Soluble oligosaccharide syrup production

Abstract

Disclosed herein are methods of producing an aqueous liquid composition comprising soluble oligosaccharides. Examples of such liquid compositions include syrups and sweeteners. Food products and precursors produced by this methodology or by using the present liquid compositions are also disclosed.

Description

[0001] TITLE

[0002] SOLUBLE OLIGOSACCHARIDE SYRUP PRODUCTION

[0003] This application claims the benefit of U.S. Provisional Appl. Nos. 63 / 734,364 and 63 / 734,362 (both filed December 16, 2024), which are incorporated herein by reference in their entirety.

[0004] FIELD

[0005] The present disclosure relates to soluble low-glycemic sweeteners, food products containing such a sweetener, and methods of their production using blends of sucrose - with or without addition of an acceptor - in the presence of an alpha-glucan-producing glucosyltransferase (sucrase).

[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 IFF10162WOPCT_SequenceListing.xml created on December 10, 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] There is a large push for sugar reduction in many food applications to provide healthier food alternatives for consumers. Since consumers are generally not willing to compromise on flavor, texture, and the overall food experience, it can be challenging to provide healthier alternatives for using sugar (sucrose) in food - indeed, sugar contributes not only to flavor, but also texture, color, and viscosity. It has therefore been necessary to attempt replacing or reducing sugar content by using artificial sweeteners along with a bulking agent such as maltodextrin or insoluble fiber.

[0010] Alternative low-glycemic sweeteners have been described using alternan sucrase or dextran sucrase in priming reactions with sucrose and corn syrup, further using maltose as an acceptor molecule (Int. Patent Appl. Publ. Nos. W02004 / 023894, W02006 / 088884, WO2011140212). For example, W02004 / 023894 described using an alternan sucrase in a priming reaction having a sucrose / maltose ratio of less than 1. The resulting syrups contained alternan, an alpha-glucan with alternating alpha-1,3 and alpha-1,6 glycosidic linkages, or dextran, which is an alpha-glucan of mainly alpha-1,6 glycosidic linkages. In other work, a commercially available syrup with alternan was produced having a reduced glycemic response in humans as compared to the glycemic response resulting from high fructose corn syrup (Grysman et al., 2008, Eur. J. Clin. Nutr. 62:1364-1371). Furthermore, the alternan-containing syrup had a lower glycemic index (53) compared to the glycemic index of sucrose (65), leading to the perception that alternan-containing syrup is a good alternative to sucrose.

[0011] Alternan-oligosaccharides have been described as prebiotic ingredients. U. S. Patent No. 7182954 discloses that oligosaccharides produced by an alternan sucrase enzyme-catalyzed reaction of sucrose with various acceptor sugars were effective as prebiotics for controlling enteric bacterial pathogens.

[0012] Despite these efforts, further work is warranted to provide syrups and related ingestible products that provide consumer satisfaction (e.g., taste, mouthfeel, etc.) and dietary benefits.

[0013] SUMMARY

[0014] In one embodiment, the present disclosure concerns a method (process) comprising:

[0015] (a) providing a reaction composition comprising at least water, sucrose, and a glucosyltransferase enzyme that is capable of synthesizing water-insoluble alpha-1,3-glucan, wherein the reaction composition comprises at least about 45% by weight of the sucrose,

[0016] (b) incubating the reaction composition, wherein water-soluble oligosaccharides are produced by the glucosyltransferase in the reaction composition (optionally referred to herein as an ' aqueous liquid composition"), and

[0017] (c) optionally isolating the water-soluble oligosaccharides, typically along with any monosaccharides that are also comprised in the reaction composition after step (b); optionally wherein there is little or no difference in the viscosity of the reaction composition resulting from step (b) as compared to the viscosity of the reaction composition initially provided in step (a).

[0018] In one embodiment, the present disclosure concerns a method (process) comprising:

[0019] (a) providing a reaction composition comprising at least water, sucrose, a primer molecule (i.e., acceptor) and a glucosyltransferase enzyme that is capable of synthesizing water-insoluble alpha-1, 3-glucan, wherein the ratio of the sucrose to the primer molecule is below about 1,3 on a weight basis,

[0020] (b) incubating the reaction composition, wherein water-soluble oligosaccharides are produced by the glucosyltransferase in the reaction composition, and

[0021] (c) optionally isolating the water-soluble oligosaccharides, typically along with any monosaccharides that are also comprised in the reaction composition after step (b); optionally wherein there is little or no difference in the viscosity of the reaction composition resulting from step (b) as compared to the viscosity of the reaction composition initially provided in step (a).

[0022] In one embodiment, the present disclosure concerns a method (process) comprising:

[0023] (a) providing a reaction composition comprising at least water, sucrose, an ingredient comprising at least one monosaccharide, disaccharide, and / or oligosaccharide, and a glucosyltransferase enzyme that is capable of synthesizing water-insoluble alpha-1,3-glucan,

[0024] (b) incubating the reaction composition, wherein water-soluble oligosaccharides are produced by the glucosyltransferase in the reaction composition, and

[0025] (c) optionally isolating the water-soluble oligosaccharides, typically along with any monosaccharides that are also comprised in the reaction composition after step (b); optionally wherein there is little or no difference in the viscosity of the reaction composition resulting from step (b) as compared to the viscosity of the reaction composition initially provided in step (a).

[0026] Any of the foregoing embodiments can further include, for example,

[0027] (i) incorporating the reaction composition resulting from step (b) in a product, optionally wherein the enzyme activity of the reaction composition has been terminated prior to the incorporating,

[0028] (ii) using the reaction composition resulting from step (b) itself as a product, optionally wherein the enzyme activity of the reaction composition has been terminated prior to the using, or

[0029] (iii) performing step (c) of isolating the water-soluble oligosaccharides, and optionally incorporating the isolated water-soluble oligosaccharides in a product.

[0030] In another embodiment, the present disclosure concerns a product (e.g., food product / precursor, pharmaceutical product, or personal care product) produced by any of the foregoing methods.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES FIG. 1: Pictures of aqueous solutions (10 wt% sucrose and 0.1-6 wt% of fructose, glucose, lactose or maltose) treated with vGTFJ (SEQ ID NO:3), Refer to Example 1. FIG. 2: Pictures of aqueous sucrose solutions (various concentrations) treated with both GTF 0768 and vGTFJ enzymes (various ratios), or vGTFJ alone (GTF 0768:vGTFJ% of 0:100%). Refer to Example 2. FIG. 3: Evaluation of pellet size post-incubation of sucrose / maltose solutions (varying sucrose to maltose ratios) with GTF0768:vGTFJ (5:95%), vGTFJ alone, or GTF 0768 atone. Refer to Example 4.

[0032] FIG. 4: Maltose level relative to the total soluble carbohydrates in product samples of reactions with or without enzyme treatment with various sucrose / maltose ratios. Refer to Example 4.

[0033] Table A. Summary of Protein SEQ ID Numbers i i Protein

[0034] i Description i SEQ ID NO.

[0035] GTF 0768. Mature form of Leuconostoc

[0036] pseudomesenteroides protein, but with two additional 1

[0037] i N-terminal amino acids.; (1449 aa)

[0038] GTF 0768. Mature, wild type form of L 2

[0039] i pseudomesenteroides protein (US2016 / 0122445).; (1447 aa)

[0040] | vGTFJ. Variant of GTF 6855 (SEQ ID NO:5 below). |aa)

[0041] 4

[0042] Variant of GTF 6855 (SEQ ID NO:5 below).

[0043] GTF 6855, derivable from Streptococcus salivarius

[0044] SK126. The first 178 amino acids of the protein are

[0045] deleted compared to GENBANK Acc. No. 5

[0046] i ZP 04061500.1; a start methionine is included.; (1341 aa)

[0047] GTF 7527, derivable from Streptococcus salivarius.

[0048] The first 178 amino acids of the protein are deleted

[0049] compared to GENBANK Acc. No. CAA77900.1; a 6

[0050] ; start methionine is included.; (1341 aa)

[0051] GTF 2678, derivable from Streptococcus salivarius

[0052] K12. The first 188 amino acids of the protein are

[0053] deleted compared to GENBANK Acc. No. 7 EJO16940.1; a start methionine is included. (1341 aa) GTF 2919, derivable from Streptococcus salivarius

[0054] PS4. The first 92 amino acids of the protein are

[0055] deleted compared to GENBANK Acc. No. 8

[0056] | EIC80898.1; a start methionine is included. | (1340 aa)

[0057] GTF 2765, derivable from unknown Streptococcus sp.

[0058] C150. The first 193 amino acids of the protein are

[0059] deleted compared to GENBANK Acc. No. 9

[0060] | ZP 08047301.1; a start methionine is included. | (1340 aa)

[0061] Wild type GTF corresponding to GTF 6855,

[0062] I Streptococcus salivarius SK126 (GENBANK Acc. No. 10

[0063] I ZP 04061500.1). § (1518 aa)

[0064] GTF 0768. N-terminal-truncated form A of the mature

[0065] form of L. pseudomesenteroides protein (SEQ ID

[0066] NO:2). The first 24 amino acids of the protein are 11

[0067]

[0068] ; deleted compared to SEQ ID NO:2.; (1423 aa) GTF 0768. N-terminal-truncated form B of the mature

[0069] form of L. pseudomesenteroides protein (SEQ ID

[0070] NO:2). The first 72 amino acids of the protein are 12

[0071] | deleted compared to SEQ ID NO:2. | (1375 aa) GTF 0974, derivable from Streptococcus salivarius

[0072] 57.1. The first 187 amino acids of the protein are

[0073] deleted compared to GENBANK Acc. No. 13 | AEJ53088.1. ( (1392aa) GTF 6831. Mature form of GTF from Streptococcus 14

[0074] | salivarius M 18, with modifications. | (1553aa) GTF 6831. Mature form of GTF from Streptococcus 15

[0075]

[0076] [ salivarius M 18. I (1557 aa)

[0077] DETAILED DESCRIPTION

[0078] The disclosures of all cited patent and non-patent literature are incorporated herein by reference in their entirety.

[0079] 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.

[0080] Where present, all ranges are inclusive and combinabie, 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.

[0081] 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.

[0082] 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 alpha-giucan 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% aipha-giycosidic linkages. Examples of alpha-giucan polymers herein include aipha-1,3-glucan and alpha-1,6-glucan (dextran).

[0083] 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.

[0084] 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 copoiymer. 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-aipha-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”.

[0085] The glycosidic linkage profile of an alpha-glucan herein can be determined using any method known in the art. For example, a linkage profile can be determined using methods using nuclear magnetic resonance (NMR) spectroscopy (e.g,,13C NMR or1H NMR). These and other methods that can be used are disclosed in, for example. Food Carbohydrates: Chemistry, Physical Properties, and Applications (S. W. Cui, Ed., Chapter 3, S. W, Cui, Structural Analysis of Polysaccharides, Taylor & Francis Group LLC, Boca Raton, FL, 2005), which is incorporated herein by reference.

[0086] 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).

[0087] 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, - 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.

[0088] 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.

[0089] The terms "glucosyltransferase'', “glucosyltransferase enzyme’’, " GTF", “glucansucrase" and the like are used interchangeably herein. The activity of a glucosyltransferase herein catalyzes the reaction of the substrate sucrose to make the products alpha-glucan and fructose. Other products (by-products) of a GTF reaction can include glucose, various soluble gluco-oligosaccharides, and leucrose. Wild type forms of glucosyltransferase enzymes generally contain (in the N-terminal to C-terminal direction) a signal peptide (which is typically removed by cleavage processes), a variable domain, a catalytic domain, and a glucan-binding domain. A glucosyltransferase herein is classified under the glycoside hydrolase family 70 (GH70) according to the CAZy (Carbohydrate-Active EnZymes) database (Cantarel et al., Nucleic Acids Res. 37; D233- 238, 2009). The term “dextransucrase” (and like terms) can optionally be used to characterize a glucosyltransferase enzyme that produces dextran.

[0090] The term “glucosyltransferase catalytic domain" herein refers to the domain of a glucosyltransferase enzyme that provides alpha-g I ucan-sy nth esizing activity to a glucosyltransferase enzyme. A glucosyltransferase catalytic domain typically does not require the presence of any other domains to have this activity.

[0091] The terms “enzymatic reaction", “glucosyltransferase reaction", “glucan synthesis reaction”, “reaction composition", “reaction formulation" and the like are used interchangeably herein and generally refer to a reaction that initially comprises water, sucrose, at least one active glucosyltransferase enzyme, and optionally other components. Components that can be further present in a glucosyltransferase reaction typically after it has commenced include fructose, glucose, leucrose, soluble glucooligosaccharides (e.g., DP2-DP7) (such may be considered as products or by-products, depending on the glucosyltransferase used), and / or insoluble alpha-glucan product(s) of DP8 or higher. It would be understood that certain glucan products, such as alpha-1,3- glucan with a degree of polymerization (DP) of at least 8 or 9, typically are waterinsoluble and thus not dissolved in a glucan synthesis reaction. The term “under suitable reaction conditions” as used herein refers to reaction conditions that support conversion of sucrose to alpha-glucan product(s) via glucosyltransferase enzyme activity. It is during such a reaction that glucosyl groups originally derived from the input sucrose are enzymatically transferred and used in alpha-glucan polymer synthesis; glucosyl groups as involved in this process can thus optionally be referred to as the glucosyl component or moiety (or like terms) of a glucosyltransferase reaction.

[0092] 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 alpha-glucan within the food product itself (or precursor). Such produced alpha-glucan (e.g., graft copolymer, alpha-1,3-glucan, and / or alpha-1, 6-glucan) can be soluble or insoluble. While an alpha-1,3-glucan product is typically insoluble and an alpha-1,6- glucan product is typically soluble, a graft copolymer product can either be soluble or insoluble, in a food product / precursor herein. In situ production of alpha-glucan in a food product / precursor typically substitutes for adding alpha-glucan herein as an ingredient in food, though such addition can be performed if desired (e.g., to supplement the alphaglucan produced in situ).

