Method to provide improved texture and stable sucrose levels in foodstuff with glucosyltransferase

Abstract

Disclosed herein are methods of producing a food product or food precursor. These methods can comprise, for example, (a) providing milk, (b) adding sucrose to the milk to form sweetened milk, (c) contacting the sweetened milk with at least one glucosyltransferase to form insoluble alpha-glucan in the milk, (d) acidifying the milk product to a pH below about 5.0, and (e) inactivating the glucosyltransferase by subjecting the acidified milk product to a temperature of about 70-110 °C. Such methodology can produce an acidified milk product having a stable sucrose level and typically also a stable level of texture. Such methodology can optionally use other types of food products / precursors instead of milk, as appropriate. One or more glucosyltransferases used in the methodology can be selected from (i) a glucosyltransferase that synthesizes alpha-1,6-glucan, and / or (ii) a glucosyltransferase that synthesizes alpha-1,3-glucan. Food products / precursors produced by this methodology are also disclosed.

Description

[0001] TITLE

[0002] METHOD TO PROVIDE IMPROVED TEXTURE AND STABLE SUCROSE LEVELS IN FOODSTUFF WITH GLUCOSYLTRANSFERASE

[0003] This application claims the benefit of U.S. Provisional Appl. No. 63 / 669,828 (filed July 11 , 2024), which is incorporated herein by reference in its entirety.

[0004] FIELD

[0005] The present disclosure relates to fresh-fermented products (dairy or non-dairy), such as a post-pasteurized fresh-fermented product, comprising stable sucrose content and texture modulation formed through the action of a glucosyltransferase in situ in a cofermentation step. Since the fresh-fermented product has been modified such that the glucosyltransferase is not active post-production - i.e. , no glucosyltransferase activity during product shelf life - stable levels of sucrose and texture of the product are maintained.

[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 IFF10173WOPCT_SequenceListing.xml created on July 1 , 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] Selected glucosyltransferase enzyme candidates have been investigated for sugar reduction, cleaner label, and the option to work as an alternative to hydrocolloids. All are pronounced consumer demands. These, glucosyltransferases have previously been described for use in dairy and fresh-fermented products, either alone or in combination with other enzymes such as glucosyltransferases, invertases, or betagalactosidases.

[0010] Dairy products

[0011] There is increasing demand for products that can be stored and transported at ambient temperature, and that have a prolonged shelf life of several months, which features simplify handling and reduce carbon footprint. To obtain such long-term shelf life at ambient temperature, the fermented dairy product is heat-treated after the fermentation process to at least inhibit further growth of the lactic acid bacteria used in the fermentation process. The heat treatment may, for example, be a pasteurization process or an ultra-high temperature (UHT) process. Such products are sometimes referred to as post-pasteurized fermented products or ambient fermented products. In particular, these products may be, for example, a post-pasteurized yogurt, also known as ambient yogurt, long shelf-life yogurt or drinking yogurt, or a plant-based analog of such.

[0012] Ambient yogurt, typically stirred or drinking yogurt, are particularly relevant in regions where consumers are challenged by lack of cold chain conditions. Ambient yogurt reduces distribution costs and complexities compared to cold chain distribution, making it easier to export. The heat treatment process creates a product that can be stored at room temperature, becoming a healthy and convenient alternative to cold yogurt, while still providing a good source of dairy protein, vitamins and minerals. Glucosyltransferase enzymes

[0013] By adding at least one glucosyltransferase enzyme as an in situ processing aid in food products containing sucrose, the enzyme cleaves sucrose and releases fructose while being covalently linked to the glucose moiety. The glucosyl-enzyme complex links the glucosyl moiety to an acceptor molecule to form glucose polymer product (i.e. , glucan) through a polymerization reaction. A glucosyltransferase enzyme of the GTFJ type transfers the glucosyl moiety to an acceptor molecule via alpha-1 ,3 linkage formation to produce linear insoluble alpha-glucan polymer (alpha-1 , 3-glucan). The glucosyl-enzyme complexes typically also produce byproducts, such as by transferring the glucose to free fructose resulting in a disaccharide sucrose isomer known as leucrose, or by transferring to water resulting in free glucose.

[0014] Some prior art documents disclose in situ use of glucosyltransferase(s) in milk and / or cold yogurt. W02020 / 010176 describes the use of glucosyltransferase(s) to produce insoluble polysaccharides in situ in a co-fermentation process, thereby providing a yogurt with increased thickness. W02023 / 055902 describes the single or combined use of a glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan and a glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan in foodstuff including fresh fermented products. The ‘902 reference further describes how the resulting texture can be further adjusted through combined use of other enzymes such as betagalactosidase or transglucosidase, or by the presence of other sugar components in the recipe.

[0015] The prior art, however, only describes examples of glucosyltransferase dosages that provide full sucrose conversion of up to 8% when added at the inoculation step of preparing fresh fermented products. Because glucosyltransferase enzyme(s) is typically not inactivated in the food production process, at sucrose levels above 8%, the enzyme(s) will generally provide extreme texture, which could be difficult to process (stirring and pumping) in a production plant, and the final texture might not be desirable for consumers. Furthermore, at high sucrose levels, the loss of sweetness could be significant, and thus is another reason why it is desirable to convert only part of the sucrose content. A lower dosage of glucosyltransferase enzyme(s) could be used to address these issues; however, this could still cause undesirable changes in texture and flavor over the shelf life of the product due to continued glucosyltransferase activity.

[0016] In conclusion, there is a need for products having stable levels of sucrose and thereby avoiding further sucrose conversion into texturizing polysaccharides. Through inactivation of added glucosyltransferase enzyme(s) as now presently described, it will be possible to produce fresh fermented products with a controlled conversion level of sucrose while avoiding either extreme texture or changes of both texture and flavor over product shelf life.

[0017] SUMMARY

[0018] In one embodiment, the present disclosure concerns a method of producing an acidified milk product having improved texture, wherein the improved texture comprises increased thickness and / or increased mouthfeel, the method comprising the steps of: (a) providing milk, (b) adding sucrose to the milk to form sweetened milk (alternatively, steps [a] and [b] can be combined as a step of providing sweetened milk), (c) contacting the sweetened milk with at least one glucosyltransferase to form insoluble alpha-glucan in the milk, thereby providing a milk product having the improved texture (alternatively, sucrose addition of step [b] can be conducted following adding the at least one glucosyltransferase), (d) following step (c), or concurrently with step (c), acidifying the milk product to a pH below about 5.0, and (e) inactivating the glucosyltransferase by subjecting the acidified milk product of step (d) to a temperature of about 70 °C to about 110 °C, thereby producing an acidified milk product having a stable sucrose level and a stable level of the improved texture.

[0019] In another embodiment, the present disclosure concerns a food product produced by the method as presently disclosed.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES FIG. 1 : Sugar content in samples. Refer to Example 3.

[0021] FIG. 2: Soluble carbohydrate content in drinking yogurt over product shelf life. Refer to Example 4.

[0022] FIG. 3: Picture of yogurt products of Trials 1-5 at day 19 of ambient storage. Refer to Example 5. FIG. 4: Percent sucrose relative to total carbohydrates in samples of Trial 1-5 products that received additional sucrose, before or after 3-hour incubation at 30 °C. Refer to Example 5.

