Method for reducing sugar in foodstuff through conversion of fructose with epimerase

Abstract

Disclosed herein are methods of producing a food product or food precursor, for example. These methods can comprise: (a) providing a food product / precursor that comprises at least water and fructose, and (b) contacting the food product / precursor with an epimerase that converts fructose to a fructose epimer, wherein at least one fructose epimer is produced in the food product / precursor. A glucosyltransferase enzyme (GTF) can be further included with the epimerase for contacting the food product / precursor, for example. Food products and food precursors produced by this methodology are also disclosed.

Description

[0001] TITLE

[0002] METHOD FOR REDUCING SUGAR IN FOODSTUFF THROUGH CONVERSION OF FRUCTOSE WITH EPIMERASE

[0003] This application claims the benefit of International Application No.

[0004] PCT / CN2024 / 131256 (filed November 11, 2024), which is incorporated herein by reference in its entirety.

[0005] FIELD

[0006] The present disclosure is in the field of polysaccharides and food products. For example, the disclosure pertains to treating food and food precursors with an epimerase enzyme in situ.

[0007] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0008] The official copy of the sequence listing is submitted electronically via EFS-Web as a file named NB42212WOPCT3_SequenceListing.xml created on November 6, 2025 and having a size of about 78 kilobytes, and is filed concurrently with the specification. The sequence listing contained in this file is part of the specification and is incorporated herein by reference in its entirety.

[0009] BACKGROUND

[0010] Today many consumers are exceedingly interested in information about the production methods and ingredients of the food products they consume. Health is a major consumer drive and translates into a request for clean label with a focus on organic, natural and “free from” artificial additives / ingredients. For decades, there has been a focus to adopt a low-fat diet that, which more recently, has shifted towards a low-carbohydrate diet. Several countries have also implemented ‘sugar-tax’ on various food products to reduce the health and economic burden due to obesity.

[0011] This has led to a large push for sugar reduction in food products. It is however a complex task to solve, as sugar contributes not only to flavor, but also texture, color, viscosity and, in some products, to reducing water activity to retard bacterial and fungal growth. It has therefore often been a necessity to replace the reduced sugar with artificial sweeteners and / or a bulking agent such as maltodextrin or insoluble fibers.

[0012] While demand is increasing for healthier foods, many difficulties arise in the fact that consumers are not willing to compromise on flavor, texture and the overall food experience. Recent discoveries (W02023 / 055902) have mitigated several of these difficulties through in situ conversion of sucrose into non-digestible alpha-glucans by using one or more glucosyltransferases, thereby reducing the amount of sucrose / sugar present while enhancing texture and eliminating the need for added stabilizers. During this in situ processing, the glucosyltransferase(s) releases fructose, a sugar that provides significant sweetness to the food. However, there is an ongoing debate of whether excessive fructose intake may contribute to certain metabolic disorders.

[0013] Allulose, also known as D-allulose or D-psicose, is a rare naturally occurring low calorie sugar having a sweetness profile similar to that of sucrose, making it a desirable alternative to higher calorie sweeteners, such as sucrose, fructose, and glucose.

[0014] Allulose is a C-3 epimer of D-fructose, and may thus be produced, e.g., commercially, by conversion of D-fructose to allulose by enzymes such as epimerases.

[0015] Epimerases capable of converting D-fructose to allulose have been found to have a variety of properties, e.g., temperature, pH, and metal cofactor requirements, that can impact their enzymatic activity. Epimerases stable at high temperature and low pH with a reduced need for supplemented metal cofactors are of particular value for in situ conversion of fructose to allulose in foodstuff.

[0016] Thus, there is a need for epimerases capable of converting D-fructose to allulose in situ at low pH and high temperature with a reduced need for added metal cofactors. The compositions and methods provided herein address these and other needs.

[0017] SUMMARY

[0018] In one embodiment, the present disclosure concerns a method of producing a food product / precursor. Such a method can comprise:

[0019] (a) providing a food product / precursor that comprises at least water and fructose, and

[0020] (b) contacting the food product / precursor with an epimerase that converts fructose to a fructose epimer, wherein at least one fructose epimer is produced in the food product / precursor.

[0021] In another embodiment, the present disclosure concerns a food product or food precursor produced by a method herein.

[0022] BRIEF DESCRIPTION OF THE SEQUENCES

[0023] Table A. Summary of Protein SEQ ID Numbersa

[0024]

[0025]

[0026]

[0027]

[0028] aSEQ ID NOs:21, 27, 30, 32-34, and 44-46 are intentionally not included in this table and merely serve as placeholders.

[0029] BRIEF DESCRIPTION OF THE DRAWING FIG. 1: Orange juice samples treated with GTF enzymes blend at Brix 30 or Brix 11.8, as compared to control. All juice samples were diluted to 11.8 Brix prior to heat treatment and time of picture. The control and middle sample (treated at 30 Brix) had a distinct separation (arrows). Refer to Example 5.

[0030] DETAILED DESCRIPTION

[0031] The disclosures of all cited patent and non-patent literature are incorporated herein by reference in their entirety.

[0032] Unless otherwise disclosed, the terms “a”, “an” and “the” as used herein are intended to encompass one or more (i.e., at least one) of a referenced feature. Where present, all ranges are inclusive and combinable, except as otherwise noted. For example, when a range of “1 to 5” (i.e., 1-5) is recited, the recited range should be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, and the like.

[0033] The terms “epimerase”, “epimerase enzyme” and the like are used interchangeably herein. In some aspects, the activity of an epimerase is of EC 5.1.3.30, reversibly catalyzing the conversion of D-fructose (“D-fructose” and “fructose” are used interchangeably herein) to D-allulose (“D-allulose”, “allulose” and “D-psicose” are used interchangeably herein). Such an epimerase can optionally be referred to as a “D-allulose 3-epimerase” (and like terms). In some aspects, the activity of an epimerase is of EC 5.1.3.31, reversibly catalyzing the conversion of D-fructose to D-tagatose. Such an epimerase can optionally be referred to as a “D-tagatose 3-epimerase” (and like terms). Allulose is a C-3 epimer of fructose, and can optionally be characterized as a “fructose epimer”. Fructose used as a substrate by an epimerase in a food product / precursor herein can be (i) fructose intrinsic to, and / or exogenously added to, the food product / precursor, and / or (ii) fructose produced in situ by a glucosyltransferase enzyme in the food / food precursor, for example.

[0034] The terms “alpha-glucan”, “alpha-glucan polymer” and the like are used interchangeably herein. An alpha-glucan is a polymer comprising glucose monomeric units linked together by alpha-glycosidic linkages. In typical embodiments, an alphaglucan herein comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% alpha-glycosidic linkages. Examples of alpha-glucan polymers herein include graft copolymers as presently disclosed, as well as alpha-1, 3-glucan and alpha-1, 6-glucan.

[0035] The terms “alpha-1, 3-glucan”, “poly alpha-1, 3-glucan”, “alpha-1, 3-glucan polymer” and the like are used interchangeably herein. Alpha-1, 3-glucan is a polymer comprising glucose monomeric units linked together by glycosidic linkages, wherein at least about 50% of the glycosidic linkages are alpha-1,3. Alpha-1, 3-glucan in certain embodiments comprises at least about 90% or 95% alpha-1,3 glycosidic linkages. Most or all of the other linkages in alpha-1, 3-glucan herein typically are alpha-1,6, though some linkages may also be alpha-1,2 and / or alpha-1,4. Alpha-1, 3-glucan as presently disclosed can characterize an alpha-1, 3-glucan side chain herein. In some aspects, alpha-1, 3-glucan can characterize an alpha-1, 3-glucan “homopolymer”, which is alpha-1, 3-glucan that is not part of a dextran-alpha-1, 3-glucan copolymer. The terms “dextran”, “dextran polymer”, “dextran molecule”, “alpha-1, 6-glucan” and the like herein refer to a water-soluble alpha-glucan comprising at least 50%, 60%, 70%, 80%, or 90% alpha-1,6 glycosidic linkages (with the balance of the linkages typically being alpha-1,3). Enzymes capable of synthesizing dextran from sucrose may be described as “dextransucrases” (EC 2.4.1.5). A “substantially linear” (“mostly linear”, and like terms) dextran has 5% or less branches, before being modified herein to have with alpha-1, 3-glucan side chains. A “linear” dextran has no branches, before being modified herein to have alpha-1, 3-glucan side chains. Branches, if present prior to modification of dextran with alpha-1, 3-glucan side chains, can be short, being one (pendant) to three glucose monomers in length. Yet, in some aspects, dextran can be “dendritic”, which is a branched structure emanating from a core in which there are chains (containing mostly or all alpha-1, 6-linkages) that iteratively branch from each other (e.g., a chain can be a branch from another chain, which in turn is a branch from another chain, and so on). Yet, in still some aspects, dextran is not dendritic, but has a branch-on-branch structure that does not emanate from a core. Dextran as used in a glucosyltransferase reaction herein for alpha-1, 3-glucan synthesis (to produce a dextran-alpha-1, 3-glucan copolymer) can optionally be characterized as a “primer” or “acceptor”. In some aspects, dextran can characterize a dextran “homopolymer”, which is dextran that is not part of a dextran-alpha-1, 3-glucan copolymer.

[0036] The term “copolymer” herein refers to a polymer comprising at least two different types of alpha-glucan, such as dextran and alpha-1, 3-glucan.

[0037] The terms “graft copolymer”, “branched copolymer” and the like herein generally refer to a copolymer comprising a “backbone” (or “main chain”) and one or more side chains branching from the backbone. The side chains are structurally distinct from the backbone.

[0038] Examples of graft copolymers herein are “dextran-alpha-1, 3-glucan graft copolymers” (and like terms) that comprise a backbone comprising dextran, and one or more side chains of alpha-1, 3-glucan. A backbone in some aspects can itself be a branched dextran as disclosed herein; the addition of alpha-1, 3-glucan side chains to such a backbone (thereby forming a graft copolymer herein) can be, for example, via enzymatic extension from non-reducing ends presented by short branches (alpha-1,2, -1,3, or -1,4 branch, each typically comprised of a single glucose monomer; i.e., pendant glucose). Short branches (that can be enzymatically extended into an alpha-1, 3-glucan side chain) can be present on an otherwise linear or mostly linear dextran, or can be present on a branching dextran. In some aspects, alpha-1, 3-glucan can also be synthesized from non-reducing ends of dextran main chains, such as in embodiments in which the dextran backbone is linear or mostly linear, or embodiments in which the dextran backbone is branching (e.g., dendritic, or not dendritic [branches do not emanate from a core] but has branch-on-branch structure); such alpha-1, 3-glucan is not, technically-speaking, a side chain to the dextran, but rather an extension from the dextran main chain(s).

[0039] The percent branching in an alpha-glucan herein refers to that percentage of all the linkages in the alpha-glucan that represent branch points. For example, the percent of alpha-1,3 branching in an alpha-glucan herein refers to that percentage of all the linkages in the glucan that represent alpha-1,3 branch points. Except as otherwise noted, linkage percentages disclosed herein are based on the total linkages of a glucan, or the portion of a glucan for which a disclosure specifically regards.

[0040] The terms “linkage”, “glycosidic linkage”, “glycosidic bond” and the like refer to the covalent bonds connecting the sugar monomers within a saccharide compound (oligosaccharides and / or polysaccharides). Examples of glycosidic linkages include 1,6-alpha-D-glycosidic linkages (herein also referred to as “alpha-1,6” linkages), 1,3-alpha-D-glycosidic linkages (herein also referred to as “alpha-1,3” linkages), 1,4-alpha-D-glycosidic linkages (herein also referred to as “alpha-1,4” linkages), and 1,2-alpha-D-glycosidic linkages (herein also referred to as “alpha-1,2” linkages). The glycosidic linkages of a glucan polymer herein can also be referred to as “glucosidic linkages”. Herein, “alpha-D-glucose” is referred to as “glucose”.

[0041] The glycosidic linkage profile of an alpha-glucan herein can be determined using any method known in the art. For example, a linkage profile can be determined using methods using nuclear magnetic resonance (NMR) spectroscopy (e.g.,13C NMR or1H NMR). These and other methods that can be used are disclosed in, for example, Food Carbohydrates: Chemistry, Physical Properties, and Applications (S. W. Cui, Ed., Chapter 3, S. W. Cui, Structural Analysis of Polysaccharides, Taylor & Francis Group LLC, Boca Raton, FL, 2005), which is incorporated herein by reference.

[0042] The “molecular weight” of an alpha-glucan herein can be represented as weightaverage molecular weight (Mw) or number-average molecular weight (Mn), the units of which are in Daltons (Da) or grams / mole. In some aspects, molecular weight can be represented as DPw (weight average degree of polymerization) or DPn (number average degree of polymerization). DPw and DPn are calculated from the corresponding Mw or Mn, respectively, by dividing by the molar mass of one monomer unit Mi. In the case of glucan polymer, Mi = 162.14. In some aspects, molecular weight can sometimes be provided as “DP” (degree of polymerization), which simply refers to the number of glucoses comprised within the alpha-glucan on an individual molecule basis. Various means are known in the art for calculating these various molecular weight measurements such as with high-pressure liquid chromatography (HPLC), size exclusion chromatography (SEC), or gel permeation chromatography (GPC).

[0043] The term “sucrose” herein refers to a non-reducing disaccharide composed of an alpha-D-glucose molecule and a beta-D-fructose molecule linked by an alpha-1, 2-glycosidic bond. Sucrose is known commonly as table sugar. Sucrose can alternatively be referred to as “alpha-D-glucopyranosyl-(1^2)-beta-D-fructofuranoside”. “Alpha-D-glucopyranosyl” and “glucosyl” are used interchangeably herein.

[0044] The terms “sugar” or “sugars”, unless used to specifically refer to sucrose only, refer to any monosaccharide (e.g., fructose, glucose, and / or galactose) and / or disaccharide (e.g., sucrose, leucrose, and / or lactose; and / or optionally DP2 glucooligosaccharide), and / or optionally any oligosaccharide (e.g., ranging from DP3 to DP4, DP5, DP6, DP7, DP8, DP9, DP10, DP12, DP14, DP15, DP16, DP18, or DP20; typically gluco-oligosaccharide) such as those disclosed herein. Sugars herein typically are water-soluble.

