Method for improving flavor in foodstuff

Abstract

Disclosed herein are methods of glucosylating limonoid compounds and / or flavonoid compounds, and / or otherwise reducing levels of limonoid compounds and / or flavonoid compounds in compositions, as well as methods of reducing off-flavor in a food product / precursor. Further disclosed are compositions (i) comprising one or more glucosylated limonoid compounds and / or glucosylated flavonoid compounds, or (ii) otherwise comprising less limonoid compounds and / or flavonoid compounds, such as a food product / precursor.

Description

[0001] TITLE

[0002] METHOD FOR IMPROVING FLAVOR IN FOODSTUFF

[0003] This application claims the benefit of U.S. Provisional Appl. Nos. 63 / 567,048 (filed March 19, 2024), 63 / 676,457 (filed July 29, 2024), and 63 / 767,703 (filed March 6, 2025), which are each incorporated herein by reference in their entirety.

[0004] FIELD

[0005] The present disclosure is in the field of glycosylation of components present in plant-based food products to improve flavor. For example, the disclosure pertains to glucosylation of limonoids and / or flavonoids in situ in food and food precursors.

[0006] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0007] The official copy of the sequence listing is submitted electronically via EFS-Web as a file named IFF101198USPSP_SequenceListing.xml created on February 27, 2025 and having a size of about 39 kilobytes, and is filed concurrently with the specification. The sequence listing contained in this file is part of the specification and is incorporated herein by reference in its entirety.

[0008] BACKGROUND

[0009] Plant-based or non-dairy protein food alternatives, such as soy-, almond-, pea-, bean-, rice- or oat-based products (e.g., milk or fresh fermented products), are one of the fastest growing segments in all food product categories worldwide (Makinen et al., 2016, Crit. Rev. Food Sci. Nutr. 56:339-349; Sethi et al., 2016, J. Food Sci. Technol. 53:3408-3423). Off-flavors, however, remain a major hurdle in the use of plant-based materials in mainstream food applications. For example, soy-derived materials contain several polyphenolic compounds, such as isoflavones, saponins and phenolic acids that impart undesirable sensory properties such as bitterness and astringency tastes. Today, very cumbersome processes, such as dispersion of material in alkaline conditions followed by membrane separation or size-exclusion chromatography, are tried and used to remove these components and hence their unwanted flavor (Damodaran et al., 2013, Annu. Rev. Food Sci. Technol. 4:327-346).

[0010] Additionally, limonoids and flavonoids are plant compounds that can impart off- flavors in foods, beverages and ingredients containing these compounds. Modes of modifying limonoid and / or flavonoid compounds to reduce their off-flavoring effects in foodstuff are sought after.

[0011] SUMMARY

[0012] In one embodiment, the present disclosure concerns a method of producing a food product / precursor, the method comprising: (a) providing a food product / precursor that comprises at least water, sucrose and a plant-based material, and

[0013] (b) contacting the food product / precursor with at least:

[0014] (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, and / or

[0015] (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, whereby the food product / precursor, after step (b), has a reduced content of one or more limonoid compounds and / or flavonoid compounds that were present in the food product / precursor of step (a), optionally wherein the reduced content of one or more limonoid and / or flavonoid compounds results in a reduction of off-flavor of the food product / precursor.

[0016] In another embodiment, the present disclosure concerns a method of glucosylating a limonoid and / or flavonoid compound, the method comprising: providing a composition that comprises at least water, sucrose, a limonoid compound and / or flavonoid compound, and at least one glucosyltransferase enzyme, wherein the glucosyltransferase enzyme is selected from:

[0017] (i) a glucosyltransferase enzyme (a dextransucrase) that synthesizes alpha-1 ,6- glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1 , 6-glucan are alpha-1 ,6 linkages, and / or

[0018] (ii) a glucosyltransferase enzyme (a glucansucrase) 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, wherein at least one glucosylated form of the limonoid compound and / or flavonoid compound is produced in the composition.

[0019] In another embodiment, the present disclosure concerns a food product / precursor produced by a method herein.

[0020] In another embodiment, the present disclosure concerns a composition, or a glucosylated limonoid compound and / or glucosylated flavonoid compound, produced by a method herein. BRIEF DESCRIPTION OF THE SEQUENCES

[0021] Table A. Summary of Protein SEQ ID Numbers

[0022] BRIEF DESCRIPTION OF THE DRAWING

[0023] FIG. 1 A: HPLC profile of clarified orange juice. Refer to Example 1 .

[0024] FIG. 1 B: HPLC profile of a limonin standard at 33 ppm concentration. Refer to Example 1 .

[0025] FIG. 2A: HPLC profile of clarified orange juice control. Refer to Example 2.

[0026] FIG. 2B: HPLC profile of the clarified orange juice that was incubated with vGTFJ (SEQ ID NO:3). Refer to Example 2.

[0027] FIG. 2C: HPLC profile of the clarified orange juice that was incubated with GTF 0768 (SEQ ID NO:1). Refer to Example 2.

[0028] FIG. 2D: HPLC profile of the clarified orange juice that was incubated with a combination of both vGTFJ (SEQ ID NO:3) and GTF 0768 (SEQ ID NO:1). Refer to Example 2.

[0029] FIG. 3A: HPLC profile of clarified orange juice control. Refer to Example 3.

[0030] FIG. 3B: HPLC profile of clarified orange juice that was incubated with a combination of both vGTFJ (SEQ ID NO:3) and GTF 0768 (SEQ ID NO:1) for 3 hours at 5 °C. Refer to Example 3.

[0031] FIG. 3C: HPLC profile of clarified orange juice that was incubated with a combination of both vGTFJ (SEQ ID NO:3) and GTF 0768 (SEQ ID NO:1) for 18 hours at 5 °C. Refer to Example 3.

[0032] FIG. 4A: HPLC profile of clarified orange juice control. Refer to Example 4.

[0033] FIG. 4B: HPLC profile of clarified orange juice that was incubated with an enzymatically inactivated combination of both vGTFJ (SEQ ID NO:3) and GTF 0768 (SEQ ID NO:1). Refer to Example 4.

[0034] FIG. 4C: HPLC profile of clarified orange juice that was incubated with two separate doses of a combination of enzymatically active vGTFJ (SEQ ID NO:3) and GTF 0768 (SEQ ID NO:1). Refer to Example 4.

[0035] FIG. 5A: HPLC profile of clarified orange juice control. Refer to Example 5. FIG. 5B: HPLC profile of the clarified orange juice that was incubated with GTF 6831 (SEQ ID NO: 14). Refer to Example 5.

[0036] FIG. 6: HPLC profile of clarified orange juice after treatment with a combination of both vGTFJ (SEQ ID NO:3) and GTF 0768 (SEQ ID NO:1). Fractions (1-7) are shown and were tested with Ehrlich’s reagent. Refer to Example 6.

[0037] FIG. 7A: HPLC profile of orange juice that was spiked with additional vicenin-2. Refer to Example 7.

[0038] FIG. 7B: HPLC profile of the orange juice after it was incubated with a combination of both vGTFJ (SEQ ID NO:3) and GTF 0768 (SEQ ID NO:1). Refer to Example 7.

[0039] FIG. 8A: The limonoid and flavonoid region of an HPLC profile of orange juice before and after treatment (various time-points) with a combination of both vGTFJ (SEQ ID NO:3) and GTF 0768 (SEQ ID NO:1). Refer to Example 8.

[0040] FIG. 8B: Time course of limonin reduction in orange juice treated with a combination of both vGTFJ (SEQ ID NO:3) and GTF 0768 (SEQ ID NO:1). Refer to Example 8.

[0041] FIG. 8C: Time course of sugar content changes in orange juice treated with a combination of both vGTFJ (SEQ ID NO:3) and GTF 0768 (SEQ ID NO:1). Refer to Example 8.

[0042] FIG. 9: Limonin reduction in orange juice treated with vGTFJ (SEQ ID NO:3) or GTF 0768 (SEQ ID NO:1), or with various combinations thereof. Refer to Example 10.

[0043] DETAILED DESCRIPTION

[0044] The disclosures of all cited patent and non-patent literature are incorporated herein by reference in their entirety.

[0045] 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.

[0046] Where present, all ranges are inclusive and combinable, except as otherwise noted. For example, when a range of “1 to 5” (i.e., 1-5) is recited, the recited range should be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, and the like. The numerical values of the various ranges in the present disclosure, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both proceeded by the word “about”. In this manner, slight variations above and below the stated ranges can typically be used to achieve substantially the same results as values within the ranges. Also, the disclosure of these ranges is intended as a continuous range including each and every value between the minimum and maximum values.

[0047] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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).

[0054] 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.

[0055] 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”.

[0056] 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 s, S. W. Cui, Structural Analysis of Polysaccharides, Taylor & Francis Group LLC, Boca Raton, FL, 2005), which is incorporated herein by reference.

[0057] 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).

[0058] 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.

[0059] 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.

[0060] 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 / or other products such as glucosylated limonoid compound or glucosylated flavonoid compound) and fructose. Other products (by-products) of a GTF reaction can include glucose, various soluble gluco-oligosaccharides, and leucrose. Wild type forms of glucosyltransferase enzymes generally contain (in the N-terminal to C-terminal direction) a signal peptide (which is typically removed by cleavage processes), a variable domain, a catalytic domain, and a glucan-binding domain. A glucosyltransferase herein is classified under the glycoside hydrolase family 70 (GH70) according to the CAZy (Carbohydrate-Active EnZymes) database (Cantarel et al., Nucleic Acids Res. 37:D233-238, 2009). The term “dextransucrase” (and like terms) can optionally be used to characterize a glucosyltransferase enzyme that produces dextran.

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

[0062] The terms “enzymatic reaction”, “glucosyltransferase reaction”, “glucan synthesis reaction”, “reaction composition”, “reaction formulation” and the like are used interchangeably herein and generally refer to a reaction that initially comprises water, sucrose, at least one active glucosyltransferase enzyme, and optionally other components (e.g., one or more limonoid compounds and / or flavonoid compounds). 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 and / or glucosylation of a limonoid compound and / or flavonoid compound; 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.

[0063] The term “in situ" as used herein typically characterizes a glucosyltransferase reaction(s) that occurs inside a food product or precursor thereof and thereby produces (i) alpha-glucan, and / or (ii) a glucosylated limonoid compound and / or glucosylated flavonoid compound, 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.

[0064] 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%.

[0065] 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.

[0066] 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).

[0067] The terms “ingestible product” and “ingestible composition” are used interchangeably herein, and refer to any substance that, either alone or together with another substance, may be taken orally (i.e., by mouth), whether intended for consumption or not. Thus, an ingestible product includes food / beverage products. “Food / beverage products” refer to any edible product intended for consumption (e.g., for nutritional purposes) by humans or animals, including solids, semi-solids, or liquids. A “food” herein can optionally be referred to as a “foodstuff”, “food 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 s / Yu-produced alpha-glucan and / or in situ- produced glucosylated limonoid compound and / or glucosylated flavonoid compound. 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 (e.g., a fruit / vegetable juice treated with one or more GTFs, after which it is added as an ingredient for preparing a food or beverage). In some aspects, a food precursor can characterize a food product or ingredient as it exists before treatment with one or more GTF enzymes in a method herein.

[0068] 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.

[0069] “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).