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

[0024] DETAILED DESCRIPTION

[0025] The disclosures of all cited patent and non-patent literature are incorporated herein by reference in their entirety.

[0026] 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.

[0027] Where present, all ranges are inclusive and combinable, except as otherwise noted. For example, when a range of “1 to 5” (i.e., 1-5) is recited, the recited range should be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, and the like. The numerical values of the various ranges in the present disclosure, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both proceeded by the word “about”. In this manner, slight variations above and below the stated ranges can typically be used to achieve substantially the same results as values within the ranges. Also, the disclosure of these ranges is intended as a continuous range including each and every value between the minimum and maximum values.

[0028] 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.

[0029] The terms “alpha-glucan”, “alpha-glucan polymer” and the like are used interchangeably herein. An alpha-glucan is a polymer comprising glucose monomeric units linked together by alpha-glycosidic linkages. In typical embodiments, an alphaglucan herein comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% alpha- glycosidic linkages. Examples of alpha-glucan polymers herein include graft copolymers as presently disclosed, as well as alpha-1 , 3-glucan and alpha-1 , 6-glucan.

[0030] 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.

[0031] The terms “dextran”, “dextran polymer”, “dextran molecule”, “alpha-1 , 6-glucan” and the like herein refer to a water-soluble alpha-glucan comprising at least 50%, 60%, 70%, 80%, or 90% alpha-1 ,6 glycosidic linkages (with the balance of the linkages typically being alpha-1 ,3). Enzymes capable of synthesizing dextran from sucrose may be described as “dextransucrases” (EC 2.4.1.5). A “substantially linear” (“mostly linear”, and like terms) dextran has 5% or less branches, before being modified herein to have with alpha-1 , 3-glucan side chains. A “linear” dextran has no branches, before being modified herein to have alpha-1 , 3-glucan side chains. Branches, if present prior to modification of dextran with alpha-1 , 3-glucan side chains, can be short, being one (pendant) to three glucose monomers in length. Yet, in some aspects, dextran can be “dendritic”, which is a branched structure emanating from a core in which there are chains (containing mostly or all alpha-1 ,6-linkages) that iteratively branch from each other (e.g., a chain can be a branch from another chain, which in turn is a branch from another chain, and so on). Yet, in still some aspects, dextran is not dendritic, but has a branch-on-branch structure that does not emanate from a core. Dextran as used in a glucosyltransferase reaction herein for alpha-1 , 3-glucan synthesis (to produce a dextran-alpha-1 , 3-glucan copolymer) can optionally be characterized as a “primer” or “acceptor”. In some aspects, dextran can characterize a dextran “homopolymer”, which is dextran that is not part of a dextran-alpha-1 , 3-glucan copolymer.

[0032] The term “copolymer” herein refers to a polymer comprising at least two different types of alpha-glucan, such as dextran and alpha-1 , 3-glucan.

[0033] The terms “graft copolymer”, “branched copolymer” and the like herein generally refer to a copolymer comprising a “backbone” (or “main chain”) and one or more side chains branching from the backbone. The side chains are structurally distinct from the backbone.

[0034] Examples of graft copolymers herein are “dextran-alpha-1 ,3-glucan graft copolymers” (and like terms) that comprise a backbone comprising dextran, and one or more side chains of alpha-1 , 3-glucan. A backbone in some aspects can itself be a branched dextran as disclosed herein; the addition of alpha-1 ,3-glucan side chains to such a backbone (thereby forming a graft copolymer herein) can be, for example, via enzymatic extension from non-reducing ends presented by short branches (alpha-1 ,2, - 1 ,3, or -1 ,4 branch, each typically comprised of a single glucose monomer; i.e., pendant glucose). Short branches (that can be enzymatically extended into an alpha-1 ,3-glucan side chain) can be present on an otherwise linear or mostly linear dextran, or can be present on a branching dextran. In some aspects, alpha-1 , 3-glucan can also be synthesized from non-reducing ends of dextran main chains, such as in embodiments in which the dextran backbone is linear or mostly linear, or embodiments in which the dextran backbone is branching (e.g., dendritic, or not dendritic [branches do not emanate from a core] but has branch-on-branch structure); such alpha-1 , 3-glucan is not, technically-speaking, a side chain to the dextran, but rather an extension from the dextran main chain(s).

[0035] The percent branching in an alpha-glucan herein refers to that percentage of all the linkages in the alpha-glucan that represent branch points. For example, the percent of alpha-1 ,3 branching in an alpha-glucan herein refers to that percentage of all the linkages in the glucan that represent alpha-1 ,3 branch points. Except as otherwise noted, linkage percentages disclosed herein are based on the total linkages of a glucan, or the portion of a glucan for which a disclosure specifically regards.

[0036] The terms “linkage”, “glycosidic linkage”, “glycosidic bond” and the like refer to the covalent bonds connecting the sugar monomers within a saccharide compound (oligosaccharides and / or polysaccharides). Examples of glycosidic linkages include 1 ,6- alpha-D-glycosidic linkages (herein also referred to as “alpha-1 ,6” linkages), 1 ,3-alpha- D-glycosidic linkages (herein also referred to as “alpha-1 ,3” linkages), 1 ,4-alpha-D- glycosidic linkages (herein also referred to as “alpha-1 ,4” linkages), and 1 ,2-alpha-D- glycosidic linkages (herein also referred to as “alpha-1 ,2” linkages). The glycosidic linkages of a glucan polymer herein can also be referred to as “glucosidic linkages”. Herein, “alpha-D-glucose” is referred to as “glucose”.

[0037] The glycosidic linkage profile of an alpha-glucan herein can be determined using any method known in the art. For example, a linkage profile can be determined using methods using nuclear magnetic resonance (NMR) spectroscopy (e.g.,13C NMR or1H NMR). These and other methods that can be used are disclosed in, for example, Food Carbohydrates: Chemistry, Physical Properties, and Applications (S. W. Cui, Ed., Chapter s, S. W. Cui, Structural Analysis of Polysaccharides, Taylor & Francis Group LLC, Boca Raton, FL, 2005), which is incorporated herein by reference.

[0038] 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).

[0039] The term “sucrose” herein refers to a non-reducing disaccharide composed of an alpha-D-glucose molecule and a beta-D-fructose molecule linked by an alpha-1 , 2- glycosidic bond. Sucrose is known commonly as table sugar. Sucrose can alternatively be referred to as “alpha-D-glucopyranosyl-(1^2)-beta-D-fructofuranoside”. “Alpha-D- glucopyranosyl” and “glucosyl” are used interchangeably herein.

[0040] 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.

[0041] 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.

[0042] The term “glucosyltransferase catalytic domain” herein refers to the domain of a glucosyltransferase enzyme that provides alpha-glucan-synthesizing activity to a glucosyltransferase enzyme. A glucosyltransferase catalytic domain typically does not require the presence of any other domains to have this activity.

[0043] 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.

[0044] 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).

[0045] The terms “percent by volume”, “volume percent”, “vol %”, “v / v %” and the like are used interchangeably herein. The percent by volume of a solute in a solution can be determined using the formula: [(volume of solute) / (volume of solution)] x 100%.