[0093] The terms "percent by volume”, "volume percent", “vol

[0094]

[0095] "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%.

[0096] The terms "percent by weight”, "weight percentage (wt%)”, “weight-weight percentage (% w / w)’’ and the iike 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.

[0097] The terms "weight'volume percent”, ‘‘w / v%’rand 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).

[0098] 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, ood / beverage products” refer to any edible product intended for consumption (e.g., for nutritional purposes) by humans or animals, including solids, semi-solids, or liquids. A “food” herein can optionally be referred to as a “foodstuff, "food product”, or other like term, for example. Herein, unless otherwise disclosed, a beverage or other ingestible liquid is an example of a food product. While the present disclosure generally regards food and food precursors that are by definition intended for ingestion or eventual ingestion (food precursor first made into food before being eaten), the disclosure likewise regards other ingestible products (e.g,, supplement, nutraceutical, pharmaceutical product) comprising in s / tu-produced alpha-glucan. A food precursor herein can be (i) a food as it exists before one or more processing steps (e.g., fermentation, aging, cooling / freezing, heating, baking, mixing) that render it to be a food product intended for direct consumption, and / or (ii) an ingredient for use in preparing a food product, for example. In some aspects, a food precursor can characterize a food product or ingredient as it exists before treatment with one or more GTF enzymes in a method herein.

[0099] " Dairy product / precursor” and like terms herein refer to a food product / precursor that contains milk and / or is made from milk. In some aspects, a dairy product / precursor contains at least about 2.5, 5, or 10 wt% milk or milk solids.

[0100] " Lactase-treated milk / dairy" and like terms herein refer to milk / dairy products or precursors treated with one or more lactase enzymes to reduce the amount of lactose sugar therein.

[0101] “Reduced lactose milk / dairy5' and like terms herein refer to milk / dairy products or precursors in which the weight percentage of lactose is about 2% or less, for example. “Lactose-free milk / dairy” and like terms herein refer to milk / dairy products or precursors in which the weight percentage of lactose is about 0.5 wt% or less, for example.

[0102] “Yogurt”, "dairy-based yogurt”, “fermented dairy product” and like terms herein generally refer to a dairy food / beverage produced by acidifying lactic fermentation of a dairy substrate such as milk. Such a product can optionally contain secondary ingredients such as fruits, vegetables, sugars, flavors, etc.

[0103] The terms "dietary fiber”, “glucan fiber” and the like herein refer to an alpha¬ glucan that is indigestible and / or that does not increase blood-glucose levels when enterally administered to a mammal. In general, a dietary fiber herein is not significantly hydrolyzed by endogenous enzymes in the upper gastrointestinal tract of mammals such as humans.

[0104] “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).

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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 130C'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.

[0110] Alpha-glucan in some aspects of the present disclosure can provide stability to a dispersion or emulsion of a food product / precursor. The “stability" (or the quality of being “stable”) of a dispersion or emulsion herein is, for example, the ability of dispersed particles of a dispersion, or liquid droplets dispersed in another liquid (emulsion), to remain dispersed (e.g., about, or at least about, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100 wt% of the particles of the dispersion or liquid droplets of the emulsion are in a dispersed state) for a period of about, or at least about, 2, 4, 6, 9, 12, 18, 24, 30, or 36 months following initial preparation of the dispersion or emulsion. A stable dispersion or emulsion can resist total creaming, sedimentation, flocculation, and / or coalescence of dispersed / emulsified material, for example.

[0111] 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-nr1-s‘1.

[0112] As used herein, the term ''poiypeptide” 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 (Val, V).

[0113] 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.

[0114] Percent identity can be readily determined by any known method, including but not limited to those described in: 1) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humana: NJ (1994); 4) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991), all of which are incorporated herein by reference.

[0115] 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 Clustai method of alignment which encompasses several varieties of the algorithm including the Clustai V method of alignment (described by Higgins and Sharp, CABIOS.

[0116] 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 Clustai method can be KTUPLE-1, GAP PENALTY =3, WINDOWS and DIAGONALS SAVED-5. For nucleic acids, these parameters can be KTUPLE=2, GAP PENALTY-5, WINDOW=4 and DIAGONALS SAVED=4. Additionally, the Clustai 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.

[0117] 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.

[0118] 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 product herein (e.g., food product or precursor) (as well as enzymatic reactions used to prepare these materials). It is believed that the embodiments disclosed herein are synthetic / man-made (could not have been made except for human intervention / involvement), and / or have properties that are not naturally occurring.

[0119] 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.

[0120] One or more glucosyltransferase enzymes used in a method as presentiy disclosed can comprise, for example:

[0121] (ii) a glucosyltransferase (GTF) enzyme that synthesizes, or is capable of synthesizing, alpha-1, 3-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1,3-glucan are alpha-1,3 linkages (or, a GTF enzyme with alpha-1,3-glucan synthesis activity), and / or

[0122] (i) a GTF enzyme that synthesizes, or is capable of synthesizing, alpha-1,6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1,6-glucan are alpha- 1,6 linkages (or, a GTF enzyme with alpha- 1,6-glucan / dextran synthesis activity). In some aspects, a GTF enzyme that is capable of synthesizing alpha-1,3-glucan herein can comprise an amino acid sequence that is about 100% identical to, or at ieast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 26, 28, 30, 34, or 59, or amino acid residues 55-960 of SEQ ID NO:4, residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:28, or residues 55- 960 of SEQ ID NO:20, and have GTF activity; these amino acid sequences are as disclosed in U. S. Patent Appl. Publ, No. 2019 / 0078063, which is incorporated herein by reference. It is noted that such a GTF enzyme comprising SEQ ID NO:2, 4, 8, 10, 14, 20, 26, 28, 30, 34, or amino acid residues 55-960 of SEQ ID NO:4, residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20, can synthesize alpha-glucan comprising at least about 90% (-100%) alpha-1,3 linkages. A GTF enzyme that is capable of synthesizing alpha-1, 3-glucan in some aspects can be that identified as GTF 0974 (SEQ ID NO: 13 herein, SEQ ID NO:110 in US2018 / 0291311), or a GTF comprising an amino acid sequence that is about 100% identical to, or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, the foregoing amino acid sequence of GTF 0974 (and having GTF activity). Any of the foregoing GTF enzyme amino acid sequences can be modified as described herein to increase product yield, modify product molecular weight, and / or enhance GTF performance and / or stability.

[0123] A GTF enzyme can, in some aspects, be capable of synthesizing 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 is capable of producing more products. Examples of a suitable modified GTF enzyme are disclosed in Tables 3-7 of U. S. Patent Appl. Publ. No. 2019 / 0078063, which is incorporated herein by reference. A modified GTF enzyme, for example, can comprise one or more amino acid substitutions corresponding with those in Tables 3-7 (ibid.) that is / are associated with an alpha-1,3-glucan yield of at least 40% (the position numbering of such at least one substitution corresponds with the position numbering of SEQ ID NO:62 as disclosed in U. S. Patent Appl. Publ. No. 2019 / 0078063). A set of amino acid modifications as listed in Tables 6 or 7 (ibid.) can be used, for example.

[0124] The amino acid sequence of a GTF enzyme for alpha-1,3-glucan synthesis in some aspects has been modified such that the enzyme is capable of producing 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.

[0125] The amino acid sequence of a GTF enzyme for alpha-1,3-glucan synthesis in some aspects has been modified such that the enzyme is capable of producing 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.

[0126] 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.

[0127] In some aspects, a modified GTF that is capable of alpha-1, 3-glucan synthesis (i) comprises at least one amino acid substitution or a set of amino acid substitutions (as described above regarding yield or molecular weight), and (ii) comprises or consists of a GTF catalytic domain that is at least about 80%. 85%, 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to amino acid residues 55-960 of SEQ ID NO:4, amino acid residues 54-957 of SEQ ID NO:65, amino acid residues 55-960 of SEQ ID NO:30, amino acid residues 55-960 of SEQ ID NO:28, or amino acid residues 55-960 of SEQ ID NO:20 (each of these sequences as disclosed in U. S. Patent Appl. Publ. No. 2019 / 0078063, which is incorporated herein by reference). Each of these subsequences are the approximate catalytic domains of each respective reference sequence, and produce alpha-1,3-glucan comprising at least about 50% (e.g., ≥90% or ≥95%) alpha-1,3 linkages. In some aspects, a modified GTF (i) comprises at least one amino acid substitution or a set of amino acid substitutions (as described above), and (ii) comprises or consists of an amino acid sequence that is at least about 80%, 85%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:62 or a subsequence thereof such as SEQ ID NO;4 (without start methionine thereof) or positions 55-960 of SEQ ID NO:4 (approximate catalytic domain) (each of these sequences as disclosed in U. S. Patent Appl. Publ. No. 2019 / 0078063).

[0128] 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 Appi. Publ. No. 2019 / 0078063. Thus, each of presently disclosed SEQ ID NOs:5, 6, 7, 8, 9 and 10 can be used in any of the disclosed aspects, as appropriate. For example, a GTF enzyme that is capable of synthesizing alpha-1,3-glucan herein can comprise an amino acid sequence that is about 100% identical to, or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, SEQ ID NO:5, 6, 7, 8, 9, or 10, or amino acid residues 55- 960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55-960 of SEQ ID NO:8, or residues 55-960 of SEQ ID NO:9. Any of these sequences can be modified as described herein to affect alpha-1, 3-glucan yield and / or molecular weight and / or stability, for example.

[0129] In some aspects, a GTF enzyme that is capable of alpha-1, 3-glucan synthesis has been modified such that the enzyme has enhanced performance and / or stability benefit(s). Such a GTF enzyme can be as disclosed, for example, in Int. Patent Appl. Publ. No. W02023 / 055902, which is incorporated herein by reference. Modification of such a GTF can be, for example, by having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid substitutions as compared to a corresponding parent GTF enzyme (e.g., a wild type mature GTF or active subsequence thereof such as a catalytic domain). Exemplary performance and / or stability benefits herein include one or more of increased thermal stability, increased storage stability, increased solubility, better pH profile, increased specific activity, modified substrate specificity, modified substrate binding, modified pH-dependent activity, modified pH-dependent stability, increased oxidative stability, increased expression, and / or increased glucan product yield (and / or decreased byproduct [e.g., leucrose] yield). In some aspects, a performance benefit is realized at a relatively low temperature (e.g., <5CC) or at a relatively high temperature (e.g., >40DC). 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.

[0130] Some examples of modified GTF enzymes capable of alpha-1, 3-glucan production 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).

[0131] In some aspects of the present disclosure, a modified GTF enzyme can comprise or consist of an amino acid sequence that is at least about 80%, 85%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:3, and have one or more of (or all of) the following amino acid residues: 8-Asn, 9-Aia, 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.

[0132] In some aspects of the present disclosure, a modified GTF enzyme can comprise or consist of an amino acid sequence that is at least about 80%, 85%. or 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:4, and have one or more of (or all of) the following amino acid residues: 336-Tyr, 430-Tyr, 431- Val, 448-Ala, 564-Ser, 1011-Glu, 1150-His, 1155-Ala, 1241-Lys, 1242-Glu, 1243-Gly, 1244-Ser, 1247-Leu, and / or 1248-Val. The valine at position 1 of SEQ ID NO:4 in any of the foregoing aspects can optionally instead be a methionine, or can be deleted.

[0133] Although it is believed that a modified GTF enzyme capable of producing alpha- 1, 3-glucan 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.

[0134] Although amino acid substitutions in a modified GTF enzyme capable of producing alpha-1, 3-glucan are generally disclosed in some aspects with respect to corresponding positions in SEQ ID NQ: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.

[0135] A GTF herein, which under typical conditions as previously disclosed is capable of producing alpha-1,3-glucan with a DPw or DPn of at least about 800 or 1600 for example, can produce water-soluble oligosaccharides or water-soluble alpha-1,3-glucan oligosaccharides under conditions as presently disclosed (e.g., elevated reaction composition sucrose content [e.g., a 45 wt% sucrose], low reaction composition sucrose- to-primer / acceptor content [e.g., less than -1.3:1 on weight basis], or presence of a monosaccharide- and / or disaccharide-containing ingredient [e.g., malt extract or whey] in a reaction composition). Water-soluble oligosaccharides or water-soluble alpha-1, 3- gfucan oligosaccharides as produced in a reaction composition herein can comprise about, or at least about, 50%. 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%.

[0136] 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% alpha-1,3-glycosidic linkages, for example. Water-soluble oligosaccharides or water-soluble alpha-1,3- glucan oligosaccharides as produced in a reaction composition herein can have a DPw, DPn, or DP of about 2, 3, 4, 5, 6, 7. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-9, 2-8, 5-20, 3-15, 3-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-9, 5-8, 10-20, 10-18, 10-16, 10-14, or 10-12, for example. Such degree of polymerization typically is with regard to the monomer content, regardless of monomer type (e.g., deg. of pol. can relate to oligosaccharides having only glucose monomers, or those having both glucose monomers other types of monomer[s]). In some aspects, the monomer units of water-soluble oligosaccharides or water-soluble alpha-1, 3-glucan oligosaccharides as produced in a reaction composition herein are about, 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%, or 100% glucose monomeric units. Water-soluble oligosaccharides or water-soluble alpha-1,3-glucan oligosaccharides herein can be linear or branched. Water-soluble oligosaccharides as produced in a reaction composition herein can be as described above, for example. In some aspects, such produced disaccharides and / or oligosaccharides 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 and / or oligosaccharides produced in a reaction composition in some aspects can include one or more of nigerose, leucrose, trehalulose, maltuiose, isomaltulose, and turanose. Examples of oligosaccharides produced in a reaction composition in some aspects can include (i) alpha-1, 3-linked glucose units (optionally also with alpha-1, 6-linked glucose units) (e.g., of any length as disclosed above, typically in terms of deg. of sub., typically linear), and (ii) a disaccharide or oligosaccharide as disclosed herein (e.g., nigerose, leucrose, trehalulose, maltuiose, isomaltulose, turanose, maltose, isomaltose, lactose, lactosucrose, malto¬ oligosaccharide [MOS], isomalto-oligosaccharide [IMO], or galacto-oligosaccharide [GOS], and / or a disaccharide or oligosaccharide present in an ingredient herein such as malt extract or whey; any of the foregoing can be used as a primer / acceptor ingredient in a reaction composition herein), wherein portion (i) is in glycosidic linkage with portion (ii), and portion (ii) is at the reducing end of the disaccharide and / or oligosaccharide molecule (e.g., by virtue of having used any of the disaccharides or oligosaccharides of (ii) as an acceptor for priming synthesis of the alpha-1, 3-linked glucose units (optionally also alpha-1, 6-linked glucose units).