[0070] The terms “limonoid”, “limonoid compound” and like terms are used interchangeably herein, and generally refer to tetranortriterpene (or tetranortriterpenoid) phytochemicals. Limonoids have a furanolactone core structure that varies between different limonoids. The prototypical structure of a limonoid has four six-membered rings and a furan ring. An example of a limonoid compound herein is limonin, which can optionally also be referred to as a type of tetracyclic triterpenoid dilactone. Limonin has the following structure:

[0071] A “glucosylated limonoid” or “glucosylated limonoid compound” (and like terms) herein refers to a limonoid that has been enzymatically modified to contain one or more glucosyl groups. It is believed that such one or more glucosyl groups can be of one or more single (pendant) glucosyl groups and / or of one more linear chains of two or more glucosyl groups. A glucosylated limonoid herein can optionally be characterized (or further characterized) as being a product of an enzymatic reaction as presently described comprising at least water, sucrose, a limonoid compound, and a glucosyltransferase enzyme.

[0072] The terms “flavonoid”, “flavonoid compound” and like terms are used interchangeably herein, and generally refer to polyphenolic secondary metabolite phytochemicals that have a 15-carbon core structure of two phenyl rings (A and B) and a heterocyclic ring (C) containing an embedded oxygen. This core structure can optionally be abbreviated as C6-C3-C6. Examples of a flavonoid compound herein include sinensetin and nobiletin, either of which can optionally also be referred to as a type of polymethoxylated flavonoid. Sinensetin has the following structure: , . .. .

[0073] (sinensetin).

[0074] Nobiletin has the following structure: (nobiletin).

[0075] Vicenin-2 has the following structure:

[0076] (vicenin-2).

[0077] Vicenin-2 is an example of a flavonoid glycoside, wherein its glycosyl groups (which are glucosyl groups) pre-exist prior to a GTF treatment herein. GTF treatment of a flavonoid glycoside such as vicenin-2 can result in further glucosylation of the compound. The term “vicenin-2 glucoside” (and like terms) herein refers to vicenin-2 that has been modified by a GTF treatment.

[0078] A “glucosylated flavonoid” or “glucosylated flavonoid compound” (and like terms) herein refers to a flavonoid that has been enzymatically modified to contain one or more glucosyl groups. It is believed that such one or more glucosyl groups can be of one or more single (pendant) glucosyl groups and / or of one more linear chains of two or more glucosyl groups. A glucosylated flavonoid herein can optionally be characterized (or further characterized) as being a product of an enzymatic reaction as presently described comprising at least water, sucrose, a flavonoid compound, and a glucosyltransferase enzyme.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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. These definitions likewise apply to a glucosylated limonoid or glucosylated flavonoid herein, for example.

[0084] 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.

[0085] 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 (lie, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), valine (Vai, V).

[0086] The terms “sequence identity”, “identity” and the like as used herein with respect to polynucleotide or polypeptide sequences refer to the nucleic acid residues or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window. Thus, “percentage of sequence identity”, “percent identity” and the like refer to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e. , gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. It would be understood that, when calculating sequence identity between a DNA sequence and an RNA sequence, T residues of the DNA sequence align with, and can be considered “identical” with, U residues of the RNA sequence. For purposes of determining “percent complementarity” of first and second polynucleotides, one can obtain this by determining (i) the percent identity between the first polynucleotide and the complement sequence of the second polynucleotide (or vice versa), for example, and / or (ii) the percentage of bases between the first and second polynucleotides that would create canonical Watson and Crick base pairs.

[0087] 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.

[0088] 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. 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, WIND0W=5 and DIAGONALS SAVED=5. For nucleic acids, these parameters can be KTUPLE=2, GAP PENALTY=5, WIND0W=4 and DIAGONALS SAVED=4. Additionally, the Clustal W method of alignment can be used (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci. 8:189-191(1992); Thompson, J.D. et al, Nucleic Acids Research, 22 (22): 4673-4680, 1994) and found in the MEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (protein / nucleic acid) can be: GAP PENALTY=10 / 15, GAP LENGTH PENALTY=0.2 / 6.66, Delay Divergent Seqs(%)=30 / 30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB.

[0089] Various polypeptide amino acid sequences and polynucleotide sequences are disclosed herein as features of certain embodiments. Variants of these sequences that are at least about 70-85%, 85-90%, or 90%-95% identical to the sequences disclosed herein can be used or referenced. Alternatively, a variant amino acid sequence or polynucleotide sequence can have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with a sequence disclosed herein. A variant amino acid sequence or polynucleotide sequence herein has the same function / activity of the disclosed sequence, or at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% of the function / activity of the disclosed sequence. Any polypeptide amino acid sequence disclosed herein not beginning with a methionine or valine can typically further comprise at least a start-methionine or start-valine at the N-terminus of the amino acid sequence. In contrast, any polypeptide amino acid sequence disclosed herein beginning with a methionine or valine can optionally lack such a methionine or valine residue. In some aspects, any polypeptide amino acid sequence disclosed herein beginning with a methionine or valine can instead have, respectively, a valine or methionine as the first amino acid residue.

[0090] The term “isolated” means a substance (or method / 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, food precursor, graft copolymer, glucosylated limonoid compound, and / or glucosylated flavonoid compound herein (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.

[0091] 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.

[0092] Some embodiments of the present disclosure concern a method of producing a food product / precursor. Such a method can comprise:

[0093] (a) providing a food product / precursor that comprises at least water, sucrose and a plant-based material, and

[0094] (b) contacting the food product / precursor with at least:

[0095] (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, and / or

[0096] (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, whereby the food product / precursor, after step (b), has a reduced content of one or more limonoid compounds and / or flavonoid compounds that were present in the food product / precursor of step (a), optionally wherein the reduced content of one or more limonoid and / or flavonoid compounds results in a reduction of off-flavor of the food product / precursor.

[0097] A food product / precursor resulting from this method typically comprises at least one glucosylated limonoid compound and / or glucosylated flavonoid compound, where the glucosylation of either compound is from the glucosyltransferase(s) activity. A food product / precursor resulting from this method typically, but not necessarily, further comprises at least one alpha-glucan (e.g., alpha-1 ,6-glucan / dextran, alpha-1 , 3-glucan, and / or a graft copolymer) as presently disclosed.

[0098] Step (b) of producing a food product / precursor can comprise contacting a food product / precursor with at least: (i) a glucosyltransferase (GTF) 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, a GTF enzyme with alpha-1 , 6-glucan I dextran synthesis activity), and / or

[0099] (ii) a GTF 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 (or, a GTF enzyme with alpha-1 , 3-glucan synthesis activity).

[0100] 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 14 or 15 (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. 2017 / 0218093, 2018 / 0282385, 2018 / 0291311 , or 2016 / 0122445, which are each incorporated herein by reference. For example, the GTF identified as GTF 8117 (SEQ ID NO:30), GTF 6831 (SEQ ID NO:32), or GTF 5604 (SEQ ID NO:33) in US2018 / 0282385 can be used, or the GTF identified as GTF 2919 (SEQ ID NO:5), GTF 2918 (SEQ ID NO:9), GTF 2920 (SEQ ID NO:13), or GTF 2921 (SEQ ID NO:17) in US2016 / 0122445 can be used, or a GTF comprising an amino acid sequence that is about 100% identical to, or at least about 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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. 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).

[0108] 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. 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 or U.S. Pat. Appl. Publ. No. 2024 / 0108021 , which are each 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.

[0109] 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).

[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: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.

[0111] 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 -Vai, 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.

[0112] 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.

[0113] 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.

[0114] 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 %,

[0115] 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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 using methods such as precipitation, filtration, and / or concentration. Alternatively, a lysate or extract comprising a GTF can be used without further isolation. If the GTF was secreted (i.e. , it is present in the fermentation broth), it can optionally be used as isolated from, or as comprised in, the fermentation broth. The activity of a GTF enzyme can be confirmed by biochemical assay, such as measuring its conversion of sucrose to glucan polymer.

[0121] Alpha-glucan produced in step (b) of producing a food product / precursor in some aspects comprises a graft copolymer comprising:

[0122] (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

[0123] (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.

[0124] 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.

[0125] One, two, three, or more different GTF enzymes that synthesize alpha-1 ,6-glucan herein can be used, for example, in step (b) of producing a food product / precursor. In some aspects, only an alpha-1 , 6-glucan-producing GTF(s) is used in step (b) (i.e., an alpha-1 , 3-glucan-producing GTF is not used). One, two, three, or more different GTF enzymes that synthesize alpha-1 , 3-glucan herein can be used, for example, in step (b) of producing a food product / precursor. In some aspects, only an alpha-1 , 3-glucan- producing GTF(s) is used in step (b) (i.e., an alpha-1 , 6-glucan-producing GTF is not used). In some aspects, an alpha-1 , 6-glucan-producing GTF(s) can be added to (made to contact) a food 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 step (b) of producing a food product / precursor 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, only dextran and / or alpha-1 , 3-glucan homopolymer(s) is / are produced with little (e.g., < 5 wt% of all glucan products) or no production of graft copolymer. Still, in some aspects, it is believed possible that alpha-1 , 6-glucan and / or alpha-1 , 3-glucan is / are not produced when using an alpha-1 , 6-glucan-producing GTF(s) and / or an alpha-1 , 3- glucan-producing GTF(s) in step (b) herein.

[0126] 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.

[0127] The content of at least one of an alpha-1 ,6-glucan-producing GTF and / or alpha- 1 ,3-glucan-producing GTF in a food product / precursor herein can be about, or at least about, 0.1 , 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.75, 1 , 1.25, 1.5, 1.75., 2, 2.5, 0.1-1 , 0.1-0.75, 0.1-0.5, 0.1-0.3, 0.2-1 , 0.2-0.75, 0.2-0.5, 0.2-0.3, 0.5-2.5, 0.5-2, 0.5-1.5, 0.5-1 , 1-2.5, 1- 2, or 1-1.5 wt%, for example. In some aspects, a single GTF is used, whereas two, three, or more GTF enzyme(s) can be used in other aspects. The foregoing enzyme contents can be with respect to one GTF enzyme, or a combination of GTF enzymes. The foregoing GTF enzyme contents are typically with respect to active enzyme(s), but in some aspects can be with respect to total protein of isolated / purified enzyme(s).

[0128] In some aspects, the ratio of a GTF enzyme that synthesizes alpha-1 , 6-glucan to a 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, 55:45, 50:50, 45:55, 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 80:20 to 60:40). The amount of each enzyme (active enzyme) for purposes of determining a ratio thereof herein can be on a molar, weight, or GTF activity (e.g., U [units], activity units per gram formulated enzyme) 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. Nos. 2014 / 0087431 or 2024 / 0108021 , or Int. Patent Appl. Publ. No. W02023 / 055902, which are each 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.

[0129] A GTF enzyme (or any other enzyme as presently disclosed) for use in a method herein is typically in purified (isolated) form. A purified enzyme can be essentially free from insoluble and / or soluble components of an organism / cell used to produce the enzyme, and / or any medium that was used for cellular fermentation of the enzyme. In some aspects, a purified enzyme denotes an enzyme preparation that contains less than 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1 % by weight of other material (e.g., polypeptide material) with which the enzyme is natively or recombinantly associated. In some aspects, a GTF and / or any other enzyme herein is not comprised in or otherwise associated with (e.g., expressed by) a microbial (e.g., bacterial, yeast, fungal, algal) cell that might be present (e.g., endogenously or purposely added) in a food product / precursor herein; however, in some aspects a GTF and / or any other enzyme herein is comprised in or otherwise associated with (e.g., expressed by) a microbial (e.g., bacterial, yeast, fungal, algal) cell such as one that heterologously expresses the enzyme(s) (i.e., recombinant cells). Contacting a food product / precursor with a GTF enzyme(s) herein typically is not performed in an oral cavity or other environment in which unpurified / non-isolated GTF enzymes can possibly be present.