[0046] The terms “percent by weight”, “weight percentage (wt%)”, “weight-weight percentage (% w / w)” and the like are used interchangeably herein. Percent by weight refers to the percentage of a material on a mass basis as it is comprised in a composition, mixture, or solution.

[0047] The terms “weight / volume percent”, “w / v%” and the like are used interchangeably herein. Weight / volume percent can be calculated as: ((mass [g] of material) / (total volume [mL] of the material plus the liquid in which the material is placed)) x 100%. The material can be insoluble in the liquid (i.e. , be a solid phase in a liquid phase, such as with a dispersion), or soluble in the liquid (i.e., be a solute dissolved in the liquid).

[0048] The terms “ingestible product” and “ingestible composition” are used interchangeably herein, and refer to any substance that, either alone or together with another substance, may be taken orally (i.e., by mouth), whether intended for consumption or not. Thus, an ingestible product includes food / beverage products. “Food / beverage products” refer to any edible product intended for consumption (e.g., for nutritional purposes) by humans or animals, including solids, semi-solids, or liquids. A “food” herein can optionally be referred to as a “foodstuff”, “food product”, or other like term, for example. Herein, unless otherwise disclosed, a beverage or other ingestible liquid is an example of a food product. While the present disclosure generally regards food and food precursors that are by definition intended for ingestion or eventual ingestion (food precursor first made into food before being eaten), the disclosure likewise regards other ingestible products (e.g., supplement, nutraceutical, pharmaceutical product) comprising in s / Yu-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.

[0049] The term “texture” as used in reference to a food product / precursor herein means the thickness of the food product / precursor and / or sensory perception of the food product / precursor by, for example, vision, touch, or oral / taste processing (e.g., mouthfeel). An “improvement” in texture means an increase in thickness and / or an increase in the sensory perception. Unless otherwise noted, as used herein the “thickness” of a food product / precursor means the apparent viscosity extracted at shear rate of about 10-13 Hz (e.g., -11.7 Hz) during a rheological analysis; an increase in apparent viscosity at such a shear rate indicates an increase in thickness. The apparent viscosity extracted at shear rate of about 230-270 Hz (e.g., -249 Hz) during a rheological analysis is correlated to “mouthfeel”; an increase in apparent viscosity at such a shear rate indicates an increase in mouthfeel.

[0050] “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.

[0051] “Lactase-treated mil k / dairy” and like terms herein refer to mi Ik / dairy products or precursors treated with one or more lactase enzymes to reduce the amount of lactose sugar therein.

[0052] “Reduced lactose mil k / dairy” and like terms herein refer to mi Ik / 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.

[0053] “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.

[0054] The terms “dietary fiber”, “glucan fiber” and the like herein refer to an alphaglucan that is indigestible and / or that does not increase blood-glucose levels when enterally administered to a mammal. In general, a dietary fiber herein is not significantly hydrolyzed by endogenous enzymes in the upper gastrointestinal tract of mammals such as humans.

[0055] “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).

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] An alpha-glucan herein that is “insoluble”, “aqueous-insoluble”, “water-insoluble” (and like terms) herein does not dissolve (or does not appreciably dissolve) in water or other aqueous conditions, optionally where the aqueous conditions are at a pH of 4-9 (e.g., pH 6-8) and / or a temperature of about 1 to 130 °C (e.g., 20-25 °C). In some aspects, less than 1.0 gram (e.g., no detectable amount) of an aqueous-insoluble alphaglucan dissolves in 1000 milliliters of such aqueous conditions (e.g., water at 23 °C). In contrast, an alpha-glucan that is “soluble”, “aqueous-soluble”, “water-soluble” and the like appreciably dissolves under the above aqueous conditions.

[0061] 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.

[0062] 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 nr1s-1.

[0063] As used herein, the term “polypeptide” is defined as a chain of amino acid residues, usually having a defined sequence. As used herein the term polypeptide is interchangeable with the terms “peptides” and “proteins”. Typical amino acids contained in polypeptides herein include (respective three- and one-letter codes shown parenthetically): alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine (Gin, Q), glycine (Gly, G), histidine (His, H), isoleucine (lie, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), valine (Vai, V).

[0064] 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.

[0065] Percent identity can be readily determined by any known method, including but not limited to those described in: 1) Computational Molecular Biology (Lesk, A.M., Ed.) Oxford University: NY (1988); 2) Biocomputinq: Informatics and Genome Projects (Smith, D.W., Ed.) Academic: NY (1993); 3) Computer Analysis of Sequence Data, Part I (Griffin, A.M., and Griffin, H.G., Eds.) Humana: NJ (1994); 4) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5) Sequence Analysis Primer (Gribskov. M. and Devereux, J., Eds.) Stockton: NY (1991), all of which are incorporated herein by reference.

[0066] Preferred methods for determining percent identity are designed to give the best match between the sequences tested. Methods of determining identity and similarity are codified in publicly available computer programs, for example. Sequence alignments and percent identity calculations can be performed using the MEGALIGN program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wl), for example. Multiple alignment of sequences can be performed, for example, using the Clustal method of alignment which encompasses several varieties of the algorithm including the Clustal V method of alignment (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values can correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method can be KTUPLE=1 , GAP PENALTY=3, WIND0W=5 and DIAGONALS SAVED=5. For nucleic acids, these parameters can be KTUPLE=2, GAP PENALTY=5, WIND0W=4 and DIAGONALS SAVED=4. Additionally, the Clustal W method of alignment can be used (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci. 8:189-191(1992); Thompson, J.D. et al, Nucleic Acids Research, 22 (22): 4673-4680, 1994) and found in the MEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (protein / nucleic acid) can be: GAP PENALTY=10 / 15, GAP LENGTH PENALTY=0.2 / 6.66, Delay Divergent Seqs(%)=30 / 30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB.

[0067] 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.

[0068] The term “isolated” means a substance (or method / process) in a form or environment that does not occur in nature. A non-limiting example of an isolated substance includes any non-naturally occurring substance such as a food product or food precursor (as well as enzymatic reactions used to prepare these materials). It is believed that the embodiments disclosed herein are synthetic / man-made (could not have been made except for human intervention / involvement), and / or have properties that are not naturally occurring.

[0069] 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.

[0070] One or more glucosyltransferase enzymes used in a method as presently disclosed can comprise, for example:

[0071] (i) a glucosyltransferase (GTF) enzyme that synthesizes alpha-1 , 6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1 , 6-glucan are alpha- 1 ,6 linkages (or, a GTF enzyme with alpha-1 , 6-glucan I dextran synthesis activity), and / or

[0072] (ii) a GTF enzyme that synthesizes alpha-1 ,3-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1 ,3-glucan are alpha-1 ,3 linkages (or, a GTF enzyme with alpha-1 , 3-glucan synthesis activity). In some aspects, a GTF enzyme (dextransucrase) that synthesizes alpha-1 , 6- glucan herein can comprise an amino acid sequence that is about 100% identical to, or at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, SEQ ID NO: 1 , 2, 11 , or 12 (GTF 0768), or 14 or 15 (GTF 6831) and have GTF activity. Yet, in some aspects, a GTF enzyme that synthesizes alpha-1 ,6- glucan can be as disclosed in any of U.S. Patent Appl. Publ. Nos. 2017 / 0218093, 2018 / 0282385, 2018 / 0291311 , or 2016 / 0122445, which are each incorporated herein by reference. For example, the GTF identified as GTF 8117 (SEQ ID NO:30), GTF 6831 (SEQ ID NO:32), or GTF 5604 (SEQ ID NO:33) in US2018 / 0282385 can be used, or the GTF identified as GTF 2919 (SEQ ID NO:5), GTF 2918 (SEQ ID NO:9), GTF 2920 (SEQ ID NO:13), or GTF 2921 (SEQ ID NO:17) in US2016 / 0122445 can be used, or a GTF comprising an amino acid sequence that is about 100% identical to, or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, the amino acid sequence of any of these GTF enzymes (and having GTF activity) can be used.