[0137] In some aspects, a GTF enzyme (dextransucrase) that is capable of synthesizing alpha-1,6-glucan herein can comprise an amino acid sequence that is about 100% identical to, or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, SEQ ID NO: 1, 2, 11, or 12 (GTF 0768), or 14 or 15 (GTF 6831) and have GTF activity. Yet, in some aspects, a GTF enzyme that is capable of synthesizing alpha-1,6-glucan can be as disclosed in any of U. S. Patent Appl. Publ. Nos. 2017 / 0218093, 2018 / 0282385, 2018 / 0291311, or 2016 / 0122445, which are each incorporated herein by reference. For example, the GTF identified as GTF 8117 (SEQ ID NO:30), GTF 6831 (SEQ ID NO:32), or GTF 5604 (SEQ ID NO:33) in US2018 / 0282385 can be used, or the GTF identified as GTF 2919 (SEQ ID NO:5), GTF 2918 (SEQ ID NO:9), GTF 2920 (SEQ ID NO:13), or GTF 2921 (SEQ ID NO:17) in US2016 / 0122445 can be used, or a GTF comprising an amino acid sequence that is about 100% identical to, or at least about 80%, 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, the amino acid sequence of any of these GTF enzymes (and having GTF activity) can be used. 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.

[0138] In some aspects, such as in a reaction composition further comprising (A) an ingredient comprising one or more primer / acceptor molecules herein (e.g., whey or malt extract), and (B) a transgalactosylating beta-galactosidase herein (and optionally the GTF component is a combination of a GTF capable of producing alpha-1, 3-glucan and a GTF capable of producing alpha-1,6-glucan), a reaction composition resulting from step (b) has a theoretical sweetness that is about, or at least about, 45%, 50%, 75%, 100%, 125%, or 150% higher than the theoretical sweetness of the primer / acceptor molecule¬ comprising ingredient (ingredient source of monosaccharides and / or disaccharides) as it existed before adding the ingredient to the reaction composition of step (a). Theoretical sweetness can be measured using any suitable means, for example, or as presently disclosed in the Examples (e.g.. using parameters within 5% of the disclosed parameter values). Sugar content in some aspects in which theoretical sweetness is increased can be decreased by about, or at least about, 20%, 25%, 30%, or 33% by weight, for example (i.e., a double benefit of achieving sugar reduction while increasing relative sweetness can be realized).

[0139] In some aspects, a reaction composition resulting from step (b) has a consistency (e.g., thin and / or pourable) that makes it amenable for facile use (i.e., easy-to-use) as an ingredient, such as in food product / precursor preparation. The viscosity of a reaction composition resulting from step (b) herein can be about, or less than about, 4000, 3750, 3500, 3250, 3000, 2500. 2000, 1500, 1000, 750, 600, 500, 400, 300, 375, 350, 200, 100, 100-500, 100-400, 100-375, 100-350, 100-300, 100-200, 200-500, 200-400, 200-375, 200-350, 200-300, 2000-4000, 2000-3500, 2000-3250, 2500-4000, 2500-3500, 2500- 3250, 3000-4000, or 3000-3500 cP, for example. Such a viscosity can be as measured under any temperature disclosed herein, for example, such as at about 20 or 25 °C (or at 4-30 °C, 15-30 °C, 15-25 °C, or 20-25 °C, e.g.). Viscosity can be measured using a viscometer or rheometer, for example, and can optionally be as measured at a shear rate (rotational shear rate) of about 0.1, 0.3, 0.5, 1.0, 3, 5, 10, 50, 100, 500, 1000, 0.1-500, 0.1-100, 1.0-500, 1.0-1000, or 1.0-100 s-1 (1 / s), or about 10 rpm (revolutions per minute) (or about 5, 10. 20, 25, 50, 100, 200, or 250 rpm), for example. In some aspects, a GTF enzyme can be any as disclosed herein and include 1-300 (or any integer there between [e.g., 10, 15, 20. 25, 30, 35, 40, 45. or 50]) residues on the N-terminus and / or C-terminus. Such additional residues can be from a corresponding wild type sequence from which the GTF enzyme is derivable, or can be a heterologous sequence such as an epitope tag (at either N~ or C~terminus) or a heterologous signal peptide (at N-terminus), for example. A GTF enzyme herein typically lacks an N~terminal signal peptide; such an enzyme can optionally be characterized as being mature if its signal peptide was removed during a secretion process.

[0140] 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. dentirousetii, S. downei, S. mutans, S. oralis, S, gallolyticus and S. sanguinis. Examples of Leuconostoc species include 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. easel, L. curvatus, L. plantarum, L. sakei, L. brevis, L buchneri, L. fermentum and L. reuteri.

[0141] 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.

[0142] One, two, three, or more different GTF enzymes that are capable of synthesizing alpha-1, 3-glucan herein can be used, for example, in a method as presently disclosed. In some aspects, only an alpha-1,3-glucan-producing GTF(s) is used (i.e., an alpha-1, 6-glucan-producing GTF is not used). One, two, three, or more different GTF enzymes that synthesize alpha- 1,6-glucan herein can be used, for example, in a method as presently disclosed. In some aspects, though less typical, only an alpha- 1,6-glucan- producing GTF(s) is used (i.e., an alpha-1, 3-glucan-producing GTF is not used; thus, optionally, any disclosure herein regarding '‘alpha-1,3 linkages’' [or like language] can be replaced with "alpha-1,6 linkages”). In some aspects, an alpha-1,3-glucan-producing GTF(s) can be added to a reaction composition herein before adding an alpha-1, 6- 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, 6-glucan-producing GTF(s) can be added to a reaction composition before adding an alpha-1,3-glucan-producing GTF(s), Still, in some aspects, a dextran optionally as disclosed herein, but produced exogenously to the reaction composition, can be added as an ingredient to a reaction composition to which an alpha-1, 3-glucan-producing GTF has already been added or will be added.

[0143] While typical aspects herein regard production of water-soluble disaccharides and / or oligosaccharides in a reaction composition, in some aspects, one or more of alpha-1, 3-glucan homopolymer(s) (e.g., DPw >20, w / any linkage profile herein, such as 3:95% or >100% alpha-1,3 linkages), alpha-1,6-glucan (dextran) homopolymer(s) (e.g., DPw >20, w / any linkage profile herein, such as £95% or £100% alpha-1,6 linkages), and / or dextran-alpha-1,3-glucan graft copolymer(s) (e.g., as disclosed in U. S. Pat. Appl. Publ. No. 2024 / 0108021 or Int. Pat. Appl. Publ. No. WO2023 / 055902, which are incorporated herein by reference) is / are additionally produced in the reaction composition. For example, (i) alpha-1,3-glucan homopolymer, (ii) alpha-1, 3-glucan homopolymer and alpha-1,6-glucan homopolymer, (iii) alpha-1, 3-glucan homopolymer, alpha-1,6-glucan homopolymer, and dextran-alpha-1,3-glucan graft copolymer, or (iv) dextran-alpha-1,3-glucan graft copolymer can additionally be produced. Such production of polysaccharide material, if it occurs in some aspects, can result in a reaction composition of the disclosure in which about, or less than about, 10, 5, 2, 1, or 0.5 wt% of all the produced saccharides are a polysaccharide product (e.g., DPw >20). In some aspects, there is 0 wt%, less than 5, 4, 3, 2, or 1 wt%, or no detectable amount of, any of the foregoing polysaccharide materials in the reaction composition (or on the basis of the weight of the saccharides produced in the reaction composition). Yet, in some aspects, a reaction composition resulting from GTF enzyme incubation herein (typically of a completed reaction) has a ratio of water-insoluble alpha- 1,3-glucan (i.e., some amount of water-insoluble alpha-1,3-glucan was produced in the reaction composition) to water-soluble oligosaccharide products (optionally in combination with any monosaccharides that are also comprised in the reaction composition resulting from the GTF incubation) that is less than about 25:75, 20:80, 15:85, 10:90, 5:95, or 3:97 on a weight basis.

[0144] The content of at least one of an alpha-1,3-glucan-producing GTF and / or alpha-1,6-glucan-producing GTF in a reaction composition 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).

[0145] In some aspects in which both GTF types herein are used, the ratio of a glucosyltransferase enzyme that is capable of synthesizing alpha-1,6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha-1,3-glucan in the reaction composition is about 90:10 to about 5:95. Yet, in some aspects, an alpha- 1,6- glucan-producing (capable) GTF to alpha-1,3-glucan-producing (capable) GTF ratio can be about 50:50 to about 5:95 (e.g., about 40:60, 30:70, 20:80, or 10:90), or a range between any two of the foregoing ratios. The amount of each enzyme (active enzyme) for purposes of determining a ratio thereof herein can be on a molar, weight, or GTF activity basis, for example. The activity of a GTF enzyme for preparing a ratio herein can optionally be determined as disclosed in U. S. Patent Appl. Publ. No. 2014 / 0087431 or Int. Patent Appl. Publ. No. WO2023 / 055902, which are incorporated herein by reference. For example. a full (e.g., “100%”) complement of a GTF enzyme for setting up a ratio herein can be that amount of enzyme that can convert most of (e.g., >95%, >98%, >99%). or all of, sucrose in a GTF reaction comprising or consisting of water, sucrose (e.g., 50 or 100 g / L), the GTF, and optionally buffer / salt in a given amount of time (e.g., 6, 12, 18, 24, 30, or 36 hours); such a measured amount can optionally be characterized as a normalized amount of GTF.

[0146] 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 reaction composition herein with a GTF enzyme(s) typically is not performed in an oral cavity or other environment in which unpurified / non-isolated GTF enzymes can possibly be present,

[0147] 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 reaction composition herein. A GTF enzyme (or any other enzyme as presently disclosed) in some aspects for use in a method herein can be comprised in a preparation that is substantially free of (e.g., <0.5, <0.1, <0.05 wt%) any other enzyme(s) such as a lipase, protease, amylase, mannanase, pectinase, cellulase, and / or p-nitrobenzylesterase; such a preparation typically has little or no detectable activity (ies) of such other enzyme(s).

[0148] A reaction composition herein can be brought into contact with one or more GTF enzymes by mixing / stirring / blending, for example. Incubation of GTF enzyme(s) in the reaction composition can be for a time sufficient, for example, for the GTF(s) to produce water-soluble oligosaccharides in the reaction composition, such as for about, or at least about. 0.5. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18. 24, 30, 36, 42, 48. 72, 96, 1-6. 1-5, 1-4, 1-3, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, or 3-4 hours, or for about, or at least about, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 days (or a range between any two of these hours and / or days). In some aspects, such incubation can be for less than about 8, 7, 6, 5, 4, 3, or 2 hours. The temperature for incubating one or more GTF enzymes in a reaction composition herein can be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 2-5, 2-10, 2-15, 2-20, 2-25, 2-30, 2-35, 2-40, 2-45, 2-50, 3-5, 3-10, 3-15, 3-20, 3-25, 3-30, 3-35, 3-40, 3-45, 3-50, 5-10, 5-15, 5- 20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 15-20, 15-25, 15-30, 15-35, 15-40, 15-45, 15-50, 20-25, 20-30, 20-35, 20-40, 20-45, 20-50, 25-30, 25-35, 25-40, 25-45, 25-50, 30-35, 30-40, 30-45, or 30-50 °C, for example. As appropriate, for example, depending on how ingredients and enzyme(s) herein are introduced to each other, steps (a) and (b) of methods herein can optionally be considered to be performed simultaneously or separately. A reaction composition, following the foregoing incubation, can optionally be terminated such by applying elevated temperature (e.g., heat-inactivation at 90-100 °C or 70-110 ’C) for a suitable amount of time (e.g., about 1, 2, 5, or 10 minutes). Such a terminated reaction composition herein still be referred to as a “reaction composition” (for ease of reference), even though it no longer has any GTF activity and optionally any additional enzyme activity. In some aspects, GTF activity termination (e.g., by elevated temperature) can be preceded by reducing the pH of a reaction composition to below about 5.0 or 4.8.

[0149] A reaction composition herein contains water (i.e., it is an aqueous composition), GTF enzyme can be added in dry form (e.g., powder, flakes, lyophilized enzyme preparation) or wet form for preparing a reaction composition. In some aspects, a reaction composition can be prepared by combining GTF enzyme(s) and other reaction ingredients (sucrose and, in some aspects, a primer / acceptor such as a monosaccharide- and / or disaccharide-containing ingredient) under dry conditions (resulting combination is dry), after which time water or an aqueous solution is added, which in tum allows GTF production of water-soluble oligosaccharides to proceed. The water content of a reaction composition herein can be about, or at least about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99 wt%, for example; in some aspects, the disclosed water content is the balance (±1, 2, 3, 4, or 5 wt%) needed to bring a reaction composition to 100 wt% in view of the desired initial sucrose content of the reaction composition (e.g., if the initial sucrose content is to be 50 wt%, then the reaction composition is set up to contain about 50 wt% water [e.g., ±1-5 wt%]). The initial sucrose content of a reaction composition herein can be about, or at least about, 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 45-70, 45-65, 45-60, 50-70, 50-65, or 50-60 wt%, for example. The pH of a reaction composition herein can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 4.0-10.0, 4.0-9.0.

