Method for improving plant-based foodstuff flavor

Abstract

Disclosed herein are methods of glucosylating isoflavone glycosides, as well as methods of reducing off-taste in food products / precursors. Further disclosed are compositions, such as food products / precursors, comprising one or more glucosylated isoflavone glycosides.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 379,883, filed Oct. 17, 2022, which is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0002] The present disclosure pertains to the field of glycosylating components present in plant-based food products to improve flavor. For example, the present disclosure relates to the in situ glucosylation of isoflavone glycosides in foods and food precursors.

[0003] References to electronically submitted sequence listings

[0004] The official copy of the Sequence Listing is submitted electronically via EFS-Web as NB42164WOPCT_SequenceListing.xml, created on Oct. 16, 2023, having a size of approximately 46 kilobytes, and is filed concurrently with this specification. The Sequence Listing contained in this document is part of this specification and is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0005] Plant-based or non-dairy protein food alternatives, such as soy, almond, pea, bean, rice, or oat-based products (e.g., milk or fresh fermented products), are among the fastest-growing segments of all food product categories globally ( et al., 2016, Crit. Rev. Food Sci. Nutr. 56:339-349; Sethi et al., 2016, J. Food Sci. Technol. 53:3408-3423). However, off-flavors remain a major barrier to the use of plant-based materials in mainstream food applications. For example, soy-derived materials contain several polyphenolic compounds (such as isoflavones, saponins, and phenolic acids), which produce adverse sensory properties such as bitterness and astringency. Currently, very cumbersome processes (such as dispersing the material in alkaline conditions followed by membrane separation or size exclusion chromatography) are attempted and used to remove these components and thus their unwanted flavors (Damodaran et al., 2013, Annu. Rev. Food Sci. Technol. 4:327-346).

[0006] Flavonoids are ubiquitous plant secondary metabolites that have received attention due to their multiple biological activities and benefits. Glycosylation has been used to modify these bioactive compounds to enhance the solubility or chemical and / or biological stability of the aglycone. Typically, Leloir glycosyltransferases have been used to glycosylate flavonoids, but these enzymes have low yields and require nucleotide-sugars as glycosyl donors. It has been reported that GH70 dextransucrases using sucrose as a glucose donor glucosylate various aglycones (Overwin et al., 2016, J. Biotechnol. 233:121-128; Li et al., 2021, Crit. Rev. Food Sci. Nutr. 1-21). However, this previous work has generally focused on synthesizing monoglycoside compounds because further glycosylation may affect biological activities such as antioxidant activity. Summary of the Invention

[0007] In one embodiment, the present disclosure relates to a method for producing a food product / precursor, the method comprising: (a) providing a food product / precursor comprising at least water, sucrose, and a plant-based material, and (b) contacting the food product / precursor with at least: (i) a glucosyltransferase that synthesizes α-1,6-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,6-glucan are α-1,6 bonds, and / or (ii) a glucosyltransferase that synthesizes α-1,3-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,3-glucan are α-1,3 bonds, typically wherein at least one α-glucan is produced in the food product / precursor, whereby the food product / food precursor after step (b) has a reduced off-flavor compared to the food product / precursor before step (b), and optionally, a reduced sugar content compared to the food product / precursor before step (b).

[0008] In another embodiment, the present disclosure relates to a food product / precursor produced by the method herein.

[0009] In another embodiment, the present disclosure relates to a method for glucosylating an isoflavone glycoside, the method comprising: providing a composition comprising at least water, sucrose, an isoflavone glycoside, and a glucosyltransferase, wherein the glucosyltransferase is selected from: (i) a glucosyltransferase that synthesizes α-1,6-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,6-glucan are α-1,6 bonds; and / or (ii) a glucosyltransferase that synthesizes α-1,3-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,3-glucan are α-1,3 bonds, wherein at least one glucosylated form of the isoflavone glycoside is produced in the composition, and typically wherein at least one α-glucan is produced in the composition.

[0010] In another embodiment, the present disclosure relates to a composition or a glucosylated isoflavone glycoside produced by the methods herein.

[0011] In another embodiment, the present disclosure relates to a composition comprising a glucosylated isoflavone glycoside, wherein the glucosylated isoflavone glycoside is produced by contacting an isoflavone glycoside with a glucosyltransferase in the presence of at least water and sucrose, wherein the glucosyltransferase is selected from: (i) a glucosyltransferase that synthesizes an α-1,6-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,6-glucan are α-1,6 bonds, and / or (ii) a glucosyltransferase that synthesizes an α-1,3-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,3-glucan are α-1,3 bonds, optionally wherein the composition is a food product / precursor.

[0012] Sequence Description

[0013] Table A. Summary of protein SEQ ID numbers

[0014]

[0015]

[0016] Detailed Description

[0017] Unless otherwise disclosed, the terms "a / an" and "the" as used herein are intended to cover one or more (i.e., at least one) of the recited features.

[0018] If present, all ranges are inclusive and combinable, unless otherwise stated. For example, when listing a range of "1 to 5" (i.e., 1 - 5), the recited range should be interpreted to include ranges such as "1 to 4", "1 to 3", "1 - 2", "1 - 2 and 4 - 5", "1 - 3 and 5", etc.

[0019] The terms "α-glucan", "α-glucan polymer", etc. are used interchangeably herein. An α-glucan is a polymer comprising glucose monomer units linked together by α-glycosidic bonds. In typical embodiments, the α-glucans herein comprise 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% α-glycosidic bonds. Examples of α-glucan polymers herein include graft copolymers as presently disclosed, as well as α-1,3-glucans and α-1,6-glucans.

[0020] The terms “α-1,3-glucan”, “poly-α-1,3-glucan”, “α-1,3-glucan polymer” and the like are used interchangeably herein. An α-1,3-glucan is a polymer comprising glucose monomer units linked together by glycosidic bonds, wherein at least about 50% of the glycosidic bonds are α-1,3. In certain embodiments, the α-1,3-glucan comprises at least about 90% or 95% α-1,3 glycosidic bonds. Most or all of the other bonds in the α-1,3-glucan herein are typically α-1,6, although some bonds can also be α-1,2 and / or α-1,4. The α-1,3-glucan as disclosed in the present invention can characterize the α-1,3-glucan side chains herein. In some aspects, the α-1,3-glucan can characterize an α-1,3-glucan “homopolymer” which is an α-1,3-glucan that is not part of a dextran-α-1,3-glucan copolymer.

[0021] The terms “dextran”, “dextran polymer”, “dextran molecule”, “α-1,6-glucan” and the like herein refer to a water-soluble α-glucan comprising at least 50%, 60%, 70%, 80% or 90% α-1,6 glycosidic bonds (the remaining bonds are typically α-1,3). An enzyme capable of synthesizing dextran from sucrose can be described as “dextransucrase” (EC 2.4.1.5). “Substantially linear” (“mostly linear” and similar terms) dextran has 5% or less branching before being modified herein to have α-1,3-glucan side chains. “Linear” dextran has no branching before being modified herein to have α-1,3-glucan side chains. If branching is present before modification of dextran with α-1,3-glucan side chains, the branches can be short, one (of the side chain) to three glucose monomers in length. However, in some aspects, dextran can be “dendritic”, which is a branched structure emanating from a core, wherein there are chains that branch iteratively from one another (containing mostly or all α-1,6-bonds) (e.g., a chain can be a branch from another chain, which in turn is a branch from another chain, and so on). However, in still other aspects, dextran is not dendritic, but has a branch-to-branch structure that does not emanate from a core. Dextran used in the glucosyltransferase reaction herein for α-1,3-glucan synthesis (to produce a dextran-α-1,3-glucan copolymer) can optionally be characterized as a “primer” or “receptor”. In some aspects, dextran can characterize an α-dextran “homopolymer” which is a dextran that is not part of a dextran-α-1,3-glucan copolymer.

[0022] The term “copolymer” herein refers to a polymer comprising at least two different types of α-glucans (such as dextran and α-1,3-glucan).

[0023] As used herein, terms such as "graft copolymer" and "branched copolymer" generally refer to copolymers that include a "backbone" (or "main chain") and one or more side chains branched from the backbone. The side chains are structurally different from the backbone.

[0024] Examples of graft copolymers herein are "dextran-α-1,3-glucan graft copolymers" (and similar terms), which copolymers include a backbone containing dextran and one or more side chains of α-1,3-glucan. In some aspects, the backbone can itself be a branched dextran as disclosed herein; addition of the α-1,3-glucan side chains to such a backbone (thereby forming the graft copolymers herein) can occur, for example, via enzymatic extension from the non-reducing end presented by short branches (α-1,2, α-1,3, or α-1,4 branches, each typically containing a single glucose monomer; i.e., side chain glucose). The short branches (which can be enzymatically extended into α-1,3-glucan side chains) can be present on otherwise linear or mostly linear dextran, or can be present on branched dextran. In some aspects, α-1,3-glucan can also be synthesized from the non-reducing end of the dextran backbone, as in embodiments where the dextran backbone is linear or mostly linear, or in embodiments where the dextran backbone is branched (e.g., dendritic or non-dendritic [branches do not emanate from a core] but having a branch-to-branch structure); technically, this α-1,3-glucan is not a side chain of dextran, but an extension from one or more dextran backbones.

[0025] The percentage of branching in the α-glucans herein refers to the percentage of all bonds in the α-glucan that represent branch points. For example, the percentage of α-1,3 branching in the α-glucans herein refers to the percentage of all bonds in the glucan that represent α-1,3 branch points. Unless otherwise indicated, the bond percentages disclosed herein are based on the total bonds of the glucan, or for the portion of the glucan to which the disclosure herein particularly pertains.

[0026] The terms "bond", "glycosidic linkage", "glycosidic bond", etc. refer to the covalent bond that connects sugar monomers within carbohydrate compounds (oligosaccharides and / or polysaccharides). Examples of glycosidic bonds include 1,6-α-D-glycosidic bond (also referred to herein as "α-1,6" bond), 1,3-α-D-glycosidic bond (also referred to herein as "α-1,3" bond), 1,4-α-D-glycosidic bond (also referred to herein as "α-1,4" bond), and 1,2-α-D-glycosidic bond (also referred to herein as "α-1,2" bond). The glycosidic linkage of the dextran polymer herein may also be referred to as "glucosidic linkage". In this article, "α-D-glucose" is called "glucose".

[0027] The glycosidic bond profile of the α-dextran herein can be determined using any method known in the art. For example, a method using nuclear magnetic resonance (NMR) spectroscopy (e.g., 13 C NMR or 1 H NMR) can be used to determine the bond profile. These and other methods that can be used are disclosed in, for example, Food Carbohydrates: Chemistry, Physical Properties, and Applications Food Carbohydrates: Chemistry, Physical Properties and Applications (edited by S.W. Cui, Chapter 3, S.W. Cui, Structural Analysis of Polysaccharides, Taylor & Francis Group LLC, Boca Raton, Florida, 2005), which is hereby incorporated by reference in its entirety.

[0028] The "molecular weight" of the α-dextran herein can be expressed as the weight-average molecular weight (Mw) or the number-average molecular weight (Mn), with the unit of Dalton (Da) or grams per mole. In some aspects, the molecular weight can be expressed as DPw (weight-average degree of polymerization) or DPn (number-average degree of polymerization). DPw and DPn are calculated by dividing the corresponding Mw or Mn by the molar mass of a monomer unit M 1 . If it is a dextran polymer, M 1 = 162.14. In some aspects, the molecular weight can sometimes be provided as "DP" (degree of polymerization), which simply refers to the number of glucoses contained in the α-dextran based on a single molecule. Various means for calculating these different molecular weight measurements are known in the art, such as using high-performance liquid chromatography (HPLC), size-exclusion chromatography (SEC), or gel permeation chromatography (GPC).

[0029] ​The term "sucrose" in this text refers to a non-reducing disaccharide composed of an α-D-glucose molecule and a β-D-fructose molecule linked by an α-1,2-glycosidic bond. Generally, sucrose is referred to as table sugar. Sucrose may alternatively be referred to as "α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside". "α-D-Glucopyranosyl" and "glucosyl" are used interchangeably in this text.

[0030] The term "sugar", unless used to specifically refer to sucrose, refers to any monosaccharide, disaccharide, or oligosaccharide (e.g., in the range of DP3 to DP4, DP5, DP6, DP7, DP8, DP9, DP10, DP12, DP14, DP15, DP16, DP18, or DP20), such as those disclosed herein. Sugars in this text are typically water-soluble.

[0031] The terms "glucosyltransferase", "glucosyltransferase enzyme", "GTF", "dextransucrase", etc. are used interchangeably in this text. The activity of the glucosyltransferase in this text catalyzes the reaction of the substrate sucrose to produce the products α-glucan and fructose. Other products (by-products) of the GTF reaction may include glucose, various soluble glucose-oligosaccharides, and levanbiose. The wild-type form of the glucosyltransferase typically contains (in the N-terminal to C-terminal direction) a signal peptide (typically removed by a cleavage process), a variable domain, a catalytic domain, and a glucan-binding domain. According to the CAZy (Carbohydrate-Active enZYmes) database (Cantarel et al., Nucleic Acids Res. [Nucleic Acids Research] 37:D233-238, 2009), the glucosyltransferases in this text are classified under glycoside hydrolase family 70 (GH70). The term "dextransucrase" (and similar terms) may optionally be used to characterize glucosyltransferases that produce dextran.

[0032] The term "glucosyltransferase catalytic domain" in this text refers to the domain of the glucosyltransferase that provides the α-glucan synthesis activity. Typically, the glucosyltransferase catalytic domain does not require the presence of any other domain to have this activity.

[0033] The terms "enzymatic reaction", "glucosyltransferase reaction", "dextran synthesis reaction", "reaction composition", "reaction formulation", etc. are used interchangeably herein and generally refer to a reaction initially comprising water, sucrose, at least one active glucosyltransferase, and optionally other components. Components that may further be present in the reaction typically after the glucosyltransferase reaction has commenced include fructose, glucose, leucrose, soluble glucose-oligosaccharides (e.g., DP2-DP7) (such sugars may be considered products or by-products depending on the glucosyltransferase used) and / or one or more insoluble α-dextran products of DP8 or higher. It is understood that certain dextran products having a degree of polymerization (DP) of at least 8 or 9 (e.g., α-1,3-dextran) are water-insoluble and thus insoluble in the dextran synthesis reaction mixture. As used herein, the term "under suitable reaction conditions" refers to reaction conditions that support the conversion of sucrose to one or more α-dextran products by glucosyltransferase activity. It is during this reaction process that the glucosyl groups initially derived from the input sucrose are enzymatically transferred and used for the synthesis of the α-dextran polymer; thus, the glucosyl groups involved in the process may optionally be referred to as the glucosyl component or moiety (or similar terms) of the glucosyltransferase reaction.