[0073] A dextransucrase herein is capable of producing dextran comprising about, or at least about, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% alpha-1 ,6 glycosidic linkages, for example. Such a percent alpha-1 ,6 linkage profile takes into account the total of all linkages in the dextran (main chains of alpha-1 ,6 glucan and, if present, branch portions therefrom). Dextran as disclosed elsewhere herein such as in a homopolymer or graftcopolymer can have any of the foregoing linkage profiles, for example.

[0074] A dextransucrase herein is capable of producing dextran having a weightaverage molecular weight (Mw) of about, at least about, or less than about, 1000, 2500, 5000, 7500, 10000, 25000, 50000, 75000, 100000, 150000, 200000, 250000, 500000, 750000, 1000000, 1000-10000, 1000-100000, 1000-1000000, 10000-100000, 10000- 1000000, or 100000-1000000 Daltons, for example. In some aspects, the Mw is about, at least about, or less than about, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 10-50, 10-70, 10-80, 10-100, 10-120, 10-130, 10-150, 10-200, 25-50, 25-70, 25-80, 25-100, 25-120, 25-130, 25-150, 25-200, 50-70, 50-80, 50-100, 50-120, 50-130, 50-150, 50-200, 70-80, 70-100, 70-120, 70-130, 70-150, 70-200, 80-100, 80-120, 80-130, 80-150, 80-200, 100-120, 100-130, 100-150, 100-200, 120-130, 120-150, 120-200, 130-150, or 130-200 million Daltons, for example. Dextran as disclosed elsewhere herein, such as in a homopolymer or graftcopolymer, can have any of the foregoing molecular weight profiles, for example.

[0075] In some aspects, a GTF enzyme that synthesizes alpha-1 , 3-glucan herein can comprise an amino acid sequence that is about 100% identical to, or at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 26, 28, 30, 34, or 59, or amino acid residues 55- 960 of SEQ ID NO:4, residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20, and have GTF activity; these amino acid sequences are disclosed in U.S. Patent Appl. Publ. No. 2019 / 0078063, which is incorporated herein by reference. It is noted that such a GTF enzyme comprising SEQ ID NO:2, 4, 8, 10, 14, 20, 26, 28, 30, 34, or amino acid residues 55-960 of SEQ ID NO:4, residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20, can synthesize alpha-glucan comprising at least about 90% (-100%) alpha-1 ,3 linkages. A GTF enzyme that synthesizes alpha-1 , 3-glucan in some aspects can be that identified as GTF 0974 (SEQ ID NO: 13 herein, SEQ ID NO:110 in US2018 / 0291311), or a GTF comprising an amino acid sequence that is about 100% identical to, or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, the foregoing amino acid sequence of GTF 0974 (and having GTF activity). Any of the foregoing GTF enzyme amino acid sequences can be modified as described herein to increase product yield, modify product molecular weight, and / or enhance GTF performance and / or stability.

[0076] A GTF enzyme for producing alpha-1 , 3-glucan herein can, in some aspects, synthesize alpha-1 , 3-glucan at a yield of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, or 96%. Yield in some aspects can be measured based on the glucosyl component of the reaction, and / or as measured using HPLC or NIR spectroscopy. Yield can be achieved in a reaction conducted for about 16-24 hours (e.g., -20 hours), for example. Examples of such a GTF enzyme are those having an amino acid sequence modified such that the enzyme produces more products (alpha-1 , 3-glucan and fructose), and less by-products (e.g., glucose, oligosaccharides such as leucrose), from a given amount of sucrose substrate. For example, one, two, three, four, or more amino acid residues of the catalytic domain of an alpha-1 , 3-glucan-producing GTF herein can be modified / substituted to obtain a GTF enzyme that produces more products. Examples of a suitable modified GTF enzyme are disclosed in Tables 3-7 of U.S. Patent Appl. Publ. No. 2019 / 0078063. A modified GTF enzyme, for example, can comprise one or more amino acid substitutions corresponding with those in Tables 3-7 (ibid.) that is / are associated with an alpha-1 , 3- glucan yield of at least 40% (the position numbering of such at least one substitution corresponds with the position numbering of SEQ ID NO:62 as disclosed in U.S. Patent Appl. Publ. No. 2019 / 0078063). A set of amino acid modifications as listed in Tables 6 or 7 (ibid.) can be used, for example.

[0077] The amino acid sequence of a GTF enzyme for alpha-1 ,3-glucan synthesis in some aspects has been modified such that the enzyme produces alpha-1 , 3-glucan with a molecular weight (DPw) that is lower than the molecular weight of alpha-1 , 3-glucan produced by its corresponding parent GTF. Examples of a suitable modified GTF enzyme are disclosed in Tables 3 and 4 of U.S. Patent Appl. Publ. No. 2019 / 0276806, which is incorporated herein by reference. A modified GTF enzyme, for example, can comprise one or more amino acid substitutions corresponding with those in Tables 3 and / or 4 (ibid.) that is / are associated with an alpha-1 ,3-glucan product molecular weight that is at least 5% less than the molecular weight of alpha-1 , 3-glucan produced by parent enzyme (the position numbering of such at least one substitution corresponds with the position numbering of SEQ ID NO:62 as disclosed in U.S. Patent Appl. Publ. No. 2019 / 0276806). A set of amino acid modifications as listed in Table 4 (ibid.) can be used, for example.

[0078] The amino acid sequence of a GTF enzyme for alpha-1 ,3-glucan synthesis in some aspects has been modified such that the enzyme produces alpha-1 , 3-glucan with a molecular weight (DPw) that is higher than the molecular weight of alpha-1 ,3-glucan produced by its corresponding parent GTF. Examples of a suitable modified GTF enzyme are disclosed in Tables 3, 4 and 5 of U.S. Patent Appl. Publ. No. 2019 / 0078062, which is incorporated herein by reference. A modified GTF enzyme, for example, can comprise one or more amino acid substitutions corresponding with those in Tables 3, 4 and / or 5 (ibid.) that is / are associated with an alpha-1 , 3-glucan product molecular weight that is at least 5% higher than the molecular weight of alpha-1 ,3-glucan produced by parent enzyme (the position numbering of such at least one substitution corresponds with the position numbering of SEQ ID NO:62 as disclosed in U.S. Patent Appl. Publ. No. 2019 / 0078062). A set of amino acid modifications as listed in Table 5 (ibid.) can be used, for example.