[0150] 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 reaction composition in some aspects can be acidic (e.g., pH < 3.0, 3.2, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5), neutral (e.g., pH 6.5- 7.5), or basic / alkaline (e.g., pH > 7.5, 8.0, 8.5, 9.0, 9.5).

[0151] In some aspects, a reaction composition as initially provided has no detectable amount of, does not comprise, or has less than 0.01 wt% of, a primer molecule (acceptor). For example, a reaction composition with a relatively high initial sucrose content herein (e.g., greater than about 45, 50, or 55 wt%) can be set up in this manner. Yet, in some aspects, a primer / acceptor is initially included in a reaction composition as presently disclosed. Examples of a primer / acceptor that can initially be included in a reaction composition herein include one or more of a monosaccharide, disaccharide, and / or oligosaccharide (e.g., DP3+). Examples of suitable disaccharide and oligosaccharide prime rs / acceptors herein include maltose and / or lactose. Examples of suitable disaccharide and oligosaccharide primers / acceptors herein include nigerose, leucrose, trehalulose, maltulose, isomaltulose, furanose, isomaltose, lactosucrose, malto-oligosaccharide [MOS], isomalto-oligosaccharide [IMO], and / or galactooligosaccharide [GOSj. An example of a suitable monosaccharide primer / acceptor herein is glucose.

[0152] In some aspects in which one or more primer / acceptor molecules are used, the ratio of the sucrose to the primer / acceptor molecule(s), as initially provided in a reaction composition herein, below 1.3:1 or 1.2:1 on a weight basis. For example, the ratio of the sucrose to the primer / acceptor molecule(s) can be about, or below about, 1.1:1, 1.05:1, 1.0:1, 0.95:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.75:1-1.1:1, 0.75:1-1.0:1, or 0.75:1-0.95:1 on a weight basis. In some aspects, the initial concentration of primer / acceptor molecule(s) in a reaction composition can be about, or at least about, 0.1, 0.3, 0.5, 0.8, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, 10, 12.5, 15, 20, 25, 0.1-6.5, 2-6.5, 3-6.5, 3-6, 5-15, 10-15, 5-20, 10-20, 5-25, or 10-25 wt% or w / v%.

[0153] In some aspects, suitable monosaccharide, disaccharide, and / or oligosaccharide primer / acceptor molecules (or disaccharide and / or oligosaccharide primer / acceptor molecules) can be provided as an ingredient that comprises one or more of these primer / acceptor molecules. Such an ingredient can comprise or consist of malt extract and / or whey (e.g., from cheese, lactic casein, skim milk, milk, or yogurt), for example. An ingredient in some aspects can comprise or consist of a soluble fraction resulting from a previous separate reaction (in water with at least sucrose) using a GTF enzyme herein that is capable of producing alpha-1,3-glucan or alpha-1,6-glucan. An ingredient in some aspects can comprise or consist of honey and / or mapie syrup. An ingredient in some aspects can comprise or consist of milk, milk powder, soy milk, almond milk, coconut milk, yogurt / yogurt drink, and / or buttermilk. An ingredient in some aspects can comprise or consist of corn syrup (e.g., light corn syrup), rice syrup, golden syrup, molasses, agave nectar, inulin syrup, yacon syrup, and / or Jerusalem artichoke syrup. One, two, or more suitable ingredients can be used in a reaction composition, for example. The amount of one or more ingredients used in a reaction composition herein can be that amount which provides a desired initial concentration (e.g., herein) of its constituent monosaccharide, disaccharide, and / or oligosaccharide primer / acceptor molecules in the reaction composition.

[0154] A monosaccharide, disaccharide, and / or oligosaccharide primer / acceptor molecule used in some aspects can be from a source physically outside of a reaction composition herein (i.e., the primer / acceptor is added an ingredient), and / or can be provided via in situ production in the reaction composition such as by one or more enzymes that are endogenous and / or exogenous to the reaction composition. An enzyme for use in a reaction composition (i.e., exogenous enzyme) for producing a monosaccharide, disaccharide, and / or oligosaccharide primer / acceptor 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 a transgalactosylating beta-galactosidase, 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, which are incorporated herein by reference. An EC 2.4.1.24 transglucosidase (also termed as "1,4-alpha-glucan 6-alpha~glucosyltransferase”) can transfer an alpha-D-glucosyl residue of an alpha-1,4 -glucan, -oligosaccharide (i.e,, MOS), or -disaccharide (i.e., maltose) to the primary hydroxy group of free glucose or glucose in an alpha-1,4 -glucan, -oligosaccharide (i.e., MOS), or -disaccharide. Thus, an EC 2.4.1.24 transglucosidase produces isomalto-oligosaccharides (IMO) (e.g., DP3-DP5 or DP3-DP6) in some aspects. Suitable transgalactosylating beta-galactosidases herein are disclosed in U. S. Patent Appl. Publ. No. 2013 / 0189746 or U. S. Patent Nos. 10531672 or 10683523, for example, which are incorporated herein by reference. A transgalactosylating beta- galactosidase is an enzyme that degrades lactose by transferring the galactose of lactose to galactose, glucose, or other acceptor thereby producing galactooligosaccharides (GOS) (e.g., GOS can also be an acceptor for forming a longer GOS). A particular example of such an enzyme is Nurica™ (IFF). A transglucosidase or transgalactosylating beta-galactosidase herein can be dosed into a reaction composition herein at about 0.1-1.5, 0.1-1.25, 0.1-1.0, 0.1-0.75, 0.1-0.5, 0.2-1.5, 0.2-1.25, 0.2-1.0, 0.2-0.75, 0.2-0, 5, 0,5-1.5, 0.5-1.25, 0.5-1, 0, 0.5-0,75, 0.75-1,5, 0,75-1.25, or 0.75-1,0 % (v / w). for example.

[0155] A GTF enzyme can optionally be provided in a method herein by introducing a recombinantly engineered cell (e.g., a microbial cell such as a bacterial or fungal / yeast cell) to a reaction composition, wherein the cell recombinantly (heterologously) expresses and secretes the GTF enzyme in the reaction composition. Such a cell can be that of a microbe that is amenable to recombinant engineering and useful in food processing (e.g,, fermentation), for example, such as a microbial cell disclosed herein (as applicable). In some aspects, a recombinantly engineered cell that is provided to the reaction composition 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 ceil, 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).

[0156] In some aspects, a method of the present disclosure further comprises:

[0157] (i) incorporating (e.g., adding as an ingredient) a reaction composition produced by the method (resulting from incubation step [b]) in a product, optionally wherein the GTF enzyme activity of the reaction composition is terminated prior to incorporating step [i],

[0158] (ii) using the reaction composition produced by the method (resulting from incubation step [b]) itself as a product (i.e., the reaction composition is a product). optionally wherein the GTF enzyme activity of the reaction composition is terminated prior to use step [iij, or

[0159] (iii) performing step (c) of isolating the water-soluble oligosaccharides (typically along with any monosaccharides that are also comprised in the reaction composition after step [b]), and incorporating (e.g., adding as an ingredient) the isolated water- soluble oligosaccharides (and typically also monosaccharides) in a product.

[0160] In some aspects, a product of any of foregoing steps (i), (ii), or (iii) can be in the form of, and / or comprised in, a food product / precursor, such as disclosed in Int. Pat. Appl. Publ. Nos. WO2023 / 055902, W02024 / 086560, or WO2024 / 206631, which are incorporated herein by reference; some non-limiting examples include a syrup, sweetener, beverage, or liquid food product / precursor. Yet, in some aspects, a product can be in the form of, and / or comprised in, a pharmaceutical product or personal care product (and / or an industrial product or household product), such as disclosed in U. S. Patent Appl. Publ. Nos. 2018 / 0022834, 2018 / 0237816, 2018 / 0230221, 20180079832, 2016 / 0311935, 2016 / 0304629, 2015 / 0232785, 2015 / 0368594, 2015 / 0368595, 2016 / 0122445, 2019 / 0202942, or 2019 / 0309096, or Int. Pat. Appl. Publ. No.

[0161] WO2016 / 133734, which are incorporated herein by reference.

[0162] A “food product / precursor* (i.e., a food product or precursor) as provided or used in a method in some aspects of the present disclosure 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) (e.g., can be characterized as being “sweetened”). The sucrose content of a food product / precursor finally provided for use in a method 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.

[0163] A food product / precursor in some aspects (such as to which an incubated / completed reaction composition herein can be added; or to which water- soluble oligosaccharides purified / isolated from an incubated / completed reaction composition can be added; or for which an incubated / completed reaction composition can be directly used as, or further processed to be) can be a syrup, sweetener, or beverage. A syrup, sweetener, or beverage can optionally have one or more attributes, beyond any as provided herein by performing the presently disclosed method, as disclosed in U. S. Pat. Appl. Publ. Nos. 2010 / 0040728, 2017 / 0006902, 2017 / 0218093, 2013 / 0216652, 20180146699, 2009 / 0123603, 2021 / 0076724, or 2017 / 0332670, which are incorporated herein by reference. A beverage in some aspects can be a juice (e.g., fruit juice such as orange juice, apple juice, mango juice, peach juice, banana juice, date juice, apricot juice, grapefruit juice, papaya juice, pineapple juice, raspberry juice, strawberry juice, pear juice, tangerine juice, cranberry juice, acai juice, grape juice, or cherry juice; vegetable juice such as carrot juice, tomato juice, or mixed- vegetable juice), sweetened beverage (soda / soft drink, sweetened tea of coffee), coffee, tea, ready-to- drink (RTD), or any other suitable beverage.

[0164] A food product / precursor in some aspects (such as to which an incubated / completed reaction composition herein can be added; or to which water-soluble oligosaccharides purified / isolated from an incubated / completed reaction composition can be added; or for which an incubated / completed reaction composition can be directly used as, or further processed to be) can be a dairy product / precursor, such as a dairy beverage or food. Suitable examples of a dairy product / precursor herein include milk, cheese, yogurt, dessert, cream, and butter. Milk herein can be whole milk (e.g., ~3% fat), ~2% fat milk, -1% fat milk (“low-fat”), or fat-free (non-fat) milk, for example. Milk, whether used directly as a beverage or as a precursor for preparing a dairy product / precursor herein, can be from a cow, goat, sheep, buffalo, yak, llama, camel, or horse, for example. A cheese herein can be, for example, hard or semi-hard cheese (e.g., Cheddar, mozzarella, Swiss, parmesan, provolone), soft or semi-soft cheese (e.g., ricotta, cottage cheese, feta, American, brie), processed, or nonprocessed. A yogurt herein can be, for example, whole milk yogurt, low-fat yogurt, fat- free yogurt, or Greek yogurt (e.g., plain, low-fat, non-fat). A yogurt herein can optionally contain fruit and / or be flavored. A yogurt herein can optionally be drinkable (i.e., yogurt beverage). A dairy dessert in some aspects can be a pudding (e.g., whole milk, 2% milk), frozen yogurt (e.g., low-fat), ice cream (e.g., low-fat), sherbet, milk shake, gelato, or custard; thus, in some aspects a dairy dessert can be a frozen dairy dessert (e.g., ice cream, sherbet, milk shake, frozen yogurt, gelato). Ice cream can be hard ice cream or soft (soft-serve) ice cream, for example. A dairy beverage in some aspects can be milk, chocolate milk, coffee milk, flavored milk, yogurt beverage, kumis, ryazhenka, ayran, lassi, cholado, licuado, or kefir. A dairy cream can be clotted cream (e.g., >55% milkfat), heavy cream (e.g., >36% milkfat), whipping cream (e.g., 30%-36% milkfat), light cream (e.g., 18%-30% milkfat), sour cream (>18% milkfat), half-and-half (e.g., 10.5%-18% milkfat), or ice cream (e.g., >10% milkfat). A dairy product / precursor herein can be lactose-free or have a reduced lactose content, for example. A dairy product / precursor herein can be a fermented dairy product / precursor (e,g., yogurt, buttermilk, creme fraiche, quark, fromage frais, soured milk, vinegar), for example. Some dairy products / precursors herein include dairy confections such as milk chocolate, white chocolate, caramel, and toffee. A dairy product / precursor in some aspects can be any as disclosed in U. S. Pat. Appl. Publ. Nos. 2021 / 0282422, 2013 / 0230623, 2005 / 0244541, 2017 / 0135360, 2009 / 0304864, 2017 / 0094987, or 2003 / 0152685, or U. S. Pat. Nos. 5482728 or 6352734, all of which are incorporated herein by reference.

[0165] A food product / precursor in some aspects (such as to which an incubated / completed reaction composition herein can be added; or to which water-soluble oligosaccharides purified / isolated from an incubated / completed reaction composition can be added; or for which an incubated / completed reaction composition can be directly used as, or further processed to be) can be a condiment (table condiment) or any other preparation (of liquid consistency or solid consistency) that is added to a food (typically cooked food) to impart a flavor and / or to enhance a flavor. Examples of condiments herein include / comprise any tomato-based condiment (e.g., ketchup, tomato sauce, salsa, marinara sauce), mustard (e.g., yellow mustard, Dijon mustard), relish, horseradish, wasabi, hot sauce, chili sauce / oil, mayonnaise, aioli, barbecue sauce, soy sauce, alfredo sauce, au jus, Bearnaise sauce, cranberry sauce, chutney (mango chutney, onion chutney, tamarind chutney), cocktail sauce, fish sauce, oyster sauce, hoisin sauce, sriracha sauce, marmalade, fruit preserves (jam / jelly).

[0166] Hollandaise sauce, hummus, guacamole, pesto sauce, miso, gochujang, Worcestershire sauce, salad dressing, salsa verde, sesame oil, sour cream, tartar sauce, or teriyaki sauce. In some additional or alternative aspects, food product / precursor can be a condiment (e.g., any of the foregoing, as appropriate, such as a fish sauce, oyster sauce, soy sauce, or other Asian-style / influenced) or similar product, or any other type of food product / precursor (as appropriate), and have a salt content of about, or up to about, 2.5, 5, 7.5, 10, 15, 20, 25, 10-25, 10-20, 10-15, 15-25, or 15-20 wt%, for example.