[0130] A GTF enzyme (or any other enzyme as presently disclosed) for use in a method herein can be comprised in a sterile-filtered preparation, for example. In some aspects, an enzyme can be sterile-filtered inline while applying the enzyme to a food product / precursor during step (b) 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. Pasteurization can be conducted by any suitable means, for example, such as by raising the temperature of the food product / precursor to about 70, 72.5, 75, 77.5, 70-75, or 70-77.5 °C, and holding this temperature for about 10, 15, 20, 10-20, 15-20, or 14-16 seconds, and then cooling the food product / precursor to a desired lower temperature. In some aspects, an enzyme can be added to a food product / precursor that has been fermented (after fermentation), or alternatively an enzyme can be added during or before fermenting the food product / precursor. A GTF enzyme (or any other enzyme as presently disclosed) in some aspects for use in a method herein can be comprised in a preparation (for adding in step [b] herein) 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).

[0131] A food product / precursor herein can be brought into contact with one or more GTF enzymes in step (b) by mixing / stirring / blending, for example. Incubation of GTF enzyme(s) in the food product / precursor can be for a time sufficient, for example, for the GTF(s) to produce alpha-glucan in the food product / precursor and / or to glucosylate a limonoid and / or flavonoid herein, such as for about, at least about, or up to about, 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 24, 30, 36, 42, 48, 72, 96, 1-6, 1-5, 1-4, 1-3, 2-6, 2-5, 2-4, 2-3, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, or 6-8 hours, or for about, at least about, or up to 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). In some aspects, such incubation can be for less than about 8, 7, 6, 5, 4, 3, or 2 hours (e.g., less than about 3, 5, or 7 hours). The temperature for incubating one or more GTF 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, 3-7, 3-10, 3-15, 3-20, 3-25, 3-30, 3-35, 3-40, 3-45, 3-50, 4-6, 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 (e.g., about 3-7, 4-6, or 5 °C).

[0132] Typically, a food product / precursor brought into contact with a GTF enzyme 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 GTF enzyme. GTF enzyme 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 a GTF enzyme under dry conditions (resulting combination is dry), after which time water or an aqueous solution is added, which in turn allows GTF production of alpha-glucan to proceed. Thus, depending on how ingredients and GTF enzyme(s) 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 as provided in step (a), or in step (b) following addition of GTF enzyme, 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 GTF 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,

[0133] 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,

[0134] 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).

[0135] A GTF enzyme herein can optionally be provided in step (b) of a method 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 GTF enzyme 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 (b) 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 a GTF 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 GTF enzyme [e.g., fused to the GTF]). 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).

[0136] In some aspects, a food product / precursor herein can be brought into contact with one or more GTF enzymes by virtue of adding the food product / precursor to an aqueous composition comprising at least sucrose and the one or more GTF enzymes. While the food product / precursor in this aspect has at least some sucrose and water, a food product / precursor in some other aspects does not comprise sucrose and / or water. Such a GTF / sucrose-containing aqueous composition can optionally be referred to herein as a “GTF / sucrose starter composition”. Typically, one or more food precursors as presently disclosed (e.g., ingredients such as a liquid food product / precursor, beverage, RTD, fruit / vegetable puree, syrup, or juice or juice concentrate) can be added to a GTF / sucrose starter composition, although one or more food products themselves as presently disclosed (e.g., fruit or vegetable matter such as pieces [e.g., slices, cubes, or other shaped pieces]) can be added (typically in conjunction with adding a food precursor). In some aspects, a GTF / sucrose starter composition can already comprise at least one food product / precursor, such as any disclosed herein. The initial sucrose concentration of a GTF / sucrose starter composition can be as presently disclosed, for example, such as 5-60%, 5-50%, 5-40%, 10-60%, 10-50%, 10-40%, 20-60%, 20-50%, 20-40%, 30-60%, 30-50%, 30-40%, 40-60%, or 40-50% by weight. In some aspects, a GTF / sucrose starter composition has few (e.g., less than about 1 , 0.5, 0.1 , 0.05, or 0.01 wt%) or no saccharide compounds (e.g., one or more monosaccharides, disaccharides, oligosaccharides and / or polysaccharides, such as presently disclosed) aside from sucrose. A GTF / sucrose starter composition can comprise at least one alpha-1 , 6- glucan-producing GTF and / or an alpha-1 , 3-glucan-producing GTF as presently disclosed, for example. The temperature, pH and / or any other condition / parameter of this methodology (before and / or after adding one or more food products / precursors to a GTF / sucrose starter composition) can be as disclosed herein, for example. In some aspects, a GTF / sucrose starter composition can be incubated for about, at least about, or up to about, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 120, 180, 240, 300, 360, 420, 480, 540, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 20-45, 20-40, 20-35, 20-30, 20-25, 25-45, 25-40, 25-35, or 25-30 minutes, for example, before adding one or more food products / precursors. Any of these foregoing time periods can also apply to the period of time allowed to proceed after adding the food product / precursor to the GTF / sucrose starter composition, until optionally terminating the GTF activity (e.g., heat-inactivation at a temperature of at least 90 °C, or at 90-100 °C) of the final food product / precursor. Typically, the one or more food products / precursors added to the GTF / sucrose starter composition comprises one or more saccharide compounds (e.g., one or more monosaccharides, disaccharides, oligosaccharides and / or polysaccharides, such as presently disclosed). In some aspects, adding one or more food product / precursors to a GTF / sucrose starter composition can be done to control or adjust the degree of thickening and / or texturization desired in the final food product / precursor being produced. The thickening and / or texturization that can be achieved by such methodology can be greater (e.g., about, or at least about, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, or 500% greater) than the thickening and / or texturization that would have been achieved if all the ingredients used to make the final food product / precursor had all been combined at about the same time. Merely as examples, the foregoing process of adding one or more food products / precursors to a GTF / sucrose starter composition can be used herein to produce a marmalade, gel / gelatin, pudding, custard, fermented product, or cream, optionally with one or more suspended solid food ingredients such as fruit or vegetable pieces.

[0137] 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 GTF enzyme to the food product / precursor.

[0138] 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 furanose. 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 a GTF enzyme herein is added (e.g., time, temperature, pH), and can be added before, during, or after the addition of GTF enzyme. Such an enzyme can be a transglucosidase (EC [enzyme code] 2.4.1 .24) or an amylase, for example. Suitable transglucosidases herein include FoodPro® TGO and those disclosed in U.S. Patent Appl. Publ. Nos. 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.

[0139] A food product / precursor herein typically comprises a plant-based material, either in its general entirety (e.g., a fruit or vegetable juice) or as an ingredient in a greater food product / precursor composition (e.g., juice added as an ingredient to a food product / precursor). In general, a plant-based material herein is a human-edible and / or animal (e.g., mammal such as a domestic pet such as a dog or cat)-edible material obtained from (or obtainable from) a plant, or optionally, though not from a plant, per se, from a Protista family member (e.g., an algae or seaweed). A plant-based material herein can be from a fruit, seed / grain, leaf, stem, flower, root, tuber, or other suitable edible part of a plant, for instance. Examples of plant-based material include juice, pulp, rind (peel) or zest, seeds, juice with pulp, juice with pulp and seed parts; any of these can be obtained from a fruit or other plant part, as appropriate.

[0140] 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. Nos. 2017 / 0218093 or 2022 / 0322685, 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 pet food (e.g., kibbles), and snacks (e.g., cheese curls, filled pillow puffs, chips [e.g., com 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.

[0141] A food product / precursor can comprise a plant-based material of a fruit, vegetable, berry, or seed / nut for example. A food product / precursor in some aspects can comprise a plant-based material from a plant of the Rutaceae family or the Cucurbitaceae family (or “a Cucurbitaceae family plant”). Suitable examples of a Rutaceae family plant herein include citrus fruit. A citrus fruit in some aspects can be an orange / sweet orange (e.g., Valencia orange, navel orange, blood orange, or a mandarin orange such as a tangerine or clementine), bitter orange / sour orange (e.g., Seville orange, bigarade orange, marmalade orange, bergamot orange), grapefruit, pomelo, lemon, lime (e.g., key lime, desert lime, persian lime), tangelo, citron, kumquat, or papeda fruit (e.g., kaffir lime). A citrus fruit in some aspects is an orange or grapefruit. Suitable examples of a Cucurbitaceae family plant herein include cucumber, water melon, bitter melon, squash, pumpkin, and zucchini. A fruit, vegetable, or berry in some aspects can be apple, mango, peach, plum, banana, date, apricot, papaya, pineapple, raspberry, cranberry, strawberry, blueberry, blackberry, acai, goji, pear, cherry, grape (green, red), raisin, currant, melon, watermelon, cantaloupe, honeydew melon, kiwi, coconut, carrot, tomato, kale, spinach, chard, beet, sweet potato, celery, parsley, or wheatgrass. A food product / precursor herein can comprise a juice of (and / or solids of) any of the foregoing plant-based materials or any others disclosed herein, for example.

[0142] A food product / precursor in some aspects can be a syrup or beverage, for example. A beverage in some aspects can be a juice (e.g., of any fruit, vegetable, berry or other suitable plant-based material herein), a beverage containing a juice or syrup as an ingredient thereof (e.g., a carbonated beverage such as a soft drink / soda, a mixed alcoholic or non-alcoholic drink, a non-carbonated beverage), sweetened beverage (soda / soft drink [carbonated beverage], sweetened tea, or coffee), ready-to-drink (RTD), or any other beverage having natural and / or added sugar (sucrose). In some aspects, a beverage comprises coffee or tea. A beverage in some aspects can be any as 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 carbonated beverage (e.g., soft drink, soda) or non-carbonated beverage as disclosed in any of U.S. Patent Appl. Publ. Nos. 2019 / 0373921 , 2012 / 0088013, 2017 / 0367374, 2008 / 0226797, 2018 / 0289042, 2008 / 0226800, 2002 / 0102331 , 20230301330, or 2015 / 0216218.

[0143] A food product / precursor can comprises fruit juice (e.g., a beverage) and / or fruit solids (e.g., pulp and / or seed pieces), for example. Such fruit juice and / or solids thereof can be of any fruit herein, such as any citrus fruit (e.g., oranges). A fruit herein can be harvested (picked) when it is ripe / ripened (generally optimal) or over-ripened, for example. However, in some aspects, a fruit herein can be harvested about, or at least about, 1 , 2, 3, 4, 5, 1-5, 1-4, 1-3, 1-2, 2-5, 2-4, 2-3, 3-5, or 3-4 weeks earlier than when the fruit is typically harvested (e.g., typical harvest time is when fruit is considered ripe, and / or at peak season). In some additional or alternative aspects, a fruit that is early- harvested can harvested between / during in its transition from green / unripe (but typically at or near [e.g., within 5% or 10%] full mature size) to fully-colored (i.e. , picked during the fruit’s color change). Thus, step (a) of providing a food product / precursor can optionally comprise such an early harvesting step, or a step of providing a food product / precursor from such an early-harvested fruit.