[0045] The terms “leucrose” and “D-glucopyranosyl-alpha(1-5)-D-fructopyranose” are used interchangeably herein. Leucrose has the following structure:

[0046]

[0047] The terms “glucosyltransferase”, “glucosyltransferase enzyme”, “GTF”, “glucansucrase” and the like are used interchangeably herein. The activity of a glucosyltransferase herein catalyzes the reaction of the substrate sucrose to make the products alpha-glucan and fructose. Other products (by-products) of a GTF reaction can include glucose, various soluble gluco-oligosaccharides, and leucrose. Wild type forms of glucosyltransferase enzymes generally contain (in the N-terminal to C-terminal direction) a signal peptide (which is typically removed by cleavage processes), a variable domain, a catalytic domain, and a glucan-binding domain. A glucosyltransferase herein is classified under the glycoside hydrolase family 70 (GH70) according to the CAZy (Carbohydrate-Active EnZymes) database (Cantarel et al., Nucleic Acids Res. 37:D233- 238, 2009). The term “dextransucrase” (and like terms) can optionally be used to characterize a glucosyltransferase enzyme that produces dextran.

[0048] The term “glucosyltransferase catalytic domain” herein refers to the domain of a glucosyltransferase enzyme that provides alpha-glucan-synthesizing activity to a glucosyltransferase enzyme. A glucosyltransferase catalytic domain typically does not require the presence of any other domains to have this activity.

[0049] The terms “glucosyltransferase reaction”, “glucosyltransferase reaction composition”, “glucan synthesis reaction”, and the like are used interchangeably herein and generally refer to a reaction that initially comprises water, sucrose, at least one active glucosyltransferase enzyme, and optionally other components. Components that can be further present in a glucosyltransferase reaction typically after it has commenced include fructose, glucose, leucrose, soluble gluco-oligosaccharides (e.g., DP2-DP7) (such may be considered as products or by-products, depending on the glucosyltransferase used), and / or insoluble alpha-glucan product(s) of DP8 or higher. It would be understood that certain glucan products, such as alpha-1, 3-glucan with a degree of polymerization (DP) of at least 8 or 9, are water-insoluble and thus not dissolved in a glucan synthesis reaction. The term “under suitable reaction conditions” as used herein refers to reaction conditions that support conversion of sucrose to alphaglucan product(s) via glucosyltransferase enzyme activity. It is during such a reaction that glucosyl groups originally derived from the input sucrose are enzymatically transferred and used in alpha-glucan polymer synthesis; glucosyl groups as involved in this process can thus optionally be referred to as the glucosyl component or moiety (or like terms) of a glucosyltransferase reaction.

[0050] The terms “fructosyltransferase”, “fructosyltransferase enzyme”, “FTF”, “fructansucrase” and the like are used interchangeably herein. In general, the activity of a fructosyltransferase herein catalyzes the reaction of the substrate sucrose to make the products fructan (polyfructose) and glucose. Examples of fructosyltransferases herein include inulosucrase (EC 2.4.1.9) and levansucrase (EC 2.4.1.10), which produce the fructans inulin and levan, respectively. Another fructosyltransferase example is sucrose: sucrose fructosyltransferase (EC 2.4.1.99).

[0051] The term “in situ" in some aspects characterizes a glucosyltransferase reaction that occurs inside a food product or precursor thereof and thereby produces alphaglucan and fructose within the food product itself (or precursor). Such produced alphaglucan (e.g., graft copolymer, alpha-1, 3-glucan, and / or alpha-1,6-glucan) can be soluble or insoluble. While an alpha-1, 3-glucan product is typically insoluble and an alpha-1,6- glucan product is typically soluble, a graft copolymer product can either be soluble or insoluble, in a food product / precursor herein. In situ production of alpha-glucan in a food product / precursor typically substitutes for adding alpha-glucan herein as an ingredient in food, though such addition can be performed if desired (e.g., to supplement the alphaglucan produced in situ). The term “in situ" in some aspects characterizes an epimerase reaction that occurs inside a food product or precursor thereof and thereby produces a fructose epimer such as allulose within the food product itself (or precursor). Both a glucosyltransferase reaction and an epimerase reaction can be conducted in situ, for example, such as by treating a sucrose-containing food product / precursor with glucosyltransferase to produce alpha-glucan and fructose in the food product / precursor, and then treating the food product / precursor with an epimerase to produce a fructose epimer such as allulose. In situ treatment of a food product / precursor with glucosyltransferase and epimerase enzymes can be conducted sequentially or simultaneously, for example. The term “in situ" in some aspects characterizes a fructosyltransferase, lactase, or glucose isomerase reaction(s) that occurs inside a food product or precursor thereof and thereby produces respective product(s) of these enzymes within the food product itself (or precursor).

[0052] The terms “percent by volume”, “volume percent”, “vol %”, “v / v %” and the like are used interchangeably herein. The percent by volume of a solute in a solution can be determined using the formula: [(volume of solute) / (volume of solution)] x 100%.

[0053] The terms “percent by weight”, “weight percentage (wt%)”, “weight-weight percentage (% w / w)” and the like are used interchangeably herein. Percent by weight refers to the percentage of a material on a mass basis as it is comprised in a composition, mixture, or solution.

[0054] The terms “weight / volume percent”, “w / v%” and the like are used interchangeably herein. Weight / volume percent can be calculated as: ((mass [g] of material) / (total volume [mL] of the material plus the liquid in which the material is placed)) x 100%. The material can be insoluble in the liquid (i.e., be a solid phase in a liquid phase, such as with a dispersion), or soluble in the liquid (i.e., be a solute dissolved in the liquid).

[0055] The terms “ingestible product” and “ingestible composition” are used interchangeably herein, and refer to any substance that, either alone or together with another substance, may be taken orally (i.e., by mouth), whether intended for consumption or not. Thus, an ingestible product includes food / beverage products.

[0056] “Food / beverage products” refer to any edible product intended for consumption (e.g., for nutritional purposes) by humans or animals, including solids, semi-solids, or liquids. A “food” herein can optionally be referred to as a “foodstuff”, “food stuff”, “food product”, or other like term, for example. Herein, unless otherwise disclosed, a beverage or other ingestible liquid is an example of a food product. While the present disclosure generally regards food and food precursors that are by definition intended for ingestion or eventual ingestion (food precursor first made into food before being eaten), the disclosure likewise regards other ingestible products (e.g., supplement, nutraceutical, pharmaceutical product) comprising in situ-produced alpha-glucan and / or fructose epimer. A food precursor herein can be (i) a food as it exists before one or more processing steps (e.g., fermentation, aging, cooling / freezing, heating, baking, mixing) that render it to be a food product intended for direct consumption, and / or (ii) an ingredient for use in preparing a food product, for example. In some aspects, a food precursor can characterize a food product or ingredient as it exists before treatment with one or more GTF enzymes and / or epimerases in a method herein.

[0057] The term “texture” as used herein in reference to a food product / precursor herein means the thickness of the food product / precursor and / or sensory perception of the food product / precursor by, for example, vision, touch, or oral / taste processing. An “improvement” in texture means an increase in thickness and / or an increase in the sensory perception. Unless otherwise noted, as used herein the “thickness” of a food product / precursor means the apparent viscosity extracted at shear rate of about 10-13 Hz (e.g., -11.7 Hz) during a rheological analysis; an increase in apparent viscosity at such a shear rate indicates an increase in thickness. The apparent viscosity extracted at shear rate of about 230-270 Hz (e.g., -249 Hz) during a rheological analysis is correlated to “mouthfeel”; an increase in apparent viscosity at such a shear rate indicates an increase in mouthfeel.

[0058] “Dairy product / precursor” and like terms herein refer to a food product / precursor that contains milk and / or is made from milk. In some aspects, a dairy product / precursor contains at least about 2.5, 5, or 10 wt% milk or milk solids.

[0059] “Lactase-treated mil k / dairy” and like terms herein refer to m ilk / dairy products or precursors treated with one or more lactase enzymes to reduce the amount of lactose sugar therein.

[0060] “Reduced lactose mil k / dairy” and like terms herein refer to mil k / dairy products or precursors in which the weight percentage of lactose is about 2% or less, for example. “Lactose-free mi Ik / dairy” and like terms herein refer to mi Ik / dairy products or precursors in which the weight percentage of lactose is about 0.5 wt% or less, for example. “Yogurt”, “dairy-based yogurt”, “fermented dairy product” and like terms herein generally refer to a dairy food / beverage produced by acidifying lactic fermentation of a dairy substrate such as milk. Such a product can optionally contain secondary ingredients such as fruits, vegetables, sugars, flavors, etc.

[0061] “Flour” and like terms herein refer to powder made by grinding (milling) grains / cereals, roots / tubers, beans / legumes, or nuts / seeds, for example. Typically, the material that is ground into flour is entered into the grinding process in raw, dried form. A flour herein that is made from grain can optionally be referred to as a “grain flour”. “Meal” and other like terms herein refer to a substance that is similar to flour, but with a grain or particle size that is larger / coarser. A meal is not ground / milled as finely as flour. A meal herein that is made from grain can optionally be referred to as a “grain meal”. Flour and meal are generally used as ingredients in various food products. Flour and meal produced from a grain can optionally be characterized as grain derivatives herein.

[0062] “Dough”, “food dough” and like terms herein refer to a mixture comprising at least (i) flour and / or meal and (ii) a liquid (e.g., water or milk), and typically is in a suitable form (stiff / firm) for kneading or rolling. A dough can optionally be referred to with reference to the grain, grain derivative, or other material from which it was derived (e.g., wheat dough, wheat flour dough, com flour dough, cornmeal dough). Since dough typically is not eaten as a food prior to further processing (e.g., baking), dough can optionally be characterized as a “food precursor”.

[0063] A “baked food” (and like terms) herein refers to a food that has been baked during its preparation. Baking herein refers to a process of applying dry heat to a food / food precursor for a period of time during preparation of the food. In general, baking is conducted in an enclosed (typically confined) space such as within an oven. Bread is an example of a food for which its preparation process comprises baking.

[0064] An “extruded food” (and like terms) herein refers to a food that has been extruded during its preparation. Food extrusion is a process by which a mix of ingredients (e.g., dough) is forced through an opening in a perforated device (e.g., plate or die), which typically is specifically designed for the food being extruded. After this step, an extruded food typically is then cut to a particular size.

[0065] The terms “dietary fiber”, “glucan fiber” and the like herein refer to an alphaglucan that is indigestible and / or that does not increase blood-glucose levels when enterally administered to a mammal. In general, a dietary fiber herein is not significantly hydrolyzed by endogenous enzymes in the upper gastrointestinal tract of mammals such as humans. “Fermentation” and like terms herein as applied to food product / precursor refer to the conversion of carbohydrates in a food product / precursor into alcohol(s) and / or acid(s) through the action of one or more microorganisms (e.g., bacteria, yeast).

[0066] A composition herein that is “dry” or “dried” typically has less than 5, 4, 3, 2, 1, 0.5, or 0.1 wt% water comprised therein.

[0067] The terms “aqueous liquid”, “aqueous fluid”, “aqueous conditions”, “aqueous setting”, “aqueous system” and the like as used herein can refer to water or an aqueous solution. An “aqueous solution” herein can comprise one or more dissolved salts, where the maximal total salt concentration can be about 3.5 wt% in some embodiments.

[0068] Although aqueous liquids herein typically comprise water as the only solvent in the liquid, an aqueous liquid can optionally comprise one or more other solvents (e.g., polar organic solvent) that are miscible in water. Thus, an aqueous solution can comprise a solvent having at least about 10 wt% water.

[0069] An “aqueous composition” herein has a liquid component that comprises about, or at least about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100 wt% water, for example. Examples of aqueous compositions include mixtures, solutions, dispersions (e.g., suspensions, colloidal dispersions) and emulsions, for example.

[0070] An alpha-glucan herein that is “insoluble”, “aqueous-insoluble”, “water-insoluble” (and like terms) herein does not dissolve (or does not appreciably dissolve) in water or other aqueous conditions, optionally where the aqueous conditions are at a pH of 4-9 (e.g., pH 6-8) and / or a temperature of about 1 to 130 °C (e.g., 20-25 °C). In some aspects, less than 1.0 gram (e.g., no detectable amount) of an aqueous-insoluble alphaglucan dissolves in 1000 milliliters of such aqueous conditions (e.g., water at 23 °C). In contrast, an alpha-glucan that is “soluble”, “aqueous-soluble”, “water-soluble” and the like appreciably dissolves under the above aqueous conditions.

[0071] The term “viscosity” as used herein refers to the resistance of a food product / precursor to deformation at a given rate. Viscosity may also be defined as a measure of the extent to which a fluid (aqueous or non-aqueous) resists a force tending to cause it to flow. Furthermore, viscosity can be defined as the shear stress resulting from an applied shear rate. Both dynamic and kinematic viscosity are meant by the term viscosity, as both parameters are directly correlated through the density of a food product / precursor. Various units of viscosity that can be used herein include centipoise (cP, cps) and Pascal-second (Pa s), for example. A centipoise is one one-hundredth of a poise; one poise is equal to 0.100 kg m-1S’1. As used herein, the term “polypeptide” is defined as a chain of amino acid residues, usually having a defined sequence. As used herein the term polypeptide is interchangeable with the terms “peptides” and “proteins”. Typical amino acids contained in polypeptides herein include (respective three- and one-letter codes shown parenthetically): alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine (Gin, Q), glycine (Gly, G), histidine (His, H), isoleucine (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).

[0072] The terms “sequence identity”, “identity” and the like as used herein with respect to polypeptide sequences refer to amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window. Thus, “percentage of sequence identity”, “percent identity” and the like refer to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity.

[0073] Percent identity can be readily determined by any known method, including but not limited to those described in: 1) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2) Biocomputinq: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humana: NJ (1994); 4) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5) Sequence Analysis Primer (Gribskov. M. and Devereux, J., Eds.) Stockton: NY (1991), all of which are incorporated herein by reference.

[0074] Preferred methods for determining percent identity are designed to give the best match between the sequences tested. Methods of determining identity and similarity are codified in publicly available computer programs, for example. Sequence alignments and percent identity calculations can be performed using the MEGALIGN program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wl), for example. Multiple alignment of sequences can be performed, for example, using the Clustal method of alignment which encompasses several varieties of the algorithm including the Clustal V method of alignment (described by Higgins and Sharp, CABIOS.

[0075] 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values can correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity 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 method of alignment can be used (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191(1992); Thompson, J. D. et al, Nucleic Acids Research, 22 (22): 4673-4680, 1994) and found in the MEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (protein / nucleic acid) can be: GAP PENALTY=10 / 15, GAP LENGTH PENALTY=0.2 / 6.66, Delay Divergent Seqs(%)=30 / 30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB.