[0034] In some aspects herein, the "yield" of the α-dextran product in the glucosyltransferase reaction represents the molar yield based on the sucrose converted. The molar yield of the α-dextran product can be calculated based on the number of moles of α-dextran product divided by the number of moles of sucrose converted. The number of moles of sucrose converted can be calculated as follows: (mass of initial sucrose - mass of final sucrose) / molecular weight of sucrose [342 g / mol]. This molar yield calculation can be considered a measure of the selectivity of the reaction towards α-dextran. In some aspects, the "yield" of the α-dextran product in the glucosyltransferase reaction can be based on the glucosyl component of the reaction. The following formula can be used to measure such a yield (yield based on glucosyl):

[0035] α-dextran yield = ((IS / 2 - (FS / 2 + LE / 2 + GL + SO)) / (IS / 2 - FS / 2)) x 100%.

[0036] The fructose balance of the glucosyltransferase reaction can be measured to ensure that HPLC data (if applicable) is not out of range (90% - 110% is considered acceptable). The following formula can be used to measure the fructose balance:

[0037] Fructose balance = ((180 / 342 x (FS + LE) + FR) / (180 / 342 x IS)) x 100%.

[0038] In the two formulas above, IS is [Initial Sucrose], FS is [Final Sucrose], LE is [Leuconobiose], GL is [Glucose], SO is [Soluble Oligomers] (glucose-oligosaccharides) and FR is [Fructose]; the concentration of each of the above substrates / products provided in double brackets is in grams / L and is measured, for example, by HPLC.

[0039] As used herein, the term "in situ" characterizes one or more glucosyltransferase reactions that occur within a food product or its precursor, thereby producing α-glucans within the food product itself (or precursor). The α-glucans so produced (e.g., graft copolymers, α-1,3-glucans, and / or α-1,6-glucans) can be soluble or insoluble. While α-1,3-glucan products are typically insoluble and α-1,6-glucan products are typically soluble, graft copolymer products can be soluble or insoluble in the food product / precursor herein. In situ production of α-glucans in the food product / precursor typically replaces the addition of α-glucans as ingredients in the food herein, but such addition can be made if desired (e.g., to supplement in situ-produced α-glucans).

[0040] The terms "percent by volume", "volume percent", "vol %", "v / v %", etc. are used interchangeably herein. The volume percent of a solute in a solution can be determined using the formula: [(volume of solute) / (volume of solution)] x 100%.

[0041] The terms "percent by weight", "weight percentage, wt %", "weight-weight percentage, %w / w", etc. are used interchangeably herein. Weight percentage refers to the percentage of a material on a mass basis when the material is included in a composition, mixture, or solution.

[0042] The terms "weight / volume percentage", "w / v %", etc. are used interchangeably herein. Weight / volume percentage can be calculated as: ((mass of material [g]) / (total volume of material plus the liquid in which the material is placed [mL])) x 100%. The material can be insoluble in the liquid (i.e., a solid phase in the liquid phase, such as in the case of a dispersion), or soluble in the liquid (i.e., a solute dissolved in the liquid).

[0043] The terms "ingestible product" and "ingestible composition" are used interchangeably herein and refer to any substance that can be taken orally (i.e., via the mouth), either alone or in combination with another substance, whether or not it is intended for consumption. Thus, ingestible products include food products / beverage products. "Food product / beverage product" refers to any edible product intended for human or animal consumption (e.g., for nutritional purposes), including solids, semi-solids, or liquids. "Food" herein may optionally be referred to as, for example, "foodstuff", "food product", or other similar terms. In this disclosure, beverages or other ingestible liquids are examples of food products unless otherwise disclosed. Although this disclosure generally relates to foods and food precursors, which by definition are intended for ingestion or ultimate ingestion (food precursors are first made into foods before being ingested), this disclosure equally relates to other ingestible products (e.g., supplements, nutraceuticals, pharmaceuticals) that contain in-situ generated α-glucans. Food precursors herein can be, for example, (i) foods that exist prior to one or more processing steps (e.g., fermentation, aging, cooling / freezing, heating, baking, mixing) that convert the food precursors into food products intended for direct consumption, and / or (ii) raw materials used to prepare food products. In some aspects, food precursors can characterize food products or raw materials, such as the food products or raw materials that exist prior to treatment with one or more GTF enzymes in the methods herein.

[0044] As used herein when referring to food products / precursors, the term "texture" refers to the consistency of the food product / precursor and / or the sensory perception of the food product / precursor through, for example, visual, tactile, or oral / taste processes. "Improvement" of texture means an increase in consistency and / or an enhancement of the sensory perception. Unless otherwise specified, the "consistency" of a food product / precursor as used herein refers to the apparent viscosity extracted at a shear rate of about 10 - 13 Hz (e.g., about 11.7 Hz) during rheological analysis; an increase in apparent viscosity at such a shear rate indicates an increase in consistency. During rheological analysis, the apparent viscosity extracted at a shear rate of about 230 - 270 Hz (e.g., about 249 Hz) is related to "mouthfeel"; an increase in apparent viscosity at such a shear rate indicates an increase in mouthfeel.

[0045] "Fermentation" herein and similar terms applied to food products / precursors refer to the conversion of carbohydrates in the food product / precursor into one or more alcohols and / or acids by the action of one or more microorganisms (e.g., bacteria, yeast).

[0046] The terms "isoflavone glycoside", "isoflavone monoglycoside" and similar terms are used interchangeably herein and refer to isoflavones having a single glycoside group (a sugar group such as glucosyl). Examples of isoflavone glycosides herein include daidzin (7-O-glucosyl-4'-hydroxyisoflavone), genistin (7-O-glucosyl-4'5-dihydroxyisoflavone) and glycitin (glycitein 7-O-β-glucoside), which are the monoglucosylated forms (having a single glucoside group) of the isoflavones daidzein, genistein and glycitein, respectively. Isoflavones such as daidzein, genistein and glycitein are examples of "aglucone" herein. The "glucosylated isoflavone glycoside" (and similar terms) herein refers to an isoflavone glycoside that contains one or more (e.g., up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11) glucosyl groups in addition to the sugar group (e.g., glucosyl group) already present in the isoflavone glycoside. It is believed that such one or more glucosyl groups can be within a straight chain of two or more glucosyl groups (i.e., a glucan), and / or in a bond at different sites (optionally in its chain) from the isoflavone glycoside (e.g., extending from a glucoside group or from another site of the isoflavone glycoside).

[0047] Compositions herein (which are "dry" or "dried") typically have less than 5, 4, 3, 2, 1, 0.5 or 0.1 wt% water contained therein.

[0048] As used herein, the terms "aqueous liquid", "aqueous fluid", "aqueous condition", "aqueous environment", "aqueous system", etc. can refer to water or an aqueous solution. The "aqueous solution" herein can contain one or more dissolved salts, where in some embodiments the maximum total salt concentration can be about 3.5 wt%. Although the aqueous liquid herein typically contains water as the sole solvent in the liquid, the aqueous liquid can optionally contain one or more other solvents miscible in water (e.g., polar organic solvents). Thus, the aqueous solution can contain a solvent having at least about 10 wt% water.

[0049] For example, an "aqueous composition" herein has a liquid component containing about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99 or 100 wt% water. Examples of aqueous compositions include, for example, mixtures, solutions, dispersions (e.g., suspensions, colloidal dispersions) and emulsions.

[0050] As used herein, "insoluble", "water-insoluble", "aqueous-insoluble" (and similar terms) α-glucans do not dissolve (or do not visibly 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 at a temperature of about 1°C to 130°C (e.g., 20°C - 25°C). In some aspects, less than 1.0 gram (e.g., an undetectable amount) of water-insoluble α-glucan dissolves in 1000 milliliters of such aqueous conditions (e.g., water at 23°C). In contrast, "soluble", "water-soluble", "aqueous-soluble", etc. α-glucans visibly dissolve under the above aqueous conditions.

[0051] As used herein, the term "viscosity" refers to the resistance of a food product / precursor to deformation at a given rate. Viscosity can also refer to a measure of the degree to which a liquid (aqueous or non-aqueous) resists forces tending to cause it to flow. Additionally, viscosity can be defined as the shear stress generated by an applied shear rate. Both dynamic viscosity and kinematic viscosity refer to the term viscosity, as these two parameters are directly related through the density of the food product / precursor. Multiple viscosity units that can be used herein include, for example, centipoise (cP, cps) and Pascal seconds (Pa·s). One centipoise is one-hundredth of a poise; one poise is equal to 0.100 kg·m -1 ·s -1 。

[0052] As used herein, the term "polypeptide" is defined as a chain of amino acid residues, typically having a defined sequence. As used herein, the term polypeptide may be used interchangeably with the terms "peptide" and "protein". Typical amino acids included in the polypeptides herein are (the corresponding three-letter and one-letter codes are shown in parentheses): alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile, 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 (Val, V).

[0053] As used herein, the terms "sequence identity", "identity", etc. with respect to polynucleotide or polypeptide sequences refer to the nucleic acid residues or amino acid residues in two sequences being the same when aligned with maximum correspondence over a specified comparison window. Thus, "percent sequence identity", "percent identity", etc. refer to the value determined by comparing two optimally aligned sequences over a comparison window, where, when comparing the best alignment of the two sequences to a reference sequence (which does not contain additions or deletions), the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps). The percentage is calculated by determining the number of positions in the two sequences that have the same nucleic acid bases or amino acid residues to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and then multiplying the result by 100 to yield the percent sequence identity. It should be understood that when calculating sequence identity between a DNA sequence and an RNA sequence, the T residue of the DNA sequence is aligned with the U residue of the RNA sequence and can be considered to be "identical" thereto. For the purpose of determining the "percent complementarity" between a first polynucleotide and a second polynucleotide, it can be obtained by determining (i) the percent identity between the first polynucleotide and the complementary sequence of the second polynucleotide (or vice versa), e.g., and / or (ii) the percentage of bases between the first polynucleotide and the second polynucleotide that give rise to canonical Watson-Crick base pairs.

[0054] Percent identity can be readily determined by any known method, including but not limited to those described in the following references: 1) Computational Molecular Biology (Lesk, A.M. Ed.) Oxford University [Oxford University Press]: New York, N.Y. (1988); 2) Biocomputing: Informatics Biocomputing: Informatics and Genome Projects (Smith, D.W. Ed.) Academic [Academic Press]: New York, N.Y. (1993); 3) Computer Analysis of Sequence Data, Part I Computer Analysis of , Part I] (Griffin, A.M. and Griffin, H.G. Eds.) Humana [Humana Press]: Totowa, N.J. (1994); 4) Sequence Analysis in Molecular Biology [Analysis] (von Heinje, G. Ed.) Academic [Academic Press] (1987); and 5) SequenceAnalysisPrimer [Primer for Sequence Analysis] (Gribskov, M. and Devereux, J. Eds.) Stockton [Stockton Press]: New York, N.Y. (1991), which are hereby incorporated by reference in their entirety.

[0055] Preferred methods for determining percent identity are designed to give the best match between test sequences. For example, methods for determining identity and similarity are incorporated into 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, Wis.). Multiple alignments of sequences can be performed, for example, using the Clustal alignment method, which encompasses several different algorithms, including the Clustal V alignment method (described by Higgins and Sharp, CABIOS. [Computer Applications in the Biosciences] 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci. [Computer Applications in the Biosciences], 8:189-191 (1992)) and is present in the MEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values may correspond to GAP PENALTY = 10 and GAP LENGTH PENALTY = 10. The default parameters for pairwise alignments and percent identity calculations of protein sequences using the Clustal method can be KTUPLE = 1, GAP PENALTY = 3, WINDOW = 5, and DIAGONALS SAVED = 5. For nucleic acids, these parameters can be KTUPLE = 2, GAP PENALTY = 5, WINDOW = 4, and DIAGONALS SAVED = 4. Additionally, the Clustal W alignment method (described by Higgins and Sharp, CABIOS. [Computer Applications in the Biosciences] 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci. [Computer Applications in the Biosciences] 8:189-191 (1992); Thompson, J.D. et al., Nucleic Acids Research [Nucleic Acids Research], 22(22):4673-4680, 1994) and is present in the MEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). The default parameters for multiple alignments (protein / nucleic acid) can be: GAP PENALTY = 10 / 15, GAP LENGTH PENALTY = 0.2 / 6.66, % of Divergent Sequences Delayed = 30 / 30, DNA Transition Weight = 0.5, Protein Weight Matrix = Gonnet Series, DNA Weight Matrix = IUB.

[0056] As a feature of certain embodiments, various polypeptide amino acid sequences and polynucleotide sequences are disclosed herein. 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, the variant amino acid sequences or polynucleotide sequences 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 to the sequences disclosed herein. The variant amino acid sequences or polynucleotide sequences herein have the same function / activity as the disclosed sequences, or have 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 sequences. Typically, any polypeptide amino acid sequence disclosed herein that does not begin with methionine or valine can further include at least one initiating methionine or initiating valine at the N-terminus of the amino acid sequence. Conversely, any polypeptide amino acid sequence disclosed herein that begins with methionine or valine can optionally lack such methionine or valine residue. In some aspects, any polypeptide amino acid sequence disclosed herein that begins with methionine or valine alternatively has methionine or valine, respectively, as the first amino acid residue.

[0057] The terms "aligned with", "corresponding to", etc. are used interchangeably herein. Some aspects of the present disclosure relate to glucosyltransferases that contain at least one amino acid substitution at a position corresponding to at least one specific amino acid residue of SEQ ID NO:10. The amino acid position of the glucosyltransferase or a subsequence thereof (e.g., the catalytic domain or the catalytic domain plus the glucan-binding domain) (such an amino acid position or sequence may be referred to as the "query" position or sequence) can be characterized as corresponding to a specific amino acid residue of SEQ ID NO:10 (such an amino acid position or sequence may be referred to as the "subject" position or sequence) if (1) the query sequence can be aligned with the subject sequence (e.g., where the alignment shows that the query sequence and the subject sequence [or a subsequence of the subject sequence] are at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90% identical), and (2) if the query amino acid position is directly aligned with the subject amino acid position under the alignment of (1) (direct alignment comparison). Generally, any alignment algorithm, tool, and / or software disclosed herein (e.g., BLASTP, ClustalW, ClustalV, Clustal-Omega, EMBOSS) can be used to align the query amino acid sequence with the subject sequence (SEQ ID NO:10 or a subsequence of SEQ ID NO:10) to determine the percentage of identity. For additional examples only, the Needleman-Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol. [Journal of Molecular Biology] 48:443-453, 1970) can be used to align the query sequence with the subject sequence herein, as performed in the Needle program of the European Molecular Biology Open Software Suite (EMBOSS [e.g., version 5.0.0 or later], Rice et al., Trends Genet. [Trends in Genetics] 16:276-277, 2000). The parameters of such an EMBOSS alignment can include, for example: a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix.

[0058] The numbering of the specific amino acid residues of SEQ ID NO:10 in this text (e.g., Tyr-185, Val-186, Leu-513, Gln-588, Phe-607, Ile-608, Lys-625, Arg-741, Val-1188, Lys-1327, Glu-1332, Asp-1418, Ala-1419, Ser-1420, Thr-1421, Arg-1424, Leu-1425, Thr-1431, Glu-1450) is relative to the full-length amino acid sequence of SEQ ID NO:10. The first amino acid of SEQ ID NO:10 (i.e., position 1, Met-1) is at the start of the signal peptide. Unless otherwise disclosed, the substitutions in this text are relative to the full-length amino acid sequence of SEQ ID NO:10 as the reference sequence.