[0144] In some aspects, the number of days between harvesting fruit and juicing the fruit can be about, or under about, 0.5, 1 , 1.5, 2, 5, 10, 15, 20, 25, 30, 40, 50, 0.5-1 , 0.5-1.5, 0.5-2, 0.5-5, 1-1.5, 1-2, or 1-5 days. Juicing of fruit herein typically is done with whole fruit (i.e., containing at least the peel / rind, flesh and seeds [if seeded]). A fruit juice can contain one type of fruit juice (e.g., only orange juice), or it can contain a blend of two, three, or more juices, for example.

[0145] A food product / precursor comprising fruit juice can be stored for about, or up to about, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, for example, before applying step (b) of reacting the food product / precursor with one or more GTF enzymes herein. In some additional or alternative aspects, a food product / precursor comprising a fruit juice, or a harvested fruit before it is used to produce such a food product / precursor, can be subjected to a temperature of at least about 15 °C (e.g., >20 °C, >25 °C) for at least 4, 6, 12, 16, or 24 hours (and / or any other conditions that can increase aglycone limonoid and / or aglycone flavonoid content) before applying step (b) of reacting the food product / precursor with one or more GTF enzymes herein.

[0146] 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 performing step (b). 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 a GTF enzyme. 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. lactis, B. longum, B. animalis, B. breve, B. infantis), Komagataeibacter ssp., Liquorilactobacillus ssp. (e.g., Liquorilactobacillus neglii, Liquorilactobacillus ghanensis), Gluconobacterssp. 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., plant-based fresh fermented product) in some aspects can be an acidic culture / strain (or mix) (e.g., (Danisco® VEGE 053, Danisco® VEGE 011 , 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., Danisco® VEGE 022, Danisco® VEGE 053, Danisco® VEGE 022, Danisco® VEGE 047, Danisco® VEGE 061 , 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, S. vitulinus), Pediococcus ssp. (e.g., Pediococcus pentosaceus), K. lactis, Dekkera ssp. (e.g., Dekkera bruxellensis), Zygosaccharomyces ssp. (e.g., Zygosaccharomyces bailii), Brettanomyces ssp., Pichia genus (e.g., P. kluyveri, P. fermentans), Geotrichum, Debaryomyces and Candida genus (e.g., C. humilis, C. famata). A yeast in some aspects can be characterized as baker’s (baking) yeast, brewing yeast, wine-making 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 plant-based fresh fermented product herein, beer, beer wort, wine, pomace, cider, miso, kimchi, sauerkraut, pickles / pickle juice, soybean curd, tofu, kombucha, soy sauce, bread, sourdough, or meat, for example.

[0147] 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.

[0148] 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. Yet, in some aspects, a food product / precursor can comprise any of the foregoing plant-based ingredient(s) and be in any suitable food / precursor form disclosed herein (i.e., the food product / precursor need not be characterized as a non- dairy food product / precursor such as plant-based milk).

[0149] 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. 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 (e.g., acetic acid), vinegar, fruit / vegetable (e.g., herein, such as orange, apple, mango, peach, plum, banana, date, apricot, grapefruit, papaya, pineapple, raspberry, strawberry, blueberry, blackberry, pear, tangerine, cherry, grape, raisin, currant, 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, blended, or 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 (each 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.

[0150] 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.

[0151] A food product / precursor in some aspects herein, typically by virtue of comprising certain plant-based material (e.g., citrus fruit material), and typically as the food product / precursor exists before being treated with one or more GTF enzymes herein (i.e. , as provided in step [a]), comprises at least one limonoid compound. A limonoid compound can be a tetracyclic triterpenoid dilactone type of limonoid, and / or can be an aglycone limonoid in some aspects. Examples of limonoid compounds herein include limonin, nomilin, obacunone, obacunoic acid, isolimonic acid, deacetylnomilin, ichigan, isoobacunoic acid, and dictomnolide. A foregoing food product / precursor can have about, or at least about, 3, 4, 5, 6, 8, 10, 12, 14, or 16 ppm, for example, of one or a combination of limonoids (optionally characterized as “initial limonoid content” [e.g., initial limonin content]). Following GTF enzyme(s) treatment, a food product / precursor can have a limonoid content (e.g., limonin content) that is less than (e.g., about, or at least about, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 60-90%, 60-80%, 70-90%, or 70- 80% less than) the initial limonoid content (e.g., initial limonin content), typically by virtue of having one or more initial limonoids being glucosylated. In some aspects, the limonoid content (e.g., limonin content) of a GTF(s)-treated food product / precursor can be about, or below about, 6, 5, 4, 3, 2, 1 , 1-6, 1-5, 1-4, 2-6, 2-5, or 2-4 ppm.

[0152] The glucosylation of an initial limonoid herein, whether it be of an aglycone limonoid or limonoid glucoside, by GTF(s) forms a glucosylated limonoid (or a further glucosylated limonoid, in the case of glucosylation of a limonoid glucoside). While glucosylated limonoids can optionally still be considered to be limonoids, per se, it would be understood that the initial limonoid composition profile of a food product / precursor is reduced / modified by GTF(s) treatment. In some aspects, a result of GTF(s) treatment herein can be a reduction (e.g., by about, or by at least about, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 60-90%, 60-80%, 70-90%, or 70-80% by weight) (e.g., by about, or by at least about, 25%, 30%, 35%, 40%, 25-40%, 25-35%, or 30-40% by weight) in the initial aglycone limonoid content or in the initial aglycone tetracyclic triterpenoid dilactone limonoid content.

[0153] A food product / precursor in some aspects herein, typically by virtue of comprising certain plant-based material, and typically as the food product / precursor exists before being treated with one or more GTF enzymes herein (i.e., as provided in step [a]), comprises at least one flavonoid compound. A flavonoid compound can be a polymethoxylated flavonoid type of flavonoid, and / or can be an aglycone flavonoid in some aspects. A polymethoxylated flavonoid can have five methoxy (methyl ether) groups (e.g., sinensetin, tangeretin) or six methoxy (methyl ether) groups (e.g., nobiletin), for example. Flavonoid compounds in some aspects include sinensetin and / or nobiletin, optionally along with tangeretin and / or naringenin. Flavonoid compounds in some aspects include flavonoid glycosides / glucosides (i.e. , flavonoids with one or more glycosyl / glucosyl groups pre-existing before GTF treatment - i.e., being part of the initial flavonoid content) such as vicenin-A, for example. A foregoing food product / precursor can have about, or at least about, 3, 4, 5, 6, 8, 10, 12, 14, or 16 ppm, for example, of one or a combination of flavonoids (optionally characterized as “initial flavonoid content” [e.g., initial sinensetin and / or nobiletin content). Following GTF enzyme(s) treatment, a food product / precursor can have a flavonoid content (e.g., sinensetin and / or nobiletin content) that is less than (e.g., about, or at least about, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 60-90%, 60-80%, 70-90%, or 70-80% less than) the initial flavonoid content (e.g., initial sinensetin and / or nobiletin content), typically by virtue of having one or more initial flavonoids being glucosylated. In some aspects, the flavonoid content (e.g., sinensetin and / or nobiletin content) of a GTF(s)-treated food product / precursor can be about, or below about, 6, 5, 4, 3, 2, 1 , 1-6, 1-5, 1-4, 2-6, 2-5, or 2-4 ppm.

[0154] The glucosylation of an initial flavonoid herein, whether it be of an aglycone flavonoid or flavonoid glucoside, by GTF(s) forms a glucosylated flavonoid (or a further glucosylated flavonoid, in the case of glucosylation of a flavonoid glucoside). While glucosylated flavonoids can optionally still be considered to be flavonoids, per se, it would be understood that the initial flavonoid composition profile of a food product / precursor is reduced / modified by GTF(s) treatment. In some aspects, a result of GTF(s) treatment herein can be a reduction (e.g., by about, or by at least about, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 60-90%, 60-80%, 70-90%, or 70-80% by weight) in the initial aglycone flavonoid content or in the initial aglycone polymethoxylated flavonoid content.

[0155] Measuring the content of one or more limonoids and / or flavonoids herein can be performed using any suitable methodology, such as with a procedure disclosed in the below Examples (or with parameters within 5% or 10% of those disclosed in the below Examples).

[0156] A food product / precursor in some aspects after step (b) of a method herein has reduced off-flavor as compared to the food product / precursor as it existed before step (b) (i.e. as it existed before being treated with one or more GTF enzymes). Off-flavor can be reduced by about, or at least about 25%, 50%, 70%, 75%, 80%, 85%, 90%, or 95%, for example. Such a reduction in off-flavor can, in some aspects, be as compared to (i) the off-flavor that existed before the contacting with GTF(s), or (ii) the off-flavor of a suitable control (e.g., only difference being no GTF treatment). Off-flavor herein can comprise bitterness, sourness, and / or astringency, for example. Bitterness can optionally be measured in International Bitterness Units (IBU). Off-flavor can be as measured using a sensory panel evaluation, for example. Reduced off-flavor herein can be, for example, that off-flavor that is reduced by virtue of reducing the initial limonoid (e.g., initial limonin) content and / or the initial flavonoid (e.g., initial sinensetin and / or nobiletin content) content.

[0157] A food product / precursor in some aspects after step (b) of a method herein optionally has one or more of the following features as compared to the food product / precursor as it existed before step (b) (i.e. as it existed before being treated with one or more GTF enzymes):

[0158] (I) reduced sugar content,

[0159] (II) increased texture, such texture optionally comprising increased thickness and / or increased mouthfeel,

[0160] (III) improved physical appearance,

[0161] (IV) reduced calories,

[0162] (V) increased dietary fiber, and / or

[0163] (VI) increased stringiness or stretchability.

[0164] In some aspects, the sugar content (e.g., wt%) in a food product / precursor after step (b) can be reduced by about, or at least about, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 20-65%, 20-60%, 25-65%, or 25-60%, as compared to the sugar content of the food product / precursor as it existed before step (b). In some aspects, this reduction is with respect to all sugars in the food product / precursor, whereas in other aspects this reduction is with respect to a particular sugar such a sucrose. Sugar can be any as presently disclosed, for example. Sugar content herein can be measured by HPLC, for example.

[0165] In some aspects, the texture of a food product / precursor after step (b) can be increased by about, or at least about, 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 750%, 1000%, 1250%, 1500%, 1750%, 2000%, 2250%, 2500%, 3000%, 3500%, 4000%, 4500%, 5000%, 5500%, 6000%, 6500%, or 7000% as compared to the texture of the food product / precursor as it existed before step (b). Texture can be in terms of thickness or mouthfeel, and / or measured in units of Pascal-seconds (Pa s) or cP, for example. In some aspects, texture (thickness) can be measured by determining food product / precursor viscosity when extracted at a shear rate of about 11 to 12 Hz (e.g., 11.7 Hz). Texture (mouthfeel) can be measured by determining food product / precursor viscosity when extracted at a shear rate of about 248-250 Hz (e.g., 249 Hz), for example.

[0166] In some aspects, a food product / precursor after step (b) has an improved physical appearance as compared to the physical appearance of the food product / precursor as it existed before step (b). Improved physical appearance can be increased homogeneity (e.g., visual homogeneity) and / or increased shininess (e.g., visual shininess), for example; such increase(s) can be by about, or at least about, 5%, 10%, 20%, 25%, 30%, 40%, or 50% in some aspects.

[0167] In some aspects, the dietary caloric content (calories that can be accessed during digestion) of a food product / precursor after step (b) can be decreased by about, or at least about, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 75% as compared to the dietary caloric content of the food product / precursor as it existed before step (b).