[0076] Various polypeptide amino acid sequences are disclosed herein as features of certain embodiments. Variants of these sequences that are at least about 70-85%, 85-90%, or 90%-95% identical to the sequences disclosed herein can be used or referenced. Alternatively, a variant amino acid sequence can have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with a sequence disclosed herein. A variant amino acid sequence herein has the same function / activity of the disclosed sequence, or at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% of the function / activity of the disclosed sequence. Any polypeptide amino acid sequence disclosed herein not beginning with a methionine or valine can typically further comprise at least a start-methionine or start-valine at the N-terminus of the amino acid sequence. In contrast, any polypeptide amino acid sequence disclosed herein beginning with a methionine or valine can optionally lack such a methionine or valine residue. In some aspects, any polypeptide amino acid sequence disclosed herein beginning with a methionine or valine can instead have, respectively, a valine or methionine as the first amino acid residue. The term “isolated” means a substance (or process) in a form or environment that does not occur in nature. A non-limiting example of an isolated substance includes any non-naturally occurring substance such as a food product or food precursor (as well as enzymatic reactions used to prepare these materials). It is believed that the embodiments disclosed herein are synthetic / man-made (could not have been made except for human intervention / involvement), and / or have properties that are not naturally occurring.

[0077] The term “increased” as used herein can refer to a quantity or activity that is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 50%, 100%, or 200% more than the quantity or activity for which the increased quantity or activity is being compared. The terms “increased”, “elevated”, “enhanced”, “greater than”, “improved” and the like are used interchangeably herein.

[0078] Some embodiments of the present disclosure concern a method of producing a food product / precursor. Such a method can comprise:

[0079] (a) providing a food product or food precursor (“food product / precursor”) that comprises at least water and fructose, and

[0080] (b) contacting the food product / precursor with an epimerase that converts fructose to a fructose epimer, wherein at least one fructose epimer is produced in the food product / precursor.

[0081] In some aspects of this method, a fructose epimer produced in the food product / precursor is allulose, in which case the epimerase used is a D-allulose 3-epimerase. Such a method can optionally be characterized herein as a fructose reduction method or an allulose production method.

[0082] In some aspects, an epimerase enzyme that converts fructose to allulose (D-allulose 3-epimerase) can comprise, or consist of, an amino acid sequence that is about, or at least about, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 100% identical to SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 35, 36, 37, 38, 39, 40, 41, 42, 43, 47, 48, 49, 50, 51, 52, 53, 54, or 55, and have epimerase activity. In some aspects, an epimerase enzyme that converts fructose to allulose (D-allulose 3-epimerase) can comprise, or consist of, an amino acid sequence that is about, or at least about, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 100% identical to SEQ ID NO: 15, 17, 18, 19, 20, 22, 23, 24, 25, 28, 29, 35, 37, 38, 39, 40, 41, 42, or 43, and have epimerase activity. In some aspects, an epimerase enzyme that converts fructose to allulose (D-allulose 3-epimerase) can comprise, or consist of, an amino acid sequence that is about, or at least about, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 100% identical to SEQ ID NO:14, 15, or 29, and have epimerase activity. In some aspects, an epimerase enzyme that converts fructose to allulose (D-allulose 3-epimerase) can comprise, or consist of, an amino acid sequence that is about, or at least about, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 100% identical to: (i) SEQ ID NO:18 or 29, (ii) SEQ ID NO:35, or (iii) SEQ ID NO:18, 29, or 35, and have epimerase activity. In some aspects, an epimerase enzyme that converts fructose to allulose (D-allulose 3-epimerase) can be as disclosed in any of U. S. Patent Appl. Publ. Nos. 2023 / 0055400, 2018 / 0112244, or 2015 / 0210996, which are each incorporated herein by reference. An epimerase can be produced, for example, by following the disclosure of Int. Pat. Appl. Publ. No. WO2023 / 114814, which is incorporated herein by reference.

[0083] An epimerase in some aspects can have thermal stability at elevated temperatures. In some aspects, an epimerase can exhibit activity of converting fructose to allulose at temperatures in which the equilibrium is shifted to produce a higher allulose conversion yield. In some aspects, an epimerase is thermostable at a temperature of about, or at least about, 40, 50, 60, 70, 80, 85, 90, 40-90, 50-90, 50-85, 50-80, 50-75, 50-70, or 60-70 °C. In some aspects, such as at any of the foregoing temperatures / ranges, an epimerase retains at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of its baseline enzymatic activity. Baseline enzymatic activity can be an activity determined for an epimerase that was not exposed to a temperature (e.g., elevated temperature) as described in this paragraph, for example. In some aspects, the retained activity is sufficient for converting fructose to allulose.

[0084] Thermostability may be determined as a residual activity measured after an epimerase has been exposed to (e.g., incubated at) an elevated temperature, for example for a particular duration. In some aspects, an epimerase retains residual activity following incubation at a temperature of about 60 °C to about 70°C for a duration of between about 5 minutes to about 120 minutes. In some aspects, the residual activity retained following incubation is at least 50%, 60%, 70%, 75%, 80,%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 50-100%, 75-100%, 80-100%, or 90-100% of a baseline activity. The baseline activity can be an activity determined for an epimerase that was not subjected to an elevated temperature (e.g., a temperature as described in this or the preceding paragraph). In some aspects, the baseline activity is an activity determined for an epimerase at a temperature of about 50 °C.

[0085] An epimerase herein can have pH stability. For example, an epimerase herein can have pH stability in a range of pHs where fructose and / or allulose are stable. In some aspects, an epimerase is pH-stable in a pH range that reduces non-enzymatic browning of the sugars via the Maillard reaction. For example, the pH may be in a range where the Maillard reaction proceeds at a slower rate compared to a neutral or basic pH such as 7, 7.5, 8.5, 9, 9.5, or 10.

[0086] In some aspects, an epimerase has a pH activity profile that is, or overlaps with, the pH range that reduces non-enzymatic browning of the sugars via the Maillard reaction (e.g., at a given temperature). In some aspects, an epimerase is pH-stable in a pH range that reduces non-enzymatic browning of the sugars via the Maillard reaction at a given temperature. In some embodiments, the pH range can be one that reduces the speed of the Maillard reaction at a specific temperature compared to a neutral or basic pH such as 7, 7.5, 8.5, 9, 9.5, or 10 pH, at the same temperature. In some aspects, an epimerase is stable at a pH in the range of about 4-10, 4.5-6, 4.5-6.5, 4.5-7, 4.5-7.5, 4.5-8, 4.5-9, 4.5-10, 4.5-10, 5-8, 5-6.5, 5-7, or 5.5-6.5. In some aspects, an epimerase retains a level of activity of at least about 25%, 30%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 50-100%, 75-100%, 80-100%, or 90-100% of a maximal activity level across a foregoing pH / range. The maximal level of epimerase activity can be determined by measuring the activity of the epimerase across a range of pHs to find minimum and maximum activity levels (e.g., characterize a dose response curve or standard curve), for example. In some aspects, the retained epimerase activity is sufficient for converting fructose to allulose.

[0087] An epimerase herein can have a reduced need for, or no need for, supplemented metal cofactors for activity. In some aspects, an epimerase retains an activity observed in the presence of a metal cofactor, e.g., when a metal cofactor is intentionally added, or when no or a reduced amount of metal cofactor is added. Thus, in some aspects, an epimerase does not require, or only requires a reduced amount, of metal cofactor to be supplemented for the epimerase to be active. In some aspects, an epimerase uses existing metal cofactors present in a fructose-containing food product / precursor to be active. In some aspects, an epimerase retains at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 50-100%, 75-100%, 80-100%, or 90-100% of baseline activity in the absence of added metal cofactor. In some aspects, baseline epimerase activity is as measured in the presence of a metal cofactor at a concentration of about 0.1-2, 0.1-1, 0.1 -0.5, 0.1-0.25, or 0.1 -0.15 mM. In some aspects, an epimerase retains a percentage of activity as described in this paragraph and / or herein when an amount of metal cofactor that is less than the concentration of metal cofactor present for the baseline activity is added. In some aspects, a reduced amount is about, or at least about, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 20-90%, 30-90%, 40-90%, or 50-90% less than the concentration of metal cofactor present for the baseline activity. In some aspects, the retained activity is sufficient for converting fructose to allulose. In some aspects, a metal cofactor is an ion. In some aspects, the metal cofactor is magnesium (magnesium ion). In some aspects, a metal cofactor is a salt (e.g., a magnesium salt).

[0088] An epimerase herein can have one or more of a thermal stability, pH stability, and / or reduced or no requirement for an added metal cofactor(s) as described above and herein, for example. In some, an epimerase is thermal stable, pH-stable, and has a reduced or no dependence on an added metal cofactor(s).

[0089] In some aspects, a fructose reduction method can further comprise contacting a food product / precursor with at least one glucosyltransferase enzyme, wherein the food product / precursor further comprises sucrose. Such contacting with the glucosyltransferase enzyme can be performed simultaneously with, or after, contacting the food product / precursor with one or more epimerase enzymes, for example.

[0090] Optionally, at least one alpha-glucan is produced in the food product / precursor by virtue of using the at least one glucosyltransferase enzyme. Typically, the leucrose in this method was all made by, or mostly made by, the glucosyltransferase enzyme(s).

[0091] Yet, in some aspects, a food product / precursor provided in step (a) is provided by contacting a food product / precursor that comprises at least water and sucrose with at least one glucosyltransferase enzyme. In other words, contacting with at least one glucosyltransferase enzyme can be performed prior to contacting the food product / precursor with at least one epimerase. All of, or a portion of (e.g., some fructose can optionally already be present independent of glucosyltransferase activity), the fructose in the food product / precursor provided in step (a) in such aspects can be produced by the glucosyltransferase enzyme. Optionally, at least one alpha-glucan is produced in the food product / precursor by virtue of using the at least one glucosyltransferase enzyme in this manner.

[0092] In some aspects of a fructose reduction method herein that employs at least one glucosyltransferase enzyme, the amount of leucrose produced in a food product / precursor is less than the amount of leucrose that would have been produced in the food product / precursor if the epimerase was not contacted with the food product / precursor. In some aspects, such a reduction in leucrose can be about, or at least about, 10%, 20%, 30%, 40%, or 50% on a weight basis. Typically, reduction in leucrose production can, at least in part, result from epimerase activity removing fructose as an available substrate for glucosyltransferase, thereby reducing the latter enzyme’s byproduction of leucrose.

[0093] In some aspects, a glucosyltransferase enzyme is:

[0094] (i) a glucosyltransferase enzyme that synthesizes alpha-1, 6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1, 6-glucan are alpha-1,6 linkages, or

[0095] (ii) a glucosyltransferase enzyme that synthesizes alpha-1, 3-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1, 3-glucan are alpha-1,3 linkages.

[0096] In some aspects, a GTF enzyme (dextransucrase) that synthesizes alpha-1,6-glucan herein can comprise an amino acid sequence that is about 100% identical to, 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, or 12 (GTF 0768), or SEQ ID NO:56 or 57 (GTF 6831), and have GTF activity. Yet, in some aspects, a GTF enzyme that synthesizes alpha-1, 6-glucan can be as disclosed in any of U. S. Patent Appl. Publ. Nos.

[0097] 2017 / 0218093, 2018 / 0282385, 2018 / 0291311, or 2016 / 0122445, which are each incorporated herein by reference. For example, the GTF identified as GTF 8117 (SEQ ID NO:30), GTF 6831 (SEQ ID NO:32), or GTF 5604 (SEQ ID NO:33) in

[0098] US2018 / 0282385 can be used, or the GTF identified as GTF 2919 (SEQ ID NO:5), GTF 2918 (SEQ ID NO:9), GTF 2920 (SEQ ID NO:13), or GTF 2921 (SEQ ID NO:17) in US2016 / 0122445 can be used, or a GTF comprising an amino acid sequence that is about 100% identical to, or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, the amino acid sequence of any of these GTF enzymes (and having GTF activity) can be used.

[0099] A dextransucrase herein is capable of producing dextran comprising about, or at least about, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% alpha-1,6-glycosidic linkages, for example. Such a percent alpha-1,6 linkage profile takes into account the total of all linkages in the dextran (main chains of alpha-1,6 glucan and, if present, branch portions therefrom). Dextran as disclosed elsewhere herein such as in a homopolymer or graftcopolymer can have any of the foregoing linkage profiles, for example.

[0100] A dextransucrase herein is capable of producing dextran having a weightaverage molecular weight (Mw) of about, at least about, or less than about, 1000, 2500, 5000, 7500, 10000, 25000, 50000, 75000, 100000, 150000, 200000, 250000, 500000, 750000, 1000000, 1000-10000, 1000-100000, 1000-1000000, 10000-100000, 10000-1000000, or 100000-1000000 Daltons, for example. In some aspects, the Mw is about, at least about, or less than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 10-50, 10-70, 10-80, 10-100, 10-120, 10-130, 10-150, 10-200, 25-50, 25-70, 25-80, 25-100, 25-120, 25-130, 25-150, 25-200, 50-70, 50-80, 50-100, 50-120, 50-130, 50-150, 50-200, 70-80, 70-100, 70-120, 70-130, 70-150, 70-200, 80-100, 80-120, 80-130, 80-150, 80-200, 100-120, 100-130, 100-150, 100-200, 120-130, 120-150, 120-200, 130-150, or 130-200 million Daltons, for example. Dextran as disclosed elsewhere herein such as in a homopolymer or graftcopolymer can have any of the foregoing molecular weight profiles, for example.

[0101] In some aspects, a GTF enzyme that synthesizes alpha-1, 3-glucan herein can comprise an amino acid sequence that is about 100% identical to, 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 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 Appl. Publ. No. 2019 / 0078063, which is incorporated herein by reference. It is noted that such a GTF enzyme comprising SEQ ID NO:2, 4, 8, 10, 14, 20, 26, 28, 30, 34, or amino acid residues 55-960 of SEQ ID NO:4, residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20, can synthesize alpha-glucan comprising at least about 90% (-100%) alpha-1,3 linkages. A GTF enzyme that synthesizes alpha-1, 3-glucan in some aspects can be that identified as GTF 0974 (SEQ ID NO:13 herein, SEQ ID NO:110 in US2018 / 0291311), or a GTF comprising an amino acid sequence that is about 100% identical to, or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, the foregoing amino acid sequence of GTF 0974 (and having GTF activity). Any of the foregoing GTF enzyme amino acid sequences can be modified as described herein to increase product yield, modify product molecular weight, and / or enhance GTF performance and / or stability.