[0079] In some aspects, a modified GTF for alpha-1 , 3-glucan synthesis (i) comprises at least one amino acid substitution or a set of amino acid substitutions (as described above regarding yield or molecular weight), and (ii) comprises or consists of a GTF catalytic domain that is at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to amino acid residues 55-960 of SEQ ID NO:4, amino acid residues 54-957 of SEQ ID NO:65, amino acid residues 55-960 of SEQ ID NO:30, amino acid residues 55-960 of SEQ ID NO:28, or amino acid residues 55-960 of SEQ ID NO:20 (each of these sequences as disclosed in U.S. Patent Appl. Publ. No. 2019 / 0078063, which is incorporated herein by reference). Each of these subsequences are the approximate catalytic domains of each respective reference sequence, and produce alpha-1 , 3-glucan comprising at least about 50% (e.g., >90% or >95%) alpha-1 ,3 linkages. In some aspects, a modified GTF (i) comprises at least one amino acid substitution or a set of amino acid substitutions (as described above), and (ii) comprises or consists of an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:62 or a subsequence thereof such as SEQ ID NO:4 (without start methionine thereof) or positions 55-960 of SEQ ID NO:4 (approximate catalytic domain) (each of these sequences as disclosed in U.S. Patent Appl. Publ. No. 2019 / 0078063).

[0080] In the present disclosure, SEQ ID NOs:5, 6, 7, 8, 9 and 10 (Table A) are the same amino acid sequences as, respectively, SEQ ID NOs:4, 65, 30, 28, 20 and 62 as disclosed in U.S. Patent Appl. Publ. No. 2019 / 0078063. Thus, each of presently disclosed SEQ ID NOs:5, 6, 7, 8, 9 and 10 can be used in any of the disclosed aspects, as appropriate. For example, a GTF enzyme that synthesizes alpha-1 , 3-glucan herein can comprise an amino acid sequence that is about 100% identical to, or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, SEQ ID NO:5, 6, 7, 8, 9, or 10, or amino acid residues 55-960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55-960 of SEQ ID NO:8, or residues 55-960 of SEQ ID NO:9. Any of these sequences can be modified as described herein to affect alpha-1 , 3-glucan yield and / or molecular weight and / or stability, for example.

[0081] In some aspects, a GTF enzyme for alpha-1 , 3-glucan synthesis has been modified such that the enzyme has enhanced performance and / or stability benefit(s). Such a GTF enzyme can be as disclosed, for example, in Int. Patent Appl. Publ. No. W02023 / 055902, which is incorporated herein by reference. Modification of such a GTF can be, for example, by having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid substitutions as compared to a corresponding parent GTF enzyme (e.g., a wild type mature GTF or active subsequence thereof such as a catalytic domain). Exemplary performance and / or stability benefits herein include one or more of increased thermal stability, increased storage stability, increased solubility, better pH profile, increased specific activity, modified substrate specificity, modified substrate binding, modified pH-dependent activity, modified pH-dependent stability, increased oxidative stability, increased expression, and / or increased glucan product yield (and / or decreased byproduct [e.g., leucrose] yield). In some aspects, a performance benefit is realized at a relatively low temperature (e.g., <5 °C) or at a relatively high temperature (e.g., >40 °C). An increase in any of the foregoing features can be by about, or at least about, 5%, 10%, 15%, 20%, 25%, or 30%, for example, as compared to the respective activity of a parent GTF enzyme that has not been modified.

[0082] Some examples of modified alpha-1 , 3-glucan-producing GTF enzymes herein having enhanced performance and / or stability benefit(s) comprise or consist of SEQ ID NO:3 (vGTFJ) or 4. It is noted that SEQ ID NOs:3 and 4 are both derivable from SEQ ID NO:5 (GTF 6855), for example (e.g., SEQ ID NO:5 can be a backbone for making substitutions to render SEQ ID NOs:3 and 4).

[0083] In some aspects of the present disclosure, a modified GTF enzyme can comprise or consist of an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:3, and have one or more of (or all of) the following amino acid residues: 8-Asn, 9-Ala, 336-Tyr, 411 -Leu, 430-Tyr, 448-Ala, 564-Ser, 1254-Gln, and / or 1273-Phe. The valine at position 1 of SEQ ID NO:3 in any of the foregoing aspects can optionally instead be a methionine, or can be deleted.

[0084] In some aspects of the present disclosure, a modified GTF enzyme can comprise or consist of an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:4, and have one or more of (or all of) the following amino acid residues: 336-Tyr, 430-Tyr, 431 -Vai, 448-Ala, 564- Ser, 1011-Glu, 1150-His, 1155-Ala, 1241-Lys, 1242-Glu, 1243-Gly, 1244-Ser, 1247-Leu, and / or 1248-Val. The valine at position 1 of SEQ ID NO:4 in any of the foregoing aspects can optionally instead be a methionine, or can be deleted.

[0085] Although it is believed that a modified alpha-1 , 3-glucan-producing GTF enzyme in some aspects need only have a catalytic domain, the modified GTF can be comprised within a larger amino acid sequence. For example, a catalytic domain may be linked at its C-terminus to a glucan-binding domain, and / or linked at its N-terminus to a variable domain and / or signal peptide.

[0086] Although amino acid substitutions in a modified alpha-1 , 3-glucan-producing GTF enzyme are generally disclosed in some aspects with respect to corresponding positions in SEQ ID NO: 10, such substitutions can alternatively be stated simply with respect to its / their position number in the amino acid sequence used to produce the modified GTF itself (e.g., SEQ ID NO:5 [optionally without start methionine thereof] or positions 55-960 of SEQ ID NO:5 [approximate catalytic domain]), as convenience may dictate. Such can be done simply by aligning the amino acid sequence with SEQ ID NO: 10 and identifying the position number(s) of interest in the amino acid sequence based on its / their direct alignment with the corresponding position(s) in SEQ ID NO: 10.

[0087] An alpha-1 , 3-glucan-producing GTF herein is capable of producing alpha-1 , 3- glucan comprising about, or at least about, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %,

[0088] 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,

[0089] 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% alpha-1 ,3-glycosidic linkages, for example. Alpha-1 ,3-glucan as disclosed elsewhere herein such as in a homopolymer or graft-copolymer can have any of the foregoing linkage profiles, for example.

[0090] An alpha-1 , 3-glucan-producing GTF herein is capable of producing alpha-1 , 3- glucan with a DPw, DPn, or DP of about, less than about, or at least about, 11 , 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, or 1650, for example. DPw, DPn, or DP can optionally be expressed as a range between any two of these values. Merely as examples, the DPw, DPn, or DP can be about 100-1650, 200- 1650, 300-1650, 400-1650, 500-1650, 600-1650, 700-1650, 100-1250, 200-1250, 300- 1250, 400-1250, 500-1250, 600-1250, 700-1250, 100-1000, 200-1000, 300-1000, 400- 1000, 500-1000, 600-1000, 700-1000, 100-900, 200-900, 300-900, 400-900, 500-900, 600-900, 700-900, 11-25, 12-25, 11-22, 12-22, 11-20, 12-20, 20-300, 20-200, 20-150, 20-100, 20-75, 30-300, 30-200, 30-150, 30-100, 30-75, 50-300, 50-200, 50-150, 50-100, 50-75, 75-300, 75-200, 75-150, 75-100, 100-300, 100-200, 100-150, 150-300, 150-200, or 200-300. Alpha-1 ,3-glucan as disclosed elsewhere herein such as in a homopolymer or graft-copolymer can have any of the foregoing molecular weight profiles, for example.