[0059] The "non-natural glucosyltransferase" in this text (the terms "mutant", "variant", "modified", and similar terms can also be used to describe such glucosyltransferase) has at least one amino acid substitution at a position corresponding to a specific amino acid residue of SEQ ID NO:10 (SEQ ID NO:3 and 4 are examples of non-natural GTFs). Such at least one amino acid substitution typically replaces one or more amino acid residues that normally (naturally) occur at the same position in the natural counterpart (parent) of the non-natural glucosyltransferase (i.e., although SEQ ID NO:10 is used as a reference for positions, the amino acid substitutions in this text are relative to the natural counterpart of the non-natural glucosyltransferase) (put another way, when aligning the sequence of the non-natural glucosyltransferase with SEQ ID NO:10, determining whether there is a substitution at a specific position does not depend on the respective amino acid residues in SEQ ID NO:10, but on what amino acid is present at the subject position in the natural counterpart of the non-natural glucosyltransferase). The amino acids that normally appear at the relevant sites of the natural counterpart glucosyltransferase are usually (but not always) the same as (or consistent with) the specific amino acid residues of SEQ ID NO:10 being aligned. Relative to its natural counterpart sequence, the non-natural glucosyltransferase optionally can have other amino acid changes (mutations, deletions, and / or insertions).

[0060] The term "isolated" means a substance (or process) in a form that does not exist in nature or in an environment that does not exist in nature. Non-limiting examples of isolated substances include any non-naturally occurring substances, such as food products, food precursors, graft copolymers, and / or glucosylated isoflavone glycosides herein (as well as the enzymatic reactions for preparing these materials). It is believed that the embodiments disclosed herein are synthetic / artificial (not possible to manufacture without human intervention / participation), and / or have non-naturally occurring properties.

[0061] As used herein, the term "increased" can mean 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 in quantity or activity compared to the quantity or activity being compared for increased quantity or activity. The terms "increased", "enhanced", "augmented", "greater than", "improved", etc. are used interchangeably herein.

[0062] Some embodiments of the present disclosure relate to methods for producing food products / precursors. Such methods may include:

[0063] (a) providing a food product / precursor comprising at least water, sucrose, and plant-based material, and

[0064] (b) contacting the food product / precursor with at least:

[0065] (i) a glucosyltransferase that synthesizes α-1,6-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,6-glucan are α-1,6 bonds; and / or

[0066] (ii) a glucosyltransferase that synthesizes α-1,3-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,3-glucan are α-1,3 bonds,

[0067] typically wherein at least one α-glucan is produced in the food product / precursor,

[0068] whereby the food product / food precursor after step (b) has a reduced off-flavor compared to the food product / food precursor before step (b),

[0069] and optionally, a reduced sugar content compared to the food product / food precursor before step (b).

[0070] Step (b) of producing the food product / precursor may include contacting the food product / precursor with at least:

[0071] (i) A glucosyltransferase (GTF) that synthesizes α-1,6-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,6-glucan are α-1,6 bonds, and / or

[0072] (ii) A GTF enzyme that synthesizes α-1,3-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,3-glucan are α-1,3 bonds.

[0073] In some aspects, the GTF enzyme (dextran sucrase) that synthesizes α-1,6-glucan herein can comprise an amino acid sequence that is about 100% identical or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO:1, 2, 11, 12, 14, 15, 16, 17 or 18 (GTF 0768), and has GTF activity. However, in some aspects, the GTF enzyme that synthesizes α-1,6-glucan can be as disclosed in any one of U.S. Patent Application Publication Nos. 2017 / 0218093, 2018 / 0282385, 2018 / 0291311 or 2016 / 0122445 (each of which is incorporated herein by reference). For example, a GTF identified as GTF 8117 (SEQ ID NO:30), GTF 6831 (SEQ ID NO:32) or GTF 5604 (SEQ ID NO:33) in US 2018 / 0282385 can be used, or a 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 US 2016 / 0122445 can be used, or a GTF comprising an amino acid sequence that is about 100% identical or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence of any one of these GTF enzymes (and having GTF activity) can be used.

[0074] The dextransucrase herein is capable of producing dextrans comprising, for example, 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% α-1,6-glycosidic linkages. This percentage α-1,6 linkage profile takes into account the sum of all linkages in the dextran (the backbone of the α-1,6 glucan and the branched portions therefrom if present). Dextrans as disclosed elsewhere herein, such as dextrans in homopolymers or graft copolymers, can have, for example, any of the above linkage characteristics.

[0075] The dextransucrase in the present text is capable of producing dextrans having a weight-average molecular weight (Mw) of, for example, 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. In some aspects, the Mw is, for example, 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. Dextrans as disclosed elsewhere herein, such as dextrans in homopolymers or graft copolymers, may have any of the above molecular weight characteristics, for example.

[0076] In some aspects, the GTF enzymes for synthesizing α-1,3-glucan herein can comprise an amino acid sequence that is about 100% identical or at least about 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 the 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 disclosed in U.S. Patent Application Publication No. 2019 / 0078063, which is incorporated herein by reference. It should be noted that such GTF enzymes comprising SEQ ID NO: 2, 4, 8, 10, 14, 20, 26, 28, 30, 34, or the 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 α-glucans comprising at least about 90% (about 100%) of α-1,3 linkages. In some aspects, the GTF enzyme for synthesizing α-1,3-glucan can be the GTF 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 or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% identical to the above amino acid sequence of GTF 0974 (and having GTF activity). Any of the above 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.

[0077] In some aspects, the GTF enzymes herein for producing α-1,3-glucan can synthesize α-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%. In some aspects, the yield can be measured based on the glucosyl component of the reaction and / or as measured using HPLC or NIR spectroscopy. For example, the yield can be obtained in a reaction carried out for about 16 - 24 hours (e.g., about 20 hours). Examples of such GTF enzymes are those with a modified amino acid sequence such that the enzyme produces more product (α-1,3-glucan and fructose) and less by-product (e.g., oligosaccharides such as glucose, levanbiose, etc.) from a given amount of sucrose substrate. For example, one, two, three, four or more amino acid residues in the catalytic domain of the GTF that produces α-1,3-glucan herein can be modified / replaced to obtain a GTF enzyme that produces more product. Examples of suitable modified GTF enzymes are disclosed in Tables 3 - 7 of U.S. Patent Application Publication No. 2019 / 0078063. The modified GTF enzyme can, for example, contain one or more amino acid substitutions corresponding to those in Tables 3 - 7 (ibid.), and the one or more amino acid substitutions are associated with an α-1,3-glucan yield of at least 40% (the position numbers of such at least one substitution correspond to the position numbers of SEQ ID NO:62 as disclosed in U.S. Patent Application Publication No. 2019 / 0078063). For example, a set of amino acid modifications listed in Table 6 or Table 7 (ibid.) can be used.

[0078] In some aspects, the amino acid sequence of the GTF enzyme for α-1,3-glucan synthesis has been modified such that the molecular weight (DPw) of the α-1,3-glucan produced by the enzyme is lower than the molecular weight of the α-1,3-glucan produced by its corresponding parental GTF enzyme. Examples of suitable modified GTF enzymes are disclosed in Tables 3 and 4 of U.S. Patent Application Publication No. 2019 / 0276806, which is incorporated herein by reference. The modified GTF enzyme can, for example, contain one or more amino acid substitutions corresponding to those in Tables 3 and / or 4 (ibid.), and the one or more amino acid substitutions are associated with the molecular weight of the α-1,3-glucan product, and the molecular weight of the α-1,3-glucan product is at least 5% smaller than the molecular weight of the α-1,3-glucan produced by the parental enzyme (the position numbers of such at least one substitution correspond to the position numbers of SEQ ID NO:62 as disclosed in U.S. Patent Application Publication No. 2019 / 0276806). For example, a set of amino acid modifications listed in Table 4 (ibid.) can be used.

[0079] In some aspects, the amino acid sequence of a GTF enzyme for α-1,3-glucan synthesis has been modified such that the molecular weight (DPw) of the α-1,3-glucan produced by the enzyme is higher than that of the α-1,3-glucan produced by its corresponding parental GTF. Examples of suitable modified GTF enzymes are disclosed in Tables 3, 4, and 5 of U.S. Patent Application Publication No. 2019 / 0078062, which is incorporated herein by reference. A modified GTF enzyme can, for example, comprise one or more amino acid substitutions corresponding to the amino acid substitutions in Tables 3, 4, and / or 5 (supra), the one or more amino acid substitutions being related to the molecular weight of the α-1,3-glucan product, which is at least 5% higher than the molecular weight of the α-1,3-glucan produced by the parental enzyme (the position numbers of such at least one substitution corresponding to the position numbers of SEQ ID NO:62 as disclosed in U.S. Patent Application Publication No. 2019 / 0078062). For example, a set of amino acid modifications as listed in Table 5 (supra) can be used.

[0080] In some aspects, a modified GTF for α-1,3-glucan synthesis (i) comprises at least one amino acid substitution or a set of amino acid substitutions (as described above with respect to yield or molecular weight), and (ii) comprises a GTF catalytic domain that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to or consists of the amino acid residues 55-960 of SEQ ID NO:4, the amino acid residues 54-957 of SEQ ID NO:65, the amino acid residues 55-960 of SEQ ID NO:30, the amino acid residues 55-960 of SEQ ID NO:28, or the amino acid residues 55-960 of SEQ ID NO:20 (each of these sequences as disclosed in U.S. Patent Application Publication No. 2019 / 0078063, which is incorporated herein by reference). Each of these subsequences is an approximate catalytic domain of each corresponding reference sequence and produces an α-1,3-glucan comprising at least about 50% (e.g., ≥90% or ≥95%) α-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 an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to or consists of SEQ ID NO:62 or a subsequence thereof (e.g., SEQ ID NO:4) (without its initiating methionine) or the amino acid residues 55-960 of SEQ ID NO:4 (approximate catalytic domain) (each of these sequences as disclosed in U.S. Patent Application Publication No. 2019 / 0078063).

[0081] In the present disclosure, SEQ ID NOs: 5, 6, 7, 8, 9, and 10 (Table A) are amino acid sequences that are identical to SEQ ID NOs: 4, 65, 30, 28, 20, and 62, respectively, as disclosed in U.S. Patent Application Publication No. 2019 / 0078063. Accordingly, each of SEQ ID NOs: 5, 6, 7, 8, 9, and 10 of the present disclosure can be used, as appropriate, in any of the disclosed aspects. For example, the GTF enzyme for synthesizing α-1,3-glucan herein can comprise an amino acid sequence that is about 100% identical or at least about 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 one of these sequences can be modified as described herein to affect, for example, α-1,3-glucan yield and / or molecular weight and / or stability.

[0082] In some aspects, the GTF enzyme for α-1,3-glucan synthesis has been modified such that the enzyme has enhanced performance and / or one or more stability benefits. Such modifications can be achieved, for example, by having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid substitutions compared to the corresponding parental GTF enzyme (e.g., wild-type mature GTF or its active subsequence, such as the catalytic domain). Exemplary performance and / or one or more stability benefits herein include increased thermal stability, increased storage stability, increased solubility, better pH profile, increased specific activity, altered substrate specificity, altered substrate binding, altered pH-dependent activity, altered pH-dependent stability, increased ability to be antioxidant against antioxidant substances, increased expression, and / or increased glucan product yield (and / or decreased byproduct [e.g., levanbiose] yield). In some aspects, the performance benefits are achieved at relatively low temperatures (e.g., <5°C) or relatively high temperatures (e.g., >40°C). The increase in any of the above characteristics can be, for example, about or at least about 5%, 10%, 15%, 20%, 25%, or 30% compared to the corresponding activity of the unmodified parental GTF enzyme.

[0083] Some examples of modified GTF enzymes that produce α-1,3-glucan with enhanced performance and / or one or more stability benefits in the present text include SEQ ID NO:3 (vGTFJ) or 4 or consist of them. Notably, for example, both SEQ ID NO:3 and 4 are derived from SEQ ID NO:5 (GTF 6855) (e.g., SEQ ID NO:5 can be the backbone for making substitutions to present SEQ ID NO:3 and 4).

[0084] Notably, compared to SEQ ID NO:5, SEQ ID NO:3 has the following amino acid substitutions: Tyr-8-Asn (i.e., substituting Asn for Tyr at position 8 compared to SEQ ID NO:5), Val-9-Ala, Leu-336-Tyr, Gln-411-Leu, Phe-430-Tyr, Lys-448-Ala, Arg-564-Ser, Thr-1254-Gln, and Glu-1273-Phe (except having valine at position 1). The positions of each of these substitutions correspond to the positions Tyr-185, Val-186, Leu-513, Gln-588, Phe-607, Lys-625, Arg-741, Thr-1431, and Glu-1450 of SEQ ID NO:10, which is used as a reference sequence herein.

[0085] Notably, compared to SEQ ID NO:5, SEQ ID NO:4 has the following amino acid substitutions: Leu-336-Tyr, Phe-430-Tyr, Ile-431-Val, Lys-448-Ala, Arg-564-Ser, Val-1011-Glu, Lys-1150-His, Glu-1155-Ala, Asp-1241-Lys, Ala-1242-Glu, Ser-1243-Gly, Thr-1244-Ser, Arg-1247-Leu, and Leu-1248-Val (except having valine at position 1). The positions of each of these substitutions correspond to the positions Leu-513, Phe-607, Ile-608, Lys-625, Arg-741, Val-1188, Lys-1327, Glu-1332, Asp-1418, Ala-1419, Ser-1420, Thr-1421, Arg-1424, and Leu-1425 of SEQ ID NO:10, which is used as a reference sequence herein.

[0086] In some aspects of the present disclosure, the modified GTF enzyme can comprise or consist of an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO:3, and has one or more (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. In any of the above aspects, the valine at position 1 of SEQ ID NO:3 can optionally be replaced by methionine or can be absent.

[0087] In some aspects of the present disclosure, the modified GTF enzyme can comprise or consist of an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO:4, and has one or more (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. In any of the above aspects, the valine at position 1 of SEQ ID NO:4 can optionally be replaced by methionine or can be absent.

[0088] Any one of the amino acids listed in the aspects related to SEQ ID NO:3 and 4 above can optionally be replaced by another amino acid selected from Table B based on amino acid conservation.