[0102] A GTF enzyme for producing alpha-1, 3-glucan herein can, in some aspects, synthesize alpha-1, 3-glucan at a yield of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%. Yield in some aspects can be measured based on the glucosyl component of the reaction, and / or as measured using HPLC or NIR spectroscopy. Yield can be achieved in a reaction conducted for about 16-24 hours (e.g., ~20 hours), for example. Examples of such a GTF enzyme are those having an amino acid sequence modified such that the enzyme produces more products (alpha-1, 3-glucan and fructose), and less by-products (e.g., glucose, oligosaccharides such as leucrose), from a given amount of sucrose substrate. For example, one, two, three, four, or more amino acid residues of the catalytic domain of an alpha-1, 3-glucan-producing GTF herein can be modified / substituted to obtain a GTF enzyme that produces more products. Examples of a suitable modified GTF enzyme are disclosed in Tables 3-7 of U. S. Patent Appl. Publ. No. 2019 / 0078063. A modified GTF enzyme, for example, can comprise one or more amino acid substitutions corresponding with those in Tables 3-7 (ibid.) that is / are associated with an alpha-1, 3-glucan yield of at least 40% (the position numbering of such at least one substitution corresponds with the position numbering of SEQ ID NO:62 as disclosed in U. S. Patent Appl. Publ. No. 2019 / 0078063). A set of amino acid modifications as listed in Tables 6 or 7 (ibid.) can be used, for example.

[0103] The amino acid sequence of a GTF enzyme for alpha-1, 3-glucan synthesis in some aspects has been modified such that the enzyme produces alpha-1, 3-glucan with a molecular weight (DPw) that is lower than the molecular weight of alpha-1, 3-glucan produced by its corresponding parent GTF. Examples of a suitable modified GTF enzyme are disclosed in Tables 3 and 4 of U. S. Patent Appl. Publ. No. 2019 / 0276806, which is incorporated herein by reference. A modified GTF enzyme, for example, can comprise one or more amino acid substitutions corresponding with those in Tables 3 and / or 4 (ibid.) that is / are associated with an alpha-1, 3-glucan product molecular weight that is at least 5% less than the molecular weight of alpha-1, 3-glucan produced by parent enzyme (the position numbering of such at least one substitution corresponds with the position numbering of SEQ ID NO:62 as disclosed in U. S. Patent Appl. Publ. No. 2019 / 0276806). A set of amino acid modifications as listed in Table 4 (ibid.) can be used, for example. The amino acid sequence of a GTF enzyme for alpha-1,3-glucan synthesis in some aspects has been modified such that the enzyme produces alpha-1, 3-glucan with a molecular weight (DPw) that is higher than the molecular weight of alpha-1,3-glucan produced by its corresponding parent GTF. Examples of a suitable modified GTF enzyme are disclosed in Tables 3, 4 and 5 of U. S. Patent Appl. Publ. No. 2019 / 0078062, which is incorporated herein by reference. A modified GTF enzyme, for example, can comprise one or more amino acid substitutions corresponding with those in Tables 3, 4 and / or 5 (ibid.) that is / are associated with an alpha-1, 3-glucan product molecular weight that is at least 5% higher than the molecular weight of alpha-1,3-glucan produced by parent enzyme (the position numbering of such at least one substitution corresponds with the position numbering of SEQ ID NO:62 as disclosed in U. S. Patent Appl. Publ. No. 2019 / 0078062). A set of amino acid modifications as listed in Table 5 (ibid.) can be used, for example.

[0104] In some aspects, a modified GTF for alpha-1, 3-glucan synthesis (i) comprises at least one amino acid substitution or a set of amino acid substitutions (as described above regarding yield or molecular weight), and (ii) comprises or consists of a GTF catalytic domain that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to amino acid residues 55-960 of SEQ ID NO:4, amino acid residues 54-957 of SEQ ID NO:65, amino acid residues 55-960 of SEQ ID NO:30, amino acid residues 55-960 of SEQ ID NO:28, or amino acid residues 55-960 of SEQ ID NO:20 (each of these sequences as disclosed in U. S. Patent Appl. Publ. No.

[0105] 2019 / 0078063, which is incorporated herein by reference). Each of these subsequences are the approximate catalytic domains of each respective reference sequence, and produce alpha-1, 3-glucan comprising at least about 50% (e.g., >90% or >95%) alpha-1,3 linkages. In some aspects, a modified GTF (i) comprises at least one amino acid substitution or a set of amino acid substitutions (as described above), and (ii) comprises or consists of an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:62 or a subsequence thereof such as SEQ ID NO:4 (without start methionine thereof) or positions 55-960 of SEQ ID NO:4 (approximate catalytic domain) (each of these sequences as disclosed in U. S. Patent Appl. Publ. No. 2019 / 0078063).

[0106] In the present disclosure, SEQ ID NOs:5, 6, 7, 8, 9 and 10 (Table A) are the same amino acid sequences as, respectively, SEQ ID NOs:4, 65, 30, 28, 20 and 62 as disclosed in U. S. Patent Appl. Publ. No. 2019 / 0078063. Thus, each of presently disclosed SEQ ID NOs:5, 6, 7, 8, 9 and 10 can be used in any of the disclosed aspects, as appropriate. For example, a GTF enzyme that synthesizes alpha-1,3-glucan herein can comprise an amino acid sequence that is about 100% identical to, 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 of these sequences can be modified as described herein to affect alpha-1,3-glucan yield and / or molecular weight and / or stability, for example.

[0107] In some aspects, a GTF enzyme for alpha-1, 3-glucan synthesis has been modified such that the enzyme has enhanced performance and / or stability benefit(s). Such a GTF enzyme can be as disclosed, for example, in Int. Patent Appl. Publ. No. W02023 / 055902, which is incorporated herein by reference. Modification of such a GTF can be, for example, by having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid substitutions as compared to a corresponding parent GTF enzyme (e.g., a wild type mature GTF or active subsequence thereof such as a catalytic domain). Exemplary performance and / or stability benefits herein include one or more of increased thermal stability, increased storage stability, increased solubility, better pH profile, increased specific activity, modified substrate specificity, modified substrate binding, modified pH-dependent activity, modified pH-dependent stability, increased oxidative stability, increased expression, and / or increased glucan product yield (and / or decreased byproduct [e.g., leucrose] yield). In some aspects, a performance benefit is realized at a relatively low temperature (e.g., <5 °C) or at a relatively high temperature (e.g., >40 °C). An increase in any of the foregoing features can be by about, or at least about, 5%, 10%, 15%, 20%, 25%, or 30%, for example, as compared to the respective activity of a parent GTF enzyme that has not been modified.

[0108] Some examples of modified alpha-1, 3-glucan-producing GTF enzymes herein having enhanced performance and / or stability benefit(s) comprise or consist of SEQ ID NO:3 (vGTFJ) or 4. It is noted that SEQ ID NOs:3 and 4 are both derivable from SEQ ID NO:5 (GTF 6855), for example (e.g., SEQ ID NO:5 can be a backbone for making substitutions to render SEQ ID NOs:3 and 4).

[0109] In some aspects of the present disclosure, a modified GTF enzyme can comprise or consist of an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:3, and have one or more of (or all of) the following amino acid residues: 8-Asn, 9-Ala, 336-Tyr, 411-Leu, 430-Tyr, 448-Ala, 564-Ser, 1254-Gln, and / or 1273-Phe. The valine at position 1 of SEQ ID NO:3 in any of the foregoing aspects can optionally instead be a methionine, or can be deleted.

[0110] In some aspects of the present disclosure, a modified GTF enzyme can comprise or consist of an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:4, and have one or more of (or all of) the following amino acid residues: 336-Tyr, 430-Tyr, 431-Val, 448-Ala, 564-Ser, 1011-Glu, 1150-His, 1155-Ala, 1241-Lys, 1242-Glu, 1243-Gly, 1244-Ser, 1247-Leu, and / or 1248-Val. The valine at position 1 of SEQ ID NO:4 in any of the foregoing aspects can optionally instead be a methionine, or can be deleted.

[0111] Although it is believed that a modified alpha-1, 3-glucan-producing GTF enzyme in some aspects need only have a catalytic domain, the modified GTF can be comprised within a larger amino acid sequence. For example, a catalytic domain may be linked at its C-terminus to a glucan-binding domain, and / or linked at its N-terminus to a variable domain and / or signal peptide.

[0112] Although amino acid substitutions in a modified alpha-1, 3-glucan-producing GTF enzyme are generally disclosed in some aspects with respect to corresponding positions in SEQ ID NO: 10, such substitutions can alternatively be stated simply with respect to its / their position number in the amino acid sequence used to produce the modified GTF itself (e.g., SEQ ID NO:5 [optionally without start methionine thereof] or positions 55-960 of SEQ ID NO:5 [approximate catalytic domain]), as convenience may dictate. Such can be done simply by aligning the amino acid sequence with SEQ ID NO: 10 and identifying the position number(s) of interest in the amino acid sequence based on its / their direct alignment with the corresponding position(s) in SEQ ID NO:10.

[0113] An alpha-1, 3-glucan-producing GTF herein is capable of producing alpha-1, 3-glucan comprising about, or at least about, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% alpha-1,3-glycosidic linkages, for example. Alpha-1,3-glucan as disclosed elsewhere herein such as in a homopolymer or graft-copolymer can have any of the foregoing linkage profiles, for example.

[0114] An alpha-1, 3-glucan-producing GTF herein is capable of producing alpha-1, 3-glucan with a DPw, DPn, or DP of about, less than about, or at least about, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, or 1650, for example. DPw, DPn, or DP can optionally be expressed as a range between any two of these values. Merely as examples, the DPw, DPn, or DP can be about 100-1650, 200-1650, 300-1650, 400-1650, 500-1650, 600-1650, 700-1650, 100-1250, 200-1250, 300-1250, 400-1250, 500-1250, 600-1250, 700-1250, 100-1000, 200-1000, 300-1000, 400-1000, 500-1000, 600-1000, 700-1000, 100-900, 200-900, 300-900, 400-900, 500-900, 600-900, 700-900, 11-25, 12-25, 11-22, 12-22, 11-20, 12-20, 20-300, 20-200, 20-150, 20-100, 20-75, 30-300, 30-200, 30-150, 30-100, 30-75, 50-300, 50-200, 50-150, 50-100, 50-75, 75-300, 75-200, 75-150, 75-100, 100-300, 100-200, 100-150, 150-300, 150-200, or 200-300. Alpha-1,3-glucan as disclosed elsewhere herein such as in a homopolymer or graft-copolymer can have any of the foregoing molecular weight profiles, for example.

[0115] In some aspects, a GTF enzyme can be any as disclosed herein and include 1-300 (or any integer there between [e.g., 10, 15, 20, 25, 30, 35, 40, 45, or 50]) residues on the N-terminus and / or C-terminus. Such additional residues can be from a corresponding wild type sequence from which the GTF enzyme is derivable, or can be a heterologous sequence such as an epitope tag (at either N- or C-terminus) or a heterologous signal peptide (at N-terminus), for example. A GTF enzyme herein typically lacks an N-terminal signal peptide; such an enzyme can optionally be characterized as being mature if its signal peptide was removed during a secretion process.

[0116] A GTF enzyme herein can typically be derived from bacteria. Examples of bacterial GTF enzymes are those derived from a Streptococcus species, Leuconostoc species, or Lactobacillus species. Examples of Streptococcus species include S. salivarius, S. sobrinus, S. dentirousetti, S. downei, S. mutans, S. oralis, S. gallolyticus and S. sanguinis. Examples of Leuconostoc species include L mesenteroides, L amelibiosum, L argentinum, L carnosum, L citreum, L cremoris, L dextranicum and L fructosum. Examples of Lactobacillus species include L. acidophilus, L. delbrueckii, L. helveticus, L. salivarius, L. casei, L. curvatus, L. plantarum, L. sakei, L. brevis, L. buchneri, L. fermentum and L. reuteri.

[0117] A GTF enzyme herein can be prepared by fermentation of an appropriately engineered microbial strain, for example. Recombinant enzyme production by fermentation can be done, for example, using microbial species such as E. coli, Bacillus strains (e.g., B. subtilis), Ralstonia eutropha, Pseudomonas fluorescens, Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, and species of Aspergillus (e.g., A. awamori) and Trichoderma (e.g., T. reesei) (e.g., see Adrio and Demain, Biomolecules 4:117-139, 2014, which is incorporated herein by reference). A nucleotide sequence encoding a GTF amino acid sequence is typically linked to a heterologous promoter sequence to create an expression cassette for the enzyme, and / or is codon-optimized accordingly. Such an expression cassette can be incorporated in a suitable plasmid or integrated into the microbial host chromosome. The expression cassette can include a transcriptional terminator nucleotide sequence following the amino acid coding sequence. The expression cassette can also include, between the promoter sequence and GTF amino acid coding sequence, a nucleotide sequence encoding a signal peptide (e.g., heterologous signal peptide) that is designed for direct secretion of the GTF enzyme. At the end of fermentation, cells can be ruptured accordingly (generally when a signal peptide for secretion is not employed) and the GTF enzyme can be isolated or purified using methods such as precipitation, filtration, and / or concentration. Alternatively, a lysate or extract comprising a GTF can be used without further isolation or purification. If the GTF was secreted (i.e., it is present in the fermentation broth), it can optionally be used as isolated / purified from, or as comprised in, the fermentation broth. The activity of a GTF enzyme can be confirmed by biochemical assay, such as measuring its conversion of sucrose to glucan polymer.

[0118] In some aspects, both of (i) a glucosyltransferase enzyme herein that synthesizes alpha-1, 6-glucan and (ii) a glucosyltransferase enzyme herein that synthesizes alpha-1,3-glucan are contacted with a food product / precursor. Such can result in producing, for example, an alpha-glucan in the food product / precursor that is a graft copolymer comprising:

[0119] (i) an alpha-1, 6-glucan (dextran) backbone, wherein at least about 50% of the glycosidic linkages of the alpha-1, 6-glucan (dextran) backbone are alpha-1,6 linkages, and

[0120] (ii) at least one alpha-1,3-glucan side chain, wherein at least about 50% of the glycosidic linkages of the alpha-1, 3-glucan chain are alpha-1,3 linkages.

[0121] Such a graft copolymer can be aqueous-soluble or aqueous-insoluble. Dextran backbone of an alpha-glucan graft copolymer herein can be dextran as presently disclosed, for example, or can be as disclosed (e.g., molecular weight, linkage / branching profile, production method) in U. S. Patent Appl. Publ. Nos. 2016 / 0122445, 2017 / 0218093, 2018 / 0282385, 2020 / 0165360, or 2019 / 0185893, which are each incorporated herein by reference. In some aspects, a dextran backbone (before being integrated into a graft copolymer) has been alpha-1,2- and / or alpha-1, 3-branched; the percent alpha-1,2 and / or alpha-1,3 branching of a backbone of a graft copolymer herein can be about, at least about, or less than about, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 2-25%, 2-20%, 2-15%, 2-10%, 5-25%, 5-20%, 5-15%, 5-10%, 7-13%, 8-12%, 9-11%, 10-25%, 10-20%, or 10-15%, for example. Alpha-1, 3-glucan side chain(s) of an alpha-glucan graft copolymer herein can be alpha-1,3-glucan as presently disclosed, for example, or can be as disclosed (e.g., molecular weight, linkage profile), in U. S. Patent Nos. 7000000, 8871474, 10301604, or 10260053, or U. S. Patent Appl. Publ. Nos. 2019 / 0112456, 2019 / 0078062, 2019 / 0078063, 2018 / 0340199, 2018 / 0021238, 2018 / 0273731, 2017 / 0002335, 2015 / 0232819, 2015 / 0064748, 2020 / 0165360, 2020 / 0131281, or 2019 / 0185893, which are each incorporated herein by reference.