[0091] 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.

[0092] A GTF enzyme herein can typically be derived from bacteria. Examples of bacterial GTF enzymes are those derived from a Streptococcus species, Leuconostoc species, or Lactobacillus species. Examples of Streptococcus species include S. salivarius, S. sobrinus, S. dentirousetti, S. downei, S. mutans, S. oralis, S. gallolyticus and S. sanguinis. Examples of Leuconostoc species include L mesenteroides, L amelibiosum, L argentinum, L carnosum, L citreum, L cremoris, L dextranicum and L fructosum. Examples of Lactobacillus species include L. acidophilus, L. delbrueckii, L. helveticus, L. salivarius, L. casei, L. curvatus, L. plantarum, L. sakei, L. brevis, L. buchneri, L. fermentum and L. reuteri.

[0093] 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.

[0094] Alpha-glucan produced in a food product / precursor in some aspects can comprise a graft copolymer comprising:

[0095] (i) an alpha-1 ,6-glucan (dextran) backbone, wherein at least about 50% of the glycosidic linkages of the alpha-1 ,6-glucan (dextran) backbone are alpha-1 ,6 linkages, and

[0096] (ii) at least one alpha-1 ,3-glucan side chain, wherein at least about 50% of the glycosidic linkages of the alpha-1 , 3-glucan chain are alpha-1 ,3 linkages.

[0097] Such a graft copolymer can be aqueous-soluble or aqueous-insoluble. Dextran backbone of an alpha-glucan graft copolymer herein can be dextran as presently disclosed, for example, or can be as disclosed (e.g., molecular weight, linkage / branching profile, production method) in U.S. Patent Appl. Publ. Nos. 2016 / 0122445, 2017 / 0218093, 2018 / 0282385, 2020 / 0165360, or 2019 / 0185893, which are each incorporated herein by reference. In some aspects, a dextran backbone (before being integrated into a graft copolymer) has been alpha-1 ,2- and / or alpha-1 , 3-branched; the percent alpha-1 ,2 and / or alpha-1 ,3 branching of a backbone of a graft copolymer herein can be about, at least about, or less than about, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 2-25%, 2-20%, 2-15%, 2-10%, 5-25%, 5-20%, 5-15%, 5-10%, 7-13%, 8-12%, 9-11 %, 10-25%, 10-20%, or 10-15%, for example. Alpha-1 ,3- glucan side chain(s) of an alpha-glucan graft copolymer herein can be alpha-1 ,3-glucan as presently disclosed, for example, or can be as disclosed (e.g., molecular weight, linkage profile), in U.S. Patent Nos. 7000000, 8871474, 10301604, or 10260053, or U.S. Patent Appl. Publ. Nos. 2019 / 0112456, 2019 / 0078062, 2019 / 0078063, 2018 / 0340199, 2018 / 0021238, 2018 / 0273731 , 2017 / 0002335, 2015 / 0232819, 2015 / 0064748, 2020 / 0165360, 2020 / 0131281 , or 2019 / 0185893, which are each incorporated herein by reference.

[0098] One, two, three, or more different GTF enzymes that synthesize alpha-1 ,6-glucan herein can be used, for example, in a method as presently disclosed. In some aspects, only an alpha-1 ,6-glucan-producing GTF(s) is used (i.e., an alpha-1 , 3-glucan-producing GTF is not used). One, two, three, or more different GTF enzymes that synthesize alpha-1 , 3-glucan herein can be used, for example, in a method as presently disclosed. In some aspects, only an alpha-1 , 3-glucan-producing GTF(s) is used (i.e., an alpha-1 ,6- glucan-producing GTF is not used). In some aspects, an alpha-1 ,6-glucan-producing GTF(s) can be added to (made to contact) a food product / precursor before adding an alpha-1 , 3-glucan-producing GTF(s), while in some aspects both these types of GTF enzymes can be added at about the same time (simultaneously). Still, in some aspects, an alpha-1 , 3-glucan-producing GTF(s) can be added to a food product / precursor before adding an alpha-1 ,6-glucan-producing GTF(s). Still, in some aspects, a dextran as disclosed herein, but produced exogenously to the food product / precursor, can be added as an ingredient to a food product / precursor to which an alpha-1 , 3-glucan- producing GTF has already been added or will be added. While not being held to any particular theory, it is believed that addition of at least one alpha-1 ,6-glucan-producing GTF (and / or exogenously produced dextran) and at least one alpha-1 , 3-glucan- producing GTF in a method of producing a food product / precursor allows for production of a dextran-alpha-1 ,3-glucan graft copolymer as presently disclosed, possibly along with production of dextran and / or alpha-1 , 3-glucan homopolymer(s) (i.e., alpha-1 ,6- glucan and / or alpha-1 , 3-glucan produced independent from the production of graft copolymer). However, it is believed possible that, in some aspects, only dextran and / or alpha-1 , 3-glucan homopolymer(s) is / are produced with little (e.g., < 5 wt% of all glucan products) or no production of graft copolymer. Still, in some aspects, it is believed possible that alpha-1 , 6-glucan and / or alpha-1 , 3-glucan is / are not produced when using an alpha-1 ,6-glucan-producing GTF(s) and / or an alpha-1 , 3-glucan-producing GTF(s) in a method herein.

[0099] The molecular weight and / or linkage profile of alpha-glucan produced by a GTF enzyme (dextransucrase or alpha-1 , 3-glucan-producing GTF) as generally disclosed above can be as observed, for example, in an isolated reaction consisting of, or essentially of, water, sucrose, GTF enzyme and optionally one or more salts and / or buffer. In some aspects, the molecular weight and / or linkage profile of alpha-glucan as produced by one or both of these types of GTF enzyme in a food product / precursor herein may be different from what is produced in the foregoing isolated reaction.

[0100] The content of at least one of an alpha-1 ,6-glucan-producing GTF and / or alpha- 1 , 3-glucan-producing GTF in a food product / precursor herein can be about, or at least about, 0.1 , 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.75, 1 , 1.25, 1.5, 1.75., 2, 2.5, 0.1-1 , 0.1-0.75, 0.1-0.5, 0.1-0.3, 0.2-1 , 0.2-0.75, 0.2-0.5, 0.2-0.3, 0.5-2.5, 0.5-2, 0.5-1.5, 0.5-1 , 1-2.5, 1- 2, or 1-1.5 wt%, for example. In some aspects, a single GTF is used, whereas two, three, or more GTF enzyme(s) can be used in other aspects. The foregoing enzyme contents can be with respect to one GTF enzyme, or a combination of GTF enzymes. The foregoing GTF enzyme contents are typically with respect to active enzyme(s), but in some aspects can be with respect to total protein of isolated / purified enzyme(s).