[0167] A food product / precursor in some aspects (such as to which an incubated / completed reaction composition herein can be added; or to which water- soluble oligosaccharides purified / isolated from an incubated / completed reaction composition can be added; or for which an incubated / completed reaction composition can be directly used as, or further processed to be) can be a fermented (or will be a fermented) food product / precursor, for example, such as any of those disclosed in Int. Pat. Appl. Publ. Nos. WO2023 / 055902, WO2024 / 086560, WO2024 / 206631. or W02002 / 034061, which are incorporated herein by reference. A food product / precursor that is fermented, or will be fermented, can be a dairy product herein (e.g., as above, such as milk or yogurt), beer, beer wort, wine, pomace, cider, miso, kimchi, sauerkraut, pickles / pickle juice, soybean curd, tofu, kombucha, soy sauce, bread, sourdough, or meat, for example.

[0168] A food product / precursor in some aspects (such as to which an incubated / completed reaction composition herein can be added; or to which water- soluble oligosaccharides purified / isolated from an incubated / completed reaction composition can be added; or for which an incubated / completed reaction composition can be directly used as, or further processed to be) can be a non-dairy food product / precursor. For example, a non-dairy food product / precursor can be a plant-based 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, mung 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,

[0169] A food product / precursor in some aspects (such as to which an incubated / completed reaction composition herein can be added; or to which water- soluble oligosaccharides purified / isolated from an incubated / completed reaction composition can be added; or for which an incubated / completed reaction composition can be directly used as, or further processed to be) 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, petfood, or animal / livestock feed.

[0170] A food product / precursor in some aspects can comprise one or more 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., orange, apple, mango, peach, plum, banana, date, apricot, grapefruit, papaya, pineapple, raspberry, strawberry, blueberry, blackberry, cranberry, pear, tangerine, cherry, grape, melon, watermelon, cantaloupe, honeydew melon, kiwi, lemon, lime, carrot, tomato), or fruit / vegetable juice (juice concentrate), puree, paste, or other processed form (e.g., sliced, cubed, chopped pieces) of a fruit / vegetable as disclosed, or any other component suitable for use as an ingredient in a food product / precursor. Such one or more additional ingredients can be as disclosed in U. S. Patent Appl. Publ. Nos. 2016 / 0122445 or 2017 / 0218093 (both incorporated herein by reference), for example, and / or can be natural or artificial. Examples of ingredients suitable as sweeteners (or for any other purpose such as flavoring) include acesulfame potassium, advantame, agave syrup, alitame, aspartame, barley malt syrup, birch syrup, brazzein, brown rice syrup, cane juice, caramel, coconut palm sugar, corn syrup, curculin, cyclamate, dextrose, erythritol, fructo-oligosaccharide, fructose (levulose), galactose, glucose (dextrose), glycerol (glycerin), glycyrrhizin, golden syrup, high fructose corn syrup (e.g., HFCS-42, -55, -90), high maltose corn syrup (HMCS), honey, hydrogenated starch 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 di hydrochaicone, neotame, palm sugar, pentadin, polydextrose, rapadura, refiners syrup, saccharin, sorbitol (glucitol), sorghum syrup, stevia / 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.

[0171] 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.

[0172] In some aspects, a food product / precursor herein such as one obtained / provided after performing step (i), (ii), or (iii), has one or more of the following features (e.g., as compared to the food product / precursor as it existed before step [I], [ii], or [iii]): (I) increased dietary fiber (e.g., increased soluble dietary fiber), (II) increased prebiotic activity, (III) reduced caloric density or reduced calories, and / or (IV) reduced glycemic index. Such beneficial feature(s) (i.e., nutritional / dietary benefits]) (which may further include reduced blood sugar and / or increased satiety), which typically can be realized with ingestion of the food product / precursor by a human or other mamma! (e.g., primate, pet such as a cat or dog, or livestock such as a pig, cow, horse, goat, sheep), can be improved by at least about 10%, 25%. 50%, or 100%, for example, as compared to when using a control food product / precursor product. A suitable control food product / precursor typically is the same as the food product / precursor, except that the control food product / precursor can be one to which an incubated / completed reaction composition herein was not added, or to which water-soluble oligosaccharides purified / isolated from an incubated / completed reaction composition were not added, or that represents the food product / precursor before GTF treatment thereof in a reaction composition.

[0173] In some aspects of a method as presently disclosed, there is little or no difference in the viscosity of a reaction composition resulting from step (b) as compared to the viscosity of the reaction composition initially provided in step (a). For example, the viscosity of the reaction composition resulting from step (b) can be increased, if at all, by no more than 3-fold, 2-fold, 1.5-fold, or 1.25 fold. Viscosity can be as measured at any temperature disclosed herein, for example, such as 20 “C or 25 °C (e.g., or 4-30 °C, 15-30 °C, 15-25 °C, or 20-25 " C). Viscosity typically is as measured at atmospheric pressure (about 760 torr) or a pressure that is ±10% thereof. Viscosity can be measured using a viscometer or rheometer, for example, and can optionally be as measured at a shear rate (rotational shear rate) of about 0.1, 0.3, 0.5, 1.0, 3, 5, 10, 50, 100, 500, 1000, 0.1-500, 0.1-100, 1.0-500, 1.0-1000, or 1.0-100 s-1 (1 / s), or about 10 rpm (e.g., or 5, 10, 20, 25, 50, 100, 200, or 250 rpm), for example.

[0174] Non-limiting examples of compositions and methods disclosed herein include: 1. A method (process) comprising:

[0175] (a) providing a reaction composition comprising at least water, sucrose, and a glucosyltransferase enzyme that is capable of synthesizing water-insoluble alpha-1,3-glucan (e.g., as disclosed presently disclosed), wherein the reaction composition comprises at least about 45% by weight of the sucrose,

[0176] (b) incubating the reaction composition (under suitable conditions), wherein water- soluble oligosaccharides are produced by the glucosyltransferase in the reaction composition (and optionally then terminating the enzyme activity of the reaction composition), and (c) optionally isolating the water-soluble oligosaccharides, typically along with (coisolating) any monosaccharides (e.g., fructose and / or glucose) that are also comprised in the reaction composition after step (b);

[0177] optionally wherein there is little (e.g., increased by no more than 3-fold or 2-fold) or no difference in the viscosity of the reaction composition resulting from step (b) as compared to the viscosity of the reaction composition initially provided in step (a) (and optionally wherein the glucosyltransferase enzyme is the only type of glucosyltransferase enzyme present in the reaction composition) (optionally, a reaction composition provided by the method can instead be referred to herein as a “liquid composition" or “aqueous liquid composition", which term can substitute for “reaction composition" in the text of the present disclosure, as suitable and appropriate).

[0178] 2. The method of embodiment 1, wherein the reaction composition provided in step (a) has no detectable amount of, or does not comprise, a primer molecule (i.e., acceptor).

[0179] 3. The method of embodiment 1 or 2, wherein the reaction composition provided in step (a) comprises at least about 50% by weight (e.g., about 50-65 or 50-60 wt%) of the sucrose.

[0180] 4. The method of embodiment 1, 2, or 3, wherein the water-soluble oligosaccharides produced in step (b) comprise at least alpha-1, 3-glucooligosaccharides and leucrose, wherein at least about 50% of the glycosidic linkages of the alpha-1, 3-glucooligosaccharides are alpha- 1,3 glycosidic linkages, and the weight-average degree of polymerization (DPw) of the alpha-1, 3-glucooligosaccharides is 2 to about 20.

[0181] 5. The method of embodiment 4, wherein at least about 75% (e.g., or ≥ ~80%, ~85%, ~90%, ~95%, or ~100%) of the glycosidic linkages of the alpha-1,3- glucooligosaccharides are alpha-1,3 glycosidic linkages.

[0182] 6. The method of embodiment 1, 2, 3, 4, or 5, wherein the reaction composition resulting from step (b) has a ratio of water-insoluble alpha-1, 3-glucan (i.e., some amount of water-insoluble alpha-1, 3-glucan was produced in the reaction composition) to the water-soluble oligosaccharides (optionally in combination with any monosaccharides that are also comprised in the reaction composition resulting from step [b]) that is less than about 25:75 (e.g., or ≥ ~10:90, ~5:95, or ~3:97) on a weight basis.

[0183] 7. The method of embodiment 1, 2, 3, 4, 5, or 6, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan comprises: (i) an amino acid sequence that is at least 80%, 85%, or 90% identical to residues 55-960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55- 960 of SEQ ID NO:8, residues 55-960 of SEQ ID NO:9, or SEQ ID NO:13 (e.g., an amino acid sequence that is at least 80%, 85%, or 90% identical to residues 55-960 of SEQ ID NO:5 or residues 54-957 of SEQ ID NO:6), or (ii) an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:3, 4, 5, 6, 7, 8, 9, or 13 (e.g., an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:3, 4, 5, or 6).

[0184] 8. The method of embodiment 1, 2, 3, 4, 5, 6, or 7, wherein the reaction composition further comprises a glucosyltransferase enzyme that is capable of synthesizing alpha- 1,6-glucan.

[0185] 9. The method of embodiment 8. wherein the glucosyltransferase enzyme that is capable of synthesizing alpha-1,6-glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:1, 2, 11, 12, 14, or 15 (e.g., an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:1, 2, 11, or 12).

[0186] 10. The method of embodiment 8 or 9, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha- 1,3-glucan in the reaction composition is about 90:10 to about 5:95.

[0187] 11. The method of embodiment 8, 9, or 10, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha-1,6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha-1,3-glucan in the reaction composition is about 50:50 to about 5:95.

[0188] 12. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, further comprising: (I) incorporating (e.g., adding as an ingredient) the reaction composition resulting from step (b) in a product (e.g., food product / precursor, pharmaceutical product, or personal care product), optionally wherein the enzyme activity of the reaction composition has been terminated prior to the incorporating,

[0189] (ii) using the reaction composition resulting from step (b) itself as a product (e.g., food product / precursor, pharmaceutical product, or personal care product) (i.e., the reaction composition is a product), optionally wherein the enzyme activity of the reaction composition has been terminated prior to the using, or

[0190] (iii) performing step (c) of isolating the water-soluble oligosaccharides (typically along with any monosaccharides that are also comprised in the reaction composition after step [b]), and optionally incorporating (e.g., adding as an ingredient) the isolated water-soluble oligosaccharides (and typically also monosaccharides) in a product (e.g., food product / precursor. pharmaceutical product, or personal care product). 13. The method of embodiment 12, wherein the product of step (i), (ii), or (iii) is a food product / precursor (e.g., as disclosed herein, such as a syrup, sweetener, or as a beverage or other liquid food product / precursor), optionally wherein the food product / precursor, after step (i), (ii), or (iii), has one or more of the following features (e.g., as compared to the food product / precursor as it existed before step [i], [ii], or [iii]); (I) increased dietary fiber (e.g., increased soluble dietary fiber), (II) increased prebiotic activity, (ill) reduced caloric density or reduced calories, and / or (IV) reduced glycemic index.

[0191] 14. A product (e.g., food product / precursor, pharmaceutical product, or personal care product) produced by the method of embodiment 12 or 13.

[0192] Non-limiting examples of compositions and methods disclosed herein include; 1a. A method (process) comprising;

[0193] (a) providing a reaction composition comprising at least water, sucrose, a primer molecuie (i.e., acceptor) and a glucosyltransferase enzyme that is capable of synthesizing water-insoluble alpha-1, 3-glucan (e.g., as disclosed presently disclosed), wherein the ratio of the sucrose to the primer molecule is below about 1.3 on a weight basis,

[0194] (b) incubating the reaction composition (under suitable conditions), wherein water-soluble oligosaccharides are produced by the glucosyltransferase in the reaction composition (and optionaiiy then terminating the enzyme activity of the reaction composition), and

[0195] (c) optionally isolating the water-soluble oligosaccharides, typically along with (co-isolating) any monosaccharides (e.g., fructose and / or glucose) that are also comprised in the reaction composition after step (b);

[0196] optionally wherein there is little (e.g., increased by no more than 3-fold or 2-fold) or no difference in the viscosity of the reaction composition resulting from step (b) as compared to the viscosity of the reaction composition initially provided in step (a) (and optionally wherein the glucosyltransferase enzyme is the only type of glucosyltransferase enzyme present in the reaction composition) (optionaiiy, a reaction composition provided by the method can instead be referred to herein as a ‘liquid composition" or “aqueous liquid composition", which term can substitute for “reaction composition” in the text of the present disclosure, as suitable and appropriate). 2a. The method of embodiment 1a, wherein the ratio of the sucrose to the primer molecuie is equal to, or below, about 1.1 (e.g., < -1.05, -1.0, -0.95, -0.9, -0.8, -0.7, -0.6, -0.5, -0.4, -0.3, or 0.75-1.1) on a weight basis.

[0197] 3a. The method of embodiment 1a or 2a, wherein the primer molecule is maltose (and, and / or one or more of a monosaccharide, other disaccharide, and / or oligosaccharide that can serve as an acceptor molecule for the glucosyltransferase), optionally wherein the maltose is provided in the reaction composition by addition of a malt extract, 4a. The method of embodiment 1a, 2a, or 3a, wherein the water-soluble oligosaccharides produced in step (b) comprise at least alpha-1, 3-glucooligosaccharides and ieucrose, wherein at least about 50% of the glycosidic linkages of the alpha-1, 3-glucooligosaccharides are alpha-1,3 glycosidic linkages, and the weight-average degree of polymerization (DPw) of the alpha-1,3-glucooligosaccharides is 2 to about 20.

[0198] 5a. The method of embodiment 4a, wherein at least about 75% (e.g., or ≥ ~80%, ~85%, ~90%, or ~95%) of the glycosidic linkages of the alpha-1, 3-glucooligosaccharides are alpha-1,3 glycosidic linkages.