[0089] Table B. Amino Acid Conservation

[0090]

[0091]

[0092] In some aspects, modified GTF enzymes that produce α-1,3-glucan with enhanced performance and / or one or more stability benefits include one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions at one or more positions corresponding to one or more of the amino acid residues Tyr-185, Val-186, Leu-513, Gln-588, Phe-607, Ile-608, Lys-625, Arg-741, Val-1188, Lys-1327, Glu-1332, Asp-1418, Ala-1419, Ser-1420, Thr-1421, Arg-1424, Leu-1425, Thr-1431 and / or Glu-1450 of SEQ ID NO:10. For example, the modified GTF enzyme that produces α-1,3-glucan may include one or more amino acid substitutions at positions corresponding to the following amino acid residues of SEQ ID NO:10:

[0093] (i) Tyr-185, Val-186, Leu-513, Gln-588, Phe-607, Lys-625, Arg-741, Thr-1431 and / or Glu-1450 (these positions correspond to those in the GTF of SEQ ID NO:3);

[0094] (ii) Leu-513, Phe-607, Ile-608, Lys-625, Arg-741, Val-1188, Lys-1327, Glu-1332, Asp-1418, Ala-1419, Ser-1420, Thr-1421, Arg-1424 and / or Leu-1425 (these positions correspond to those in the GTF of SEQ ID NO:4);

[0095] (iii) Tyr-185, Val-186, Lys-625, Thr-1431 and / or Glu-1450;

[0096] (iv) Ile-608, Lys-625, Lys-1327, Glu-1332, Asp-1418, Ala-1419, Ser-1420, Thr-1421, Arg-1424 and / or Leu-1425;

[0097] (v) Leu-513, Gln-588, Phe-607, Lys-625 and / or Arg-741;

[0098] (vi) Leu-513, Phe-607, Ile-608, Lys-625 and / or Arg-741; and / or

[0099] (vii) Leu-513, Phe-607, Lys-625 and / or Arg-741.

[0100] In some aspects, with respect to a modified GTF enzyme that produces α-1,3-glucan and has enhanced performance and / or one or more stability benefits,

[0101] (a) The amino acid substitution at the position corresponding to amino acid residue Tyr-185 of SEQ ID NO:10 can be an Asn residue or any residue substitution that is consistent with Asn (e.g., Table B);

[0102] (b) The amino acid substitution at the position corresponding to amino acid residue Val-186 of SEQ ID NO:10 can be an Ala residue or any residue substitution that is consistent with Ala (e.g., Table B);

[0103] (c) The amino acid substitution at the position corresponding to amino acid residue Leu-513 of SEQ ID NO:10 can be a Tyr, Phe or Trp residue or any residue substitution that is consistent with Tyr, Phe or Trp (e.g., Table B);

[0104] (d) The amino acid substitution at the position corresponding to amino acid residue Gln-588 of SEQ ID NO:10 can be a Leu residue or any residue substitution that is consistent with Leu (e.g., Table B);

[0105] (e) The amino acid substitution at the position corresponding to amino acid residue Ile-608 of SEQ ID NO:10 can be a Val or Tyr residue or any residue substitution that is consistent with Val or Tyr (e.g., Table B);

[0106] (f) The amino acid substitution at the position corresponding to amino acid residue Lys-625 of SEQ ID NO:10 can be an Ala residue or any residue substitution that is consistent with Ala (e.g., Table B);

[0107] (g) The amino acid substitution at the position corresponding to amino acid residue Arg-741 of SEQ ID NO:10 can be a Ser residue or any residue substitution that is consistent with Ser (e.g., Table B);

[0108] (h) An amino acid substitution at the position corresponding to amino acid residue Val-1188 of SEQ ID NO:10 can be a Glu residue or any residue substitution that is consistent with Glu (e.g., Table B);

[0109] (i) An amino acid substitution at the position corresponding to amino acid residue Lys-1327 of SEQ ID NO:10 can be a His residue or any residue substitution that is consistent with His (e.g., Table B);

[0110] (j) An amino acid substitution at the position corresponding to amino acid residue Glu-1332 of SEQ ID NO:10 can be an Ala residue or any residue substitution that is consistent with Ala (e.g., Table B);

[0111] (k) An amino acid substitution at the position corresponding to amino acid residue Asp-1418 of SEQ ID NO:10 can be a Lys residue or any residue substitution that is consistent with Lys (e.g., Table B);

[0112] (l) An amino acid substitution at the position corresponding to amino acid residue Ala-1419 of SEQ ID NO:10 can be a Glu residue or any residue substitution that is consistent with Glu (e.g., Table B);

[0113] (m) An amino acid substitution at the position corresponding to amino acid residue Ser-1420 of SEQ ID NO:10 can be a Gly residue or any residue substitution that is consistent with Gly (e.g., Table B);

[0114] (n) An amino acid substitution at the position corresponding to amino acid residue Thr-1421 of SEQ ID NO:10 can be a Ser residue or any residue substitution that is consistent with Ser (e.g., Table B);

[0115] (o) An amino acid substitution at the position corresponding to amino acid residue Arg-1424 of SEQ ID NO:10 can be a Leu residue or any residue substitution that is consistent with Leu (e.g., Table B);

[0116] (p) An amino acid substitution at the position corresponding to amino acid residue Leu-1425 of SEQ ID NO:10 can be a Val residue or any residue substitution that is consistent with Val (e.g., Table B);

[0117] (q) An amino acid substitution at the position corresponding to amino acid residue Thr-1431 of SEQ ID NO:10 can be a Gln residue or any residue substitution that is consistent with Gln (e.g., Table B); and / or

[0118] (r) An amino acid substitution at the position corresponding to amino acid residue Glu-1450 of SEQ ID NO:10 can be a Phe residue or any residue substitution that is consistent with Phe (e.g., Table B).

[0119] Although it is believed that in some aspects the modified GTF enzyme that produces α-1,3-glucan only needs to have a catalytic domain, the modified GTF can be included within a larger amino acid sequence. For example, the catalytic domain can be linked to a glucan-binding domain at its C-terminus, and / or to a variable domain and / or a signal peptide at its N-terminus.

[0120] Although in some aspects the amino acid substitutions in the modified GTF enzyme that produces α-1,3-glucan are generally disclosed for the corresponding positions in SEQ ID NO:10, for convenience, alternatively such substitutions can also simply be expressed in terms of their position numbers in the amino acid sequence used to produce the modified GTF itself (e.g., SEQ ID NO:5 [optionally without its starting methionine] or positions 55 - 960 of SEQ ID NO:5 [approximate catalytic domain]). This can be done simply by aligning the amino acid sequence with SEQ ID NO:10 and identifying the position numbers of one or more target positions in the amino acid sequence based on their direct alignment with one or more corresponding positions in SEQ ID NO:10.

[0121] The GTF that produces α-1,3-glucan herein is capable of producing α-1,3-glucan containing, for example, 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% α-1,3-glycosidic linkages. The α-1,3-glucan as disclosed elsewhere herein, such as the glucan in a homopolymer or a graft copolymer, can have, for example, any of the above bond characteristics.

[0122] For example, the GTFs that produce α-1,3-glucan herein are capable of producing α-1,3-glucan, where DPw, DPn, or DP is about, at least about, or less than 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. DPw, DPn, or DP can optionally be expressed as a range between any two of these values. By way of example only, 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. The α-1,3-glucan as disclosed elsewhere herein, such as the glucan in a homopolymer or a graft copolymer, can have, for example, any of the above molecular weight characteristics.

[0123] In some aspects, the GTF enzyme can be any enzyme as disclosed herein and includes 1-300 (or any integer therebetween [e.g., 10, 15, 20, 25, 30, 35, 40, 45, or 50]) residues at the N-terminus and / or C-terminus. For example, such additional residues can be from the corresponding wild-type sequence from which the GTF enzyme is derived, or can be heterologous sequences, such as an epitope tag (at the N-terminus or C-terminus) or a heterologous signal peptide (at the N-terminus). The GTF enzyme herein typically lacks an N-terminal signal peptide; if its signal peptide is removed during secretion, this enzyme can optionally be characterized as being mature.

[0124] The GTF enzymes herein can typically be derived from bacteria. Examples of bacterial GTF enzymes are those derived from species of the genus Streptococcus, Leuconostoc or Lactobacillus. Examples of species of the genus Streptococcus include Streptococcus salivarius, Streptococcus sobrinus, Streptococcus mutans, Streptococcus oralis, Streptococcus gallolyticus and Streptococcus sanguinis. Examples of species of the genus Leuconostoc include Leuconostoc mesenteroides, Leuconostoc amelibiosum, Leuconostoc argentinum, Leuconostoc carnosum, Leuconostoc citreum, Leuconostoc cremoris, Leuconostoc dextranicum and Leuconostoc fructosum. Examples of species of the genus Lactobacillus include Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus salivarius, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus sakei, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus fermentum and Lactobacillus reuteri.

[0125] The GTF enzyme herein can be prepared by fermentation of, for example, a suitably engineered microbial strain. Recombinant enzyme production can be carried out by fermentation, for example, using microbial species such as Escherichia coli (E. coli), Bacillus strains (e.g., Bacillus subtilis), Ralstonia eutropha, Pseudomonas fluorescens, Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, and species of Aspergillus (e.g., Aspergillus awamori) and Trichoderma (e.g., Trichoderma reesei) (see, e.g., Adrio and Demain, Biomolecules 4:117-139, 2014, which is incorporated herein by reference). The nucleotide sequence encoding the GTF amino acid sequence is typically ligated to a heterologous promoter sequence to produce an expression cassette for the enzyme, and / or is thus codon-optimized. Such an expression cassette can be incorporated into 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 a nucleotide sequence encoding a signal peptide (e.g., a heterologous signal peptide) between the promoter sequence and the GTF amino acid coding sequence, which signal peptide is designed to direct the secretion of the GTF enzyme. At the end of fermentation, the cells can be lysed accordingly (usually when a signal peptide for secretion is not used), and the GTF enzyme can be separated using methods such as precipitation, filtration, and / or concentration. Alternatively, a lysate or extract containing GTF can be used without further separation. If GTF is secreted (i.e., it is present in the fermentation broth), it can optionally be separated from the fermentation broth for use or included in the fermentation broth for use. The activity of the GTF enzyme can be confirmed by biochemical assays, such as measuring its conversion of sucrose into dextran polymers.

[0126] In some aspects, the α-dextran produced in step (b) of producing the food product / precursor comprises a graft copolymer comprising:

[0127] (i) an α-1,6-dextran (dextran) backbone, wherein at least about 50% of the glycosidic bonds of the α-1,6-dextran (dextran) backbone are α-1,6 bonds, and

[0128] (ii) at least one α-1,3-dextran side chain, wherein at least about 50% of the glycosidic bonds of the α-1,3-dextran chain are α-1,3 bonds.

[0129] Such graft copolymers can be water-soluble or water-insoluble in water. The dextran backbone of the α-dextran graft copolymer herein can be, for example, dextran as currently disclosed, or can be dextran as disclosed in U.S. Patent Application Publication Nos. 2016 / 0122445, 2017 / 0218093, 2018 / 0282385, 2020 / 0165360 or 2019 / 0185893 (each incorporated herein by reference) (e.g., molecular weight, bond / branch characteristics, production method). In some aspects, the dextran backbone (before being incorporated into the graft copolymer) has been α-1,2- and / or α-1,3-branched; the α-1,2 and / or α-1,3 branch percentages of the backbone of the graft copolymer herein can be, for example, 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%. One or more α-1,3-dextran side chains of the α-dextran graft copolymer herein can be, for example, α-1,3-dextran as currently disclosed, or can be α-1,3-dextran as disclosed in U.S. Patent Nos. 7000000, 8871474, 10301604 or 10260053, or U.S. Patent Application Publication 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 (each incorporated herein by reference) (e.g., molecular weight, bond characteristics).

[0130] One, two, three, or more different GTF enzymes that synthesize α-1,6-glucan in the present disclosure can be used, for example, in step (b) of producing a food product / precursor. Similarly, one, two, three, or more different GTF enzymes that synthesize α-1,3-glucan in the present disclosure can be used, for example. In some aspects, one or more GTFs that produce α-1,6-glucan can be added to (contact) the food product / precursor before adding one or more GTFs that produce α-1,3-glucan, and in some aspects, these two types of GTF enzymes can be added almost simultaneously (simultaneously). In some aspects, one or more GTFs that produce α-1,3-glucan can still be added to the food product / precursor before adding one or more GTFs that produce α-1,6-glucan. In some aspects, dextran as disclosed herein (but exogenously produced for the food product / precursor) can still be added as a raw material to the food product / precursor to which GTFs that produce α-1,3-glucan have been added or will be added. Although not bound by any particular theory, it is believed that adding at least one GTF that produces α-1,6-glucan (and / or exogenously produced dextran) and at least one GTF that produces α-1,3-glucan in step (b) of producing the food product / precursor allows for the production of dextran-α-1,3-glucan graft copolymers as currently disclosed, possibly simultaneously producing dextran and / or one or more α-1,3-glucan homopolymers (i.e., the production of α-1,6-glucan and / or α-1,3-glucan is independent of the production of the graft copolymer). However, it is believed that in some aspects, only dextran and / or one or more α-1,3-glucan homopolymers may be produced, with little (e.g., <5 wt% of all glucan products) or no production of the graft copolymer.

[0131] The molecular weight and / or bond characteristics of α-glucan produced by GTF enzymes (dextransucrase or GTFs that produce α-1,3-glucan) as generally disclosed above can be as observed, for example, in a separate reaction consisting of or consisting essentially of water, sucrose, GTF enzyme, and optionally one or more salts and / or buffers. In some aspects, the molecular weight and / or bond characteristics of α-glucan produced by one or two of these types of GTF enzymes in the food product / precursor in the present disclosure can be different from the molecular weight and / or bond characteristics of α-glucan produced in the above separate reaction.

[0132] In some aspects, the ratio of the GTF enzyme that synthesizes α-1,6-glucan to the GTF enzyme that synthesizes α-1,3-glucan in step (b) is from about 85:15 to about 95:5. However, in some aspects, the ratio of the GTF that produces α-1,6-glucan to the GTF that produces α-1,3-glucan 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 a range between any two of these ratios (e.g., from about 82.5:17.5 to 97.5:2.5, 87.5:12.5 to 92.5:7.5, 89:11 to 91:9, 17.5:82.5 to 2.5:97.5, 12.5:87.5 to 7.5:92.5, 11:89 to 9:91). For the purposes of determining the ratio of each enzyme (active enzyme) herein, the amount of each enzyme (active enzyme) can be based, for example, on moles, weight, or GTF activity. The activity of the GTF enzyme used to prepare the ratios herein can optionally be determined as disclosed in U.S. Patent Application Publication No. 2014 / 0087431, which is incorporated herein by reference, and / or as disclosed in the following examples. For example, the total (e.g., "100%") complement of the GTF enzyme used to set the ratios herein can be the amount of enzyme that can convert most (e.g., >95%, >98%, >99%) or all of the sucrose in a GTF reaction containing water, sucrose (e.g., 50 or 100 g / L), GTF, and optionally a buffer / salt or consisting thereof, over a given time period (e.g., 6, 12, 18, 24, 30, or 36 hours); the amount so measured can optionally be characterized as a normalized amount of GTF.