[0101] In some aspects, the ratio of a GTF enzyme that synthesizes alpha-1 , 6-glucan to a GTF enzyme that synthesizes alpha-1 , 3-glucan in a method herein is about 85:15 to about 95:5. Yet, in some aspects, an alpha-1 , 6-glucan-producing GTF to alpha-1 , 3- glucan-producing GTF ratio can be about 97.5:2.5, 95:5, 92.5:7.5, 91 :9, 90:10, 89:11 , 87.5:12.5, 85:15, 82.5:17.5, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 5:95, or 2.5:97.5, or range between any two of these ratios (e.g., about 80:20 to 60:40). The amount of each enzyme (active enzyme) for purposes of determining a ratio thereof herein can be on a molar, weight, or GTF activity basis, for example. The activity of a GTF enzyme for preparing a ratio herein can optionally be determined as disclosed in U.S. Patent Appl. Publ. No. 2014 / 0087431 or I nt. Patent Appl. Publ. No. W02023 / 055902, which are incorporated herein by reference. For example, a full (e.g., “100%”) complement of a GTF enzyme for setting up a ratio herein can be that amount of enzyme that can convert most of (e.g., >95%, >98%, >99%), or all of, sucrose in a GTF reaction comprising or consisting of water, sucrose (e.g., 50 or 100 g / L), the GTF, and optionally buffer / salt in a given amount of time (e.g., 6, 12, 18, 24, 30, or 36 hours); such a measured amount can optionally be characterized as a normalized amount of GTF.

[0102] A GTF enzyme (or any other enzyme as presently disclosed) for use in a method herein is typically in purified (isolated) form. A purified enzyme can be essentially free from insoluble and / or soluble components of an organism / cell used to produce the enzyme, and / or any medium that was used for cellular fermentation of the enzyme. In some aspects, a purified enzyme denotes an enzyme preparation that contains less than 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1 % by weight of other material (e.g., polypeptide material) with which the enzyme is natively or recombinantly associated. In some aspects, a GTF and / or any other enzyme herein is not comprised in or otherwise associated with (e.g., expressed by) a microbial (e.g., bacterial, yeast, fungal, algal) cell that might be present (e.g., endogenously or purposely added) in a food product / precursor herein; however, in some aspects a GTF and / or any other enzyme herein is comprised in or otherwise associated with (e.g., expressed by) a microbial (e.g., bacterial, yeast, fungal, algal) cell such as one that heterologously expresses the enzyme(s) (i.e., recombinant cells). Contacting a food product / precursor with a GTF enzyme(s) herein typically is not performed in an oral cavity or other environment in which unpurified / non-isolated GTF enzymes can possibly be present.

[0103] A GTF enzyme (or any other enzyme as presently disclosed) for use in a method herein can be comprised in a sterile-filtered preparation, for example. In some aspects, an enzyme can be sterile-filtered inline while applying the enzyme to a food product / precursor during a contacting step. In some aspects, an enzyme can be added to a food product / precursor that has been pasteurized (after pasteurization), or alternatively an enzyme can be added before pasteurizing the food product / precursor. In some aspects, an enzyme can be added to a food product / precursor that has been fermented (after fermentation), or alternatively an enzyme can be added during or before fermenting the food product / precursor. A GTF enzyme (or any other enzyme as presently disclosed) in some aspects for use in a method herein can be comprised in a preparation that is substantially free of (e.g., <0.5, <0.1 , <0.05 wt%) any other enzyme(s) such as a lipase, protease, amylase, mannanase, pectinase, cellulase, and / or p- nitrobenzylesterase; such a preparation typically has little or no detectable activity(ies) of such other enzyme(s).

[0104] A food product / precursor 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 food product / precursor can be for a time sufficient, for example, for the GTF(s) to produce alpha-glucan in the food product / precursor, 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 food product / precursor herein can be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 2-5, 2-10, 2-15, 2-20, 2-25, 2-30, 2- 35, 2-40, 2-45, 2-50, 3-5, 3-10, 3-15, 3-20, 3-25, 3-30, 3-35, 3-40, 3-45, 3-50, 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 15-20, 15-25, 15-30, 15-35, 15-40, 15-45, 15- 50, 20-25, 20-30, 20-35, 20-40, 20-45, 20-50, 25-30, 25-35, 25-40, 25-45, 25-50, 30-35, 30-40, 30-45, or 30-50 °C, for example.

[0105] Typically, a food product / precursor brought into contact with a GTF enzyme herein contains water (i.e., it is an aqueous composition), and / or water is introduced to the food product / precursor before or during contacting with GTF enzyme. GTF enzyme can be added to a food product / precursor in dry form (e.g., powder, flakes, lyophilized enzyme preparation) (typically to an aqueous food product / precursor) or wet form. In some aspects, a food product / precursor can be combined with a GTF enzyme under dry conditions (resulting combination is dry), after which time water or an aqueous solution is added, which in turn allows GTF production of alpha-glucan to proceed. The water content of a food product / precursor following addition of GTF enzyme, can be about, or at least about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99 wt%, for example. The pH of a food product / precursor herein, and / or the pH for incubating one or more GTF enzymes in a food product / precursor herein, can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,

[0106] 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 4.0-10.0, 4.0-9.0, 4.0-8.0, 4.5-10.0, 4.5-9.0, 4.5-8.0, 5.0-10.0, 5.0-9.0, 5.0-8.0, 5.5-10.0, 5.5-9.0, 5.5-8.0, 6.0-10.0, 6.0-9.0, or 6.0-8.0, for example. A food product / precursor in some aspects can be acidic (e.g., pH < 3.0, 3.2,

[0107] 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 >

[0108] 7.5, 8.0, 8.5, 9.0, 9.5). In some aspects, such as when a milk is used as a food product / precursor herein, the pH of the food / milk is about, or at least about, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0; this pH characterizes the food / milk upon its provision, when it is sweetened, and / or when it is contacted with at least one GTF enzyme, for example. The pH of the food / milk following acidification herein is below 5.0, or is at, or below, 4.8, 4.7,

[0109] 4.6, 4.5, 4.4, or 4.2, for example.

[0110] A GTF enzyme herein 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 food product / precursor, wherein the cell recombinantly (heterologously) expresses and secretes the GTF enzyme in and / or around the food product / precursor. Such a cell can be that of a microbe that is amenable to recombinant engineering and useful in food processing (e.g., fermentation), such as a microbial cell disclosed herein (as applicable). In some aspects, a recombinantly engineered cell that is provided to the food product / precursor can be inactive and / or non-viable in some manner, such as by having been killed (but preferably in a manner that otherwise retains cellular shape / structure). For example, a cell can be rendered inactive and / or non-viable by being irradiated or being treated with a sterilizing agent / chemical (e.g., ethylene oxide). Typically, the means for cell inactivation and / or killing preserves at least some of the three-dimensional shape / structure of the cell, and / or ensures that a GTF enzyme(s) that had been expressed by the cell remains active and typically remains associated with the inactive / non-viable cell (e.g., such as by being associated with a cellular membrane via an optional transmembrane domain or membrane-binding domain of the GTF enzyme [e.g., fused to the GTF]). An inactive / non-viable cell typically is porous, and 1 optionally can be immobilized on a support (e.g., an inert, water-insoluble material, such as of a particle or surface).