[0199] 6a. The method of embodiment 1a, 2a, 3a, 4a, or 5a, wherein the reaction composition resulting from step (b) has a ratio of water-insoluble alpha-1, 3-glucan (i.e., some amount of water-insoluble alpha-1, 3-glucan was produced in the reaction composition) to the water-soluble oligosaccharides (optionally in combination with any monosaccharides that are also comprised in the reaction composition resulting from step [b]) that is less than about 10:90 (e.g., or ≥ ~5:95 or ~2:98) on a weight basis.

[0200] 7a. The method of embodiment 1a, 2a, 3a, 4a, 5a, or 6a, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan comprises: (i) an amino acid sequence that is at least 80%, 85%, or 90% identical to residues 55- 960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55-960 of SEQ ID NO:8, residues 55-960 of SEQ ID NO:9, or SEQ ID NO: 13 (e.g., an amino acid sequence that is at least 80%, 85%, or 90% identical to residues 55-960 of SEQ ID NO:5 or residues 54-957 of SEQ ID NO:6), or (ii) an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:3, 4, 5, 6, 7, 8, 9, or 13 (e.g., an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:3,4, 5, or 6).

[0201] 8a. The method of embodiment 1a, 2a, 3a, 4a, 5a, 6a, or 7a, wherein the reaction composition further comprises a glucosyltransferase enzyme that is capable of synthesizing alpha-1,6-glucan. 9a. The method of embodiment 8a, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha-1,6-glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:1, 2, 11, 12, 14, or 15 (e.g., an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:1, 2, 11, or 12).

[0202] 10a. The method of embodiment 8a or 9a, wherein the ratio of the glucosyitransferase enzyme that is capable of synthesizing alpha-1, 6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan in the reaction composition is about 90:10 to about 5:95.

[0203] 11a. The method of embodiment 8a, 9a, or 10a, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha-1,6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan in the reaction composition is about 50:50 to about 5:95.

[0204] 12a. The method of embodiment 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, or 11a, further comprising:

[0205] (i) incorporating (e.g., adding as an ingredient) the reaction composition resulting from step (b) in a product (e.g., food product / precursor, pharmaceutical product, or personal care product), optionally wherein the enzyme activity of the reaction composition has been terminated prior to the incorporating,

[0206] (ii) using the reaction composition resulting from step (b) itself as a product (e.g., food product / precursor, pharmaceutical product, or personal care product) (i.e., the reaction composition is a product), optionally wherein the enzyme activity of the reaction composition has been terminated prior to the using, or

[0207] (iii) performing step (c) of isolating the water-soluble oligosaccharides (typically along with any monosaccharides that are also comprised in the reaction composition after step [b]), and optionally incorporating (e.g., adding as an ingredient) the isolated water-soluble oligosaccharides (and typically also monosaccharides) in a product (e.g., food product / precursor, pharmaceutical product, or personal care product).

[0208] 13a. The method of embodiment 12a, wherein the product of step (i), (ii), or (iii) is a food product / precursor (e.g., as disclosed herein, such as a syrup, sweetener, or as a beverage or other liquid food product / precursor), optionally wherein the food product / precursor, after step (i), (ii), or (iii), has one or more of the following features (e.g., as compared to the food product / precursor as it existed before step [i], [ii], or [iii]): (I) increased dietary fiber (e.g., increased soluble dietary fiber), (II) increased prebiotic activity, (III) reduced caloric density or reduced calories, and / or (IV) reduced glycemic index. 14a. A product (e.g., food product / precursor, pharmaceutical product, or personal care product) produced by the method of embodiment 12a or 13a.

[0209] 15a. A method (process) comprising:

[0210] (a) providing a reaction composition comprising at least water, sucrose, an ingredient (added ingredient) comprising at least one monosaccharide, disaccharide, and / or oligosaccharide (or a “monosaccharide-, disaccharide-, and / or oligosaccharide-containing ingredient", or like terms, where the monosaccharide, disaccharide, and / or oligosaccharide can serve as an acceptor molecule for the glucosyltransferase) (e.g., an ingredient comprising at least a disaccharide and / or oligosaccharide), and a glucosyltransferase enzyme that is capable of synthesizing water-insoluble alpha-1, 3-glucan (e.g., as disclosed presently disclosed),

[0211] (b) incubating the reaction composition (under suitable conditions), wherein water- soluble oligosaccharides are produced by the glucosyltransferase in the reaction composition (and optionally then terminating the enzyme activity of the reaction composition), and

[0212] (c) optionally isolating the water-soluble oligosaccharides, typically along with (coisolating) any monosaccharides (e.g., fructose, glucose, and / or galactose) that are also comprised in the reaction composition after step (b);

[0213] optionally wherein there is little (e.g., increased by no more than 3-fold or 2-fold) or no difference in the viscosity of the reaction composition resulting from step (b) as compared to the viscosity of the reaction composition initially provided in step (a) (and optionally wherein the glucosyltransferase enzyme is the only type of glucosyltransferase enzyme present in the reaction composition) (optionally, a reaction composition provided by the method can instead be referred to herein as a ‘liquid composition" or “aqueous liquid composition’’, which term can substitute for “reaction composition” in the text of the present disclosure, as suitable and appropriate).

[0214] 16a. The method of embodiment 15a, wherein at least one of the monosaccharide, disaccharide, and / or oligosaccharide (or the ingredient comprising the disaccharide and / or oligosaccharide) of the ingredient serves as a primer molecule (i.e., acceptor) for the glucosyltransferase in step (b),

[0215] 17a. The method of embodiment 15a or 16a, wherein the ingredient comprising the monosaccharide, disaccharide, and / or oligosaccharide (or the ingredient comprising the disaccharide and / or oligosaccharide) comprises or consists of whey. 18a. The method of embodiment 15a. 16a, or 17a, wherein the ingredient comprising the monosaccharide, disaccharide, and / or oligosaccharide (or the ingredient comprising the disaccharide and / or oligosaccharide) comprises or consists of mait extract.

[0216] 19a. The method of embodiment 15a, 16a, 17a, or 18a, wherein the water-soluble oligosaccharides produced in step (b) comprise at least alpha-1,3-glucooligosaccharides and leucrose, wherein at least about 50% of the glycosidic linkages of the alpha-1,3- glucooligosaccharides are alpha- 1,3 glycosidic linkages, and the weight-average degree of polymerization (DPw) of the alpha-1, 3-glucooligosaccharides is 2 to about 20.

[0217] 20a. The method of embodiment 19a, wherein at least about 75% (e.g., or > -80%, ~85%, -90%, -95%, or -100%) of the glycosidic linkages of the alpha-1,3-glucooligosaccharides are alpha-1,3 glycosidic linkages.

[0218] 21a. The method of embodiment 15a, 16a, 17a, 18a, 19a, or 20a, wherein the reaction composition resulting from step (b) has a ratio of water-insoluble alpha-1, 3-glucan (i.e., some amount of water-insoluble alpha-1, 3-glucan was produced in the reaction composition) to the water-soluble oligosaccharides (optionally in combination with any monosaccharides that are also comprised in the reaction composition resulting from step [b]) that is less than about 10:90 (e.g., or < -5:95 or -2:98) on a weight basis.

[0219] 22a. The method of embodiment 15a, 16a, 17a, 18a, 19a, 20a, or 21a, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan comprises: (i) an amino acid sequence that is at least 80%, 85%, or 90% identical to residues 55-960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55-960 of SEQ ID NO:8, residues 55-960 of SEQ ID NO:9, or SEQ ID NO: 13 (e.g., an amino acid sequence that is at least 80%, 85%, or 90% identical to residues 55-960 of SEQ ID NO:5 or residues 54-957 of SEQ ID NO:6), or (ii) an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:3, 4, 5, 6, 7, 8, 9, or 13 (e.g., an amino acid sequence that is at least 80%, 85%. or 90% identical to SEQ ID NO:3, 4, 5, or 6).

[0220] 23a. The method of embodiment 15a, 16a, 17a, 18a, 19a, 20a, 21a, or 22a, wherein the reaction composition further comprises a glucosyltransferase enzyme that is capable of synthesizing alpha-1, 6-glucan.

[0221] 24a. The method of embodiment 23a, wherein the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 6-glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:1, 2, 11, 12, 14, or 15 (e.g., an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:1, 2, 11, or 12). 25a. The method of embodiment 23a or 24a, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha-1,6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha-1,3-glucan in the reaction composition is about 90:10 to about 5:95.

[0222] 26a. The method of embodiment 23a, 24a, or 25a, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha-1,6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha-1,3-glucan in the reaction composition is about 50:50 to about 5:95,

[0223] 27a. The method of embodiment 15a, 16a, 17a, 18a, 19a, 20a, 21a, 22a, 23a, 24a, 25a, or 26a, wherein the reaction composition further comprises a transgalactosylating beta-galactosidase, and the ingredient source of monosaccharides and / or disaccharides comprises lactose, wherein the water-soluble oligosaccharides produced in step (b) comprise at least alpha-1, 3-glucooligosaccharides, leucrose, and galactooligosaccharides.

[0224] 28a. The method of embodiment 15a, 16a, 17a, 18a, 19a, 20a, 21a, 22a, 23a, 24a, 25a, 26a, or 27a, wherein the reaction composition resulting from step (b) has a theoretical sweetness that is at least about 45% higher than the theoretical sweetness of the ingredient source of monosaccharides and / or disaccharides as it existed before adding it to the reaction composition of step (a),

[0225] 29a. The method of embodiment 15a, 16a, 17a, 18a, 19a, 20a, 21a, 22a, 23a, 24a, 25a, 26a, 27a, or 28a, further comprising:

[0226] (i) incorporating (e.g., adding as an ingredient) the reaction composition resulting from step (b) in a product (e.g., food product / precursor, pharmaceutical product, or personal care product), optionally wherein the enzyme activity of the reaction composition has been terminated prior to the incorporating,

[0227] (ii) using the reaction composition resulting from step (b) itself as a product (e.g,, food product / precursor, pharmaceutical product, or personal care product) (i.e., the reaction composition is a product), optionally wherein the enzyme activity of the reaction composition has been terminated prior to the using, or

[0228] (iii) performing step (c) of isolating the water-soluble oligosaccharides (typically along with any monosaccharides that are also comprised in the reaction composition after step [b]), and optionally incorporating (e.g., adding as an ingredient) the isolated water-soluble oligosaccharides (and typically also monosaccharides) in a product (e.g., food product / precursor, pharmaceutical product, or personal care product). 30a. The method of embodiment 29a. wherein the product of step (i), (ii), or (iii) is a food product / precursor (e.g., as disclosed herein, such as a syrup, sweetener, or as a beverage or other liquid food product / precursor), optionally wherein the food product / precursor, after step (i), (ii), or (iii), has one or more of the following features (e.g., as compared to the food product / precursor as it existed before step [i], [ii], or [iii]); (I) increased dietary fiber (e.g., increased soluble dietary fiber), (II) increased prebiotic activity, (ill) reduced caloric density or reduced calories, and / or (IV) reduced glycemic index.

[0229] 31a. A product (e.g., food product / precursor, pharmaceutical product, or personal care product) produced by the method of embodiment 29a or 30a.

[0230] EXAMPLES

[0231] 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.

[0232] Summary

[0233] The following Examples demonstrate, for instance, a method of producing a low-glycemic sweetener composition containing alpha-1,3-glucan oligosaccharides by treating an aqueous solution of >47.6 wt% sucrose with an alpha-glucan sucrase, which enzymatic reaction did not require any added priming ingredients (such as high fructose corn syrup [HFCS]). Surprisingly, this result was achieved without a significant increase in viscosity, which has previously been observed and sought after when using this type of enzymatic treatment in food applications (I nt. Pat. Appl. Publ. No. W02023 / 055902, incorporated herein by reference).

[0234] It was also demonstrated that a soluble low-glycemic sweetener product can be produced in an in situ environment, such as in the presence of a priming ingredient (e.g., maltose or malt extract), when the sucrose-to-maltose ratio was approximately

[0235]

[0236] (weight basis). This approach can be a beneficial way of reducing the digestibility of malt extract carbohydrates (e.g., thereby potentially reducing calorie content, while increasing dietary fiber and / or prebiotic function) without otherwise altering the flavor or beneficial nutrient profile of the malt extract. It was further shown that other sugars aside from maltose can act as acceptor molecules (primers) for several alpha-1,3-glucan sucrases, thereby expanding the potential for reducing the digestibility of other food ingredients. For example; when whey was combined with sucrose and treated with glucan sucrase and a galactooligosaccharide (GOS -producing beta-galactosidase, it was possible to both reduce total sugar content as well as increase the theoretical sweetness of the final product.

[0237] Materials / Methods

[0238] HPLC sugar content analysis

[0239] Sugar composition was measured by high performance liquid chromatography (HPLC) with a Waters® 2695 Separations module or a ThermoScientific Dionex™ UltiMate 3000 HPLC, equipped with a Phenomonex Rezex™ RPM-Monosaccharide Pb2+column (300 mm x 7.8 mm), and an Rl-detector. Water was used as the mobile phase at a flow rate of 0.400 mL / min. The column temperature was 70 °C. Samples were prepared for HPLC injection by (1 ) precipitating polysaccharides (by adding ethanol to a final concentration of 30 wt%), (2) centrifuging (10 minutes at 13000 rpm), (3) appropriately diluting the supernatant in water, and (4) a conducting sterile-filtration. Signals from the HPLC were quantified against calibration standards of sugars eluting at the same time. Sugar reduction was calculated by subtracting the total sum of mono-and di-saccharides in a test sample from the total sum of mono- and di-saccharides in a control / reference sample in which no enzyme(s) had been added.

[0240] Theoretical sweetness

[0241] The theoretical sweetness of a composition was calculated by taking the relative sweetness of individual mono- and di-saccharides and multiplying this value by its respective concentration (g / 100 g) in the composition. The individual calculations were summed to give the theoretical sweetness. Relative sweetness values for lactose, glucose, galactose, sucrose, fructose, nigerose and leucrose of 0.16, 0.74, 0.32, 1.73, 0.45 and 0.5 respectively was used.