[0168] In some aspects, the dietary fiber content (e.g., weight percent) of a food product / precursor after step (b) can be increased by about, or at least about, 5%, 10%, 25%, 50%, 75%, 100%, 200%, 300%, 400%, or 500% as compared to the dietary fiber content of the food product / precursor as it existed before step (b).

[0169] Some aspects of the present disclosure concern a food product / precursor as produced by a GTF treatment 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 / modified limonoid and / or flavonoid content, reduced sugar content, increased texture, increased stringiness (stretchability), 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 GTF enzyme as presently disclosed, and / or an alpha-glucan as presently disclosed. In some aspects, the activity of a GTF enzyme(s) used in a treatment herein can later have been terminated by suitable means (e.g., heat-inactivation at 90-100 °C).

[0170] In some aspects, a limonoid and / or flavonoid herein serves as an acceptor / primer for a GTF enzyme in a food / precursor in situ. A product of the GTF enzyme can be a glucosylated limonoid and / or glucosylated flavonoid, for example. Yet, in some additional or alternative aspects, a product of the GTF enzyme is contemplated to be, or further include, an alpha-glucan molecule comprising at least:

[0171] (i) alpha-1 ,6-glucan and / or alpha-1 , 3-glucan as disclosed herein, and (ii) a limonoid or flavonoid that has been used to prime synthesis of the alpha-1 ,6- glucan and / or alpha-1 ,3-glucan of (i).

[0172] Some aspects of the present disclosure concern a method (process) of glucosylating a limonoid and / or flavonoid compound, which method can optionally be characterized as a limonoid and / or flavonoid compound glucosylation method. Such a method can comprise a step of providing a composition that comprises at least water, sucrose, a limonoid compound and / or flavonoid compound, and a glucosyltransferase enzyme herein, and wherein the glucosyltransferase enzyme is selected from:

[0173] (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, and / or

[0174] (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, wherein at least one glucosylated form of the limonoid compound and / or flavonoid compound (glucosylated limonoid compound and / or glucosylated flavonoid compound) is produced in the composition, and / or wherein at least one alpha-glucan (above, having components [i] and [ii]) is produced in the composition. The providing step of such a method can optionally be characterized as a step of contacting a limonoid compound and / or flavonoid compound with at least one glucosyltransferase enzyme in the presence of at least water and sucrose. Typically, a limonoid compound and / or flavonoid compound glucosylation method also produces at least one alpha-glucan as presently disclosed (where the limonoid and / or flavonoid did not prime synthesis of the alpha-glucan), such as an alpha-1 , 6-glucan, an alpha-1 , 3-glucan, and / or a graft copolymer herein.

[0175] An alpha-1 , 6-glucan-synthesizing glucosyltransferase enzyme in a limonoid and / or flavonoid glucosylation method can be any as presently disclosed herein. An alpha-1 , 3-glucan-synthesizing glucosyltransferase enzyme in a limonoid and / or flavonoid glucosylation method can be any as presently disclosed herein. Any condition and / or parameter for using any of these enzymes in this method can be as presently disclosed herein (e.g., temperature, time, water content, sucrose content, GTF enzyme content).

[0176] A glucosylated limonoid and / or flavonoid herein (e.g., produces] of a limonoid and / or flavonoid glucosylation method) is believed to comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glucose monomeric units (typically in a chain) added to the original limonoid and / or flavonoid, for example. A limonoid and / or flavonoid can optionally be characterized herein to serve as an acceptor / primer for alpha-glucan synthesis by a GTF enzyme in a composition of the disclosure. Thus, some aspects of the present disclosure concern an alpha-glucan molecule comprising at least:

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

[0178] (ii) a limonoid or flavonoid that has been used to prime synthesis of the alpha-1 ,6- glucan and / or alpha-1 , 3-glucan of (i); wherein portion (i) is in glycosidic linkage with portion (ii), and portion (ii) is at the reducing end of the alpha-glucan molecule (e.g., by virtue of having used the limonoid or flavonoid of (ii) as an acceptor for priming synthesis of the alpha-1 ,6-glucan or alpha-1 ,3- glucan). The DP or DPw of the alpha-glucan can be any DP or DPw value disclosed herein, for example.

[0179] In some aspects, a composition in which a glucosylated limonoid and / or flavonoid (and / or an alpha-glucan primed by a limonoid or flavonoid) can be produced in an limonoid or flavonoid glucosylation method can be any as presently disclosed herein, such as a food precursor / product. Yet, in some aspects, such a composition 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. WO201 6 / 133734, which are all incorporated herein by reference. In some aspects, a composition 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) as disclosed in any of the foregoing publications and / or as presently disclosed. While a glucosylated limonoid or flavonoid (and / or an alpha-glucan primed by a limonoid or flavonoid) can be produced in situ in a composition, it can optionally be produced in a separate or isolated GTF reaction composition and then introduced as an ingredient for making a composition.

[0180] Several aspects herein are disclosed with respect to glucosylation of limonoid and / or flavonoid compounds. Any of these particular disclosures can, in some additional or alternative aspects, instead (or additionally) be considered with respect to reducing the amount of the referenced limonoid(s) and / or flavonoid(s), as appropriate (i.e., a disclosure herein of glucosylation of a limonoid and / or flavonoid compound can instead, and / or additionally, be considered a disclosure of reduction of the limonoid and / or flavonoid compound). Such reduction can be by a percent level disclosed herein, for example.

[0181] Non-limiting examples of compositions and methods / processes disclosed herein include:

[0182] 1. A method (process) of producing a food product / precursor, the method comprising: (a) providing a food product / precursor that comprises at least water, sucrose and a plant-based material (typically, plant-based material comprises at least one limonoid compound and / or at least one flavonoid compound) (typically, the plantbased material is human-edible and / or household pet [e.g., dog / cat]-edible), and (b) contacting the food product / precursor with at least: (i) a glucosyltransferase enzyme (a dextransucrase) that synthesizes (is capable of synthesizing) alpha-1 , 6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1 , 6-glucan are alpha-1 ,6 linkages, and / or (ii) a glucosyltransferase enzyme (a glucansucrase) that synthesizes (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, whereby the food product / precursor, after step (b), has a reduced content of one or more limonoid compounds and / or flavonoid compounds that were present in (e.g., natural to) the food product / precursor of step (a) (i.e., the limonoid and / or flavonoid compound profile in the food product / precursor entered into the method has been modified by the glucosyltransferase treatment of step [b]), optionally wherein the reduced content of one or more limonoid and / or flavonoid compounds results in a reduction of off-flavor of the food product / precursor (as compared to the off-flavor of the food product / precursor of step [a]), typically / optionally wherein at least one alpha-glucan (e.g., the alpha-1 ,6- glucan, the alpha-1 , 3-glucan, and / or a graft copolymer herein) is produced in the food product / precursor.

[0183] 2. The method of embodiment 1 , wherein the food product / food precursor, after step (b), has an increased content of at least one glucosylated limonoid and / or at least one glucosylated flavonoid (typically, where such at least one glucosylated limonoid and / or at least one glucosylated flavonoid is not present in the food product / precursor of step [a]), as compared to the food product / precursor before step (b). 3. The method of embodiment 1 or 2, wherein the food product / precursor, after step (b), has a reduced content of the one or more limonoid compounds, optionally wherein the one or more limonoid compounds is a tetracyclic triterpenoid dilactone (i.e. , optionally, the tetracyclic triterpenoid dilactone content in the food product / precursor entered into the method is reduced in step [b], typically by virtue of having been glucosylated by the glucosyltransferase enzyme[s]) and / or a limonoid glycoside / glucoside (i.e., optionally, the limonoid glycoside / glucoside content in the food product / precursor entered into the method is reduced in step [b], typically by virtue of having been glucosylated by the glucosyltransferase enzyme[s]).

[0184] 4. The method of embodiment 3, wherein the one or more limonoid compounds is limonin and / or nomilin (i.e., the limonin and / or nomilin [and / or a precursor of one or both of these such as a monolactone form] content in the food product / precursor entered into the method is reduced in step [b], typically by virtue of having been glucosylated by the glucosyltransferase enzyme[s]).

[0185] 5 The method of embodiment 1 , 2, 3, or 4, wherein the food product / precursor, after step (b), has a reduced content of the one or more flavonoid compounds, optionally wherein the one or more flavonoid compounds is a polymethoxylated flavonoid (i.e., optionally, the polymethoxylated flavone content in the food product / precursor entered into the method is reduced in step [b], typically by virtue of having been glucosylated by the glucosyltransferase enzyme[s]) and / or a flavonoid glycoside / glucoside (i.e., optionally, the flavonoid glycoside / glucoside content in the food product / precursor entered into the method is reduced in step [b], typically by virtue of having been glucosylated by the glucosyltransferase enzyme[s]).

[0186] 6. The method of embodiment 5, wherein the one or more flavonoid compounds is sinensetin and / or nobiletin and / or vicenin-2 (i.e., the sinensetin and / or nobiletin and / or vicenin-2 content in the food product / precursor entered into the method is reduced in step [b], typically by virtue of having been glucosylated by the glucosyltransferase enzyme[s]).

[0187] 7. The method of embodiment 1 , 2, 3, 4, 5, or 6, wherein the off-flavor comprises bitterness, sourness, and / or astringency.

[0188] 8. The method of embodiment 1 , 2, 3, 4, 5, 6, or 7, wherein: the glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan comprises an amino acid sequence that is at least 90% (or 95%) identical to SEQ ID NO:1 , 2, 11 , 12, 14, or 15 (e.g., the alpha-1 ,6- glucan-producing GTF comprises an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO:1 or 14), and / or the glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan comprises an amino acid sequence that is at least 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 (e.g., the alpha-1 , 3-glucan-producing GTF comprises an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO:3, 4, or 5).

[0189] 9. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, or 8, wherein the (i) glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan is used in the contacting of step (b) (e.g., only the alpha-1 , 6-glucan-producing GTF is used, without using the alpha-1 , 3-glucan-producing GTF).

[0190] 10. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, or 8, wherein both of the (i) glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan and the (ii) glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan are used in the contacting of step (b).

[0191] 10a. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, or 8, wherein the (i) glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan is used in the contacting of step (b) (e.g., only the alpha-1 , 3-glucan-producing GTF is used, without using the alpha-1 , 6-glucan-producing GTF).

[0192] 11 . The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10a, wherein the plantbased material is from a plant of the Rutaceae family (or “a Rutaceae family plant”) or the Cucurbitaceae family (or “a Cucurbitaceae family plant”).

[0193] 12. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 10a, or 11 , wherein the food product / precursor comprises fruit juice (e.g., a beverage) and / or fruit solids (e.g., pulp and / or seed pieces).

[0194] 13. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 10a, 11 , or 12, wherein the food product / precursor comprises citrus fruit juice and / or citrus fruit solids.

[0195] 14. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 10a, 11 , 12, or 13, wherein the food product / precursor comprises orange juice and / or orange solids (e.g., Valencia orange juice and / or solids).

[0196] 15. The method of embodiment 12, 13, or 14, wherein the fruit (of the fruit juice or the fruit solids) is harvested (picked) at least 1 week (e.g., at least 2 weeks or at least 3 weeks) earlier than when the fruit is typically harvested (e.g., typical harvest time is when fruit is considered ripe, and / or at peak season).