[0133] The GTF enzyme (or any other enzyme as currently disclosed) for the methods herein is typically in purified form. The purified enzyme can be substantially free of insoluble and / or soluble components of the organism / cell from which the enzyme is produced, and / or any medium used for the cell fermentation of the enzyme. In some aspects, the purified enzyme represents an enzyme preparation containing less than 10 wt%, 8 wt%, 6 wt%, 5 wt%, 4 wt%, 3 wt%, 2 wt%, 1 wt%, 0.5 wt% or 0.1 wt% of other materials (e.g., polypeptide materials) that are naturally or recombinantly associated with the enzyme. In some aspects, the GTF and / or any other enzyme herein is not contained in or otherwise associated with microorganisms (e.g., bacteria, yeast, fungi, algae) that may be present (e.g., endogenously or intentionally added) in the food products / precursors herein; however, in some aspects, the GTF and / or any other enzyme herein is contained in or otherwise associated with (e.g., expressed by) microbial (e.g., bacteria, yeast, fungi, algae) cells, such as cells that heterologously express one or more enzymes (i.e., recombinant cells). Contacting the food product / precursor with one or more GTF enzymes herein is typically not carried out in the oral cavity or other environments where unpurified / unisolated GTF enzymes may be present.

[0134] The GTF enzyme (or any other enzyme as currently disclosed) for the methods herein can be contained in, for example, a sterile-filtered preparation. In some aspects, the enzyme can be on-line sterile-filtered when applied to the food product / precursor during step (b) herein. In some aspects, the enzyme can be added to a food product / precursor that has been pasteurized (after pasteurization), or alternatively, the enzyme can be added before pasteurizing the food product / precursor. In some aspects, the enzyme can be added to a food product / precursor that has been fermented (after fermentation), or alternatively, the enzyme can be added during or before fermenting the food product / precursor. In some aspects, the GTF enzyme (or any other enzyme as currently disclosed) for the methods herein can be contained in a preparation (for addition to step [b] herein) that is substantially free of (e.g., <0.5, <0.1, <0.05 wt%) any other enzymes (e.g., lipase, protease, amylase, mannanase, pectinase, cellulase, and / or p-nitrobenzyl esterase); such a preparation typically has little or no detectable activity of such other one or more enzymes.

[0135] The food product / precursor herein can be contacted with one or more GTF enzymes in step (b) by, for example, mixing / stirring / blending. Incubation of one or more GTF enzymes in the food product / precursor can, for example, continue for a time sufficient to produce α-glucan by one or more GTFs in the food product / precursor, such as about or at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 24, 30, 36, 42, 48, 72 or 96 hours, or about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 days (or the range between any two of these hours and / or days). The temperature for incubating one or more GTF enzymes in the food product / precursor herein can be, for example, about 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 2°C - 5°C, 2°C - 10°C, 2°C - 15°C, 2°C - 20°C, 2°C - 25°C, 2°C - 30°C, 2°C - 35°C, 2°C - 40°C, 2°C - 45°C, 2°C - 50°C, 3°C - 5°C, 3°C - 10°C, 3°C - 15°C, 3°C - 20°C, 3°C - 25°C, 3°C - 30°C, 3°C - 35°C, 3°C - 40°C, 3°C - 45°C, 3°C - 50°C, 5°C - 10°C, 5°C - 15°C, 5°C - 20°C, 5°C - 25°C, 5°C - 30°C, 5°C - 35°C, 5°C - 40°C, 5°C - 45°C, 5°C - 50°C, 15°C - 20°C, 15°C - 25°C, 15°C - 30°C, 15°C - 35°C, 15°C - 40°C, 15°C - 45°C, 15°C - 50°C, 20°C - 25°C, 20°C - 30°C, 20°C - 35°C, 20°C - 40°C, 20°C - 45°C, 20°C - 50°C, 25°C - 30°C, 25°C - 35°C, 25°C - 40°C, 25°C - 45°C, 25°C - 50°C, 30°C - 35°C, 30°C - 40°C, 30°C - 45°C or 30°C - 50°C.

[0136] Typically, a food product / precursor contacted with a GTF enzyme herein comprises water (i.e., it is an aqueous composition), and / or water is introduced into the food product / precursor before or during contact with the GTF enzyme. The GTF enzyme can be added to the food product / precursor in dry form (e.g., powder, flakes, freeze-dried enzyme preparation) (typically added to an aqueous food product / precursor) or in wet form. In some aspects, the food product / precursor can be combined with the GTF enzyme under dry conditions (the resulting combination is dry), and then water or an aqueous solution is added, followed by allowing GTF to produce α-glucan. The water content of the food product / precursor provided in step (a) or step (b) after addition of the GTF enzyme can be, for example, about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99 wt%. The pH of the food product / precursor herein and / or the pH used to incubate one or more GTF enzymes in the food product / precursor herein can be, for example, about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 4.0 - 10.0, 4.0 - 9.0, 4.0 - 8.0, 4.5 - 10.0, 4.5 - 9.0, 4.5 - 8.0, 5.0 - 10.0, 5.0 - 9.0, 5.0 - 8.0, 5.5 - 10.0, 5.5 - 9.0, 5.5 - 8.0, 6.0 - 10.0, 6.0 - 9.0 or 6.0 - 8.0. In some aspects, the food product / precursor can be acidic (e.g., pH ≤ 3.0, 3.2, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 or 6.5), neutral (e.g., pH 6.5 - 7.5) or basic (e.g., pH > 7.5, 8.0, 8.5, 9.0, 9.5).

[0137] The GTF enzyme in the present text can optionally be provided in step (b) of the method by introducing a recombinant engineered cell (e.g., a microbial cell, such as a bacterial or fungal / yeast cell) into the food product / precursor provided in step (a), wherein the cell recombinantly (heterologously) expresses the GTF enzyme and secretes the GTF enzyme in and / or around the food product / precursor. Such a cell can be a cell of a microorganism suitable for recombinant engineering and useful in food processing (e.g., fermentation), such as the microbial cells disclosed herein (if applicable). In some aspects, the recombinant engineered cell provided to the food product / precursor in step (b) can be inactivated and / or rendered non-viable in some manner (such as by killing (but preferably in a manner that still preserves the cell shape / structure)). For example, the cell can be inactivated and / or rendered non-viable by irradiation or treatment with a sterilizing agent / chemical (e.g., ethylene oxide). Typically, the means used to inactivate the cell and / or kill the cell preserves at least some of the three-dimensional shape / structure of the cell, and / or ensures that one or more GTF enzymes expressed by the cell remain active and typically remain associated with the inactivated / non-viable cell (e.g., as by association with the cell membrane via an optional transmembrane domain or membrane-binding domain of the GTF enzyme [e.g., fused with the GTF]). The inactivated / non-viable cells are typically porous and can optionally be immobilized on a support (e.g., an inert, water-insoluble material, such as a particle or surface).

[0138] In some aspects, the food product / precursor herein can be contacted with one or more GTF enzymes by adding the food product / precursor to an aqueous composition comprising at least sucrose and one or more GTF enzymes. While the food product / precursor in this aspect has at least some sucrose and water, the food product / precursor in some other aspects does not contain sucrose and / or water. Such a GTF / sucrose-containing aqueous composition can optionally be referred to herein as a "GTF / sucrose starter composition". Typically, one or more food precursors as currently disclosed (e.g., raw materials such as liquid food products / precursors, beverages, RTDs, fruit / vegetable purees, syrups or juices or juice concentrates, etc.) can be added to the GTF / sucrose starter composition, although one or more food products themselves as currently disclosed (e.g., fruit or vegetable substances such as slices [such as sliced, cubed or other shaped slices]) can be added (typically simultaneously with the addition of the food precursor). In some aspects, the GTF / sucrose starter composition may already contain at least one food product / precursor, such as any food product / precursor disclosed herein. The initial sucrose concentration of the GTF / sucrose starter composition can be, for example, as currently disclosed, such as 5 wt% - 60 wt%, 5 wt% - 50 wt%, 5 wt% - 40 wt%, 10 wt% - 60 wt%, 10 wt% - 50 wt%, 10 wt% - 40 wt%, 20 wt% - 60 wt%, 20 wt% - 50 wt%, 20 wt% - 40 wt%, 30 wt% - 60 wt%, 30 wt% - 50 wt%, 30 wt% - 40 wt%, 40 wt% - 60 wt%, or 40 wt% - 50 wt%. In some aspects, in addition to sucrose, the GTF / sucrose starter composition has little (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 as currently disclosed). The GTF / sucrose starter composition can comprise, for example, at least one GTF that produces α-1,6-glucan and / or a GTF that produces α-1,3-glucan. The temperature, pH and / or any other conditions / parameters of this method (before and / or after adding one or more food products / precursors to the GTF / sucrose starter composition) can be, for example, as disclosed herein.In some aspects, the GTF / sucrose starter composition can be incubated for example 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 one or more food products / precursors. Any of these time periods can also be applied to the period of time allowed to continue after adding the food product / precursor to the GTF / sucrose starter composition until optionally terminating the GTF activity of the final food product / precursor (e.g., heat inactivation at 90°C - 100°C). Typically, one or more food products / precursors added to the GTF / sucrose starter composition comprise one or more saccharide compounds (e.g., one or more monosaccharides, disaccharides, oligosaccharides, and / or polysaccharides, as currently disclosed). In some aspects, one or more food products / precursors can be added to the GTF / sucrose starter composition to control or regulate the desired thickening and / or texturization in the final food product / precursor produced. The thickening and / or texturization achieved by such a method can be greater than (e.g., about or at least about 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, or 500% more) the thickening and / or texturization achieved by mixing all the ingredients used to make the final food product / precursor at approximately the same time. By way of example only, the above process of adding one or more food products / precursors to the GTF / sucrose starter composition can be used herein to produce orange marmalade, gel / gelatin, pudding, custard, fermented products, or creams, optionally containing one or more suspended solid food ingredients (such as fruit slices or vegetable slices).

[0139] The "food product / precursor" as provided in step (a) of the method in some aspects of the present disclosure (i.e., food product or precursor) may contain endogenous sucrose of the food product / precursor (e.g., its sucrose is natural), and / or may contain sucrose that has been added to the food product / precursor (added as a raw material during or after its preparation). Thus, step (a) may optionally include adding sucrose to the food product / precursor. Regardless of the original source of the sucrose, the sucrose content of the food product / precursor finally provided in step (a) herein may be, for example, about, at least about, or less than about 0.1, 0.5, 1, 2.5, 5, 7.7, 10, 15, 20, 25, 30, 40, 50, 60, or 70 wt%. In some aspects, the sucrose may be provided, for example, as white refined sucrose or in an unrefined form, as disclosed in U.S. Patent No. 9,719,121, which is incorporated herein by reference. When adding the GTF enzyme to the food product / precursor, sucrose may optionally be added to the food product / precursor.

[0140] In some aspects, the food product / precursor as provided in step (a) of the method herein further contains at least one disaccharide and / or at least one oligosaccharide other than sucrose. The oligosaccharide may have, for example, 3 - 15 or 3 - 20 monomer units (i.e., DP3 - DP15 or DP3 - DP20) (e.g., DP3 - DP5, DP3 - DP6); thus, in some aspects, the polysaccharides herein have more than 15 or 20 monomer units. The disaccharides and / or oligosaccharides herein may contain only, for example, glucose monomer units, and / or one or more other types of monosaccharides (e.g., galactose, fructose, mannose) as monomer units. Examples of the disaccharides (other than sucrose) herein include maltose, isomaltose, lactose, lactulose, aspergillus sugar, leuconostoc disaccharide, trehalose, maltulose, isomaltulose, and turanose. Examples of the oligosaccharides herein include glucooligosaccharides (glucose oligomers), such as maltooligosaccharides (MOS) and isomaltooligosaccharides (IMO), and galactooligosaccharides (GOS). The disaccharides and / or oligosaccharides may be added to the food product / precursor during or after the preparation of the food product / precursor. Such addition may be from a source physically outside the food product / precursor (i.e., as a raw material), and / or may be produced in situ in the food / precursor, such as by one or more endogenous and / or exogenous enzymes of the food / precursor. The enzyme (e.g., exogenous enzyme) added to the food product / precursor to produce the disaccharides and / or oligosaccharides may be added, for example, in the same or a similar manner (e.g., time, temperature, pH) as the GTF enzyme added herein, and may be added before, during, or after the addition of the GTF enzyme. Such an enzyme may be, for example, a transglucosidase (EC [enzyme code] 2.4.1.24) or an amylase. Suitable transglucosidases herein include TGO and those disclosed in U.S. Patent Application Publication No. 2008 / 0229514 or 2015 / 0240279, or U.S. Patent No. 4689296, which are hereby incorporated by reference in their entirety. The EC 2.4.1.24 glucosyltransferase (also known as "1,4-α-glucan 6-α-glucosyltransferase") can transfer the α-D-glucosyl residue of an α-1,4-glucan, -oligosaccharide (i.e., MOS) or -disaccharide (i.e., maltose) to the primary hydroxyl group of free glucose or glucose in an α-1,4-glucan, -oligosaccharide (i.e., MOS) or -disaccharide. Thus, in some aspects, the EC 2.4.1.24 glucosyltransferase produces isomaltooligosaccharides (IMO) (e.g., DP3-DP5 or DP3-DP6).

[0141] In some aspects, the food product / precursor provided in step (a) of the methods herein has (except for sucrose) little (e.g., less than 0.5, 0.25, 0.1, 0.05, 0.025 or 0.01 wt%, or undetectable) or no disaccharides and / or oligosaccharides (or little or no specific disaccharides or oligosaccharides). The food product / precursor produced in step (b) of the methods herein can similarly, for example, have little or no disaccharides and / or oligosaccharides (or little or no specific disaccharides or oligosaccharides), and also have little (e.g., as described above) or no sucrose. The disaccharides or oligosaccharides in such aspects can be any disaccharides or oligosaccharides as disclosed herein (e.g., lactose, maltose, isomaltose, MOS, IMO, GOS). One or more glycosidases (glycoside-active enzymes) can be used, for example, in the food product / precursor to reduce or eliminate the presence of one or more disaccharides and / or one or more oligosaccharides, and can be added, for example, in the same or a similar manner (e.g., time, temperature, pH) as the GTF enzyme described herein, and can be added before, during, or after the addition of the GTF enzyme. The glycosidases herein can be, for example, α-glucosidase (EC 3.2.1.20). Suitable α-glucosidases herein include those disclosed in U.S. Patent Application Publication No. 2015 / 0240278, which is incorporated herein by reference. In some aspects, the α-glucosidase used in the disclosed methods is capable of hydrolyzing α-1,4 or α-1,6 glucosidic bonds, and / or is not capable of hydrolyzing α-1,3 glucosidic bonds. The glycosidases herein can be added, for example, at about 0.1%-1.5%, 0.1%-1.25%, 0.1%-1.0%, 0.1%-0.75%, 0.1%-0.5%, 0.2%-1.5%, 0.2%-1.25%, 0.2%-1.0%, 0.2%-0.75%, 0.2%-0.5%, 0.5%-1.5%, 0.5%-1.25%, 0.5%-1.0%, 0.5%-0.75%, 0.75%-1.5%, 0.75%-1.25% or 0.75%-1.0% (v / w) to the food product / precursor herein.