[0242] Glucosyltransferase enzymes

[0243] 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 alphaglucan 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).

[0244] Example 1

[0245] Producing a Soluble Oligosaccharide Syrup from Sucrose Using a Primer (Acceptor Molecule)

[0246] Aqueous solutions having (i) 10 wt% sucrose and (ii) 0.1-6 wt% of fructose, glucose, lactose, or maltose were prepared in a 0.1 M sodium phosphate buffer pH 6.4. To each solution was added an amino acid-substituted GTF 6855 variant (SEQ ID NO:3, “vGTFJ” herein) to 0.5 wt%, and the solution was incubated 24 hours at 5 °C. At the end of the incubation, each sample was heated to 95 °C for 10 minutes to inactivate the enzyme and then cooled to room temperature. A picture was taken of reaction samples after cooling of each reaction (see FIG. 1), and then the samples were analyzed for their sugars composition according to the Materia Is / Methods.

[0247] It was clear that presence of an additional sugar aside from sucrose had an impact on the resulting texture. In all cases the formation of insoluble polysaccharides decreased with increasing concentration of the additional sugar (as can be observed in FIG. 1 ). The effect was most pronounced if the additional sugar was either glucose or maltose.

[0248] The sugar analysis results revealed that with increasing glucose addition in the reaction, there was increased formation of nigerose and oligosaccharides, and increased release of fructose (Table 1). Hence, the glucose was an acceptor molecule in the glucosyltransferase reaction causing formation of nigerose and shorter oligosaccharides supporting the conclusion from FIG. 1 that a reduced amount of insoluble polysaccharides had formed. Table 1. Soluble carbohydrate profile (% w / w)

[0249] % added

[0250] glucose Oligosaccharide Sucrose Nigerose Glucose | Leucrose J Fructose 0.1% 1.30 0.00 0.17 0.26; 080 f 4.14 0.3% 1.54 0.00 0.20 0.30 0.72 409 0.8% 2.46 0.00 0.40 0.46 071 4.75 1.6% 2.99 0.00 0.64 0.73 0.54 4.67 2.5% 3.47 0.00 1.00 1.17 0.46 4.68 3.5% 3.37 0.00 1.22 1.47; 0.37 4.47 4.5% 4.39 0.00 1.91 2.59 0.40 5.54

[0251] 4.41 0.00 2.52 3.42 0.38 5.42

[0252]

[0253] 6.0%

[0254] Similarly, it was shown that with increasing maltose addition in the reaction, there was increased formation of oligosaccharides (Table 2) in which the maltose had acted as an acceptor molecule in the glucosyltransferase reaction. Again, this result supports the conclusion from FIG. 1 that a reduced amount of insoluble polysaccharides had formed. A similar effect was observed with increasing lactose, galactose, or fructose, although to a much lower extent (Table 3). For the galactose and fructose treatments, mainly a disaccharide formed that was not elongated to an oligosaccharide.

[0255] Table 2. Soluble carbohydrate profile (% w / w)

[0256] % added

[0257] maltose Oligosaccharide Sucrose Maltose Glucose Leucrose Fructose 1.57

[0258] 0.3% 1.49 0.00 0.19 0.24 0.75 3.95 0.8% 2.20 0.00 0.23 0.25 0.76 4.34 1.6% 2.60 0.00 0.25 0.22 0.57 3.49 2.5% 3.46 0.00 0.40 0.21 0.52 3.71 3.5% 3.86 0.00 0.53 0.17 0.41 3.37 4.5% 4,69 0.00 0.81 0.17 0.38 3.56

[0259]

[0260] 6.0% 5.28 0.00 1.12 0.17 0.34 3.57 Table 3. Oligosaccharide content when increasing lactose, galactose. or fructose (% w / w)

[0261] % added

[0262] sugar Lactose W GMSWaAlSaWAcWtAoVAsWeAW Fructose i

[0263] 0.1% 1.28 1.05 1.13

[0264] 0.3% 1.89 1.07 1.11

[0265] 0.8% 1.82 0.98 1.20

[0266] 1.6% 1.51 0.99 1.30

[0267] 2.5% 2.09 0.92 1.32 i

[0268] 3.5% 2.34 0.88 1.28

[0269] 4.5% 2.51 1.01 1.27 i

[0270]

[0271] 6.0% 3.02 0.90 1.31

[0272] It was therefore ciear from the data that an increase in alternative acceptor molecuies such as glucose, maltose, or iactose causes a decrease in insoiubie polysaccharide formation, whiie increasing soluble oligosaccharide levels.

[0273] Example 2

[0274] Producing a Soluble Oiiqosaccharide Syrup from Sucrose without a Primer It was found in this Example that the effects observed in Example 1 could be similarly obtained by increasing the substrate (sucrose) concentration in the glucosyltransferase reaction. GTF 0768 (SEQ ID NO: 1) and vGTFJ (SEQ ID NO:3) enzymes were added, in various enzyme:enzyme ratios (or vGTFJ alone), to aqueous solutions of 4.9, 9.5, 19, 28.6, 38.1, 42.9, 47.6, 52.4, or 57.1 wt% sucrose. Each glucosyltransferase (GTF) reaction was incubated 24 hours at 5 °C before heatinactivating the GTF enzymes at 95 °C for 10 minutes. The individual enzyme dosages of the GTF enzymes in a blend were normalized at which a 100% dosage was the dosage necessary for an enzyme to provide full sucrose conversion during the 24 hours at 5 °C (e.g., as described in I nt. Pat. Appl. Publ. No. W02023055902, which is incorporated herein by reference).

[0275] It was surprisingly observed that GTF reactions with high sucrose concentrations (>47.6 wt%) remained transparent (no white precipitated polysaccharide) and had less viscous liquid (FIG. 2). All samples were analyzed for their sugars composition according to the Materials / Methods. However, only sugars composition data from the 0:100% GTF 0768:vGTFJ% enzyme ratio (i.e., vGTFJ only) reactions are listed in Table 4 as an example. It was clear that, aside from obtaining more soluble carbohydrates, there was a particular increase in leucrose concentration and thereby fructose was significantly reduced relative to the total soluble carbohydrates. This trend was observed for all the tested GTF 0768:vGTFJ% enzyme ratios.

[0276] Table 4. Relative distribution (%) of soluble carbohydrates resulting in GTF reactions with 0:100% GTF 0768:vGTFJ% enzyme ratio and increasing initial sucrose concentrations

[0277] initial

[0278] sucrose | Oligosaccharide | Nigerose | Glucose | Leucrose | Fructose % % % % % % 4.8 16 16 9 5 53 9.5 23 21 8 8 40 19.0 33 12 6 14 35 28.6 23 12 8 24 32 38.1 24 10 6 30 30

[0279] 42.9 25 11 6 36 23 47.6 26 11 5 37 21 52.4 23 10 6 40 22 57.1 23 10 5 42 20

[0280]

[0281] Example 3

[0282] In situ Use of Different Primers (Maltose, Lactose) in Addition to Sucrose Modulates the Ratio of Oligosaccharide Products

[0283] GTF reactions were performed in sodium acetate buffer pH 5.0 at 37 °C for 16 hours. The starting sucrose, maltose and lactose concentrations were each 100 g / L. The sugar compositions of the reactions were measured by HPLC (Agilent 1260, equipped with two Bio-Rad Aminex HPX-87P columns [300 mm x 7.8 mm] and an Rl- detector JG1362A]). Water was used as a mobile phase at a flow rate of 0.700 mL / min. The column temperature was 80 °C, Samples for HPLC injection were prepared first by heat inactivation of the reaction enzyme at 70 °C for 2 hours, then centrifugation (30 minutes at 13000 rpm), appropriate dilution of the supernatant in water, and filtration through a 0.22-μm membrane filter. Signals from the HPLC analyses were quantified against calibration standards of sugars eluting at the same time. The detailed carbohydrate distribution of reactions with selected enzymes is shown in Table 5. Table 5. Relative soluble carbohydrate product distribution (%) in GTF reactions that included maltose or lactose primer

[0284] Sum of Oligosaccharides Sucrose Maltose Lactose Glucose Leucrose Galactose Fructose Enzyme Substrate* % % | % % % | % % | %. vGTFJ S 7.54 18.68 | 6.0 | 7.10 | 11.20 | 0 | 55.49 S + M 41.54 0 J 29.21 0 2.78 4.43 0 22.06 S + L 15.13 0 0 52.18 3.03 4.86 0 24.82 GTF 6831 S 37.30 0 1.10 0 2.71 8.94 0 49.96 S + M 50.33 0 24.70 0 0.99 1.96 0 22.05 S + L 25.50 0?37 0 46.28 1.90 4.49 0.38 21?lb " GTF 0768 S 13.96 | 20.88 | 2.58 | 0

[0285] S + M 47.99 0 26.95 0 0.71 3.00 0 21.38 S + L 22.71 0 j 0 49.20 0.89 4.39 0 22.83 LEI2183** S 0 | 69.56 | 0 | 0 | 15.44 | 0 | 0 | 15.01 S + M 0.36 33.18 52.13 0 7.04 0 0 7.11 S + L 0.41 33.24 | 0 51.85 7.23 j 0 0 | 7.28 FOODPRO

[0286] S 0 0 | 0 0 53.36 | 0 0 | 46.64 S + M 0 0 | 50.55 0 26.35 0 0 | 22.65

[0287]

[0288] S + L 0 | 0 | 0 | 50.72 | 26.53 | 0 | 0 | 22.75 *Abbreviations: S, sucrose. M, maltose. L, lactose.

[0289] **LEI2183 is a sucrose phosphorylase disclosed in U. S. Pat. Appl. Publ. No. 20200163902, which is incorporated herein by reference.

[0290] ***FOODPRO I is an invertase available from International Flavors & Fragrances Inc. (IFF). We found that vGTFJ (SEQ ID NOS), GTF 6831 (SEQ ID N0:14) and GTF 0768 (SEQ ID NO:1), all alpha-glucan sucrase enzymes, produced a large quantity of soluble oligosaccharides in the presence of maltose or lactose as a primer, as compared to their respective reactions that only contained sucrose without any added primer molecule. Sucrose-using enzymes LEI2183 and FOODPRO I, to the contrary, did not significantly produce oligosaccharides in their respective reactions (Table 5). The resulting sugar profile for each enzyme was affected by the primer molecule used. For instance, GTF 6831 consumed almost all the sucrose introduced in the reaction media, and produced a high content of oligosaccharides (Table 5).

[0291] In summary, the activity of GTF enzymes can be modulated by adding a competing primer (e.g.:maltose or lactose) that shifts the polysaccharide formation process towards oligosaccharide formation. The profile of oligosaccharide products can be altered by using a different enzyme.

[0292] Example 4

[0293] Producing a Soluble Oligosaccharide Syrup from Sucrose Using Malt Extract as Primer It was investigated whether a malt extract such as BARLEX 7203 light malt extract (Harboe Brewery, Item no. 72030024) could be used in a priming reaction with giucosyltransferase.

[0294] BARLEX 7203 light malt extract has a 74-80% carbohydrate and 46% sugars (30% maltose and 12% glucose) content. GTF enzymes at either a 5:95% GTF 0768:vGTFJ% ratio or 5:95% vGTFJ: GTF 0768% ratio were added to 1-mL samples of 4X~diluted BARLEX 7203 light malt extract with varying levels of sucrose in 0.1 M MES buffer pH 5.5, Reference samples with varying levels of sucrose, but without addition of enzyme, were also included in the analysis. The w / w ratio of sucrose to maltose, which was the primary acceptor molecule in these reactions, ranged from 0.55-2.82.

[0295] The materials were incubated at 38 *C for 6.5 hours before inactivating the enzymes for 10 minutes at 95 °C. All the samples were centrifuged 10 minutes at 13000 rpm after which a picture was taken of the resulting pellets to determine pellet size (FIG.

[0296] 3). It was clear that only the incubations with the 5:95% GTF 0768:vGTFJ% and vGTFJ-only enzyme regimens formed insoluble polysaccharides as manifested by an increase in pellet size (as compared to no enzyme controls). It was also evident that, if the w / w ratio of sucrose to maltose was below or close to 1, then no increase in pellet (insoluble polysaccharide) was observed. Samples were then homogenized and subjected to sugars composition analysis according to the Materials / Methods. All samples with a sucrose to maltose ratio of.81 were excluded from further analysis due to a handling error.

[0297] The final distribution of sugars and soluble oligosaccharides in the reaction sample products is displayed in Table 6. In all samples treated with enzyme, an increase in the product soluble oligosaccharide portion was observed as compared to the sample without enzyme addition. A higher sucrose / maltose ratio resulted in a higher oligosaccharide portion. Although extra sugar had been added in the form of sucrose, it was evident that, at the sucrose / maltose ratio of 0.76 in reactions with all three enzyme treatment types, there was roughly the same percent soluble product distribution of oligosaccharides to sugar as seen when only original BARLEX was used without added sucrose (40%:60%) (Table 6). At a sucrose / maltose ratio of ~1, it was possible to increase the percent product oligosaccharide portion to >40% with each of the 5:95% (GTF 0768:vGTFJ%) and vGTFJ enzyme treatments without forming significant levels of insoluble polysaccharides (Table 6, FIG, 3). However, at a sucrose / maltose ratio above ~1.2, a large amount of insoluble polysaccharide formed in each of the 5;95% (GTF 0768:vGTFJ%) treatment and vGTFJ treatment reactions (Table 6, FIG. 3). Similar trends were observed with GTF 0768, although the reactions with this enzyme did not form insoluble polysaccharides. Table 6. Final distribution of sugar and soluble oligosaccharide products past-incubation with 5:95% GTF0768:vGTFJ, vGTFJ, or GTF 0768 enzymes at varying sucrose to maltose ratios

[0298] j Enzyme Treatment Sucrose / Maltose Ratio % Sugar % Oligosaccharides 0.55 65.0 35.0

[0299] 5:95% (GTF 0.76 58.1 41.9 0768:vGTFJ%) 1.17 50.4 49.6

[0300] 2.82 45.3 54.7

[0301] 0.55 63,9 36.1

[0302] 0.76 59.2 40.8

[0303] vGTFJ

[0304] 1.17 54.3 45.7

[0305] 2.82 51.5 48.5

[0306] 0.55 63.0 37.0

[0307] 0.76 55.6 44.4

[0308] GTF 0768

[0309] _ 1.17 _ ^_ 50.8 _ 49.2 _ _ 2.82 _ _ 53.6 _

[0310] 0.55 74.4 25.6

[0311] 0.76 76.3 23.7

[0312] no enzyme

[0313] 1.17 79.3 20.7

[0314] 2.82 87.3 12.7 BARLEX 7203 Light only

[0315]

[0316] | no enzyme (no sucrose), 4X-diluted 60.0 40.0

[0317] It was furthermore confirmed that maltose was indeed an acceptor molecule in these GTF reactions. This was evident through comparison of maltose values of product samples from reactions with and without enzyme treatment (FIG. 4). It was furthermore found that the majority of the maltose was glycosylated and hence reduced at sucrose / maltose ratios above 1.