[0197] 16. The method of embodiment 12, 13, 14, or 15, wherein the fruit juice or the fruit solids has been subjected to a temperature of at least about 15 °C (e.g., >20 °C, >25 °C) for at least 1 day. 17. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 10a, 11 , 12, 13, 14, 15, or 16, wherein the food product / precursor provided in step (a) comprises at least about 3 parts-per-million (ppm) limonin (e.g., at least about 6 or 7 ppm limonin) as an initial limonin content.

[0198] 18. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 10a, 11 , 12, 13, 14, 15, 16, or 17, wherein step (a) comprises adding sucrose to the food product / precursor. 18a. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 10a, 11 , 12, 13, 14, 15, 16, 17, or 18, wherein step (b) is conducted for up to about 3, 5, or 7 hours (e.g., about 3-7, 3-6, 3-5, 3-4, 4-7, 4-6, or 4-5 hours) (i.e. , step [b] is ended after about 3, 5, or 7 hours), optionally wherein step (b) is ended by (I) terminating the activity of the glucosyltransferase(s) (e.g., by heat-inactivation such as by heating the food product / precursor at, or above, 90 °C or 90-100 °C) (e.g., freezing the food product / precursor) and / or (II) separating the glucosyltransferase(s) and food product / precursor from each other (e.g., removing support-immobilized glucosyltransferase(s) from the food product / precursor), optionally wherein step (b) is conducted at a temperature of about 3-10 °C (e.g., about 3-7, 4-6, or 5 °C) (and optionally wherein the limonin content of the food product / precursor is reduced by about, or at least about, 25%, 30%, 35%, 40%, 25-40%, 25-35%, or 30-40% by weight as compared to the limonin content of the food product / precursor that existed before step [b]).

[0199] 18b. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 10a, 11 , 12, 13, 14, 15, 16, 17, 18, or 18a, wherein the food product / precursor provided in step (a) is pasteurized (has been pasteurized) (e.g., heating to about 70-77.5 °C or 75 °C and holding at this temperature for about 10-15, 10-20, 15-20, 14-16, or 15 seconds before cooling).

[0200] 18c. The method of embodiment 10, 11 , 12, 13, 14, 15, 16, 17, 18, 18a, or 18b, wherein the ratio of the (i) glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan to the (ii) glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan in the contacting of step (b) is about 40:60 to about 60:40 (e.g., about 45:55, 50:50, or 55:45), typically wherein the ratio is based on the activity of each glucosyltransferase enzyme (e.g., activity units per gram formulated enzyme).

[0201] 19. A food product / precursor (e.g., a beverage) produced by the method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 10a, 11 , 12, 13, 14, 15, 16, 17, 18, 18a, 18b, or 18c.

[0202] 20. A method (process) of glucosylating a limonoid and / or flavonoid compound, the method comprising: providing a composition that comprises at least water, sucrose, a limonoid compound and / or flavonoid compound, and at least one glucosyltransferase enzyme (or contacting a limonoid compound and / or flavonoid compound with at least one glucosyltransferase enzyme in the presence of at least water and sucrose), wherein the glucosyltransferase enzyme is selected from: (i) a glucosyltransferase enzyme (a dextransucrase) that synthesizes (is capable of synthesizing) alpha-1 , 6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1 , 6-glucan are alpha-1 ,6 linkages, and / or (ii) a glucosyltransferase enzyme (a glucansucrase) that synthesizes (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, wherein at least one glucosylated form of the limonoid compound and / or flavonoid compound is produced in the composition, and typically / optionally wherein at least one alpha-glucan (e.g., the alpha-1 , 6-glucan, the alpha-1 , 3-glucan, and / or a graft copolymer herein) is produced in the composition.

[0203] 21 . The method of embodiment 20, wherein the limonoid compound is limonin or nomilin (i.e. , a glucosylated form of limonin or nomilin is produced in the composition, typically by virtue of having been glucosylated by the glucosyltransferase enzyme[s]). 21a. The method of embodiment 20, wherein the limonoid compound is a tetracyclic triterpenoid dilactone (i.e., a glucosylated form of tetracyclic triterpenoid dilactone is produced in the composition, typically by virtue of having been glucosylated by the glucosyltransferase enzyme[s]).

[0204] 22. The method of embodiment 20, 21 , or 21a, wherein the flavonoid compound is sinensetin or nobiletin (i.e., a glucosylated form of sinensetin and / or nobiletin is produced in the composition, typically by virtue of having been glucosylated by the glucosyltransferase enzyme[s]).

[0205] 22a. The method of embodiment 20, 21 , or 21a, wherein the flavonoid compound is a polymethoxylated flavonoid (i.e., a glucosylated form of polymethoxylated flavonoid is produced in the composition, typically by virtue of having been glucosylated by the glucosyltransferase enzyme[s]).

[0206] 22b. The method of embodiment 20, 21 , or 21a, wherein the flavonoid compound is a flavonoid glycoside / glucoside (i.e., a further glucosylated form of the flavonoid glycoside / glucoside is produced in the composition, typically by virtue of having been glucosylated by the glucosyltransferase enzyme[s]).

[0207] 22c. The method of embodiment 22b, wherein the flavonoid glycoside / glucoside is vicenin-2 (i.e., a glucosylated form of vicenin-2 is produced in the composition, typically by virtue of having been glucosylated by the glucosyltransferase enzyme[s]; the vicenin- 2 can optionally be characterized to have been further glucosylated with respect to its original structure).

[0208] 23. The method of embodiment 20, 21 , 21a, 22, 22a, 22b, or 22c, wherein: the glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan comprises an amino acid sequence that is at least 90% (or 95%) identical to SEQ ID NO:1 , 2, 11 , 12, 14, or 15 (e.g., the alpha-1 ,6-glucan-producing GTF comprises an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO:1 or 14), and / or the glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan comprises an amino acid sequence that is at least 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 (e.g., the alpha-1 , 3-glucan- producing GTF comprises an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO:3, 4, or 5).

[0209] 24. The method of embodiment 20, 21 , 21a, 22, 22a, 22b, 22c, or 23, wherein the (i) glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan is comprised in the composition (e.g., only the alpha-1 ,6-glucan-producing GTF is used, without using the alpha-1 , 3-glucan-producing GTF).

[0210] 25. The method of embodiment 20, 21 , 21a, 22, 22a, 22b, 22c, or 23, wherein both of the (i) glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan and the (ii) glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan are comprised in the composition.

[0211] 25a. The method of embodiment 20, 21 , 21a, 22, 22a, 22b, 22c, or 23, wherein the (i) glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan is comprised in the composition (e.g., only the alpha-1 , 3-glucan-producing GTF is used, without using the alpha-1 , 6-glucan-producing GTF).

[0212] 26. The method of embodiment 20, 21 , 21a, 22, 22a, 22b, 22c, 23, 24, 25, or 25a, wherein the composition is a food product / precursor, typically further comprising a plantbased material that typically is human-edible and / or household pet [e.g., dog / cat]-edible.

[0213] 27. The method of embodiment 26, wherein the food product / precursor comprises fruit juice (e.g., a beverage) and / or fruit solids (e.g., pulp and / or seed pieces).

[0214] 28. The method of embodiment 26 or 27, wherein the food product / precursor comprises citrus fruit juice and / or citrus fruit solids.

[0215] 29. The method of embodiment 26, 27, or 28, wherein the food product / precursor comprises orange juice and / or orange solids (e.g., Valencia orange juice and / or solids). 29a. The method of embodiment 26, 27, 28, or 29, wherein the food product / precursor comprises at least about 3 parts-per-million (ppm) limonin (e.g., at least about 6 or 7 ppm limonin) as an initial limonin content.

[0216] 29b. The method of embodiment 20, 21 , 21a, 22, 22a, 22b, 22c, 23, 24, 25, 25a, 26, 27, 28, 29, or 29a, wherein the composition is held (incubated) for up to about 3, 5, or 7 hours (e.g., about 3-7, 3-6, 3-5, 3-4, 4-7, 4-6, or 4-5 hours) (i.e., step [b] is ended after about 3, 5, or 7 hours), optionally wherein the activity of the glucosyltransferase(s) is terminated upon the hours having elapsed (e.g., by heat-inactivation such as by heating the composition at, or above, 90 °C or 90-100 °C) (e.g., freezing the composition) and / or the glucosyltransferase(s) is / are separated out of the composition upon the hours having elapsed (e.g., removing support-immobilized glucosyltransferase(s) from the composition), optionally wherein the composition is held at a temperature of about 3-10 °C (e.g., about 3-7, 4-6, or 5 °C) during the hours of holding (incubating) the composition (and optionally wherein the limonin content of the composition is reduced by about, or at least about, 25%, 30%, 35%, 40%, 25-40%, 25-35%, or 30-40% by weight as compared to the limonin content of the composition that existed before the holding / incubating the composition).

[0217] 29c. The method of embodiment 25, 26, 27, 28, 29, 29a, or 29b, wherein the ratio of the (i) glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan to the (ii) glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan in the composition is about 40:60 to about 60:40 (e.g., about 45:55, 50:50, or 55:45), typically wherein the ratio is based on the activity of each glucosyltransferase enzyme (e.g., activity units per gram formulated enzyme).

[0218] 30. A composition, or a glucosylated limonoid compound and / or glucosylated flavonoid compound, produced by the method of embodiment 20, 21 , 21a, 22, 22a, 22b, 22c, 23, 24, 25, 25a, 26, 27, 28, 29, 29a, 29b, or 29c.

[0219] 31 . A composition comprising a glucosylated limonoid compound and / or glucosylated flavonoid compound, wherein the glucosylated limonoid compound and / or glucosylated flavonoid compound is produced by contacting a limonoid compound and / or flavonoid compound with a glucosyltransferase enzyme in the presence of at least water and sucrose, wherein the glucosyltransferase enzyme is selected from: (i) a glucosyltransferase enzyme (a dextransucrase) that synthesizes (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 (e.g., as recited in embodiment 23, and / or (ii) a glucosyltransferase enzyme (a glucansucrase) that synthesizes (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 (e.g., as recited in embodiment 23), optionally wherein the composition is a food product / precursor, and / or optionally wherein the glucosylated limonoid compound and / or glucosylated flavonoid compound is produced in situ in the composition or is produced in an isolated GTF reaction and then added as an ingredient for producing the composition, typically / optionally wherein at least one alpha-glucan (e.g., the alpha-1 , 6-glucan, the alpha-1 , 3-glucan, and / or a graft copolymer herein) is produced in the composition.

[0220] 32. The composition of embodiment 31 , wherein the limonoid compound is limonin (i.e. , the composition comprises glucosylated limonin).

[0221] 33. The composition of embodiment 31 or 32, wherein the flavonoid compound is vicenin-2 (i.e., the composition comprises glucosylated vicenin-2).

[0222] 34. The composition of embodiment 19 (food product / precursor) or 30, or the method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 10a, 11 , 12, 13, 14, 15, 16, 17, 18, 18a, 18b, 18c, 20, 21 , 21a, 22, 22a, 22b, 22c, 23, 24, 25, 25a, 26, 27, 28, 29, 29a, 29b, or 29c, wherein, instead of (or in addition to) being considered with respect to glucosylation of a limonoid compound(s) and / or flavonoid compound(s), the composition or method is considered with respect to reducing the amount of the recited limonoid and / or flavonoid compound(s), as appropriate (i.e., a disclosure herein of glucosylation of a limonoid and / or flavonoid compound can instead be, and / or additionally be, considered a disclosure of reduction of the limonoid and / or flavonoid compound).

