 30% of flour (Reform flour, Lantmannen Cerealia, Vejle, Denmark) and sugar for 30 s on a Diosna Spiral mixer, water was added and mixed for 60 s, and then GTF 0768 (SEQ ID NO: 1) was added and mixed for 60 s. Next, the batter was rested for 20 min at ambient temperature. The remaining 70% of the flour, yeast (SAF instant yeast, S. I. Lesaffre, France), salt, ascorbic acid and GRIND AMYL™ A1000 (diluted to 16.2 FAU / g fungal alpha-amylase guided by the falling number of the flour (International Flavors & Fragrances Inc., DK-8220 Brabrand, Denmark)) were added and mixed for 2 min at slow speed and then 5.5 minutes at high speed. All the ingredients are listed in Table 15 (Trials 1-3). The dough was rested for 10 minutes at 30 °C. Four dough pieces (each 650 g) were scaled and rested 5 minutes at ambient temperature. Then, the dough pieces were molded on a Bernier MS 500 with the following settings: Preform: -18, Drum press.: 3, Pressure board: 4.0 cm front, 3.5 cm back, Width: 370 mm front, 330 mm back, and put into tins (DK toast tins). The dough was proofed for 70 minutes at 33 °C and 85% relative humidity (RH). Half of the loaves were shocked by dropping the dough- containing tin on a table twice from a height of 6.5 cm. The dough was then baked for 30 minutes at 205 °C with damper open (Miwe prog 2). After baking, the breads were cooled for 70 minutes at ambient temperature before weighing and measuring of volume using a rapeseed displacement method.

[0315] The specific volumes of the unshocked breads with GTF 0768 from Trials 2 and 3 (FIG. 22) increased to 5.95 and 5.77 cm3 / g, respectively, as compared to the control having a specific volume of 5.49 cm3 / g. The specific volume of the shocked breads with GTF 0768 in Trials 2 and 3 (FIG. 22) increased to 5.86 and 5.69 cm3 / g, respectively, as compared to the control having a specific volume of 4.77 cm3 / g. These volume increases corresponded to 108% and 105%, respectively, for the unshocked breads, and 123% and 119%, respectively, for the shocked breads as compared to the respective control (100%). Thus, the addition of GTF enzyme for in situ alpha- 1,6-glucan generation had a very beneficial influence on the specific volume of the baked bread as well as the shock stability.

[0316] Table 15, Relative distribution (% weight of flour) of ingredients for white toast bread with autolyze and GTF enzyme

[0317] Example 16. Extraction of high molecular weight (BMW) alpha-1, 6-glucan (dextran) from bread crumb and analysis by HPLC-SEC

[0318] To extract HMW dextran polymer from bread crumb, 5 g of bread crumb was weighed into a glass beaker and then 20 g of ultrapure water was added. A magnet was added to the beaker, and it was placed on a magnetic stirrer. To reduce background effects (noise) from starch on the HPLC-SEC chromatograms, 14.5 U of glucoamylase was added per g of bread crumb. The resulting slurry was stirred at 800 rpm for 3 hours at room temperature. After incubation, the slurry was transferred to 50-mL centrifuge tubes and centrifuged at 4500 rpm for 10 min. About 1.8 mL of the supernatant was transferred to an EPPENDORF tube and the tube was left for 1 hour at room temperature to further reduce the amount of starch fragments. Subsequently, the glucoamylase was inactivated by heating the tube at 90 °C for 10 min. To remove any solids still in the sample, the tubes were centrifuged at 14000 rpm for 10 min, and about 1 mL of the supernatant was filtered through a 0.2-pm PTFE syringe filter. The filtrate was placed in an HPLC vial and capped. HPLC-SEC analysis was carried out as described in Example 8.

[0319] In FIG. 23, one sees an HPLC overlay with chromatograms showing composition of some extracted crumbs. All breads were made using the autolyze process, as described in Example 11, with 3.2% sucrose in the autolyze. The solid line is a reference made without the use of enzyme in the autolyze process. The dotted line shows the extract composition of bread crumb made with 188 U of GTF 0768 (SEQ ID NO: 1) in the autolyze process. The dashed line shows extract composition of a bread crumb made with 378 U of GTF 0768 in the autolyze process. It was observed that the bread crumb made with the highest dose of GTF 0768 in the autolyze process contained the most HMW dextran polymer. Using pullulan standards, the molecular weight of the extracted polymer was calculated to be about 2000 kDa, which was consistent with the result described in Example 8. This result reflects that the dextran polymers are stable and do not degrade in the baking process.