[0142] In some aspects, the food product / precursor can be a flour- or meal-based dough, a baked product (baked good) or an extruded product, such as any of those disclosed in WO 2021 / 034561 or U.S. Patent Application Publication No. 2017 / 0218093 or 2022 / 0322685 (which are hereby incorporated by reference). Examples of baked products or their doughs (precursors) include breads (e.g., buns, sourdough, rye bread, whole wheat bread, pita, flatbread, tortilla, cornbread, brioche, white bread, baguette, bagel, banana bread, ciabatta, brown bread, challah, focaccia, multigrain bread, breadsticks, soda bread, pumpernickel, potato bread, crackers, English muffins, whole grain bread, matzo, lavash, croutons, pizza crust) (fermented or unfermented), cakes (e.g., carrot cake, red velvet cake, angel food cake, pound cake, chocolate cake, white cake, black forest cake, tiramisu, coffee cake, cheesecake, devil's food cake, upside-down cake, Boston cream pie, Swiss roll, lemon cake, butter cake, chiffon cake, butter cake, spice cake, rum cake, sponge cake, marble cake, coconut cake, pandan cake), muffins, brownies, scones, cookies, bars, custards, pies, soda crackers, pretzels, pastries, puddings and tarts. Examples of extruded products include pasta (e.g., spaghetti, rotini, fusilli, penne, bucatini, macaroni / maccheroni, rigatoni, fettuccine, linguine, vermicelli, ziti, farfalle, gomiti / elbow, rotelle), cereals (e.g., directly expanded cereals, filled cereals, flaked cereals, breakfast cereals), certain bread products (e.g., croutons, breadsticks, flatbreads), pre-made cookie doughs, dry and semi-moist pet foods (e.g., kibble) and snacks (e.g., cheese curls, filled pillow puff snacks, chips [such as tortilla chips, pita chips, processed potato chips, corn chips], snack bars [such as vegetable bars], puffed formed products (e.g., rolls [such as cheese curls], balls, tubes, bananas, cups, bowls, plates, baby puffs)).The pasta in the present text can be, for example, extruded (e.g., see above) and / or flat / 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.

[0143] In some aspects, the food product / precursor can be a syrup or a beverage, for example, any of those disclosed in U.S. Patent Application Publication Nos. 2010 / 0040728, 2017 / 0006902, 2017 / 0218093, 2013 / 0216652, 20180146699, 2009 / 0123603, 2021 / 0076724, or 2017 / 0332670 (which are hereby incorporated by reference in their entirety). In some aspects, the beverage can be a juice (e.g., fruit juice such as orange juice, apple juice, mango juice, peach juice, banana juice, date juice, apricot juice, grapefruit juice, papaya juice, pineapple juice, raspberry juice, strawberry juice, pear juice, citrus juice or cherry juice; vegetable juice such as carrot juice, tomato juice or mixed vegetable juice), a sweetened beverage (soda / soft drink, sweet tea or coffee), a ready-to-drink (RTD) beverage or any other beverage having natural and / or added sugar (sucrose). In some aspects, the beverage is coffee or tea.

[0144] In some aspects, the food product / precursor provided in step (a) of the methods herein is fermented, while in some aspects, the food product / precursor is fermented (or further fermented) during or after step (b). Thus, step (a) of the methods herein can optionally include a step of fermenting the food product / precursor (e.g., before or after adding sucrose, as applicable). Thus, step (b) of the methods herein can optionally include fermenting the food product / precursor while contacting it with the GTF enzyme. Thus, the methods herein can optionally include a step of fermenting the food product / precursor after step (b). One or more bacterial and / or yeast cultures can be used for the fermentation of the food product / precursor herein. Suitable bacteria for the food fermentation herein include, for example, lactic acid bacteria, such as those of the family Lactobacillaceae, such as those of the following: Pediococcus (such as P. acidilactici, P. pentosaceus), Lactobacillus (such as Lactobacillus sakei, Lactobacillus fermentum [formerly known as Lactobacillus cellobiosus], Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus kefir, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus buchneri, Lactobacillus helveticus, Lactobacillus reuteri, Lactobacillus johnsonii, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus delbrueckii (such as Lactobacillus delbrueckii subsp. bulgaricus)), Lactococcus (such as Lactococcus lactis, such as Lactococcus lactis subsp. cremoris), Leuconostoc (such as Leuconostoc citreum, Leuconostoc mesenteroides) and Streptococcus (such as Streptococcus thermophilus).In some aspects, suitable bacteria for food fermentation can be species from the following: Bifidobacterium (e.g., B. bifidum, B. lactis, B. longum, B. animalis, B. breve, B. infantis), Komagataeibacter subspecies, Liquorilactobacillus subspecies (e.g., Liquorilactobacillus neglii, Liquorilactobacillus ghanensis), Gluconobacter subspecies or Propionibacterium (e.g., P. freudenreichii, such as P. shermanii subspecies) (propionic bacteria). In some aspects, the bacteria used for food fermentation herein can be characterized as Gram-positive, spherical, rod-shaped, anaerobic, aerobic, acid-tolerant, non-spore-forming, GRAS (Generally Recognized as Safe) and / or probiotic. In some aspects, the bacteria for food fermentation (e.g., plant-based fresh fermented products) can be acid cultures / strains (or mixtures) (e.g., available from International Flavors & Fragrances Inc. (IFF)). VEGE 053、 VEGE 011), which produce food with a pH of about e.g. 2.5 - 4.5, 3.0 - 4.5, 4.2 - 4.4 or 4.3, or can be mild cultures / strains (or mixtures) (e.g., available from International Flavors & Fragrances Inc.) VEGE 022、 VEGE 053、 VEGE 022、 VEGE 047、 VEGE061), the pH of the food produced thereby being about, for example, 4.6 - 5.5, 4.6 - 6.0, 4.5 - 4.7 or 4.6. The bacteria herein (e.g., mild or acidic) can optionally be mesophilic (optimal growth temperature typically at 20°C to 25°C [room temperature]). The bacterial mixture in the culture for food fermentation can comprise, for example, one, two, three, four, five, six or more different bacterial species and / or bacterial subspecies. In some aspects, suitable yeasts for food fermentation include species from the following: Saccharomyces (e.g., Saccharomyces cerevisiae, S. pastorianus, S. boulardii, S. kluyveri, S. vitulinus), Pediococcus subspecies (e.g., Pediococcus pentosaceus), Kluyveromyces lactis, Dekkera subspecies (e.g., Dekkera bruxellensis), Zygosaccharomyces subspecies (e.g., Zygosaccharomyces bailii), Brettanomyces subspecies, Pichia (e.g., P. kluyveri, P. fermentans), Geotrichum, Debaryomyces, and Candida (e.g., C. humilis, C. famata). In some aspects, the yeast can be characterized as baker's (baking) yeast, brewing yeast, wine yeast, probiotic yeast, budding / fission yeast or GRAS. The yeast mixture in the culture for food fermentation can comprise, for example, one, two, three, four, five, six or more different yeast species and / or yeast subspecies. The food product / precursor to be fermented or being fermented can be, for example, a plant-based fresh fermented product herein, beer, wort, wine, pomace, cider, miso, kimchi, sauerkraut, pickles / pickle juice, soybean curd, tofu, kombucha, soy sauce, bread, sourdough or meat.

[0145] In some aspects, the food product / precursor can be, for example, confectionery. Examples of confectionery herein include boiled sweets (hard-boiled sweets [i.e., hard candies]), sugar-coated sweets, jelly confectionery, gums, liquorice, quesadillas, caramels, toffees, fudges, chewing gums, bubble gums, nougats, chewy pastes, halawa, tablets, lozenges, icings, caster sugars, puddings, gels (e.g., fruit gels, gelatin desserts), aerated confectionery, marshmallows, baked confectionery.

[0146] In some aspects, the food product / precursor can be a non-dairy food product / precursor. For example, the non-dairy food product / precursor can be a plant-based milk (milk alternative), or contain a plant-based milk (and lack, or have very little [e.g., <0.5 wt%] of any dairy ingredients, such as lactose, whey, casein, and / or milk fat). In some aspects, the non-dairy food product / precursor is fermented (e.g., non-dairy yogurt product / precursor, such as a plant-based yogurt product / precursor). The plant-based raw materials that form the basis of the non-dairy food product / precursor herein can be from, for example, nuts / seeds (e.g., almonds, cashews, macadamias, hemp seeds, quinoa, flaxseeds), cereals / grains (e.g., oats, rice), fruits (e.g., coconuts, bananas), or vegetables (e.g., legumes, such as beans [e.g., soybeans, mung beans] and peas). In some aspects, the non-dairy food product / precursor is the milk of any one of the above nuts / seeds, cereals / grains, fruits, or vegetables; the fermented form of any one of these milks can be, for example, yogurt. However, in some aspects, the food product / precursor can contain any one of the above one or more plant-based raw materials and be in any suitable food / precursor form disclosed herein (i.e., the food product / precursor does not need to be characterized as a non-dairy food product / precursor, such as a plant-based milk).

[0147] In some aspects, the food product / precursor can be a cream soup, gravy, sauce (e.g., ketchup), salad dressing, mayonnaise, jam, jelly, marmalade, syrup, pie filling, batter for fried foods, batter for pancakes / waffles, cake icing and glaze, whipped toppings, pet food, or animal / livestock feed.

[0148] In some aspects, the food product / precursor can include one or more additional ingredients such as vegetable components (e.g., vegetable oils, vegetable proteins, vegetable carbohydrates), enzymes, fats, oils, flavorings, microbial cultures (e.g., probiotic cultures), salts, sweeteners, acids, fruits / vegetables (e.g., oranges, apples, mangoes, peaches, plums, bananas, dates, apricots, grapefruits, papayas, pineapples, raspberries, strawberries, blueberries, blackberries, pears, citrus fruits, cherries, grapes, raisins, currants, melons, watermelons, cantaloupes, honeydews, kiwis, lemons, limes, carrots, tomatoes) or fruit / vegetable juices (concentrates), purees or other processed forms of the disclosed fruits / vegetables (e.g., sliced, diced, shredded pieces), or any other component suitable for use as an ingredient in the food product / precursor. Such one or more additional ingredients can be as disclosed, for example, in U.S. Patent Application Publication Nos. 2016 / 0122445 or 2017 / 0218093 (both incorporated herein by reference), and / or can be natural or artificial. Examples of ingredients suitable as sweeteners (or for any other purpose, e.g., flavoring) include acesulfame potassium, advantame, agave syrup, alitame, aspartame, barley malt syrup, birch syrup, brazzein, brown rice syrup, cane juice, caramel, coconut palm sugar, corn syrup, curculin, cyclamate, dextrose, erythritol, fructooligosaccharides, fructose (levulose), galactose, glucose (dextrose), glycerol (glycerin), glycyrrhizin, golden syrup, high fructose corn syrup (e.g., HFCS-42, -55, -90), high maltose corn syrup (HMCS), honey, hydrogenated starch hydrolysates (HSH), isomaltooligosaccharides (IMO), inulin, invert sugar, isomalt, lactitol, lactose, maltitol, maltodextrin, maltose, mannitol, maple syrup, miraculin, molasses (e.g., blackstrap molasses), monatin, monellin, monk fruit, neohesperidin dihydrochalcone, neotame, palm sugar, pentadin, polydextrose, rapadura sugar, refined syrup, saccharin, sorbitol (glucitol), sorghum syrup, stevia / steviol glycosides (e.g., rebaudioside, such as rebaudioside A, rebaudioside D or rebaudioside M), sucralose, sugar alcohols, tagatose, thaumatin, trehalose, xylitol, and yacon syrup.

[0149] In some aspects, the food product / precursor comprises at least one isoflavone glycoside herein. Examples of such food products / precursors include legumes (e.g., soybeans, beans, green beans, pinto beans, black beans, peas [green or yellow peas], chickpeas, fava beans, pistachios, peanuts), currants, raisins, coffee beans and coffee, or any product / derivative thereof (e.g., powder, flour, meal, yogurt, tofu, miso, natto, tempeh or as disclosed herein).

[0150] In some aspects, the food product / precursor produced by the methods of the present disclosure can be concentrated, dried (e.g., dried into a powder), reconstituted (after concentration or drying) or processed (e.g., frozen) in any other manner. Examples of such products include sweetened milk, condensed milk, evaporated milk (e.g., sweetened condensed milk), condensed milk, skim milk, milk powder, frozen dairy products (e.g., ice cream), concentrated juice or dried juice powder.

[0151] In some aspects, the methods herein can further comprise the step of freezing the food product / precursor (e.g., a dairy food product / precursor, or a plant-based food product / precursor) after step (b). For example, such methods can produce plant-based ice cream or frozen yogurt. Freezing can be carried out at, for example, about -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, -40°C, -20°C to -40°C, -25°C to -35°C. In some aspects, where method step (b) comprises using at least one GTF that produces α-1,3-glucan herein, the frozen product can have an improved melting curve (e.g., melts more slowly) compared to a suitable control (e.g., a frozen product made using the same raw materials and process steps but not treated with a GTF that produces α-1,3-glucan). In some aspects, the slower melting of the frozen product can be a reduction of about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% in melting compared to the melting of a suitable control. Melting can refer to melting that occurs within about 45, 60, 75, 90, 100, 120, 150 or 60 - 120 minutes after the frozen product (from frozen conditions) is placed at, for example, ambient temperature (e.g., about 20°C, 25°C, 18°C - 25°C or 20°C - 25°C) or an elevated temperature (e.g., about 25°C - 38°C, 25°C - 35°C, 25°C - 32°C or 25°C - 30°C). Melting can optionally be measured according to, for example, the following examples or as disclosed by Granger et al. (2005, Int. Dairy J. 15(3):255 - 262, incorporated herein by reference).

[0152] In some aspects, the food product / precursor after step (b) of the methods herein has a reduced off-flavor compared to the food product / precursor that existed prior to step (b) (i.e., the food product / precursor that existed prior to treatment with one or more GTF enzymes). The off-flavor can be reduced by, for example, about or at least about 25%, 50%, 70%, 75%, 80%, 85%, 90%, or 95%. In some aspects, such a reduction in off-flavor can be compared to: (i) the off-flavor that existed prior to contact with one or more GTFs, or (ii) the off-flavor of a suitable control (e.g., the only difference being no GTF treatment). The off-flavor herein can include, for example, bitterness and / or astringency. Bitterness can optionally be measured in International Bitterness Units (IBU). The off-flavor can be measured according to the following examples, e.g., by sensory evaluation.

[0153] In some aspects, the food product / precursor after step (b) of the methods herein has a reduced content of at least one isoflavone glycoside and / or an increased content of at least one glucosylated isoflavone glycoside compared to the food product / precursor prior to step (b). The content of one isoflavone glycoside or a combination of one or more isoflavone glycosides herein can be reduced by, for example, about or at least about 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 40 wt%, 50 wt%, 60 wt%, 65 wt%, 20 wt%-65 wt%, 20 wt%-60 wt%, 25 wt%-65 wt%, or 25 wt%-60 wt% compared to the content of one or more isoflavone glycosides of the food product / precursor that existed prior to step (b). The content of one glucosylated isoflavone glycoside or a combination of one or more isoflavone glycosides herein can be increased by, for example, about or at least about 10 wt%, 25 wt%, 50 wt%, 100 wt%, 250 wt%, 500 wt%, or 1000 wt% compared to the content of one or more glucosylated isoflavone glycosides of the food product / precursor that existed prior to step (b).