[0223] EXAMPLES

[0224] 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 the 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. Materials

[0225] Substrate: Tropicana Brands group early-season orange juice, Brix 9.23. Enzymes: Glucosyltransferase (GTF) enzymes 0768 (SEQ ID NO:1 , also represented by SEQ ID NOs:2, 11 and 12), 6831 (SEQ ID NO: 14, also represented by SEQ ID NO:15) and an amino acid-substituted GTF 6855 variant (SEQ ID NO:3, “vGTFJ” herein) were used. 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 GTF 6831 produces a soluble alpha-glucan having about 100% alpha-1 ,6 linkages (i.e., a linear dextran) and a DPw typically of about 1000 to 1300. 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, if desired, to stably produce at high yield insoluble alpha-glucan having about 100% alpha-1 ,3 linkages).

[0226] HPLC column: Agilent Advanced Biopeptide 2.7 m (2.1x150mm).

[0227] HPLC: Agilent 1260.

[0228] Mobile phase: Initial mobile phase w / 0.1 % formic acid, gradient run eluted with 0.1% formic acid + 100% acetonitrile.

[0229] Ehrlich’s reagent: 0.2 mL peracetic acid + 0.3 ml_ acetic acid + 0.02 g 4- (dimethylamino) benzaldehyde (DMAB).

[0230] Methods

[0231] A limonin standard was pre-injected as a reference standard at 33 ppm concentration.

[0232] The orange juice was clarified by centrifugation.

[0233] The GTF enzymes used in the Examples below were applied according to the following table:

[0234] The GTF blend used in Examples 2, 3, 4, 6 and 7 below was prepared by combining 7.92 g as-formulated GTF 0768 (SEQ ID NO:1) and 3.52 g as-formulated vGTFJ (SEQ ID NO:3) to provide a 69%:31% by weight ratio of GTF 0768 to vGTFJ (equivalent to a 90:10 ratio on an active protein basis [see above Methods table, activity unit I g formulated]). The ratio of these enzymes in terms of activity: activity can be calculated based on values in the above table. The GTF enzyme blend was reacted with the clarified orange juice (GTF blend content in juice at 0.3 wt%) and incubated at 5 °C for 3 hours and 18 hours. Samples were collected and centrifuged, and 5 pL volumes of the supernatant was injected into the LC.

[0235] Fractions were collected from the HPLC for spectral analysis and reacted with Ehrlich’s reagent to detect the presence of free indole groups, which could indicate the formation of a limonin glycoside. A speed vacuum was used to concentrate each fraction, and then the concentrated fractions were re-suspended in 50% acetonitrile. A 1 :1 mix of the re-suspended fractions were combined with the Ehrlich’s reagent, incubated for 30 minutes at room temperature, and then the absorbance was read at 470 nm.

[0236] Example 1 Identification and Quantification of Limonin and Other Limonoids / Flavonoids in Citrus Juice

[0237] FIG. 1 A shows the HPLC profile of clarified early-season orange juice (Tropicana) and FIG. 1 B shows the HPLC profile of a limonin standard at 33 ppm concentration. Limonin was positively identified as the middle peak in FIG. 1 A with a retention time between 7-7.5 minutes. The area under the peak indicated that the concentration of limonin in the orange juice was approximately 30 ppm. Two additional unidentified limonoids / flavonoids appeared directly to the left and right of the limonin peak, as shown in FIG. 1A.

[0238] Example 2

[0239] Reduction of Limonin and Other Limonoids / Flavonoids in Citrus Juice by Glucosyltransferase Treatment

[0240] FIG. 2A shows the HPLC profile of the clarified early-season orange juice control. FIG. 2B shows the HPLC profile of the clarified orange juice that was incubated for 18 hours at 5 °C with a glucosyltransferase enzyme (vGTFJ, SEQ ID NO:3) that synthesizes alpha-1 ,3-glucan (enzyme added to a final concentration of 0.3 wt%). FIG. 2C shows the HPLC profile of the clarified orange juice that was incubated for 18 hours at 5 °C with a glucosyltransferase enzyme (GTF 0768, SEQ ID NO:1) that synthesizes dextran (enzyme added to a final concentration of 0.3 wt%). FIG. 2D shows the HPLC profile of the clarified orange juice that was incubated for 18 hours at 5 °C with a blend of these two glucosyltransferase enzymes (vGTFJ + GTF 0768, see Methods) (enzyme blend added to a final concentration of 0.3 wt%). In comparing FIG. 2B with FIG. 2A, it was apparent that little or no reduction of limonoids / flavonoids (including limonin) was observed in orange juice treated with vGTFJ alone. However, in comparing FIG. 2C with FIG. 2A, some reduction of the limonoids / flavonoids was observed in orange juice treated with GTF 0768 alone. The greatest reduction of the limonoids / flavonoids as compared to control was observed in the orange juice that was treated with both vGTFJ and GTF 0768 (FIG. 2D). As further appreciated when overlaying the HPLC graphs of FIG. 2A-2D (data not shown), it was apparent that, although vGTFJ did not exert a significant effect on limonoids / flavonoids levels when used alone in the juice, the combination of vGTFJ with GTF 0768 substantially boosted reduction of limonoids / flavonoids as compared to when using GTF 0768 alone.

[0241] Example 3 Time Course of Reduction of Limonin and Other Limonoids / Flavonoids in Citrus Juice by Glucosyltransferase Treatment

[0242] FIG. 3A shows the HPLC profile of the clarified early-season orange juice control. FIGs. 3B and 3C show, respectively, the HPLC profiles of the clarified orange juice after 3 hours, or 18 hours, of incubation at 5 °C with 0.3 wt% concentration of a blend of the glucosyltransferases vGTFJ (SEQ ID NO:3) and GTF 0768 (SEQ ID NO:1). As shown in FIG. 3B (in comparison to FIG. 3A), the limonin content of the juice was reduced by about 60% after the 3-hour incubation, while the content of all limonoids / flavonoids was reduced by 67%. No further changes of these compounds in the juice were observable after the 18-hour incubation, as shown in FIG. 3C, thereby indicating that the glucosyltransferase reactions on limonoids / flavonoids in the juice were generally complete by about the 3-hour timepoint under the foregoing conditions.

[0243] Example 4 Effects of Using Inactivated Glucosyltransferase or Additional Glucosyltransferase Dosage on Limonin and Other Limonoids / Flavonoids in Citrus Juice

[0244] FIG. 4A shows the HPLC profile of the clarified early-season orange juice control. FIG. 4B shows the HPLC profile of the clarified orange juice after 3 hours of incubation at 5 °C with 0.3 wt% concentration of a glucosyltransferase blend (vGTFJ [SEQ ID NO:3] and GTF 0768 [SEQ ID NO: 1 ]) that had been inactivated at pH 12 before adding the blend to the juice. FIG. 4C shows the HPLC profile of the clarified orange juice after two dosages of a glucosyltransferase blend: an initial dose at 0.3 wt% concentration followed by a 3-hour incubation at 5 °C, and then a second dose at 0.3 wt% at the 3- hour time point followed by an overnight incubation at 5 °C (the enzyme blend in each dose was active, having not been inactivated). No change in limonoids / flavonoids content was observed in the HPLC profile of the orange juice that was treated with the inactivated glucosyltransferase blend (compare FIG. 4B with the control profile of FIG. 4A). However, as observed in comparing FIG. 4C with the control profile of FIG. 4A, limonin content of juice treated with the active glucosyltransferase blend doses was reduced by about 78%, while all limonoids / flavonoids were reduced by about 80%.

[0245] Example 5

[0246] Reduction of Limonin and Other Limonoids / Flavonoids in Citrus Juice by Glucosyltransferase Treatment

[0247] FIG. 5A shows the HPLC profile of the clarified early-season orange juice control. FIG. 5B shows the HPLC profile of the clarified orange juice that was incubated for 18 hours at 5 °C with a glucosyltransferase enzyme (GTF 6831 , SEQ ID NO: 14) that synthesizes alpha-1 ,6-glucan (the enzyme was added to a final concentration of 0.3 wt%). In comparing FIG. 5B with the control profile of FIG. 5A, about a 59% reduction of limonin was observed in juice treated with glucosyltransferase, while all limonoids / flavonoids were reduced in content.

[0248] As further appreciated by comparing FIG. 2C (GTF 0768 activity) with FIG. 2A (control), and comparing FIG. 5B (GTF 6831 activity) with FIG. 5A (control), it was apparent that dextransucrase GTF 6831 had a significantly greater effect on reducing limonoids / flavonoids levels when used alone in juice as compared to using dextransucrase GTF 0768 alone.

[0249] Example 6

[0250] Positive Ehrlich Test Indicating Presence of a Glycoside

[0251] FIG. 6 shows the HPLC profile of clarified early-season orange juice after treatment with a blend of the glucosyltransferases vGTFJ (SEQ ID NO:3) and GTF 0768 (SEQ ID NO: 1) for 18 hours at 5 °C (the enzyme blend was added to a final concentration of 0.3 wt%). Fractions (1-7) were collected, concentrated, and reacted with Ehrlich’s reagent as described in the Methods section. Peak / fraction 1 developed a pink-red color upon treatment with the Ehrlich’s reagent, while all the other fractions remained colorless. This result indicated the presence of a glycoside in the peak 1 fraction.

[0252] Example 7

[0253] Glucosylation of Vicenin-2 in Citrus Juice by Glucosyltransferase

[0254] FIG. 7A shows the HPLC profile of 100% orange juice containing both limonin and a compound that was identified as isovitexin 8-C-beta-glucoside, otherwise known as vicenin-2. Vicenin-2 is a C-glycosyl compound that is isovitexin in which the hydrogen at position 8 is replaced by a beta-D-glucosyl residue. It is a flavone glycoside that is naturally occurring in citrus juices. Additional vicenin-2 was spiked into the orange juice at 100 ppm concentration to amplify the peaks.

[0255] FIG. 7B shows the HPLC profile of the orange juice as treated with a blend of the glucosyltransferases vGTFJ (SEQ ID NO:3) and GTF 0768 (SEQ ID NO: 1) for 90 minutes at ambient temperature (the enzyme blend was added to a final concentration of 0.3 wt%). In comparing FIGs. 7A and 7B, an approximately 30% reduction of vicenin-2 was observed and the formation of a new peak appeared directly to the left of the vicenin-2 peak (FIG. 7B). The fraction containing the new peak was collected and confirmed by MALDI-MS analysis to contain a mix of vicenin-2 glycosides, thereby indicating that vicenin-2 likely served as an acceptor for glucosylation by one or both of the glucosyltransferases. A reduction of limonin was also observed in FIG. 7B (as compared to FIG. 7A).

[0256] Example 8 Reduction of Limonin and Sucrose over Time in Citrus Juice by Glucosyltransferase T reatment

[0257] FIG. 8A illustrates the limonoid and flavonoid region of an HPLC profile of early season orange juice that was treated with 1 % w / w of a GTF enzyme blend over a period of 24 hours at a temperature of 5 °C. The blend consisted of a 40:60 ratio of GTF 0768 to vGTFJ (active protein basis [see Methods table, activity unit I g formulated]). HPLC profiles of the juice were taken at 0, 2, 5, 8, 10, 14 and 24 hours of GTF treatment. The treatment resulted in a noticeable reduction in six peaks within this region. Among these, four peaks were successfully identified as the compounds limonin, sinensetin, nomilin, and nobiletin (FIG. 8A). This reduction likely indicates that the enzyme blend is effective at altering the juice’s flavor profile (e.g., less bitter).