[0320] Example 17. Analysis of alpha-glucan linkages produced in GTF-treated bread

[0321] The glucan formed by the addition of GTF 0768 (SEQ ID NO: 1) to a brioche formulation was extracted (post-baking) and the linkage of the formed glucan was analyzed by1H NMR.

[0322] The ingredients of the brioche formulations, either with the addition of GTF 0768 or without GTF 0768 addition (negative control), can be found in Table 16.

[0323] Table 16, Relative distribution* of brioche bread ingredients with or without GTF enzyme

[0324] * Each ingredient, except for GTF 0768, is provided as percent weight by flour.

[0325] All the dry ingredients, egg, water, milk, and for trial 2, GTF 0768, were mixed using a KITCHEN AID (Artisan 5KSM125, Kitchen Aid, MI, USA) mixer at low speed for 2 minutes followed by high speed for 6 minutes. Butter was added and the dough was mixed for an additional 2.5 minutes at high speed. The doughs were proofed at room temperature for 30 minutes, then rolls punched were out as described in Example 5. The rolls were proofed for 90 minutes at 30 °C, 85% RH, and baked for 7 minutes at 200 °C.

[0326] Five g of the crumb of each of the resulting brioche rolls was first extracted with 4x volume of ultrapure water with added glucoamylase (GRIND AMYL® PLUSWEET G, 9 U / mL) on a rotating incubator at room temperature for 3 hours, followed by centrifugation for 30 minutes at *4800 g. The supernatant, which contained the water-soluble fraction of glucoamylase-treated brioche crumb, was then heat-treated for 15 minutes at 90 °C for enzyme inactivation, and centrifuged for 10 minutes at *4800 g. The supernatant was then subjected to precipitation with 30% ethanol at a ratio of 7 parts sample to three parts ethanol for 1 hour at room temperature. The sample was centrifuged for 10 minutes at *4800 g and the resulting pellet was dried for 12 hours at room temperature, followed by freeze-drying for 24 hours.

[0327] The pellet was dissolved in 700 L 3 wt% LiCl / DMSO-d6. The sample was placed on a thermomixer at 25 °C overnight. After dissolving, 50 pL D2O was added for deuterium exchange.1H NMR spectra were recorded at 343 K on a 600 MHz Bruker Avance III NMR spectrometer (Bruker Biospins, Rheinstetten, Germany) equipped with a 5 -mm Prodigy CryoProbe. Standard1H NMR spectra were acquired using a single-pulse experiment with a 30° flip angle, 128 scans collected into 64k data points with a spectral width of 19.84 ppm, a recycle delay of 20 s and an acquisition time of 2.75 s. For assignment of H-l position peaks, 2DJH-13C Heteronuclear Single Quantum Coherence (HSQC) NMR experiments were acquired using 64 scans, a spectral width of 11.9 ppm in the 'H dimension and 50 ppm in the13C dimension, a matrix size of 4096 x 128 complex data points and a relaxation delay of 1 s. In FIG. 24, the H-l region of the 'H NMR spectra shows that making brioche with GTF enzyme ingredient (dark-line spectra) resulted in a major peak from alpha-1, 6-glucan in the water-soluble fraction of the brioche. TheJH-13C HSQC experiment identified the peak as alpha- 1, 6-glucan by aJH chemical shift at 5.01 ppm and a13C chemical shift at 101 ppm. No other linkages of alpha-glucan were found in the NMR spectra. The grey -line spectra in FIG. 24 (making brioche without GTF enzyme) did not show any peak for alpha- 1, 6-glucan. These results clearly show that the added GTF 0768 enzyme synthesized alpha- 1, 6-glucan during the process of preparing brioche.

[0328] Example 18. Analysis of alpha-glucan linkages produced in GTF-treated cake

[0329] The glucan formed by the addition of GTF 0768 (SEQ ID NO: 1) and vGTFJ (SEQ ID NO:3) to a pound cake formulation was extracted (post-baking) and the linkage of the formed glucan was analyzed byJH NMR.