[0122] One, two, three, or more different GTF enzymes that synthesize alpha-1,6-glucan herein can be used, for example, in a fructose reduction method. Similarly, one, two, three, or more different GTF enzymes that synthesize alpha-1, 3-glucan herein can be used, for example, in a fructose reduction method. In some aspects, an alpha-1,6-glucan-producing GTF(s) can be added to (made to contact) a food product / precursor before adding an alpha-1, 3-glucan-producing GTF(s), while in some aspects both these types of GTF enzymes can be added at about the same time (simultaneously). Still, in some aspects, an alpha-1, 3-glucan-producing GTF(s) can be added to a food product / precursor before adding an alpha-1, 6-glucan-producing GTF(s). Still, in some aspects, a dextran as disclosed herein, but produced exogenously to the food product / precursor, can be added as an ingredient to a food product / precursor to which an alpha-1, 3-glucan-producing GTF has already been added or will be added. While not being held to any particular theory, it is believed that addition of at least one alpha-1,6-glucan-producing GTF (and / or exogenously produced dextran) and at least one alpha-1, 3-glucan-producing GTF in a food product / precursor herein allows for production of a dextran-alpha-1, 3-glucan graft copolymer as presently disclosed, possibly along with production of dextran and / or alpha-1, 3-glucan homopolymer(s) (i.e., alpha-1, 6-glucan and / or alpha-1, 3-glucan produced independent from the production of graft copolymer). However, it is believed possible that, in some aspects of using both an alpha-1, 6-glucan-producing GTF and an alpha-1, 3-glucan-producing GTF, only dextran and / or alpha-1, 3-glucan homopolymer(s) is / are produced with little (e.g., < 5 wt% of all glucan products) or no production of graft copolymer. The molecular weight and / or linkage profile of alpha-glucan produced by a GTF enzyme (dextransucrase or alpha-1, 3-glucan-producing GTF) as generally disclosed above can be as observed, for example, in an isolated reaction consisting of, or essentially of, water, sucrose, GTF enzyme and optionally one or more salts and / or buffer. In some aspects, the molecular weight and / or linkage profile of alpha-glucan as produced by one or both of these types of GTF enzyme in a food product / precursor herein may be different from what is produced in the foregoing isolated reaction.

[0123] In some aspects in which both an alpha-1,6-glucan-producing GTF and an alpha-1, 3-glucan-producing GTF are used, the ratio of the GTF enzyme that synthesizes alpha-1, 6-glucan to the GTF enzyme that synthesizes alpha-1, 3-glucan in step (b) is about 85:15 to about 95:5. Yet, in some aspects, an alpha-1,6-glucan-producing GTF to alpha-1, 3-glucan-producing GTF ratio can be about 97.5:2.5, 95:5, 92.5:7.5, 91:9, 90:10, 89:11, 87.5:12.5, 85:15, 82.5:17.5, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 5:95, or 2.5:97.5, or range between any two of these ratios (e.g., about 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). The amount of each enzyme (active enzyme) for purposes of determining a ratio thereof herein can be on a molar, weight, or GTF activity basis, for example. The activity of a GTF enzyme for preparing a ratio herein can optionally be determined as disclosed in U. S. Patent Appl. Publ. No. 2014 / 0087431 or Int. Patent Appl. Publ. No. W02023 / 055902, which are incorporated herein by reference. For example, a full (e.g., “100%”) complement of a GTF enzyme for setting up a ratio herein can be that amount of enzyme that can convert most of (e.g., >95%, >98%, >99%), or all of, sucrose in a GTF reaction comprising or consisting of water, sucrose (e.g., 50 or 100 g / L), the GTF, and optionally buffer / salt in a given amount of time (e.g., 6, 12, 18, 24, 30, or 36 hours); such a measured amount can optionally be characterized as a normalized amount of GTF.

[0124] An epimerase enzyme (and / or any other enzyme as presently disclosed such as a GTF or FTF) for use in a method herein is typically in purified form. A purified enzyme can be essentially free from insoluble and / or soluble components of an organism / cell used to produce the enzyme, and / or any medium that was used for cellular fermentation of the enzyme. In some aspects, a purified enzyme denotes an enzyme preparation that contains less than 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% by weight of other material (e.g., polypeptide material) with which the enzyme is natively or recombinantly associated. In some aspects, an epimerase, GTF, FTF, and / or any other enzyme herein is not comprised in or otherwise associated with (e.g., expressed by) a microbial (e.g., bacterial, yeast, fungal, algal) cell that might be present (e.g., endogenously or purposely added) in a food product / precursor herein; however, in some aspects any foregoing enzyme(s) is comprised in or otherwise associated with (e.g., expressed by) a microbial (e.g., bacterial, yeast, fungal, algal) cell such as one that heterologously expresses the enzyme(s) (i.e., recombinant cells). Contacting a food product / precursor with a foregoing enzyme(s) herein typically is not performed in an oral cavity or other environment in which unpurified / non-isolated enzyme can possibly be present.

[0125] An epimerase enzyme (and / or any other enzyme as presently disclosed such as a GTF or FTF) for use in a method herein can be comprised in a sterile-filtered preparation, for example. In some aspects, an enzyme can be sterile-filtered inline while applying the enzyme to a food product / precursor herein. In some aspects, an enzyme can be added to a food product / precursor that has been pasteurized (after pasteurization), or alternatively an enzyme can be added before pasteurizing the food product / precursor. In some aspects, an enzyme can be added to a food product / precursor that has been fermented (after fermentation), or alternatively an enzyme can be added during or before fermenting the food product / precursor. An epimerase enzyme (and / or any other enzyme as presently disclosed such as a GTF or FTF) in some aspects for use in a method herein can be comprised in a preparation that is substantially free of (e.g., <0.5, <0.1, <0.05 wt%) any other enzyme(s) such as a lipase, protease, amylase, mannanase, pectinase, cellulase, and / or p-nitrobenzylesterase; such a preparation typically has little or no detectable activity(ies) of such other enzyme(s).

[0126] A food product / precursor herein can be brought into contact, in step (b), with one or more epimerase enzymes (and / or any other enzyme as presently disclosed such as a GTF or FTF) herein by mixing / stirring / blending, for example. Incubation of enzyme(s) in the food product / precursor can be for about, or at least about, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 24, 30, 36, 42, 48, 72, 96, 0.5-3, 0.5-2.5, 0.5-2, 0.5-1.5, 1-3, 1-2.5, 1-2, 1-1.5, 1.5-3, or 1.5-2 hours, or for about, or at least about, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 days (or a range between any two of these hours and / or days). The temperature for incubating one or more enzymes in a food product / precursor herein can be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 2-5, 2-10, 2-15, 2-20, 2-25, 2-30, 2-35, 2-40, 2-45, 2-50, 3-5, 3-10, 3-15, 3-20, 3-25, 3-30, 3-35, 3-40, 3-45, 3-50, 5-10, 5-15, 5-20, 5-25, 5-30, 5- 35, 5-40, 5-45, 5-50, 15-20, 15-25, 15-30, 15-35, 15-40, 15-45, 15-50, 20-25, 20-30, 20-35, 20-40, 20-45, 20-50, 25-30, 25-35, 25-40, 25-45, 25-50, 30-35, 30-40, 30-45, or 30-50 °C, for example.

[0127] Typically, a food product / precursor brought into contact with an epimerase enzyme (and / or any other enzyme as presently disclosed such as a GTF or FTF) herein contains water (i.e., it is an aqueous composition), and / or water is introduced to the food product / precursor before or during contacting with one or more of these enzymes.

[0128] Enzyme(s) can be added to a food product / precursor in dry form (e.g., powder, flakes, lyophilized enzyme preparation) (typically to an aqueous food product / precursor) or wet form. In some aspects, a food product / precursor can be combined with enzyme(s) under dry conditions (resulting combination is dry), after which time water or an aqueous solution is added. Thus, depending on how ingredients and enzyme(s) herein are introduced to each other, steps (a) and (b) can optionally be considered to be performed simultaneously or separately. The water content of a food product / precursor can be about, or at least about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99 wt%, for example. The pH of a food product / precursor herein, and / or the pH for incubating one or more enzymes in a food product / precursor herein, can be 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, for example. A food product / precursor in some aspects 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 / alkaline (e.g., pH > 7.5, 8.0, 8.5, 9.0, 9.5).

[0129] An epimerase enzyme (and / or any other enzyme as presently disclosed such as a GTF or FTF) herein can optionally be provided by introducing a recombinantly engineered cell (e.g., a microbial cell such as a bacterial or fungal / yeast cell) to the food product / precursor provided in step (a), wherein the cell recombinantly (heterologously) expresses and secretes the enzyme(s) in and / or around the food product / precursor. Such a cell can be that of a microbe that is amenable to recombinant engineering and useful in food processing (e.g., fermentation), such as a microbial cell disclosed herein (as applicable). In some aspects, a recombinantly engineered cell that is provided to the food product / precursor in step (a) can be inactive and / or non-viable in some manner, such as by having been killed (but preferably in a manner that otherwise retains cellular shape / structure). For example, a cell can be rendered inactive and / or non-viable by being irradiated or being treated with a sterilizing agent / chemical (e.g., ethylene oxide). Typically, the means for cell inactivation and / or killing preserves at least some of the three-dimensional shape / structure of the cell, and / or ensures that enzyme(s) that had been expressed by the cell remains active and typically remains associated with the inactive / non-viable cell (e.g., such as by being associated with a cellular membrane via an optional transmembrane domain or membrane-binding domain of the enzyme(s) [e.g., fused to the enzyme]). An inactive / non-viable cell typically is porous, and optionally can be immobilized on a support (e.g., an inert, water-insoluble material, such as of a particle or surface).

[0130] A “food product / precursor” (i.e., a food product or precursor) as provided in step (a) of a method in some aspects of the present disclosure method can comprise sucrose that is endogenous to the food product / precursor (e.g., its sucrose is native), and / or can comprise sucrose that has been added to the food product / precursor (either during or after its preparation as an ingredient). Step (a) can thus optionally comprise adding sucrose to the food product / precursor. The sucrose content of a food product / precursor finally provided in step (a) herein, regardless of the original source of the sucrose, can be 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%, for example. In some aspects, sucrose can be provided as white refined sucrose, or in an unrefined form such as disclosed in U. S. Patent No. 9719121, for example, which is incorporated herein by reference. Sucrose can optionally be added to a food product / precursor when adding an epimerase and / or another enzyme (e.g., GTF or FTF) to the food product / precursor.

[0131] In some aspects, a food product / precursor as provided in step (a) of a method herein further comprises at least one disaccharide in addition to sucrose, and / or at least one oligosaccharide. An oligosaccharide can have 3-15 or 3-20 monomeric units (i.e., DP3-DP15 or DP3-DP20), for example (e.g., DP3-DP5, DP3-DP6); thus, in some aspects, a polysaccharide herein has more than 15 or 20 monomeric units. A disaccharide and / or oligosaccharide herein can comprise only glucose monomeric units, for example, and / or one or more other types of monosaccharides (e.g., galactose, fructose, mannose) as monomeric units. Examples of disaccharides herein (in addition to sucrose) include maltose, isomaltose, lactose, lactosucrose, nigerose, leucrose, trehalulose, maltulose, isomaltulose, and turanose. Examples of oligosaccharides herein include gluco-oligosaccharides (gluco-oligomers) such as malto-oligosaccharides (MOS) and isomalto-oligosaccharides (IMO), and galacto-oligosaccharides (GOS). A disaccharide and / or oligosaccharide can be added to a food product / precursor either during or after preparation of the food product / precursor. Such addition can be from a source physically outside of the food product / precursor (i.e., as an ingredient), and / or can be via in situ production in the food / precursor such as by one or more enzymes that are endogenous and / or exogenous to the food / precursor. An enzyme that is added to a food product / precursor (i.e., exogenous enzyme) for producing a disaccharide and / or oligosaccharide can be added, for example, in the same or similar manner in which an epimerase or other enzyme herein (e.g., GTF or FTF) is added (e.g., time, temperature, pH), and can be added before, during, or after the addition of the enzyme. Such an enzyme can be a transglucosidase (EC [enzyme code] 2.4.1.24) or a transgalactosylating beta-galactosidase, for example. Suitable transglucosidases herein include FoodPro® TGO and those disclosed in U. S. Patent Appl. Publ. Nos.

[0132] 2008 / 0229514 or 2015 / 0240279, or U. S. Patent No. 4689296, all of which are incorporated herein by reference. An EC 2.4.1.24 transglucosidase (also termed as “1,4-alpha-glucan 6-alpha-glucosyltransferase”) can transfer an alpha-D-glucosyl residue of an alpha-1,4 -glucan, -oligosaccharide (i.e., MOS), or -disaccharide (i.e., maltose) to the primary hydroxy group of free glucose or glucose in an alpha-1,4 -glucan, -oligosaccharide (i.e., MOS), or -disaccharide. Thus, an EC 2.4.1.24 transglucosidase produces isomalto-oligosaccharides (IMO) (e.g., DP3-DP5 or DP3-DP6) in some aspects. Suitable transgalactosylating beta-galactosidases herein are disclosed in U. S. Patent Appl. Publ. No. 2013 / 0189746 or U. S. Patent Nos. 10531672 or 10683523, for example, which are incorporated herein by reference. A transgalactosylating betagalactosidase is an enzyme that degrades lactose by transferring the galactose of lactose to galactose, glucose, or other acceptor thereby producing galactooligosaccharides (GOS) (e.g., GOS can also be an acceptor for forming a longer GOS). A particular example of such an enzyme is Nurica™ (IFF). A transglucosidase or transgalactosylating beta-galactosidase herein can be dosed into a food product / precursor herein 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), for example.