[0258] FIG. 8B illustrates the time course of limonin reduction in the juice as observed with the above GTF enzyme blend (-15:85) incubation. The majority of the limonin reduction occurred within the first five hours of GTF treatment, with approximately 35% of the limonin being reduced during this period. Beyond this initial phase, the rate of reduction slowed down significantly.

[0259] FIG. 8C depicts the changes in sugar concentrations in the juice over time as observed with the above GTF enzyme blend (40:60) incubation. The data indicate that there were notable fluctuations in sugar levels throughout the incubation period, which may correlate with enzymatic activity and limonin reduction. Initially, there was a rapid decrease in sucrose and increase in fructose concentration as the GTF treatment ensued. As the treatment progressed, the sugar levels began to stabilize, suggesting that the GTF enzymatic activity reached a steady state. This stabilization phase appears crucial under the tested conditions as it indicates the point at which the enzyme blend had maximized its efficiency in converting substrates. Additionally, the correlation between the reduction of limonin levels and the changes in sugar levels suggests a possible interaction or co-dependency between these two processes.

[0260] Table 1 provides a detailed comparison of the reduction levels of limonin and sucrose in the juice at various time points during the above GTF enzyme blend (40:60) incubation. Notably, at the 5-hour mark, when limonin reduction reached its peak (FIG. 8B), only 51% of the sucrose had been reduced (FIG. 8C). This indicates a significant difference in the reduction rates of these two compounds under the same conditions. The data highlight the efficiency of the GTF enzymes in reducing limonin levels compared to sucrose, which could have important implications for optimization of this enzymatic treatment regimen in related applications. Further analysis of the reaction kinetics and the factors influencing these rates could provide deeper insights into the GTF enzymes’ specificity and potential other uses.

[0261] Table 1

[0262] Example 9 Comparing Glucosyltransferase Treatment-Mediated Reduction of Limonin and Sucrose in Freshly Squeezed Orange Juice and Pasteurized Orange Juice

[0263] Samples of freshly squeezed early season orange juice and recently pasteurized early season orange juice were treated with 1 % w / w of a GTF enzyme blend over a period of 6 hours at a temperature of 10 °C. The blend consisted of a 40:60 ratio of GTF 0768 to vGTFJ (active protein basis [see Methods table, activity unit I g formulated]). Pasteurization was done by heating orange juice to 75 °C and holding at this temperature for 15 seconds before cooling. Table 2 shows the reduction in limonin and sucrose levels in the freshly squeezed early season orange juice sample. Table 3 shows the reduction in limonin and sucrose levels in the recently pasteurized early season orange juice sample. These tables provide a comparative analysis of the impact of pasteurization on the chemical composition of early season orange juice, highlighting the differences in limonin and sucrose content between the two processing methods.

[0264] T able 2. GTF-T reatment of Freshly Squeezed Orange Juice

[0265] Table 3. GTF-Treatment of Pasteurized Orange Juice

[0266] It was apparent that GTF treatment-mediated limonin reduction was greatly enhanced in the pasteurized juice versus the freshly squeezed juice. Similar GTF- mediated reduction profiles were observed for sinensetin, nomilin, and nobiletin levels between the freshly squeezed and pasteurized juice samples (data not shown). These results were unexpected, as it was contemplated that GTF treatment would have had a greater impact on freshly squeezed juice in which limonin is predominantly in its monolactone form.

[0267] Example 10

[0268] Treating Orange Juice with Various Glucosyltransferase Blend Ratios Leads to Differences in Limonin Reduction and Overall Limonoid / Flavonoid Content Table 3 demonstrates the changes in the reduction of limonin and the overall limonin / flavonoid profile of orange juice when different ratios of two GTF enzymes (GTF 0768 to vGTFJ) were used to treat the juice. In particular, three GTF blends (A-C) were tested of GTF 0768 and vGTFJ in the following ratios: (A) 60:40, (B) 50:50 and (C) 40:60 (active protein basis [see Methods table, activity unit I g formulated]). Samples of orange juice were individually treated with 0.5% w / w of each GTF enzyme blend over a period of 15 hours at a temperature of 5 °C. Notably, Blend B achieved the greatest reduction in limonin and the highest total reduction of limonoids and flavonoids after the 15-hour incubation period. This result suggests that adjusting the enzyme ratio can significantly impact the efficiency of limonin and flavonoid reduction, potentially leading to improved outcomes in related applications.

[0269] Table 4. Treating Orange Juice with Different GTF Blends FIG. 9 provides a detailed illustration of how various combinations of the two GTF enzymes (GTF 0768 to vGTFJ) can influence the levels of limonin in orange juice. In particular, four GTF blends were tested of GTF 0768 and vGTFJ in the following ratios: 80:20, 60:40, 40:60 and 20:80 (active protein basis [see Methods table, activity unit I g formulated]). Samples of orange juice were individually treated with 1 % w / w of each of these blends, or with a single GTF, over a period of 24 hours at a temperature of 5 °C.

[0270] Notably, the 40:60 GTF blend demonstrated a significant reduction in limonin content, with approximately 32% of the limonin being reduced after the 24-hour incubation period. This result highlights the effectiveness of the 40:60 GTF blend in reducing limonin levels under the specified conditions. Overall, based on the results of Table 3 and FIG. 9, blends of roughly egual levels (enzyme activity) of each GTF enzyme (e.g., between about 40% to 60% each with respect to another activity-wise [adding up to 100%]) could be most useful for reducing levels of limonin and / or limonoids / flavonoids in orange juice under conditions similar to those tested here.

Claims

CLAIMSWhat is claimed is:

1. A method (process) of producing a food product / precursor, said method comprising:(a) providing a food product / precursor that comprises at least water, sucrose and a plant-based material, and(b) contacting the food product / precursor with at least:(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, and / or(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, whereby the food product / precursor, after step (b), has a reduced content of one or more limonoid compounds and / or flavonoid compounds that were present in the food product / precursor of step (a), optionally wherein said reduced content of one or more limonoid and / or flavonoid compounds results in a reduction of off-flavor of the food product / precursor.

2. The method of claim 1 , wherein the food product / food precursor, after step (b), has an increased content of at least one glucosylated limonoid and / or at least one glucosylated flavonoid, as compared to the food product / precursor before step (b).

3. The method of claim 1 , wherein the food product / precursor, after step (b), has a reduced content of said one or more limonoid compounds, optionally wherein said one or more limonoid compounds is a tetracyclic triterpenoid dilactone.

4. The method of claim 3, wherein said one or more limonoid compounds is limonin and / or nomilin.5 The method of claim 1 , wherein the food product / precursor, after step (b), has a reduced content of said one or more flavonoid compounds, optionally wherein said one or more flavonoid compounds is a polymethoxylated flavonoid or a flavonoid glycoside / glucoside.

6. The method of claim 5, wherein said one or more flavonoid compounds is sinensetin and / or nobiletin and / or vicenin-2.

7. The method of claim 1 , wherein said off-flavor comprises bitterness, sourness, and / or astringency.

8. The method of claim 1 , wherein: said glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1 , 2, 11 , 12, 14, or 15, and / or said glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan comprises an amino acid sequence that is at least 90% identical to residues 55- 960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55-960 of SEQ ID NO:8, residues 55-960 of SEQ ID NO:9, or SEQ ID NO:13.

9. The method of claim 1 , wherein said (i) glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan is used in said contacting of step (b).

10. The method of claim 1 , wherein both of said (i) glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan and said (ii) glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan are used in said contacting of step (b).11 . The method of claim 1 , wherein the plant-based material is from a plant of the Rutaceae family or the Cucurbitaceae family.

12. The method of claim 1 , wherein the food product / precursor comprises fruit juice and / or fruit solids.

13. The method of claim 12, wherein the food product / precursor comprises citrus fruit juice and / or citrus fruit solids.

14. The method of claim 13, wherein the food product / precursor comprises orange juice and / or orange solids.

15. The method of claim 12, wherein the fruit is harvested at least 1 week earlier than when the fruit is typically harvested.

16. The method of claim 12, wherein the fruit juice and / or fruit solids have been subjected to a temperature of at least about 15 °C for at least 1 day.

17. The method of claim 1 , wherein the food product / precursor provided in step (a) comprises at least about 3 parts-per-million (ppm) limonin as an initial limonin content.

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

19. The method of claim 1 , wherein step (b) is conducted for up to about 3, 5, or 7 hours, optionally wherein step (b) is ended by (I) terminating the activity of the glucosyltransferase(s) and / or (II) separating the glucosyltransferase(s) and food product / precursor from each other, optionally wherein step (b) is conducted at a temperature of about 3-10 °C.

20. The method of claim 1 , wherein the food product / precursor provided in step (a) is pasteurized.21 . The method of claim 10, wherein the ratio of the (i) glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan to the (ii) glucosyltransferase enzyme that synthesizes alpha-1 ,3-glucan in said contacting of step (b) is about 40:60 to about 60:40, typically wherein the ratio is based on the activity of each glucosyltransferase enzyme.

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

23. A method of glucosylating a limonoid and / or flavonoid compound, said method comprising:providing a composition that comprises at least water, sucrose, a limonoid compound and / or flavonoid compound, and at least one glucosyltransferase enzyme, wherein the glucosyltransferase enzyme is selected from:(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, and / or(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, wherein at least one glucosylated form of the limonoid compound and / or flavonoid compound is produced in the composition.

24. The method of claim 23, wherein said limonoid compound is limonin or nomilin.

25. The method of claim 23, wherein said flavonoid compound is sinensetin, nobiletin, or vicenin-2.

26. The method of claim 23, wherein: said glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1 , 2, 11 , 12, 14, or 15, and / or said glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan comprises an amino acid sequence that is at least 90% identical to residues 55- 960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55-960 of SEQ ID NO:8, residues 55-960 of SEQ ID NO:9, or SEQ ID NO:13.

27. The method of claim 23, wherein said (i) glucosyltransferase enzyme that synthesizes alpha-1 ,6-glucan is comprised in said composition.

28. The method of claim 23, wherein both of said (i) glucosyltransferase enzyme that synthesizes alpha-1 ,6-glucan and said (ii) glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan are comprised in said composition.

29. The method of claim 23, wherein the composition is a food product / precursor.

30. The method of claim 29, wherein the food product / precursor comprises fruit juice and / or fruit solids.31 . The method of claim 30, wherein the food product / precursor comprises citrus fruit juice and / or citrus fruit solids.

32. The method of claim 31 , wherein the food product / precursor comprises orange juice and / or orange solids.

33. The method of claim 23, wherein the composition is held for up to about 3, 5, or 7 hours, optionally wherein the activity of the glucosyltransferase(s) is terminated upon said hours having elapsed and / or the glucosyltransferase(s) is / are separated out of the composition upon said hours having elapsed, optionally wherein the composition is held at a temperature of about 3-10 °C during said hours of holding the composition.

34. The method of claim 28, wherein the ratio of the (i) glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan to the (ii) glucosyltransferase enzyme that synthesizes alpha-1 ,3-glucan in the composition is about 40:60 to about 60:40, typically wherein the ratio is based on the activity of each glucosyltransferase enzyme.

35. A composition, or a glucosylated limonoid compound and / or glucosylated flavonoid compound, produced by the method of claim 23.

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