[0318] Having established that the oligosaccharide fraction could be increased, it was also of interest to identify whether the oligosaccharides showed slow-digesti bility or nondigestibility profiles and therefore might have an improved glycemic response following consumption.

[0319] Samples of the completed reactions were treated with ethanol to a final concentration of 30% to precipitate polysaccharides, centrifuged to remove pellets, and the saved supernatants were diluted 10-fold. 200 pL of each diluted supernatant sample was added to 400 L water and 200 pL digestive enzyme mixture to mimic digestion of the small intestine according to Englyst et al. (1999, Am. J. Clin. Nutr. 69:448-454, incorporated herein by reference). The digestive enzyme mixture contained glucoamylase at 12.8 AGU / mL (PLUSWEET G, IFF) and invertase at 14.4 SUK / mL (FOODPRO I, IFF). All the samples were incubated for either 20 minutes or 120 minutes at 37 °C before inactivating the digestive enzymes for 10 minutes at 95 °C. All the samples were then subjected to the carbohydrate analysis according to the Materia Is / Methods, though without further ethanol treatment.

[0320] The distribution between sugars and oligosaccharides at the various timepoints of digestion is displayed in Table 7. Reactions with the 5:95% (GTF 0768:vGTFJ%) or vGTFJ enzymes treatments resulted in an increased portion of slow- or non-digestible oligosaccharides. This was evident through the significantly higher level of remaining oligosaccharides of 13-34% after 120 minutes of digestion (independent of sucrose / maltose ratio). In contrast, there were only about 3-6% oligosaccharides left if there had been no conversion of the sucrose with a glucosyltransferase (after 120 minutes). Slow- or non-digestible oligosaccharides were also found in the samples treated with GTF 0768; however, the remaining oligosaccharide levels after digestion were low at sucrose / maltose levels below 1, whereas the levels increased significantly in samples with sucrose / maltose levels above 1. This was due to DP3-4 products formed with GTF 0768 at low sucrose / maltose ratios being relatively easily digestible, whereas the DP3-4 formed at low sucrose / maltose ratios with vGTFJ is less digestible (determined by visual evaluation of peaks on chromatogram). At sucrose / maltose ratios >1, a larger portion of DP4+ was formed by all enzymes that was less digestible.

[0321] Although GTF 0768 produced DP4+ oligosaccharides at sucrose / maltose ratios above 1, it was still clear the these were faster digested than the counterparts produced by vGTFJ. Table 7. Distribution between sugars and oligosaccharides at various time points of digestion with qlucoamylase and invertase

[0322] Time of digestion: _ 0 minutes _ 20 mi lutes 120 m nutes —ETS7M*1 |

[0323] treatment ratio % sugar % oligo % sugar % oligo % sugar % oligo | 0.55 | 65.0 | 35.0 [ 82.9 17.1 86.8 13.2 TIB 5:95% UTTIM 584 417 787 217 83.2 16.8 (GTF - - - - - - - - 0768:vGTFJ%) 1-17 50.4 49.6 71.8 287 79.3 20.7

[0324] ~~~~

[0325] | 272 j 433 547 603 30.7 r^55 j 637 331 842 157 857 147

[0326] 592 | 438 | 797 18.4Jj 1.17 54.3 45.7 72.6 27.4 j 75.4 24.6 2.82 51.5 48.5 | 63.3 36.7 j 65.4 34.6 | 0.55 | 63.0 | 37.0 86.9 13.1 j 93.1 6.9 076 55.6.. 44.4 82.8 17.2 } 92. i 7.9uh-1 / J 50.8 49.2 76.6 23.4 87.2 12.8 _ j 2.82 46.4 j 53.6 j 61.7 387 70.3 297 rp-551 74.4 25.6 91.8 8.2 947 57 1 076 1 762 j 237 j 924 4.2 no enzyme j1 1?j79 3j207 j 93.3 67 95.1 4.9

[0327]

[0328] j 2.82 j 87.3 j 12.7 96.2 3.8 j 96.7 3.3 * Abbreviations: S, sucrose. M, maltose.

[0329] In previous methods using either dextran sucrase or alternan sucrase, the ratio between sucrose and maltose was preferred to be >1 for the preparation of soluble oligosaccharides (e.g., EP1545243, EP2575498, EP3182981). However, we show here that a ratio of sucrose / maltose of 1 or below is preferred for vGTFJ to prevent formation of insoluble polysaccharides. We furthermore show that the resulting linear alpha-1,3-glucan oligosaccharides are less digestible compared to original oligosaccharides in the malt extract, and also less digestible than the alpha-1,6-glucan oligosaccharides formed with GTF 0768.

[0330] Example 5. Producing a Soluble Oligosaccharide Syrup from Sucrose Using Whey as a Primer Source

[0331] An alternative low-sugar, sweet oligosaccharide syrup can be prepared from whey (rich in lactose, which can serve as a GTF primer) with the addition of sucrose and use of glucosyltransferase enzymes (10:90% GTF0768:vGTFJ%) blend in combination with a trans-gaiactosylating beta-galactosidase enzyme. The whey can be from separation of cheese, lactic casein, skim milk, or similar material. The whey typically has less than 15 wt% protein and more than 65 wt% lactose relative to dry solids. In this Example, a GTF reaction was performed at a concentration of about 20% lactose w / w at 5 ”0, but it could be optimized for other temperatures up to about 50 °C by adjusting the enzyme dosages.

[0332] Three aqueous samples with 20 wt% lactose at pH 5.5, and three samples with 20 wt% lactose and 6 wt% sucrose at pH 5.5, were prepared and aliquoted in 10 ml portions. Enzymes were added according to Tabie 8, and then all the samples were incubated at 5 °C for 24 hours before heat-inactivation at 95 °C for 10 minutes.

[0333] Table 8. Trial ingredient overview

[0334] Lactose Sucrase Enzyme

[0335] Trial % % Enzyme* dosage 1 20

[0336] 20 BONLACTA 60 pL

[0337] 3 20 NURICA 120 pL 4. 20.. 6. GTF blend 40 pL

[0338] GTF blend + 40 pL +

[0339] 5 20 6 NURICA 120 pL

[0340]

[0341] 6 20 6 - * BONLACTA (lactase from IFF)

[0342] * NURICA (transgalactosylating beta-galactosidase from IFF) * GTF blend: 10:90% (GTF 0768:vGTFJ%) blend

[0343] All the samples were analyzed for the carbohydrate composition, before and after incubation with enzyme(s), according to the Materials / Methods. Results are presented in Table 9. The data confirm that the majority of the carbohydrates stay soluble (comparing soluble carbohydrates pre- vs. post-incubation) independent of which enzyme(s) was added.

[0344] Furthermore, it was clear from the data that BONLACTA primarily split lactose to glucose and galactose, whereas NURICA performed a transgalactosylation reaction forming galactooligosaccharides (GOS) while releasing glucose. The GTF blend hydrolyzed the sucrose thereby forming oligosaccharides and leucrose while releasing fructose. The highest level of oligosaccharides was, however, formed when combining NURICA and the GTF blend, having formed both galacto- and gluco-oligosaccharides. Table 9. Soluble carbohydrate composition in % (w / w)

[0345] Sblbloe caru. O

[0346] £ Pibitrencuaon- i

[0347] tz o <z \

[0348] +• ‘j, i 2 “ji i o Q ‘J b i

[0349] i <3 Q < D

[0350] a CL i w c. z a. i _i o. j O m: _J a. i 0 c.

[0351] ............

[0352] i 2 21.27 3.51 i 0.00 i 0.00 6.40 i 6.24 0.00 5.36 0 54 i 22.05 i i 3 21.62 7.34 0.00 i 0.00 i 6.67 j 4.66 ) 0.28 i 1.36 0.60 i 20.91 4 2800 4.28 1.00 0.00 17.96 0.00 0.76 0.25 3.30 27.55 i 5 27.67 9.84 i 0.00 \ 1.33 6.43 i 4.21 0.80 0.00 2.95 i 25.56 i

[0353]

[0354] i 6 28.11 0.00 5.83 i 0.00 21.37 i 0.00? 0.00 0.48 0.42 i 28.11

[0355] When calculating sugar reduction relative to the reference "whey” trial 1, as well as the relative sweetness for each sample (Table 10), it was clear that NURICA provided the highest sugar reduction (trial 3), but this was with a limited increase in sweetness. The GTF blend in contrast provided high theoretical sweetness (trial 4), but due to sucrose addition, it had an increase in sugar rather than a reduction. Only in trial 5 when combining NURICA and the GTF blend was it possible to achieve more than 25% sugar reduction while also significantly increasing the theoretical swee Itn P Ftorucseess.

[0356] Pibittoncaosnu w- Table 10.

[0357] % Sugar Theoretical Slblboue car.

[0358] Trial %

[0359] Sugar Reduction sweetness Pibittoncaosun- 1 21.40 0.00 4.13

[0360] 2 18.54 13.36 8.30

[0361] 3 13.57 36.59 6.13

[0362] 4 23.27 -8.74 10.04

[0363] 5 15.72 26.53 10.24

[0364]

[0365] 6 28.11 -31.35 10.14

Claims

CLAIMSWhat is claimed is:

1. A method comprising:(a) providing a reaction composition comprising at least water, sucrose, and a glucosyitransferase enzyme that is capable of synthesizing water-insoluble alpha- 1,3-glucan, wherein the reaction composition comprises at least about 45% by weight of said sucrose,(b) incubating the reaction composition, wherein water-soluble oligosaccharides are produced by the glucosyltransferase in the reaction composition, and(c) optionally isolating the water-soluble oligosaccharides, typically along with any monosaccharides that are also comprised in the reaction composition after step (b):optionally wherein there is little or no difference in the viscosity of the reaction composition resulting from step (b) as compared to the viscosity of the reaction composition initially provided in step (a).

2. The method of claim 1, wherein the reaction composition provided in step (a) has no detectable amount of, or does not comprise, a primer molecule.

3. The method of claim 1, wherein the reaction composition provided in step (a) comprises at ieast about 50% by weight of said sucrose.

4. The method of claim 1, wherein the water-soluble oligosaccharides produced in step (b) comprise at least alpha-1,3-glucooligosaccharides and leucrose, wherein at ieast about 50% of the glycosidic linkages of the alpha-1,3- glucooligosaccharides are alpha-1,3 glycosidic linkages, and the weight-average degree of polymerization (DPw) of the alpha-1, 3-glucooligosaccharides is 2 to about 20.

5. The method of claim 4, wherein at least about 75% of the glycosidic linkages of the alpha-1, 3-glucooligosaccharides are alpha-1,3 glycosidic linkages.

6. The method of ciaim 1, wherein the reaction composition from step (b) has a ratio of water-insoluble aipha-1, 3-glucan to said water-soluble oligosaccharides that is less than about 25:75 on a weight basis.

7. The method of claim 1, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan comprises:(i) an amino acid sequence that is at least 80%, 85%, or 90% identical to residues 55-960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55- 960 of SEQ ID NOT, residues 55-960 of SEQ ID NO:8, residues 55-960 of SEQ ID NO:

9. or SEQ ID NO:13, or(ii) an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO:3, 4, 5, 6, 7, 8, 9, or 13.

8. The method of claim 1, wherein the reaction composition further comprises a glucosyltransferase enzyme that is capable of synthesizing alpha-1,6-glucan,9. The method of claim 8, wherein said glucosyltransferase enzyme that is capable of synthesizing alpha-1,6-glucan comprises an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 1, 2, 11, 12, 14, or 15.

10. The method of claim 8, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha-1,6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan in the reaction composition is about 90:10 to about 5:95.

11. The method of claim 8, wherein the ratio of the glucosyltransferase enzyme that is capable of synthesizing alpha-1,6-glucan to the glucosyltransferase enzyme that is capable of synthesizing alpha-1, 3-glucan in the reaction composition is about 50:50 to about 5:95.

12. The method of claim 1, further comprising:(i) incorporating the reaction composition resulting from step (b) in a product, optionally wherein the enzyme activity of the reaction composition has been terminated prior to said incorporating,(ii) using the reaction composition resulting from step (b) itself as a product, optionally wherein the enzyme activity of the reaction composition has been terminated prior to said using.or(iii) performing step (c) of isolating the water-soluble oligosaccharides, and optionally incorporating the isolated water-soluble oligosaccharides in a product.

13. The method of claim 12, wherein the product of step (i). (ii), or (iii) is a food product / precursor,optionally wherein the food product / precursor, after step (i), (ii), or (iii), has one or more of the following features:(I) increased dietary fiber,(II) increased prebiotic activity,(III) reduced caloric density or reduced calories, and / or(IV) reduced glycemic index.

14. A product produced by the method of claim 12.

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