[0133] In some aspects, a food product / precursor as provided in step (a) of a method herein has, aside from the sucrose, little (e.g., less than 0.5, 0.25, 0.1, 0.05, 0.025, or 0.01 wt%, or not detectable) or no disaccharides and / or oligosaccharides (or little or no particular disaccharide or oligosaccharide). A food product / precursor as produced in step (b) of a method herein can likewise have, for example, little of no disaccharides and / or oligosaccharides (or little or no particular disaccharide or oligosaccharide), and also have little (e.g., as above) or no sucrose. A disaccharide or oligosaccharide in such aspects can be any as disclosed herein (e.g., lactose, maltose, isomaltose, MOS, IMO, GOS). One or more glycosidase enzymes (glycosidic-active enzyme) can be used, for example, in a food product / precursor to reduce or eliminate the presence of disaccharide(s) and / or oligosaccharide(s), and can be added, for example, in the same or similar manner in which an epimerase or other enzyme herein (e.g., GTF or FTF) herein is added (e.g., time, temperature, pH), and can be added before, during, or after the addition of one or more of these enzymes. A glycosidase herein can be, for example, a beta-galactosidase (EC 3.2.1.23; e.g., lactase [EC 3.2.1.108]) or alphaglucosidase (EC 3.2.1.20). Suitable beta-galactosidases herein include Bonlacta™ lactase (IFF) and lactases disclosed in U. S. Patent No. 10531672, which is incorporated herein by reference. A lactase herein is a type of beta-galactosidase enzyme that catalyzes hydrolysis of lactose to glucose and galactose. Suitable alpha-glucosidases herein include those disclosed in U. S. Patent Appl. Publ. No. 2015 / 0240278, which is incorporated herein by reference. In some aspects, an alpha-glucosidase that is used in the disclosed method is able to hydrolyze an alpha-1,4 or alpha-1,6 glucosidic linkage, or and / or is unable to hydrolyze an alpha-1,3 glucosidic linkage. A glycosidase herein can be dosed into a food product / precursor herein 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), for example.

[0134] A food product / precursor in some aspects can be a dairy product / precursor, such as a dairy beverage or food. Suitable examples of a dairy product / precursor herein include milk, cheese, yogurt, dessert, cream, and butter. Milk herein can be whole milk (e.g., ~3% fat), ~2% fat milk, ~1% fat milk (“low-fat”), or fat-free (non-fat) milk, for example. Milk, whether used directly as a beverage or as a precursor for preparing a dairy product / precursor herein, can be from a cow, goat, sheep, buffalo, yak, llama, camel, or horse, for example. Milk can optionally be pasteurized before, or after, contacting it with one or more GTF and second enzymes herein. A cheese herein can be, for example, hard or semi-hard cheese (e.g., Cheddar, mozzarella, Swiss, parmesan, provolone), soft or semi-soft cheese (e.g., ricotta, cottage cheese, feta, American, brie), processed, or non-processed. A yogurt herein can be, for example, whole milk yogurt, low-fat yogurt, fat-free yogurt, or Greek yogurt (e.g., plain, low-fat, non-fat). A yogurt herein can optionally contain fruit and / or be flavored. A yogurt herein can optionally be drinkable (i.e., yogurt beverage). A dairy dessert in some aspects can be a pudding (e.g., whole milk, 2% milk), frozen yogurt (e.g., low-fat), ice cream (e.g., low-fat), sherbet, milk shake, gelato, or custard; thus, in some aspects a dairy dessert can be a frozen dairy dessert (e.g., ice cream, sherbet, milk shake, frozen yogurt, gelato). Ice cream can be hard ice cream or soft (soft-serve) ice cream, for example. A dairy beverage in some aspects can be milk, chocolate milk, coffee milk, flavored milk, yogurt beverage, kumis, ryazhenka, ayran, lassi, cholado, licuado, or kefir. A dairy cream can be clotted cream (e.g., >55% milkfat), heavy cream (e.g., >36% milkfat), whipping cream (e.g., 30%-36% milkfat), light cream (e.g., 18%-30% milkfat), sour cream (>18% milkfat), half-and-half (e.g., 10.5%-18% milkfat), or ice cream (e.g., >10% milkfat). A dairy product / precursor herein can be lactose-free or have a reduced lactose content, for example. A dairy product / precursor herein can be a fermented dairy product / precursor (e.g., yogurt, buttermilk, creme fraiche, quark, fromage frais, soured milk, vinegar), for example. Some dairy products / precursors herein include dairy confections such as milk chocolate, white chocolate, caramel, and toffee. A dairy product / precursor in some aspects can be any as disclosed in W02020 / 010176, U. S. Patent Appl. Publ. Nos.

[0135] 2013 / 0230623, 2005 / 0244541, 2017 / 0135360, 2009 / 0304864, 2017 / 0094987, or 2003 / 0152685, or U. S. Patent Nos. 5482728 or 6352734, all of which are incorporated herein by reference.

[0136] A food product / precursor in some aspects can be a flour-based or meal-based dough, baked product (bakery product), or extruded product, such as any of those disclosed in WO2021 / 034561 or U. S. Patent Appl. Publ. No. 2017 / 0218093, which are incorporated herein by reference. Examples of baked products, or a dough (precursor) thereof, include bread (e.g., buns, sourdough, rye, whole wheat, pita, flatbread, tortilla, cornbread, brioche, white, baguette, bagels, banana, ciabatta, brown, challah, focaccia, multigrain, bread sticks, soda bread, pumpernickel, potato bread, biscuits, English muffins, whole grain, matzo, lavash, croutons, pizza crust) (leavened or unleavened), cake (e.g., carrot cake, red velvet, angle food, pound cake, chocolate, white, black forest, tiramisu, coffee cake, cheesecake, devil’s food, upside-down cake, Boston cream pie, Swiss roll, lemon cake, short cake, chiffon cake, butter cake, spice cake, rum cake, sponge cake, marble cake, coconut cake, pandan cake), muffins, brownies, scones, cookies, bars, custards, pies, crackers, pretzels, pastries, pudding and tarts. Examples of an extruded product include pasta (e.g., spaghetti, rotini, fusilli, penne, bucatini, macaroni / maccheroni, rigatoni, fettuccine, linguine, vermicelli, ziti, farfalle, gomiti / elbow, rotelle), cereal (e.g., direct expanded cereal, filled cereal, flakes, breakfast cereal), some bread products (e.g., croutons, bread sticks, flat breads), pre-made cookie-dough, dry and semi-moist petfood (e.g., kibbles), and snacks (e.g., cheese curls, filled pillow puffs, chips [e.g., corn chips, pita chips, processed potato chips, tortilla chips], snack sticks [e.g., vegetable sticks], puffed shaped products such as curls [e.g., cheese curls], balls, tubes, bananas, cups, bowls, disks, baby food puffs). Pasta herein can be extruded (e.g., see above) and / or flattened / rolled (e.g., lasagna), fresh or dried, long or short, minute / soup pasta (pastina), filled (e.g., tortellini, ravioli, agnolotti, tortelli), stretched (e.g., cencioni, corzetti, foglie d’ulivo, orecchiette), and / or egg pasta, for example.

[0137] A food product / precursor in some aspects can be a syrup or beverage, for example, such as any of those disclosed in U. S. Patent Appl. Publ. Nos. 2010 / 0040728, 2017 / 0006902, 2017 / 0218093, 2013 / 0216652, 20180146699, 2009 / 0123603, 2021 / 0076724, or 2017 / 0332670, all of which are incorporated herein by reference. A beverage in some aspects 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, tangerine juice, or cherry juice; vegetable juice such as carrot juice, tomato juice, or mixed-vegetable juice), sweetened beverage (soda / soft drink, sweetened tea of coffee), ready-to-drink (RTD), or any other beverage having natural and / or added sugar (sucrose).

[0138] A food product / precursor in some aspects can be a fermented food product / precursor, for example, such as any of those disclosed in W02002 / 034061. In some aspects, a food product / precursor provided in step (a) of a method herein is fermented, while in some aspects a food product / precursor is fermented (or further fermented) during or after steps of adding an epimerase and / or a GTF or FTF. Thus, step (a) of a method herein can optionally comprise a step of fermenting a food product / precursor (e.g., before or after adding sucrose, if applicable). Thus, step (b) of a method herein can optionally comprise fermenting the food product / precursor while contacting it with an epimerase and / or GTF or FTF. Thus, a method herein can optionally comprise, following step (b), a step of fermenting the food product / precursor. One or more bacterial and / or yeast cultures can be used for fermentation of a food product / precursor herein. Suitable bacteria for food fermentation herein include lactic acid bacteria, for example, such as Lactobacillaceae family species such as those of the Pediococcus genus (e.g., P. acidilactici, P. pentosaceus), Lactobacillus genus (e.g., L sakei, L fermentum [formerly L cellobiosus], L rhamnosus, L plantarum, L brevus, L kefir, L casei, L paracasei, L acidophilus, L salivarius, L buchneri, L helveticus, L reuteri, L johnsonii, L crispatus, L gasseri, L delbruecki such as subsp. L bulgaricus), Lactococcus genus (e.g., L lactis such as subsp. L cremoris), Leuconostoc genus (e.g., L citreum, L mesenteroides), and Streptococcus genus (e.g., S. thermophilus). Suitable bacteria for food fermentation in some aspects can be species from the Bifidobacterium genus (e.g., B. bifidum, B. longum, B. animalis, B. breve, B. infantis) or Propionibacterium genus (e.g., P. freudenreichii such as subsp. P. shermanii) (propionic acid bacteria). In some aspects, a bacteria for food fermentation herein can be characterized as Gram-positive, sphere-shaped, rod-shaped, anaerobic, aerobic, acid-tolerant, non-sporulating, GRAS (generally regarded as safe), and / or probiotic. A bacteria for food fermentation (e.g., yogurt or other dairy fermentation) in some aspects can be an acidic culture / strain (or mix) (e.g., (YO-MIX 863, YO-MIX 410, or YO-MIX T42, available from IFF) that produces food with a pH of about, for example, 2.5-4.5, 3.0-4.5, 4.2-4.4, or 4.3, or it can be a mild culture / strain (or mix) (e.g., YO-MIX PRIME 900 or YO-MIX M01, available from IFF) that produces food with a pH of about, for example, 4.6-5.5, 4.6-6.0, 4.5-4.7, or 4.6. A bacteria herein (e.g., mild of acidic) can optionally be mesophilic (temperature for optimal growth typically at 20 to 25 °C [room temperature]). A mix of bacteria in a culture for food fermentation can comprise one, two, three, four, five, six or more different species and / or sub-species of bacteria, for example. Suitable yeast for food fermentation in some aspects include species from the Saccharomyces genus (e.g., S. cerevisiae, S. pastorianus, S. boulardii, S. kluyveri), Pichia genus (e.g., P. kluyveri, P. fermentans), and Candida genus (e.g., C. humilis, C. famata). A yeast in some aspects can be characterized as baker’s (baking) yeast, brewing yeast, winemaking yeast, probiotic yeast, budding / fission yeast, or GRAS. A mix of yeast in a culture for food fermentation can comprise one, two, three, four, five, six or more different species and / or sub-species of yeast, for example. A food product / precursor that is fermented or will be fermented can be a dairy product herein (e.g., as above, such as yogurt), beer, beer wort, wine, pomace, cider, miso, kimchi, sauerkraut, pickles / pickle juice, soybean curd, tofu, kombucha, soy sauce, bread, sourdough, or meat, for example.

[0139] A food product / precursor in some aspects can be a confectionary, for instance. Examples of confectioneries herein include boiled sugars (hard boiled candies [i.e., hard candy]), dragees, jelly candies, gums, licorice, chews, caramels, toffee, fudge, chewing gums, bubble gums, nougat, chewy pastes, halawa, tablets, lozenges, icing, frosting, pudding, gels (e.g., fruit gels, gelatin dessert), aerated confectioneries, marshmallows, baked confectioneries.

[0140] A food product / precursor in some aspects can be a non-dairy food product / precursor. For example, a non-dairy food product / precursor can be a plantbased milk (milk substitute) or comprise a plant-based milk (and lack, or have little of [e.g., < 0.5 wt%], any dairy ingredients] such as lactose, whey, casein, and / or milk fat). In some aspects, a non-dairy food product / precursor is fermented (e.g., a non-dairy yogurt product / precursor such as a plant-based yogurt product / precursor). Plant-based ingredient(s) forming the basis for a non-dairy food product / precursor herein can be from nuts / seeds (e.g., almonds, cashews, macadamias, hemp seed, quinoa, flax seed), grains / cereal (e.g., oats, rice), fruit (e.g., coconut, banana), or vegetables (e.g., legumes such as beans [e.g., soybeans, mug beans] and peas), for example. In some aspects, a non-dairy food product / precursor is a milk of any of the foregoing nuts / seeds, grains / cereal, fruit, or vegetables; a fermented form of any of these milks can be a yogurt, for example.

[0141] A food product / precursor in some aspects can be a cream soup, gravy, sauce (e.g., tomato sauce), salad dressing, mayonnaise, jam, jelly, marmalade, syrup, pie filling, batter for fried foods, batter for pancakes / waffles, cake icing and glazes, whipped topping, pet food, or animal / livestock feed.

[0142] A food product / precursor in some aspects can comprise one or more additional ingredients such as a vegetable component (e.g., vegetable oil, vegetable protein, vegetable carbohydrates), enzyme, fat, oil, flavoring agent, microbial culture (e.g., probiotic culture), salt, sweetener, acid, fruit / vegetable (e.g., orange, apple, mango, peach, plum, banana, date, apricot, grapefruit, papaya, pineapple, raspberry, strawberry, blueberry, blackberry, pear, tangerine, cherry, grape, melon, watermelon, cantaloupe, honeydew melon, kiwi, lemon, lime, carrot, tomato), or fruit / vegetable juice (juice concentrate), puree, or other processed form (e.g., sliced, cubed, chopped pieces) of a fruit / vegetable as disclosed, or any other component suitable for use as an ingredient in a food product / precursor. Such one or more additional ingredients can be as disclosed in U. S. Patent Appl. Publ. Nos. 2016 / 0122445 or 2017 / 0218093 (both incorporated herein by reference), for example, and / or can be natural or artificial. Examples of ingredients suitable as sweeteners (or for any other purpose such as flavoring) include acesulfame potassium, advantame, agave syrup, alitame, aspartame, barley malt syrup, birch syrup, brazzein, brown rice syrup, cane juice, caramel, coconut palm sugar, com syrup, curculin, cyclamate, dextrose, erythritol, fructo-oligosaccharide, fructose (levulose), galactose, glucose (dextrose), glycerol (glycerin), glycyrrhizin, golden syrup, high fructose com syrup (e.g., HFCS-42, -55, -90), high maltose com syrup (HMCS), honey, hydrogenated starch hydrolysate (HSH), isomalto-oligosaccharide (IMO), inulin, inverted 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, refiners syrup, saccharin, sorbitol (glucitol), sorghum syrup, stevia I steviol glycoside (e.g., a rebaudioside such as rebaudioside A, rebaudioside D, or rebaudioside M), sucralose, sugar alcohol, tagatose, thaumatin, trehalose, xylitol, and yacon syrup.