[0330] The ingredients of the poundcake formulations, either with addition of the GTFs or without GTF addition (negative control), can be found in Table 17.

[0331] Table 17, Relative distribution* of pound cake ingredients with or without GTF enzymes

[0332] * Each ingredient, except for the GTF enzymes, is provided as percent weight by flour.

[0333] Margarine and sugar were mixed for 5.5 minutes on a Hobart N50 mixer (Hobart Scandinavia, Greve, Denmark), with eggs, and for trial 2, GTF enzymes were added during further 2 minutes of mixing. Then, the dry ingredients were added and mixed for 2.5 minutes. 350 g of batter was filled into pound cake tins and cakes were baked for 45 minutes at 180 °C.

[0334] The crumb of each of the resulting cakes was freeze-dried, and then extracted as described in Example 17, with the following modifications: the ratio of sample to ultrapure water with glucoamylase was changed to 1 parts sample to 8 parts extractant, and samples were extracted for 4 hours using a magnetic stirrer at 800 rpm.1H NMR analysis was carried out as described in Example 17.

[0335] In FIG. 25, the H-l region of theJH NMR spectra shows that making pound cake with GTF enzymes (dark-line spectra) ingredient resulted in a major peak from alpha- 1,3 -glucan in the water-soluble fraction of the cake. No other linkages of alpha-glucan were found in the NMR spectra. The grey-line spectra in FIG. 25 (making pound cake without any GTF enzyme) only showed a minor peak for alpha- 1,3 -glucan, which indicated an 87% increase in alpha-1,3 linkage content in the pound cake made using the GTF enzymes. These results clearly show that the added GTF enzymes synthesized alpha- 1,3 -glucan during the process of preparing cake.

Claims

CLAIMSWhat is claimed is:

1. A method of producing a food dough, said method comprising: mixing at least(a) flour and / or meal,(b) water or aqueous composition,(c) sucrose, and(d) at least one glucosyltransferase enzyme, thereby producing a food dough, wherein said at least one glucosyltransferase enzyme is selected from:(i) a glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6- glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,6- glucan are alpha- 1,6 linkages, and / or(ii) a glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3- glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1, 3- glucan are alpha- 1,3 linkages, typically wherein at least one alpha-glucan is produced in the food dough, optionally wherein:(I) the food dough has a shock stability that is increased as compared to the shock stability of a control food dough that lacks said at least one glucosyltransferase enzyme,(II) the food dough has a water-binding capacity that is increased as compared to the water-binding capacity of a control food dough that lacks said at least one glucosyltransferase enzyme,(III) the food dough has a viscosity that is increased as compared to the viscosity of a control food dough that lacks said at least one glucosyltransferase enzyme, and / or(IV) the food dough has a volume that is increased as compared to the volume of a control food dough that lacks said at least one glucosyltransferase enzyme.

2. The method of claim 1, wherein said at least one glucosyltransferase enzyme is the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 6-glucan.

3. The method of claim 1, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, 2, 11, 12, 14, or 15.

4. The method of claim 1, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, 2, 11, or 12.

5. The method of claim 1, wherein said at least one glucosyltransferase enzyme is the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan.

6. The method of claim 1, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to residues 55-960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55-960 of SEQ ID NO:8, or residues 55-960 of SEQ ID NO:9.

7. The method of claim 1, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to residues 55-960 of SEQ ID NO:5.

8. The method of claim 1, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:3, 4, 5, 6, 7, 8, 9, or 13.

9. The method of claim 1, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:5.

10. The method of claim 1, wherein both of the (i) glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan and (ii) glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan are used in said mixing step.

11. The method of claim 10, wherein the alpha-glucan produced in the food dough comprises a graft copolymer comprising: (i) an alpha- 1,6-glucan backbone, wherein at least about 50% of the glycosidic linkages of the alpha- 1,6-glucan backbone are alpha- 1,6 linkages, and (ii) at least one alpha- 1,3 -glucan side chain, wherein at least about 50% of the glycosidic linkages of the alpha- 1,3 -glucan chain are alpha-1,3 linkages, wherein the graft copolymer is aqueous-soluble or aqueous-insoluble.

12. The method of claim 10, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan in said mixing step is about 95:5 to about 5:95.

13. The method of claim 10, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan in said mixing step is about 95:5 to about 50:50.