[0154] In some aspects, the food product / precursor after step (b) of the methods herein optionally has one or more of the following characteristics compared to the food product / precursor that existed prior to step (b) (i.e., the food product / precursor that existed prior to treatment with one or more GTF enzymes):

[0155] (I) Reduced sugar content,

[0156] (II) Increased texture, such texture optionally includes increased consistency and / or increased mouthfeel,

[0157] (III) Improved physical appearance,

[0158] (IV) Reduced calories,

[0159] (V) Added dietary fiber, and / or

[0160] (VI) Increased extensibility or stretchability.

[0161] In some aspects, compared to the sugar content of the food product / precursor that existed prior to step (b), the sugar content (e.g., wt%) in the food product / precursor after step (b) can be reduced by about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 20%-65%, 20%-60%, 25%-65% or 25%-60%. In some aspects, this reduction is for all sugars in the food product / precursor, while in other aspects, this reduction is for specific sugars, such as sucrose. For example, the sugar can be any sugar as currently disclosed. The sugar content herein can be measured by, for example, HPLC as disclosed in the following examples.

[0162] In some aspects, compared to the texture of the food product / precursor that existed prior to step (b), the texture of the food product / precursor after step (b) can be increased by about or at least about 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 750%, 1000%, 1250%, 1500%, 1750%, 2000%, 2250%, 2500%, 3000%, 3500%, 4000%, 4500%, 5000%, 5500%, 6000%, 6500% or 7000%. Texture can refer to aspects of consistency or mouthfeel, and / or be measured in units such as Pascal-seconds (Pa·s) or cP, either of which can optionally be measured according to the following examples. In some aspects, the texture (consistency) can be measured by determining the viscosity of the food product / precursor when extracted at a shear rate of about 11 - 12 Hz (e.g., 11.7 Hz). The texture (mouthfeel) can be measured by determining the viscosity of the food product / precursor when extracted at a shear rate of, for example, about 248 - 250 Hz (e.g., 249 Hz).

[0163] In some aspects, compared to the physical appearance of the food product / precursor that existed prior to step (b), the food product / precursor after step (b) has an improved physical appearance. The improved physical appearance can be, for example, increased homogeneity (e.g., visual homogeneity) and / or increased glossiness (e.g., visual glossiness); in some aspects, such an increase can be about or at least about 5%, 10%, 20%, 25%, 30%, 40% or 50%.

[0164] In some aspects, the dietary calorie content of the food product / precursor after step (b) (calories available during digestion) can be reduced by about or at least about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or 75% compared to the dietary calorie content of the food product / precursor that existed prior to step (b).

[0165] In some aspects, the dietary fiber content of the food product / precursor after step (b) (e.g., weight percentage) can be increased by about or at least about 5%, 10%, 25%, 50%, 75%, 100%, 200%, 300%, 400% or 500% compared to the dietary fiber content of the food product / precursor that existed prior to step (b).

[0166] In some aspects, such as when a food product / precursor containing maltose (and / or IMO) (in addition to sucrose) is contacted with the α-1,3-glucan-producing GTF and / or the α-1,6-glucan-producing GTF herein, a substantial reduction in sugar (e.g., at least about 35%, 40%, 45%, 50%, 55% or 60%) can be achieved without significantly altering the texture of the food product / precursor (e.g., changing by less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%; e.g., consistency or mouthfeel).

[0167] Some aspects of the present disclosure relate to food products / precursors produced by the GTF treatment methods herein. Examples of such products / precursors are any food products / precursors as disclosed herein. Typically, such food products / precursors can have any of the characteristics disclosed herein (e.g., reduced sugar content, increased texture, increased extensibility (stretchability), improved physical appearance, reduced calorie content, increased dietary fiber, pH, temperature, age, hardness, reduced melting rate) (as appropriate / where applicable). Typically, such food products / precursors contain at least one GTF enzyme as currently disclosed and / or an α-glucan as currently disclosed.

[0168] In some aspects, the isoflavone glycosides herein are used as receptors / primers for the in situ synthesis of α-glucan by GTF enzymes in the food / precursor. Accordingly, some aspects of the present disclosure relate to polysaccharides or α-glucan molecules that comprise at least:

[0169] (i) an α-1,6-glucan or an α-1,3-glucan as disclosed herein, and

[0170] (ii) an isoflavone glycoside (e.g., genistin or daidzin).

[0171] Some aspects of the present disclosure relate to methods for glucosylating isoflavone glycosides, which may optionally be characterized as isoflavone glycoside glucosylation methods. Such methods may include the step of providing a composition that comprises at least water, sucrose, an isoflavone glycoside, and a glucosyltransferase herein, and wherein the glucosyltransferase is selected from:

[0172] (i) a glucosyltransferase that synthesizes an α-1,6-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,6-glucan are α-1,6 bonds, and / or

[0173] (ii) a glucosyltransferase that synthesizes an α-1,3-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,3-glucan are α-1,3 bonds,

[0174] wherein at least one glucosylated form of the isoflavone glycoside (glucosylated isoflavone glycoside) is produced in the composition. The providing step of such a method may optionally be characterized as the step of contacting the isoflavone glycoside with the glucosyltransferase in the presence of at least water and sucrose. Typically, the isoflavone glycoside glucosylation method also produces at least one α-glucan as currently disclosed herein, such as an α-1,6-glucan, an α-1,3-glucan, and / or a graft copolymer herein.

[0175] The α-1,6-glucan-synthesizing glucosyltransferase in the isoflavone glycoside glucosylation method may be any α-1,6-glucan-synthesizing glucosyltransferase currently disclosed herein. The α-1,3-glucan-synthesizing glucosyltransferase in the isoflavone glycoside glucosylation method may be any α-1,3-glucan-synthesizing glucosyltransferase currently disclosed herein. Any conditions and / or parameters for using any one of these enzymes in such a method may be as currently disclosed herein (e.g., temperature, time, water content, sucrose content, GTF enzyme content).

[0176] It is believed that the glucosylated isoflavone glycosides herein (e.g., one or more products of the isoflavone glycoside glucosylation method) contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more glucose monomer units (typically in a chain) added to, for example, the original isoflavone glycoside. The isoflavone glycoside may optionally be characterized herein as a receptor / primer for α-glucan synthesis by a GTF enzyme in the composition of the present disclosure. Thus, some aspects of the present disclosure relate to polysaccharides or α-glucan molecules that comprise at least:

[0177] (i) an α-1,6-glucan or an α-1,3-glucan as disclosed herein, and

[0178] (ii) an isoflavone glycoside herein (e.g., daidzin or genistin);

[0179] Part (i) and part (ii) are linked by a glycosidic bond, and part (ii) is located at the reducing end of the α-glucan molecule (for example, due to the use of the isoflavone glycoside of (ii) as an acceptor for initiating the synthesis of α-1,6-glucan or α-1,3-glucan). The DP or DPw of the α-glucan can be, for example, any DP or DPw value disclosed herein.

[0180] In some aspects, the composition in which glucosylated isoflavone glycosides can be produced in the isoflavone glycoside glucosylation method can be any composition currently disclosed herein, such as a food precursor / product. However, in some aspects, such a composition can be in the form of and / or contain, for example, a home care product, a personal care product, an industrial product, an ingestible product (e.g., a food product / precursor, such as any food product / precursor disclosed herein), or a pharmaceutical, as described in any of the following: U.S. Patent Application Nos. 2018 / 0022834, 2018 / 0237816, 2018 / 0230241, 20180079832, 2016 / 0311935, 2016 / 0304629, 2015 / 0232785, 2015 / 0368594, 2015 / 0368595, 2016 / 0122445, 2019 / 0202942, or 2019 / 0309096, or International Patent Application Publication No. WO 2016 / 133734, which are hereby incorporated by reference in their entirety. In some aspects, the composition can contain at least one component / ingredient of a home care product, a personal care product, an industrial product, a pharmaceutical product, or an ingestible product (e.g., a food product) as disclosed in any of the foregoing publications and / or as disclosed in the present invention. The glucosylated isoflavone glycosides can be produced in situ in the composition, and / or can be produced in a separate or isolated GTF reaction composition and then introduced as a raw material for preparing the composition.

[0181] In some aspects, glucosylated isoflavone glycosides can provide stability for emulsification or dispersion. Thus, glucosylated isoflavone glycosides can optionally be characterized as an emulsifying aid or a dispersing aid. The emulsification or dispersion stabilized by the glucosylated isoflavone glycosides herein can be, for example, the emulsification or dispersion of any household care product, personal care product, industrial product, ingestible product (e.g., food product / precursor, such as any food product / precursor disclosed herein), or pharmaceutical product. The "stability" (or the trait of being "stable") of the dispersion or emulsion herein is, for example, after the initial preparation of the dispersion or emulsion, the ability of the dispersed particles of the dispersion or the liquid droplets (emulsion) dispersed in another liquid 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 the 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. In some aspects, the stable dispersion or emulsion can resist the complete deposition, flocculation, and / or coalescence of the dispersed / emulsified materials.

[0182] In some aspects, for example, in the case of an aqueous dispersion or aqueous emulsion stabilized by the glucosylated isoflavone glycosides herein, its particles or liquid (e.g., oil) droplets are dispersed within about or at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the volume of the aqueous composition. In some aspects, such a level of dispersion or emulsification is expected to last for about, at least about, or up to about 0.5, 1, 2, 4, 6, 8, 10, 20, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, or 360 days, or 1, 2, or 3 years (typically starting from the initial preparation of the dispersion or emulsion). Based on the above dispersion and / or emulsion stability characteristics of the aqueous composition, the aqueous composition is expected to be suitable for use in, for example, applications / products where the stabilization of the dispersion or emulsion improves the performance of the application / product. Examples of such applications / products can be, as disclosed herein, milk / dairy products (e.g., yogurt, ice cream, cream), mayonnaise, salad dressing, beverages / tonics as carriers for delivering non-polar bioactive ingredients, cosmetics or pharmaceutical lotions / creams / foams / serums, or pharmaceutical carriers or encapsulation systems.

[0183] Some aspects of the method for glucosylating isoflavone glycosides include fermenting the food product / precursor after the step of providing a food product / precursor comprising at least water, sucrose, isoflavone glycosides, and glucosyltransferase. Thus, in such a method, the process for fermenting the food product / precursor begins only after at least a portion (e.g., about or at least about 20, 30, 40, 50, 60, 70, 80, 90, 95, 98, 99, or 99.5 weight percent) or all of the isoflavone glycosides initially present in the food product / precursor have been glucosylated by one or more glucosyltransferases.

[0184] Non-limiting examples of the compositions and methods disclosed herein include:

[0185] 1. A method for producing a food product / precursor, the method comprising: (a) providing a food product / precursor comprising at least water, sucrose, and plant-based material (e.g., leguminous food product / precursor), and (b) contacting the food product / precursor with at least: (i) a glucosyltransferase that synthesizes α-1,6-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,6-glucan are α-1,6 bonds, and / or (ii) a glucosyltransferase that synthesizes α-1,3-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,3-glucan are α-1,3 bonds, typically wherein at least one α-glucan is produced in the food product / precursor, whereby the food product / food precursor after step (b) has a reduced off-flavor compared to the food product / precursor before step (b), and optionally, a reduced sugar content compared to the food product / precursor before step (b).

[0186] 2. The method according to Example 1, wherein the food product / food precursor after step (b) has a reduced content of at least one isoflavone glycoside and / or an increased content of at least one glucosylated isoflavone glycoside compared to the food product / precursor before step (b), optionally wherein the reduced off-flavor is caused by the reduced content of at least one isoflavone glycoside.

[0187] 3. The method according to Example 2, wherein the at least one isoflavone glycoside is daidzin or genistin.

[0188] 4. The method according to Example 1, 2, or 3, wherein the off-flavor comprises bitterness and / or astringency.

[0189] 5. The method according to any one of Examples 1, 2, 3 or 4, wherein: the glucosyltransferase for synthesizing α-1,6-glucan comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, 2, 11, 12, 14, 15, 16, 17 or 18, and / or the glucosyltransferase for synthesizing α-1,3-glucan comprises an amino acid sequence that is at least 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, residues 55-960 of SEQ ID NO: 9 or SEQ ID NO: 13.

[0190] 6. The method according to any one of Examples 1, 2, 3, 4 or 5, wherein step (a) comprises adding sucrose to the food product / precursor.

[0191] 7. The method according to any one of Examples 1, 2, 3, 4, 5 or 6, wherein the food product / precursor in step (a) is fermented, or the method further comprises fermenting the food product / precursor during or after step (b).

[0192] 8. The method according to any one of Examples 1, 2, 3, 4, 5, 6 or 7, wherein the food product / precursor is a non-dairy food product / precursor (e.g., a plant-based yogurt product / precursor), optionally wherein the food product / precursor produced by the method is a fresh fermented product / precursor (e.g., a plant-based yogurt product / precursor).

[0193] 9. A food product / precursor produced by the method according to any one of Examples 1, 2, 3, 4, 5, 6, 7, 8 or 19.

[0194] 10. A method for glucosylating an isoflavone glycoside, the method comprising: providing a composition comprising at least water, sucrose, an isoflavone glycoside and a glucosyltransferase (or contacting the isoflavone glycoside with the glucosyltransferase in the presence of at least water and sucrose), wherein the glucosyltransferase is selected from: (i) a glucosyltransferase for synthesizing α-1,6-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,6-glucan are α-1,6 bonds, and / or (ii) a glucosyltransferase for synthesizing α-1,3-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,3-glucan are α-1,3 bonds, wherein at least one glucosylated form of the isoflavone glycoside (glucosylated isoflavone glycoside) is produced in the composition, and typically wherein at least one α-glucan (e.g., α-1,6-glucan herein, the α-1,3-glucan and / or a graft copolymer) is produced in the composition.

[0195] 11. The method according to embodiment 10, wherein the isoflavone glycoside is daidzin or genistin.

[0196] 12. The method according to embodiment 10 or 11, wherein: the glucosyltransferase for synthesizing α-1,6-glucan comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, 2, 11, 12, 14, 15, 16, 17 or 18, and / or the glucosyltransferase for synthesizing α-1,3-glucan comprises an amino acid sequence that is at least 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, residues 55-960 of SEQ ID NO: 9 or SEQ ID NO: 13.

[0197] 13. The method according to embodiment 10, 11 or 12, wherein the composition is a food product / precursor.

[0198] 14. The method according to embodiment 13, wherein the food product / precursor is a plant-based food product / precursor (e.g., a legume food product / precursor), optionally wherein the food product / precursor is a fresh fermented product / precursor (e.g., a plant-based yogurt product / precursor).

[0199] 15. The method according to embodiment 13 or 14, further comprising: fermenting the food product / precursor after the providing step.

[0200] 16. A composition or a glucosylated isoflavone glycoside produced by the method according to embodiment 10, 11, 12, 13, 14 or 15.