[0143] In some aspects, a food product / precursor as produced by a method of the present disclosure can be concentrated, dried (e.g., to a powder), reconstituted (following concentration or drying), or processed (e.g., frozen) in any other manner. Examples of such products include sweetened milk, concentrated milk, condensed milk (e.g., sweetened condensed milk), evaporated milk, dried milk powder, frozen dairy product (e.g., ice cream) concentrated juice, or dried juice powder.

[0144] In some aspects, a method herein can further comprise a step of freezing a food product / precursor (e.g., a dairy food product / precursor, or a plant-based food product / precursor) after contacting it with an epimerase and / or a GTF or FTF. This method can produce a frozen dairy product herein such as ice cream or frozen yogurt, or a plant-based ice cream or frozen yogurt, for example. Freezing can be done at about -10, -15, -20, -25, -30, -35, -40, -20 to -40, -25 to -35 °C, for example.

[0145] Some aspects of the present disclosure concern a food product / precursor as produced by a fructose reduction method herein. Examples of such products / precursors are any food product / precursor as disclosed herein. Typically, such a food product / precursor can have any feature as disclosed herein (e.g., reduced sugar content, increased texture, improved physical appearance, reduced caloric content, increased dietary fiber, pH, temperature, age), as appropriate / applicable. Typically, such a food product / precursor comprises at least one epimerase and / or GTF or FTF as presently disclosed, and / or an alpha-glucan as presently disclosed.

[0146] Some aspects of the present disclosure are drawn to isolated compositions / products that comprise at least (I) an isolated epimerase, and / or (II) at least one of an (i) isolated glucosyltransferase enzyme that synthesizes alpha-1,6-glucan and / or an (ii) isolated glucosyltransferase enzyme that synthesizes alpha-1,3-glucan. Such a composition / product can optionally further comprise at least one fructose epimer (e.g., allulose) and / or alpha-glucan herein. An isolated composition / product can be in the form of and / or comprised in a household care product, personal care product, industrial product, ingestible product (e.g., food product / precursor such as any disclosed herein), or pharmaceutical product, for example, such as described in any of U. S. Patent Appl. Publ. 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 Int. Patent Appl. Publ. No. WO2016 / 133734, which are all incorporated herein by reference. In some aspects, a composition / product can comprise at least one component / ingredient of a household care product, personal care product, industrial product, pharmaceutical product, or ingestible product (e.g., food product / precursor) as disclosed in any of the foregoing publications and / or as presently disclosed.

[0147] Yet, in some aspects, a composition herein can be an isolated glucosyltransferase reaction composition that contains, or did contain (i.e., the reaction is complete and there is little [e.g., <5 wt% of original sucrose amount] or no sucrose present) at least water, sucrose, an isolated epimerase, and an (i) isolated glucosyltransferase enzyme that synthesizes alpha-1, 6-glucan and / or an (ii) isolated glucosyltransferase enzyme that synthesizes alpha-1, 3-glucan. Typically, such a reaction composition can produce one or more of an alpha-1, 6-glucan, alpha-1, 3-glucan, or a graft copolymer thereof (e.g., as disclosed herein), and further produce a fructose epimer such as allulose (optionally, fructose is also present, along with any remaining [unreacted] sucrose). Such a reaction composition can be one that is prepared and performed outside of a food product or precursor herein, for example. Such a reaction composition typically is under isolated conditions herein.

[0148] Non-limiting examples of compositions and methods disclosed herein include: 1. A method (process) of producing a food product / precursor, said method comprising: (a) providing a food product / precursor that comprises at least water and fructose, and (b) contacting the food product / precursor with an epimerase that converts fructose to a fructose epimer, wherein at least one fructose epimer is produced in the food product / precursor.

[0149] 2. The method of embodiment 1, wherein the fructose epimer is D-allulose (i.e., the epimerase is a D-allulose 3-epimerase) (i.e., D-allulose is produced in the food product / precursor).

[0150] 3. The method of embodiment 1 or 2, wherein the epimerase comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, 90%, or 95%) identical to SEQ ID NO:14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 35, 36, 37, 38, 39, 40, 41, 42, 43, 47, 48, 49, 50, 51, 52, 53, 54, or 55 (or to [i] SEQ ID NO:15, 17, 18, 19, 20, 22, 23, 24, 25, 28, 29, 35, 37, 38, 39, 40, 41, 42, or 43, [ii] SEQ ID NO: 14, 15, or 29, [iii] SEQ ID NO: 18 or 29, [iv] SEQ ID NO:35, or[v] 18, 29, or 35).

[0151] 4. The method of embodiment 1, 2, or 3, wherein the contacting occurs under conditions comprising a temperature of about 30 °C to about 90 °C.

[0152] 5. The method of embodiment 1, 2, 3, or 4, wherein the contacting occurs under conditions comprising a pH of about 3.5 to about 8.0.

[0153] 6. The method of embodiment 1, 2, 3, 4, or 5, wherein no metal cofactor has been added to the food product / precursor, or a metal cofactor has been added to the food product / precursor in an amount that is insufficient to allow activity of the epimerase. 7. The method of embodiment 1, 2, 3, 4, 5, or 6, further comprising contacting the food product / precursor with at least one glucosyltransferase enzyme, wherein the food product / precursor further comprises sucrose, wherein said contacting with the glucosyltransferase enzyme is performed simultaneously with, or after, contacting the food product / precursor with the epimerase, optionally wherein at least one alpha-glucan is produced in the food product / precursor, and / or optionally wherein the amount of leucrose produced (all or mostly by the glucosyltransferase enzyme) in the food product / precursor is less than the amount of leucrose that would have been produced in the food product / precursor if the epimerase was not (had not been) contacted with the food product / precursor.

[0154] 8. The method of embodiment 1, 2, 3, 4, 5, 6, or 7, wherein the food product / precursor provided in step (a) is provided by contacting a food product / precursor that comprises at least water and sucrose with at least one glucosyltransferase enzyme (i.e., all, or a portion of [e.g., some fructose can optionally already be present independent of GTF activity], the fructose in the food product / precursor in step [a] is produced by the glucosyltransferase enzyme) (i.e., contacting with the glucosyltransferase enzyme is performed prior to contacting the food product / precursor with the epimerase), optionally wherein at least one alpha-glucan is produced in the food product / precursor.

[0155] 9. The method of embodiment 7 or 8, wherein the glucosyltransferase enzyme is: (i) a glucosyltransferase enzyme that is capable of synthesizing alpha-1,6-glucan, wherein at least about 50% (e.g., > -90%, -95%, or -100%) of the glycosidic linkages of the alpha-1, 6-glucan are alpha-1,6 linkages, or (ii) a glucosyltransferase enzyme that is capable of synthesizing alpha-1,3-glucan, wherein at least about 50% (e.g., > -90%, -95%, or -100%) of the glycosidic linkages of the alpha-1, 3-glucan are alpha-1,3 linkages. 10. The method of embodiment 9, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha-1,6-glucan comprises an amino acid sequence that is at least 90% (e.g., > -95%) identical to SEQ ID NO: 1, 2, 11, 12, 56, or 57.

[0156] 11. The method of embodiment 9 or 10, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha-1,3-glucan comprises an amino acid sequence that is at least 80% (e.g., at least about 85%, 90%, or 95%) identical to residues 55-960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55-960 of SEQ ID NO:8, residues 55-960 of SEQ ID NO:9, or SEQ ID NO:13.

[0157] 11 A. The method of embodiment 9, 10, or 11, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha-1,3-glucan comprises an amino acid sequence that is at least 80% (e.g., at least about 85%, 90%, or 95%) identical to SEQ ID NO:3, 4, 5, 6, 7, 8, 9, or 13 (e.g., an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:5).

[0158] 12. The method of embodiment 9, 10, 11, or 11a, wherein both of said (i) glucosyltransferase enzyme that is capable of synthesizing alpha-1, 6-glucan and (ii) glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan are contacted with the food product / precursor.

[0159] 13. The method of embodiment 12, wherein an alpha-glucan is produced in the food product / precursor that is a graft copolymer comprising: (i) an alpha-1,6-glucan backbone, wherein at least about 50% (e.g., > -90%, -95%, or -100%) of the glycosidic linkages of the alpha-1, 6-glucan backbone are alpha-1,6 linkages, and (ii) at least one alpha-1, 3-glucan side chain, wherein at least about 50% (e.g., > -90%, -95%, or -100%) of the glycosidic linkages of the alpha-1, 3-glucan chain are alpha-1,3 linkages, wherein said graft copolymer (alpha-glucan graft copolymer) is aqueous-soluble or aqueous-insoluble.

[0160] 14. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12, or 13, further comprising contacting the food product / precursor with at least one fructosyltransferase enzyme, wherein the food product / precursor further comprises sucrose, optionally wherein at least one fructan is produced in the food product / precursor, and / or optionally wherein said contacting with the fructosyltransferase enzyme is performed prior to, simultaneously with, or after contacting the food product / precursor with the epimerase.

[0161] 15. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12, 13, or 14, further comprising contacting the food product / precursor with at least one lactase enzyme and / or glucose isomerase enzyme, optionally wherein said contacting with the lactase enzyme and / or glucose isomerase enzyme is performed prior to, simultaneously with, or after contacting the food product / precursor with the epimerase.

[0162] 16. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12, 13, 14, or 15, wherein step (a) comprises adding sucrose to the food product / precursor.

[0163] 17. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12, 13, 14, 15, or 16, wherein the food product / precursor is a dairy food product / precursor.

[0164] 18. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12, 13, 14, 15, or 16, wherein the food product / precursor is a non-dairy food product / precursor.

[0165] 19. The method of embodiment 18, wherein the non-dairy food product / precursor is plant-based.

[0166] 20. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12, 13, 14, 15, 16, 17, 18, or 19, wherein the food product / precursor of step (a) is fermented, or the method further comprises, during or after step (b), fermenting the food product / precursor.

[0167] 21. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12, 13, 14, 15, 16, 17, 18, 19, or 20, wherein the food product / precursor is a yogurt.

[0168] 22. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21, wherein the food product / precursor is a beverage or a juice. 23. A food product / precursor produced by the method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or22.

[0169] 24. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12, 13, 14, 15, or 16, but wherein the term “food product / precursor” is replaced with the term “composition” or “product”, optionally wherein the composition or product is not a food product / precursor.

[0170] 25. The method of embodiment 24, wherein the composition is an isolated glucosyltransferase (GTF) reaction composition having at least water, sucrose and a GTF herein (e.g., with features of embodiment 7, 8, 9, 10, 11, 11a, 12, or 13), wherein the GTF reaction composition is typically used for synthesizing alpha-glucan.

[0171] 26. The method of embodiment 24, wherein the composition or product is a household care product, personal care product, industrial product, ingestible product, pharmaceutical product, or medical product.

[0172] EXAMPLES

[0173] The present disclosure is further exemplified in the following Examples. It should be understood that these Examples, while indicating certain aspects herein, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain essential characteristics of the disclosed embodiments, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt the disclosed embodiments to various uses and conditions.

[0174] Materials / Methods

[0175] High performance liquid chromatography (HPLC) sugar analysis

[0176] Sugar composition was measured by HPLC with a Waters® 2695 Separations module or a ThermoScientific Dionex™ UltiMate 3000 HPLC, equipped with a Phenomonex Rezex™ RPM-Monosaccharide Pb2+ column (300 mm x 7.8 mm) and an Rl-detector. Water was used as the mobile phase at a flow rate of 0.400 mL / min. The column temperature was 70 °C. Samples were prepared for HPLC injection by mixing 0.7 g of sample with 0.3 g of 96% ethanol, followed by heat treatment for 10 minutes at 83 °C, cooling to room temperature and centrifugation (10 minutes at 15,000 rpm). The samples were then further diluted 1:10 with demineralized water and sterile-filtered before analysis. Signals from the HPLC were quantified against calibration standards of sugars eluting at the same time except for leucrose and oligosaccharides that were calculated against the sucrose standard. Sugar reduction was calculated by subtracting the total sum of mono- and di-saccharides in the test sample from the total sum of mono-and di-saccharides in the reference sample without enzymes added. The allulose product was not quantified and was not considered a sugar. Although leucrose and the DP2 isomers are likely to be metabolized differently in the body than traditional sugars, they are classified here as sugars. If they were not considered as sugars, then this would result in attaining an even further reduction in sugar.

[0177] Glucosyltransferase enzymes

[0178] Glucosyltransferase (GTF) enzymes 0768 (SEQ ID NO:1, also represented by SEQ ID NOs:2, 11 and 12) and an amino acid-substituted GTF 6855 variant (SEQ ID NO:3, “vGTFJ” herein) were used. Both these GTF enzymes use sucrose as a substrate to produce fructose and glucan (i.e., they are glucansucrases). GTF 0768 (a dextransucrase) produces a soluble alpha-glucan with a high alpha-1,6 linkage content (i.e., a type of dextran; refer to U. S. Patent Appl. Publ. No. 20160122445, which is incorporated herein by reference), whereas vGTFJ stably produces at high yield an insoluble alpha-glucan having about 100% alpha-1,3 linkages (the variant GTF of SEQ ID NO:4 similarly can be used herein to stably produce at high yield insoluble alphaglucan having about 100% alpha-1,3 linkages). Example 1

[0179] Assessment of D-Allulose 3-Epimerase Activity in Diluted Apple Juice Concentrate An apple concentrate containing 9% (w / w) sucrose, 15% (w / w) glucose and 30% (w / w) fructose was diluted 1:1 with demineralized water (MILLIQ) and adjusted to pH 5 using 5 M NaOH. There was little or no allulose present. It was then aliquoted in 1-mL portions and tempered to 50 °C. Epimerase samples (500 ppm) - known / benchmark D-allulose 3-epimerases and D-allulose 3-epimerase candidates - were prepared in 20% glycerol. Each enzyme sample (10 pL) was transferred into a new microtiter plate (Agilent 5042-1385, PP) containing 90 pL of the diluted apple concentrate solution. The incubations were held at 50 °C for 2 hours with shaking (650 rpm) in an iEMS incubator (ThermoFisher). The reactions were quenched by adding 100 pL of 100 mM pH 3.5 sodium acetate buffer. Each quenched reaction mixture was diluted 10-fold in water (MILLIQ) and filtered for allulose analysis by HPLC using an Agilent 1200 series system with a SHODEX SP0810 HPLC column. An allulose standard curve was generated for the calculation of allulose release from each enzymatic reaction. The detected allulose amount (mM) was used to indicate the epimerase activity of the sample enzyme.