14. The method of claim 1, wherein said flour is used in the method, optionally wherein the flour is wheat flour.

15. The method of claim 1, wherein said mixing comprises(A) first combining said (b) water or aqueous composition, (c) sucrose, and (d) at least one glucosyltransferase enzyme, and then (B) combining the combination of (b), (c) and (d) with said (a) flour and / or meal.

16. The method of claim 1, wherein said mixing comprises(A) first combining(a) a portion of the total amount of the flour and / or meal to be used in said mixing,(b) most of or all of the total amount of the water or aqueous composition to be used in said mixing,(c) optionally the sucrose, and(d) optionally the at least one glucosyltransferase enzyme, thereby producing a flour slurry or pre-mix,typically wherein at least one alpha-glucan is produced in the flour slurry / pre-mix if said sucrose and at least one glucosyltransferase enzyme were included in the flour slurry / pre-mix, and then (B) combining the flour slurry / pre-mix with the remaining amount of the flour and / or meal to be used in said mixing, and any remaining amount of water or aqueous composition, sucrose and at least one glucosyltransferase enzyme to be used in said mixing, thereby producing said food dough.

17. The method of claim 1, further comprising: processing the food dough into a food.

18. The method of claim 17, wherein said processing comprises at least baking, frying, boiling, drying, chilling, extruding, and / or flattening the food dough.

19. The method of claim 14, wherein the food dough comprises a leavening agent, optionally wherein the leavening agent is yeast.

20. The method of claim 19, wherein the yeast has reduced invertase activity or no detectable invertase activity.

21. The method of claim 14, wherein the food dough is a bread dough.

22. The method of claim 14, further comprising allowing the food dough to rise, thereby providing risen food dough.

23. The method of claim 22, wherein the risen food dough is risen bread dough, optionally wherein the volume of the risen bread dough is at least 5% greater than it would be if the risen bread dough did not comprise said at least one glucosyltransferase enzyme, optionally wherein the risen bread dough has not been shocked.

24. The method of claim 23, further comprising dropping the risen bread dough at least one time over a distance of at least 1 cm, wherein, following said dropping, thevolume of the risen bread dough is at least 2% greater than it would be if the risen bread dough did not comprise said at least one glucosyltransferase enzyme.

25. The method of claim 14, wherein said mixing exhibits a torque that is at least 2% greater than it would be if the food dough did not comprise said enzyme.

26. The method of claim 14, wherein the flour has a gluten content that is less than about 12 wt%.

27. The method of claim 1, further comprising adding at least one of a lipase, alphaamylase, xylanase, non-maltogenic exoamylase, glucoamylase, glucose oxidase, or hexose oxidase during production of the food dough.

28. A food dough produced by the method of claim 1.

29. A food product produced by the method of claim 17.

30. A food dough comprising(a) flour and / or meal,(b) water or aqueous composition,(c) sucrose, and(d) at least one glucosyltransferase enzyme, selected from:(i) a glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6- glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,6- glucan are alpha- 1,6 linkages, and / or(ii) a glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3- glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1, 3- glucan are alpha- 1,3 linkages, typically wherein the food dough comprises at least one alpha-glucan product of said at least one glucosyltransferase enzyme, optionally wherein(I) the food dough has a shock stability that is increased as compared to the shock stability of a control food dough that lacks said at least one glucosyltransferase enzyme,(II) the food dough has a water-binding capacity that is increased as compared to the water-binding capacity of a control food dough that lacks said at least one glucosyltransferase enzyme,(III) the food dough has a viscosity that is increased as compared to the viscosity of a control food dough that lacks said at least one glucosyltransferase enzyme, and / or(IV) the food dough has a volume that is increased as compared to the volume of a control food dough that lacks said at least one glucosyltransferase enzyme.

31. The food dough of claim 30, wherein said at least one glucosyltransferase enzyme is the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 6-glucan.

32. The food dough of claim 30, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1, 6-glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:1, 2, 11, 12, 14, or 15.

33. The food dough of claim 30, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1, 6-glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:1, 2, 11, or 12.

34. The food dough of claim 30, wherein said at least one glucosyltransferase enzyme is the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan.

35. The food dough of claim 30, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to residues 55-960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55-960 of SEQ ID NO:8, or residues 55-960 of SEQ ID NOV.