[0201] 17. A composition comprising a glucosylated isoflavone glycoside, wherein the glucosylated isoflavone glycoside is produced by contacting an isoflavone glycoside with a glucosyltransferase in the presence of at least water and sucrose, wherein the glucosyltransferase is selected from: (i) a glucosyltransferase for synthesizing α-1,6-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,6-glucan are α-1,6 bonds (e.g., as described in embodiment 5 or 12), and / or (ii) a glucosyltransferase for synthesizing α-1,3-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,3-glucan are α-1,3 bonds (e.g., as described in embodiment 5 or 12), optionally wherein the composition is a food product / precursor, and / or optionally wherein the glucosylated isoflavone glycoside is produced in situ in the composition or produced in a separate GTF reaction and then added as a raw material for producing the composition.

[0202] 18. The composition according to embodiment 17, wherein the isoflavone glycoside is daidzin or genistin.

[0203] 19. The method according to embodiment 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14 or 15, but wherein the glucosyltransferase is a modified (non-natural) glucosyltransferase that synthesizes α-1,3-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,3-glucan are α-1,3 bonds, wherein the modified glucosyltransferase contains one or more amino acid substitutions at one or more positions corresponding to one or more amino acid residues Tyr-185, Val-186, Leu-513, Gln-588, Phe-607, Ile-608, Lys-625, Arg-741, Val-1188, Lys-1327, Glu-1332, Asp-1418, Ala-1419, Ser-1420, Thr-1421, Arg-1424, Leu-1425, Thr-1431 and / or Glu-1450 of SEQ ID NO:10, and optionally wherein the modified glucosyltransferase contains an amino acid sequence that is at least about 90% identical to SEQ ID NO:5, 6, 7, 8 or 9, or the amino acid residues 55-960 of SEQ ID NO:5, the amino acid residues 54-957 of SEQ ID NO:6, the amino acid residues 55-960 of SEQ ID NO:7, the amino acid residues 55-960 of SEQ ID NO:8 or the amino acid residues 55-960 of SEQ ID NO:9, optionally wherein the modified glucosyltransferase has enhanced performance and / or one or more stability benefits, and optionally wherein the method further comprises using a glucosyltransferase that synthesizes α-1,6-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,6-glucan are α-1,6 bonds.

[0204] Examples

[0205] The present disclosure is further illustrated in the following examples. It should be understood that although these examples indicate certain aspects herein, they are given by way of illustration only. From the above discussion and these examples, those skilled in the art can determine the essential features of the disclosed embodiments, and various changes and modifications can be made without departing from the spirit and scope of the disclosed embodiments to adapt the disclosed embodiments to various uses and conditions.

[0206] Materials / Methods

[0207] HPLC sugar content analysis

[0208] Adopt 2695 Serrations module or Thermo Scientific Dionex TM UltiMate 3000 HPLC (equipped with Phenomonex Rezex TM RPM - monosaccharide Pb 2+ The sugar composition was measured by high performance liquid chromatography (HPLC) using a TM UltiMate 3000 HPLC (equipped with Phenomonex Rezex RPM - monosaccharide Pb column (300 mm x 7.8 mm) and a refractive index detector). Water was used as the mobile phase at a flow rate of 0.400 mL / min. The column temperature was 70 °C. Samples for HPLC injection were prepared by appropriate dilution in water, optionally centrifugation (10 min at 15,000 rpm), and sterile filtration. The signals from the HPLC were quantified by comparison with calibration standards of sugars eluting simultaneously. The sugar reduction was calculated by subtracting the sum of monosaccharides and disaccharides in the test sample from the sum of monosaccharides and disaccharides in a reference sample without added enzyme.

[0209] Yogurt preparation

[0210] The pre - pasteurized (72 °C for 15 s) bulk blended skim milk (0.1% fat) (Arla Foods) stored at 4 °C - 6 °C was standardized to the desired protein (% w / w), fat (% w / w), and sucrose (% w / w) contents by adding skim milk powder (33% protein, 1.2% fat, 54% carbohydrate) from BBA Lactalis, Laval, Mayenne, France, cream (38% fat) from Arla Foods, Denmark, and sucrose (sugar sand 500, Nordic Sugar A / S, Denmark). The thus - prepared standardized milk was then pasteurized and homogenized in a plate heat exchanger pasteurizer. Homogenization was carried out at 200 bar at 65 °C, and pasteurization was carried out at 95 °C for 6 minutes, after which the milk was cooled to 43 °C. The milk was inoculated with a thermophilic starter culture at an inoculation rate of 20 DCU / 100 L; all cultures were from International Flavors & Fragrances Inc., USA. Fermentation was carried out until pH 4.60, after which the product was cooled to 24 °C. The resulting yogurt was stored at 4 °C - 6 °C for viscosity measurement.

[0211] Method for measuring apparent viscosity

[0212] Rotational rheological tests were employed to evaluate the viscosity of the produced samples. A flow curve was obtained using an Anton Paar MCR302 rheometer (Anton Paar GmbH, Ostfildern, Germany) with a ST22-4V-40 vane geometry system for aluminum cups. The samples were filled into C-CC27 aluminum cups and stored at 5 °C for at least 5 hours before analysis. The shear rate interval applied to the samples was 0.1 - 350 s -1 , which defined the ascending curve, while the reverse operation illustrated the descending curve (350 - 0.1 s -1 ). The value of the measurement point duration was chosen to be at least as long as the value of the reciprocal shear rate valid for the ascending curve. The tests were carried out at a constant temperature of 10 °C and each sample was analyzed in duplicate. A water bath was connected to the rheometer to ensure isothermal conditions.

[0213] The apparent viscosity was evaluated from the flow curve, which is applicable to fluids where the ratio of shear stress to shear rate varies with shear rate. The apparent viscosity was extracted at shear rates of 11.7 Hz or 249 Hz. The apparent viscosity extracted at a shear rate of 11.7 Hz indicates the "consistency" of the sample. The apparent viscosity extracted at a shear rate of 249 s -1 (249 Hz) is related to the sensory perception of "mouthfeel".

[0214] LC-MS

[0215] Chromatographic conditions:

[0216] The chromatographic system was an UPLC system operating at 400 μl / min. Solvent A was water / formic acid (1000 / 1) and solvent B was acetonitrile / formic acid (1000 / 1). The gradient started at 1% B (0 min), ended at 25% B at 34 min, followed by a wash step to reach 95% B, and then an equilibration step back to 1% B (5 min). The column was a reversed-phase UPLC C18 column (100 x 2.1 mm inner diameter).

[0217] Mass spectrometry conditions:

[0218] The mass spectrometer was a high-resolution orbitrap-type instrument operating in positive ion mode. The interface was electrospray. MS scans were recorded at 120 k resolution with an m / z range of 150 - 1500. MS2 scans were recorded at 15 k resolution in data-dependent mode with a cycle time of 1.5 seconds. The activation type was HCD.

[0219] Data processing:

[0220] Genedata was used Processing data. All identified features were retrieved in a library of substances representing a series of isoflavones and their glucosylated isomers (including the core structures daidzein, genistein, glycitein, daidzin, genistin, glycitin, and glucosylated forms with 2 - 10 hexoses).

[0221] Example 1

[0222] In situ glucosylation of isoflavone glycosides in fresh fermented preparations for off-flavor reduction

[0223] In plant - based fresh fermented products, the sugar - reducing and flavor effects of GTF 0768 (SEQ ID NO:1) and vGTFJ (SEQ ID NO:3) alone or in combination were studied. To produce each fresh - fermented soy product, a soy matrix was prepared by mixing 276 g of a commercially available soy beverage (Naturli’ Soya Drik ) which contains 2.1% fat, 3.7% protein, and 0.6% carbohydrates; Naturli’ Foods A / S, Vejen, Denmark) with 24 g of sucrose (equivalent to 8 wt% sucrose in the final matrix) at 50 °C for 10 minutes. Subsequently, the matrix was pasteurized at 95 °C for 3 minutes. After heat treatment, each matrix was cooled to 43 °C (fermentation temperature). Then all samples were fermented with VEGE033 culture (20 DCU / 100 mL). Three sample variants were produced. Sample 1 was used as a reference and did not include added GTF enzyme. For sample 2, GTF 0768 and vGTFJ were added to the culture at normalized ratios of 75% and 25% respectively. For sample 3, only vGTFJ was added at the time of culture inoculation. The single dose of GTF 0768 and / or vGTFJ enzyme was normalized, where 100% dose is the dose required to provide complete sucrose conversion (by GTF 0768 or vGTFJ alone) at the inoculation step. All samples were fermented until a pH of 4.8 was reached; the time for all samples to reach this pH was within 7.5 - 8.5 hours. Fermentation was stopped by shearing the samples for 20 seconds at the lowest speed setting using a handheld blender (IdeenWelt, Burgwedel, Germany), and then immediately cooling the samples to 5 °C.

[0224] The sugar content (by HPLC) and texture of each yogurt sample were evaluated according to the materials / methods. This work confirmed the conversion of sucrose to polysaccharide components by GTF 0768 and vGTFJ enzymes in samples 2 and 3. Sensory evaluation was performed on the fresh fermented product samples. It was noted that sample 1 had the worst texture and highest sweetness, but also had distinct off-flavors of bitterness and astringency. Compared to sample 1, samples 2 and 3 both had increased texture and were less sweet due to the formation of dextran-α-1,3-glucan graft copolymer (sample 2) or linear α-1,3-glucan (sample 3). Interestingly, samples 2 and 3 both had a more neutral flavor without the strong bitterness and astringency observed in sample 1.

[0225] The presence of isoflavones (daidzein, genistein), isoflavone glycosides (daidzin, genistin), and glucosylated isoflavone glycosides (daidzin and genistin modified with one or more additional glucosyl groups) in each sample was evaluated according to the materials / methods (by LC-MS). In this article, isoflavone glycosides contain a single glucosyl side chain moiety, while glucosylated isoflavone glycosides have two or more glucosyl side chain moieties; one or more additional glucosyl groups of glucosylated isoflavone glycosides are generated in this article by the activity of one or more added GTF enzymes. The isoflavones, isoflavone glycosides, and glucosylated isoflavone glycosides detected in the samples are presented in Table 1. The glucosylated forms of the isoflavone glycoside daidzin (e.g., DaidzinHex2, DaidzinHex3, DaidzinHex4, DaidzinHex5, DaidzinHex6, and DaidzinHex7 presented in Table 1) and genistin (e.g., GenistinHex2, GenistinHex3, GenistinHex4, GenistinHex5, and GenistinHex6 presented in Table 1) were found to be present only in Samples 2 and 3 and contain 2 - 11 glucosyl groups. Merely as an example to illustrate the nomenclature in Table 1, "DaidzinHex2" means that one glucosyl group has been added to daidzin, so this species has a total of two (2) glucosyl groups. It is possible that each detected glucosylated species (DaidzinHex2+, GenistinHex2+) consists of glucosylation isomers (glucosyl added at different sites on the glycoside substrate), or all glucosyls are linearly added at one site on the glycoside substrate (i.e., no isomers). Thus, it is clear that GTF 0768 and vGTFJ are capable of glucosylating isoflavone glycosides in situ. This GTF activity is associated with a reduction in the bitterness and astringency of the fresh fermentation preparation.

[0226] Table 1

[0227]

[0228]

[0229] Note: The values listed are all chromatographic peak areas.

Claims

1. A method for producing a food product / precursor, the method comprising: (a) providing a food product / precursor comprising at least water, sucrose, and plant-based material, and (b) contacting the food product / precursor with at least: (i) a glucosyltransferase that synthesizes α-1,6-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,6-glucan are α-1,6 bonds, and / or (ii) a glucosyltransferase that synthesizes α-1,3-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,3-glucan are α-1,3 bonds, typically wherein at least one α-glucan is produced in the food product / precursor, whereby the food product / food precursor after step (b) has a reduced off-flavor compared to the food product / precursor before step (b), and optionally has a reduced sugar content compared to the food product / precursor before step (b).

2. The method according to claim 1, wherein the food product / food precursor after step (b) has a reduced content of at least one isoflavone glycoside and / or an increased content of at least one glucosylated isoflavone glycoside compared to the food product / precursor before step (b), optionally wherein the reduced off-flavor is caused by the reduced content of at least one isoflavone glycoside.

3. The method according to claim 2, wherein the at least one isoflavone glycoside is daidzin or genistin.

4. The method according to claim 1, wherein the off-flavor comprises bitterness and / or astringency.

5. The method according to claim 1, wherein: the glucosyltransferase that synthesizes α-1,6-glucan comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, 2, 11, 12, 14, 15, 16, 17, or 18, and / or the glucosyltransferase that synthesizes α-1,3-glucan comprises an amino acid sequence that is at least 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, residues 55-960 of SEQ ID NO: 9, or SEQ ID NO:

13.

6. The method according to claim 1, wherein step (a) comprises adding sucrose to the food product / precursor.

7. The method according to claim 1, wherein the food product / precursor of step (a) is fermented, or the method further comprises fermenting the food product / precursor during or after step (b).

8. The method according to claim 1, wherein the food product / precursor is a non-dairy food product / precursor, optionally wherein the food product / precursor produced by the method is a fresh fermented product / precursor.

9. A food product / precursor produced by the method according to claim 1.

10. A method for glucosylating an isoflavone glycoside, the method comprising: Provided is a composition comprising at least water, sucrose, an isoflavone glycoside, and a glucosyltransferase, wherein the glucosyltransferase is selected from: (i) a glucosyltransferase that synthesizes an α-1,6-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,6-glucan are α-1,6 bonds, and / or (ii) a glucosyltransferase that synthesizes an α-1,3-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,3-glucan are α-1,3 bonds, wherein at least one glucosylated form of the isoflavone glycoside is produced in the composition, and typically at least one α-glucan is produced in the composition.

11. The method according to claim 10, wherein the isoflavone glycoside is daidzin or genistin.

12. The method according to claim 10, wherein: the glucosyltransferase that synthesizes an α-1,6-glucan comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, 2, 11, 12, 14, 15, 16, 17, or 18, and / or the glucosyltransferase that synthesizes an α-1,3-glucan comprises an amino acid sequence that is at least 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, residues 55-960 of SEQ ID NO: 9, or SEQ ID NO:

13.

13. The method according to claim 10, wherein the composition is a food product / precursor.

14. The method according to claim 13, wherein the food product / precursor is a plant-based food product / precursor, optionally wherein the food product / precursor is a fresh fermented product / precursor.

15. The method according to claim 13, which further comprises: fermenting the food product / precursor after the providing step.

16. A composition or a glucosylated isoflavone glycoside produced by the method according to claim 10.

17. A composition comprising a glucosylated isoflavone glycoside, wherein the glucosylated isoflavone glycoside is produced by contacting an isoflavone glycoside with a glucosyltransferase in the presence of at least water and sucrose, wherein the glucosyltransferase is selected from: (i) a glucosyltransferase that synthesizes an α-1,6-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,6-glucan are α-1,6 bonds, and / or (ii) a glucosyltransferase that synthesizes an α-1,3-glucan, wherein at least about 50% of the glycosidic bonds of the α-1,3-glucan are α-1,3 bonds, optionally wherein the composition is a food product / precursor.

18. The composition according to claim 17, wherein the isoflavone glycoside is daidzin or genistin.

Images