[0180] Table 1 summarizes the epimerase activity exhibited by the benchmark epimerases and candidate epimerases in the diluted apple juice concentrate. Compared with the benchmark epimerases (AglEpi, CscEpi, CjiEpi), all the epimerase candidates showed higher activity. Among them, LspEpi4 outperformed, with 30% conversion of fructose to allulose, while the benchmark, CPA-Epi3, only showed 18% conversion.

[0181] Table 1

[0182]

[0183]

[0184] Any of the D-allulose 3-epimerases listed in Table 1 should be useful in a method of the present disclosure of treating a food or food precursor with both epimerase and glucosyltransferase enzymes.

[0185] Example 2

[0186] In situ Sugar Reduction in Fresh Fermented Dairy Using Epimerase with Glucosyltransferase.

[0187] A skimmed milk base was prepared by dissolving 9% of skimmed milk powder (33% protein, 1.2% fat, 54% carbohydrate) from BBA Lactalis (Laval, Mayenne, France) and 8% sucrose (Granulated Sugar 500, Nordic Sugar A / S, Denmark) in tap water. The skimmed milk base was tempered to 43 °C and then inoculated with a thermophilic starter culture (YO-MIX PRIME 900) at an inoculation rate of 20 DCU / 100 L. An amino acid-substituted GTF 6855 variant (SEQ ID NO:3, “vGTFJ” herein) was added at 0.1 wt% in the culture-inoculated skimmed milk base, either alone or in combination with an epimerase (BsuEpil, SEQ ID NO: 14). Fermentation of each aliquot was conducted at 43 °C until pH 4.60 was reached, after which the product was cooled to 4 °C. The sugar / oligosaccharide composition of each product was measured according to the Materials / Methods.

[0188] Results of measuring carbohydrate content (%w / w) are presented in Table 2. The percent total sugar reduction was calculated for each sample relative to a reference sample that did not receive any enzyme. It was confirmed that vGTFJ converted the sucrose to polysaccharides and oligosaccharides, and released fructose, providing an overall -38% reduction in sugar. It was furthermore shown that when epimerase was included with vGTFJ, the epimerase reduced the fructose content (borne from vGTFJ activity) by roughly 30% (by virtue of the fructose being converted to allulose), and resulted in an overall sugar reduction of -47%. Table 2

[0189]

[0190] Concentrations (%w / w) are as me asured in each liquid sample.

[0191] Example 3

[0192] In situ Sugar Reduction in Appl e Concentrate Using Epimerase Alone or in Combination with Glucosyltransferase

[0193] An apple concen trate containing 9% (w / w) sucrose, 15% (w / w) glucose and 30% (w / w) fructose was d i luted 1:1 with demineralized water (MILLIQ) and adjusted to pH 5. It was then aliquoted in 1-mL portions and tempered to 50 °C. To the aliquots were added eithe r 100 pL demineralized water or 100 pL of epimerase BsuEpil (SEQ ID NO:14) or RgnEpil (SEQ ID NO:15), and then the aliquots were incubated for 3 hours at 50 °C. An additional aliquot was tempered at 40 °C, to which was simultaneously added 100 pL BsuEpil (SEQ ID NO:14) and 10 pL of a combined solution of vGTFJ (SEQ ID NO:3) and GTF 0768 (SEQ ID NO:1), and then the aliquot was incubated for 3 hours at 40 °C. The samples that were incubated at 50 °C were cooled to 5 °C, and then 10 pL of demineralized water, GTF 0768, or combined solution of vGTFJ and GTF 0768 was added; these aliquots were then incubated for 20 hours at 5 °C. All the samples were heat-inactivated for 10 minutes at 95 °C before being analyzed for sugar / oligosaccharide composition according to the Materials / Methods.

[0194] Results of measuring carbohydrate content (%w / w) in the samples are presented in Table 3. It was confirmed that the glucosyltransferases converted the sucrose to polysaccharides and oligosaccharides, providing about 12-14% sugar reduction. It was furthermore shown that when epimerase was included, it reduced roughly 25% of the fructose (i.e. converted to allulose) resulting in about 11-14% of sugar reduction. When both epimerase and glucosyltransferase(s) were used either combined or sequentially, it resulted in sugar reduction of about 20-30% (up to 36% reduction if the leucrose and DP2 maltose isomer were not considered as sugars). The reduction in leucrose obtained when using both epimerase and glucosyltransferase(s) was notable. In this regard, ensuring that an epimerase is added before adding a GTF can aid in reducing the amount of fructose available for GTF byproduction of leucrose.

[0195] Table 3

[0196]

[0197] *Concentrations (%w / w) are as measured in each liquid sample.

[0198] **Sample was incubated with epimerase and GTF enzymes simultaneously at 40 °C for 3 hours. Example 4

[0199] In Situ Sugar Reduction in Apple Concentrate Using Epimerase in Combination with Fructosyltransferase

[0200] An apple concentrate containing 9% sucrose, 15% glucose and 30% fructose was diluted 1:1 with demineralized water (MILLIQ) and adjusted to pH 5. It was then aliguoted in 1-mL portions and tempered to 50 °C. To the aliguots were added either 100 pL demineralized water (trial 1) or 100 pL of a fructosyltransferase (“INUO” enzyme as disclosed in U. S. Pat. Appl. Publ. No. 2017 / 0267981, which is incorporated herein by reference) either alone (trial 2) or in combination with epimerase BsuEpil (SEQ ID NO: 14) (trial 3). After a 5-hour incubation at 50 °C, a 200-pL sample from each preparation was extracted and mixed with 115 pL ethanol for heat-inactivation at 83 °C for 10 minutes, after which each sample was measured for its sugar / oligosaccharide composition according to the Materials / Methods.

[0201] Results of measuring carbohydrate content (%w / w) in the samples are presented in Table 4. It was confirmed that the fructosyltransferase had converted the sucrose to fructooligosaccharides, providing about 3.7% sugar reduction. An unidentified DP2 sugar was also guantified and assumed to be a fructose-fructose disaccharide that was counted as sugar. It was furthermore shown, that when both fructosyltransferase and epimerase enzymes were used together, a total sugar reduction of about 21.2% was obtained, and less disaccharide and more oligosaccharides were formed.

[0202] Table 4

[0203]

[0204] Concentrations (%w / w) are as measured in each liquid sample.

[0205] Example 5

[0206] Treating Juice with Glucosyltransferase Materials / Methods

[0207] Orange juice concentrate was received from Northwest Naturals LLC (Bothell, WA, USA). Details of orange juice concentrate attributes are listed in Table 5. HPLC grade monosaccharide and disaccharide standards (sucrose, glucose, fructose) were obtained from Sigma Aldrich (St. Louis, MO, USA). HPLC column (Rezex™ RPM- Monosaccharide Pb+2, 00H-0135-K0) and guard column (Carbo Pb, AJO-44929) were obtained from Phenomenex (Torrance, CA, USA).

[0208] Table 5. Attributes of orange juice concentrate

[0209]

[0210] 200 mL of juice concentrate was mixed with 800 mL of deionized water in centrifuge tubes. The samples were vortexed and centrifuged at 15000 rpm. 200 mL of the supernatant was removed and mixed with an additional 800 mL of deionized water. The samples were filtered using 0.45 µm PTFE filters prior to injection on the HPLC.

[0211] Each sugar profile analysis was carried out using Waters H-Class Acquity UPLC coupled with an Rl detector. The data analysis was carried out using Empower software. The mobile phase consisted of deionized water with a flow rate of 0.4 mL / min and an injection volume of 5 µL with a total run time of 30 minutes. Each separation was carried out using a Rezex™ RPM-Monosaccharide Pb+2column coupled with a guard column at 70 °C. The Rl detector temperature was maintained at 35 °C. The total sugar amount was calculated based on the sum of monosaccharides and disaccharides.

[0212] Experimental - The effect of modified processing conditions and enzyme addition on sugar reduction and product stability

[0213] The effect of modified processing conditions in which GTF enzymes were added at a final Brix of 11.8 was compared against GTF enzymes addition at 30 Brix. Prior to preparing the juice samples, 20 mL of enzyme blend was prepared by combining 13.2 mL of GTF 0768 (SEQ ID NO:1), 5.9 mL of vGTFJ (SEQ ID NO:3) and 0.9 mL of distilled water. For the 30-Brix sample, the orange juice concentrate (63.7 Brix) was first diluted to 30 Brix with distilled water. Then, the enzyme blend was added at 0.89% of the juice sample. For the 11,8-Brix sample, the concentrate was diluted to 11.8 Brix using distilled water. The enzyme blend was then added at 0.35% of the juice sample. Both the 30-Brix and 11,8-Brix samples were then incubated at 5 °C for 22 hours. The 30-Brix sample was further diluted to theoretical 11.8 Brix based on the initial concentration just prior to enzyme heat inactivation. Then, the samples were heat-treated at 85 °C for 10 minutes prior to sugar analysis. The sugar reduction of the sample dosed at 30 Brix seemed similar to the sugar reduction of the sample dosed at 11.8 Brix. Interestingly, the sample dosed at 11.8 Brix seemed to have less separation and better stability compared to the control and the sample dosed at 30 Brix (FIG. 1). The difference in stability was most likely linked to the type of oligosaccharides and polysaccharides formed from the different modes of enzyme treatment. Lower levels of soluble oligosaccharides were formed with the 11.8 Brix treatment (Table 6). Additionally, the sugar analysis results also showed that, in the 30 Brix treatment, there was more free glucose and fructose being glycosylated by the enzyme treatment resulting in higher levels of leucrose and nigerose (DP2), as well as higher levels of shorter oligosaccharides that do not contribute to the polysaccharides that are responsible for the texture / stability (Table 6 and data not shown). Hence, treating orange juice concentrate with GTF enzymes at 11.8 Brix will result in higher levels of polysaccharide production. This result is in agreement with the improved product stability shown in FIG. 1 (11.8 Brix GTF dosage).

[0214] Table 6*

[0215] >

[0216] _ Average sugar content, i / ) _

[0217] Total average OS Sucrose DP2 Lactose Glucose LeucroseGa ctcse Fructose sugars reduction Control 0.00 442 0,00 0.00 2.27 0.00 0.00 237 9.02

[0218] 30BX Blend 2 (0.35%) 2.13 0.00 0.66 0.00 1.48 0.85 0.00 3.87 6.85 24.05 11BxBien 2m) 143 0.00 044 GOO 1.80 0,58 0.00 4.18

[0219]

[0220] 2248 *OS represents oligosaccharides. DP2 is nigerose. Total sugar is the sum of DP1 and DP2.

Claims

CLAIMSWhat is claimed is:

1. A method of producing a food product / precursor, said method comprising:(a) providing a food product / precursor that comprises at least water and fructose, and(b) contacting the food product / precursor with an epimerase that converts fructose to a fructose epimer, wherein at least one fructose epimer is produced in the food product / precursor.

2. The method of claim 1, wherein the fructose epimer is D-allulose.

3. The method of claim 1, wherein the epimerase comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 35, 36, 37, 38, 39, 40, 41, 42, 43, 47, 48, 49, 50, 51, 52, 53, 54, or 55.

4. The method of claim 1, wherein the contacting occurs under conditions comprising a temperature of about 30 °C to about 90 °C.

5. The method of claim 1, wherein the contacting occurs under conditions comprising a pH of about 3.5 to about 8.0.

6. The method of claim 1, wherein no metal cofactor has been added to the food product / precursor, or a metal cofactor has been added to the food product / precursor in an amount that is insufficient to allow activity of the epimerase.

7. The method of claim 1, further comprising contacting the food product / precursor with at least one glucosyltransferase enzyme,wherein the food product / precursor further comprises sucrose,wherein said contacting with the glucosyltransferase enzyme is performed simultaneously with, or after, contacting the food product / precursor with the epimerase,optionally wherein at least one alpha-glucan is produced in the food product / precursor, and / oroptionally wherein the amount of leucrose produced in the food product / precursor is less than the amount of leucrose that would have been produced in the food product / precursor if the epimerase was not contacted with the food product / precursor.

8. The method of claim 1, wherein the food product / precursor provided in step (a) is provided by contacting a food product / precursor that comprises at least water and sucrose with at least one glucosyltransferase enzyme,optionally wherein at least one alpha-glucan is produced in the food product / precursor.

9. The method of claim 7, wherein the glucosyltransferase enzyme is:(i) a glucosyltransferase enzyme that is capable of synthesizing alpha-1,6- glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,6-glucan are alpha-1,6 linkages, or(ii) a glucosyltransferase enzyme that is capable of synthesizing alpha-1,3- glucan, wherein at least about 50% of the glycosidic linkages of the alpha- 1,3-glucan are alpha-1,3 linkages.

10. The method of claim 9, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha-1, 6-glucan comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:1, 2, 11, 12, 56, or 57.

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

12. The method of claim 9, wherein both of said (i) glucosyltransferase enzyme that is capable of synthesizing alpha-1, 6-glucan and (ii) glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan are contacted with the food product / precursor.

13. The method of claim 12, wherein an alpha-glucan is produced in the food product / precursor that is a graft copolymer comprising:(i) an alpha-1,6-glucan backbone, wherein at least about 50% of the glycosidic linkages of the alpha-1,6-glucan backbone are alpha-1,6 linkages, and (ii) at least one alpha-1,3-glucan side chain, wherein at least about 50% of the glycosidic linkages of the alpha-1, 3-glucan chain are alpha-1,3 linkages, wherein said graft copolymer is aqueous-soluble or aqueous-insoluble.

14. The method of claim 1, further comprising contacting the food product / precursor with at least one fructosyltransferase enzyme,wherein the food product / precursor further comprises sucrose,optionally wherein at least one fructan is produced in the food product / precursor, and / oroptionally wherein said contacting with the fructosyltransferase enzyme is performed prior to, simultaneously with, or after contacting the food product / precursor with the epimerase.

15. The method of claim 1, further comprising contacting the food product / precursor with at least one lactase enzyme and / or glucose isomerase enzyme, optionally wherein said contacting with the lactase enzyme and / or glucose isomerase enzyme is performed prior to, simultaneously with, or after contacting the food product / precursor with the epimerase.

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

17. The method of claim 1, wherein the food product / precursor is a dairy food product / precursor.

18. The method of claim 1, wherein the food product / precursor is a non-dairy food product / precursor.

19. The method of claim 18, wherein the non-dairy food product / precursor is plantbased.

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

21. The method of claim 1, wherein the food product / precursor is a yogurt.

22. The method of claim 1, wherein the food product / precursor is a beverage or a juice.

23. A food product / precursor produced by the method of claim 1.

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