36. The food dough of claim 30, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to residues 55-960 of SEQ ID NO:5.

37. The food dough of claim 30, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:3, 4, 5, 6, 7, 8, 9, or 13.

38. The food dough of claim 30, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:5.

39. The food dough of claim 30, wherein the food dough comprises both of the (i) glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan and (ii) glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan.

40. The food dough of claim 39, wherein the food dough comprises said at least one alpha-glucan product, and said alpha-glucan product comprises a graft copolymer comprising: (i) an alpha- 1,6-glucan backbone, wherein at least about 50% of the glycosidic linkages of the alpha- 1,6-glucan backbone are alpha-1,6 linkages, and (ii) at least one alpha- 1,3 -glucan side chain, wherein at least about 50% of the glycosidic linkages of the alpha- 1,3 -glucan chain are alpha-1,3 linkages, wherein the graft copolymer is aqueous-soluble or aqueous-insoluble.

41. The food dough of claim 39, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan is about 95:5 to about 5:95.

42. The food dough of claim 39, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan is about 95:5 to about 50:50.

43. The food dough of claim 30, wherein the food dough comprises said flour, optionally wherein the flour is wheat flour.

44. The food dough of claim 43, wherein the food dough comprises a leavening agent, optionally wherein the leavening agent is yeast.

45. The food dough of claim 43, wherein the food dough is a bread dough.

46. The food dough of claim 43, wherein food dough is a risen food dough.

47. A food product comprising(a) flour and / or meal,(b) water or aqueous composition,(c) optionally sucrose, and(d) at least one glucosyltransferase enzyme, selected from:(i) a glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6- glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,6- glucan are alpha- 1,6 linkages, and / or(ii) a glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3- glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1, 3- glucan are alpha- 1,3 linkages, typically wherein the food product comprises at least one alpha-glucan product of said at least one glucosyltransferase enzyme, optionally wherein(I) the food product has a water-binding capacity that is increased as compared to the water-binding capacity of a control food product that lacks said at least one glucosyltransferase enzyme,(II) the food product has a volume that is increased as compared to the volume of a control food product that lacks said at least one glucosyltransferase enzyme,(III) the food product has a crumb that is increased as compared to the crumb of a control food product that lacks said at least one glucosyltransferase enzyme,(IV) the food product has a softness that is increased as compared to the softness of a control food product that lacks said at least one glucosyltransferase enzyme, and / or(IV) the food product has a resilience that is increased as compared to the resilience of a control food product that lacks said at least one glucosyltransferase enzyme.

48. The food product of claim 47, wherein said at least one glucosyltransferase enzyme is the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 6-glucan.

49. The food product of claim 47, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:1, 2, 11, 12, 14, or 15.

50. The food product of claim 47, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:1, 2, 11, or 12.

51. The food product of claim 47, wherein said at least one glucosyltransferase enzyme is the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan.

52. The food product of claim 47, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to residues 55-960 of SEQ ID NO:5, residues 54- 957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55-960 of SEQ ID NO:8, or residues 55-960 of SEQ ID NO:9.

53. The food product of claim 47, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to residues 55-960 of SEQ ID NO:5.

54. The food product of claim 47, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:3, 4, 5, 6, 7, 8, 9, or 13.

55. The food product of claim 47, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:5.

56. The food product of claim 47, wherein the food product comprises both of the (i) glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan and (ii) glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan.

57. The food product of claim 56, wherein the food product comprises said at least one alpha-glucan product, and said alpha-glucan product comprises a graft copolymer comprising: (i) an alpha- 1,6-glucan backbone, wherein at least about 50% of the glycosidic linkages of the alpha- 1,6-glucan backbone are alpha-1,6 linkages, and (ii) at least one alpha- 1,3 -glucan side chain, wherein at least about 50% of the glycosidic linkages of the alpha- 1,3 -glucan chain are alpha-1,3 linkages, wherein the graft copolymer is aqueous-soluble or aqueous-insoluble.

58. The food product of claim 56, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan is about 95:5 to about 5:95.

59. The food product of claim 56, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3 -glucan is about 95:5 to about 50:50.

60. The food product of claim 47, wherein the food product comprises said flour, optionally wherein the flour is wheat flour.

61. The food product of claim 47, wherein the food product is a baked food product.

62. The food product of claim 47, wherein the food product is a bread product.

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