[0111] In some aspects, a food product / precursor herein can be brought into contact with one or more GTF enzymes by virtue of adding the food product / precursor to an aqueous composition comprising at least sucrose and the one or more GTF enzymes. While the food product / precursor in this aspect has at least some sucrose and water, a food product / precursor in some other aspects does not comprise sucrose and / or water. Such a GTF / sucrose-containing aqueous composition can optionally be referred to herein as a “GTF / sucrose starter composition”. Typically, one or more food precursors as presently disclosed (e.g., ingredients such as a liquid food product / precursor, beverage, RTD, fruit / vegetable puree, syrup, or juice or juice concentrate) can be added to a GTF / sucrose starter composition, although one or more food products themselves as presently disclosed (e.g., fruit or vegetable matter such as pieces [e.g., slices, cubes, or other shaped pieces]) can be added (typically in conjunction with adding a food precursor). In some aspects, a GTF / sucrose starter composition can already comprise at least one food product / precursor, such as any disclosed herein. The initial sucrose concentration of a GTF / sucrose starter composition can be as presently disclosed, for example, such as 5-60%, 5-50%, 5-40%, 10-60%, 10-50%, 10-40%, 20-60%, 20-50%, 20-40%, 30-60%, 30-50%, 30-40%, 40-60%, or 40-50% by weight. In some aspects, a GTF / sucrose starter composition has few (e.g., less than about 1 , 0.5, 0.1 , 0.05, or 0.01 wt%) or no saccharide compounds (e.g., one or more monosaccharides, disaccharides, oligosaccharides and / or polysaccharides, such as presently disclosed) aside from sucrose. A GTF / sucrose starter composition can comprise at least one alpha-1 , 6- glucan-producing GTF and / or an alpha-1 , 3-glucan-producing GTF as presently disclosed, for example. The temperature, pH and / or any other condition / parameter of this methodology (before and / or after adding one or more food products / precursors to a GTF / sucrose starter composition) can be as disclosed herein, for example. In some aspects, a GTF / sucrose starter composition can be incubated for about, at least about, or up to about, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 120, 180, 240, 300, 360, 420, 480, 540, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 20-45, 20-40, 20-35, 20-30, 20-25, 25-45, 25-40, 25-35, or 25-30 minutes, for example, before adding one or more food products / precursors. Any of these foregoing time periods can also apply to the period of time allowed to proceed after adding the food product / precursor to the GTF / sucrose starter composition, until optionally terminating the GTF activity (e.g., heat-inactivation at 90-100 °C or 70-110 °C) of the final food product / precursor. Typically, the one or more food products / precursors added to the GTF / sucrose starter composition comprises one or more saccharide compounds (e.g., one or more monosaccharides, disaccharides, oligosaccharides and / or polysaccharides, such as presently disclosed). In some aspects, adding one or more food product / precursors to a GTF / sucrose starter composition can be done to control or adjust the degree of thickening and / or texturization desired in the final food product / precursor being produced. The thickening and / or texturization that can be achieved by such methodology can be greater (e.g., about, or at least about, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, or 500% greater) than the thickening and / or texturization that would have been achieved if all the ingredients used to make the final food product / precursor had all been combined at about the same time. Merely as examples, the foregoing process of adding one or more food products / precursors to a GTF / sucrose starter composition can be used herein to produce a marmalade, gel / gelatin, pudding, custard, fermented product, or cream, optionally with one or more suspended solid food ingredients such as fruit or vegetable pieces.

[0112] A “food product / precursor” (i.e. , a food product or precursor) as provided 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. Sucrose can optionally be added to a food product / precursor when adding a GTF(s) to the food product / precursor.

[0113] In some aspects, a food product / precursor as provided for use in a method herein further comprises at least one disaccharide in addition to sucrose, and / or at least one oligosaccharide. An oligosaccharide can have 3-15 or 3-20 monomeric units (i.e., DP3- DP15 or DP3-DP20), for example (e.g., DP3-DP5, DP3-DP6); thus, in some aspects, a polysaccharide herein has more than 15 or 20 monomeric units. A disaccharide and / or oligosaccharide herein can comprise only glucose monomeric units, for example, and / or one or more other types of monosaccharides (e.g., galactose, fructose, mannose) as monomeric units. Examples of disaccharides herein (in addition to sucrose) include maltose, isomaltose, lactose, lactosucrose, nigerose, leucrose, trehalulose, maltulose, isomaltulose, and furanose. Examples of oligosaccharides herein include glucooligosaccharides (gluco-oligomers) such as malto-oligosaccharides (MOS) and isomaltooligosaccharides (IMO), and galacto-oligosaccharides (GOS).

[0114] A disaccharide and / or oligosaccharide can be added to a food product / precursor either during or after preparation of the food product / precursor. Such addition can be from a source physically outside of the food product / precursor (i.e., as an ingredient), and / or can be via in situ production in the food / precursor such as by one or more enzymes that are endogenous and / or exogenous to the food / precursor. An enzyme that is added to a food product / precursor (i.e., exogenous enzyme) for producing a disaccharide and / or oligosaccharide can be added, for example, in the same or similar manner in which a GTF enzyme herein is added (e.g., time, temperature, pH), and can be added before, during, or after the addition of GTF enzyme. Such an enzyme can be a transglucosidase (EC [enzyme code] 2.4.1.24) or a transgalactosylating betagalactosidase. Suitable transglucosidases herein include FoodPro® TGO and those disclosed in U.S. Patent Appl. Publ. Nos. 2008 / 0229514 or 2015 / 0240279, or U.S. Patent No. 4689296, all of which are incorporated herein by reference. An EC 2.4.1.24 transglucosidase (also termed as “1 ,4-alpha-glucan 6-alpha-glucosyltransferase”) can transfer an alpha-D-glucosyl residue of an alpha-1 ,4 -glucan, -oligosaccharide (i.e., MOS), or -disaccharide (i.e., maltose) to the primary hydroxy group of free glucose or glucose in an alpha-1 ,4 -glucan, -oligosaccharide (i.e., MOS), or -disaccharide. Thus, an EC 2.4.1.24 transglucosidase produces isomalto-oligosaccharides (IMO) (e.g., DP3-DP5 or DP3-DP6) in some aspects. 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 galacto-oligosaccharides (GOS) (e.g., GOS can also be an acceptor for forming a longer GOS). A particular example of such an enzyme is Nurica™ (IFF). A transglucosidase or transgalactosylating beta-galactosidase herein can be dosed into a food product / precursor herein at about 0.1 -1.5, 0.1-1.25, 0.1 -1.0, 0.1-0.75, 0.1 -0.5, 0.2- 1.5, 0.2-1.25, 0.2-1.0, 0.2-0.75, 0.2-0.5, 0.5-1.5, 0.5-1.25, 0.5-1.0, 0.5-0.75, 0.75-1.5, 0.75-1.25, or 0.75-1.0 % (v / w), for example. In some aspects, a food product / precursor as provided for use in a method herein has, aside from the sucrose, little (e.g., less than 0.5, 0.25, 0.1 , 0.05, 0.025, or 0.01 wt%, or not detectable) or no disaccharides and / or oligosaccharides (or little or no particular disaccharide or oligosaccharide). A food product / precursor as produced in a step herein of contacting it with one or more GTF enzymes can likewise have, for example, little of no disaccharides and / or oligosaccharides (or little or no particular disaccharide or oligosaccharide), and also have little (e.g., as above) or no sucrose. A disaccharide or oligosaccharide in such aspects can be any as disclosed herein (e.g., lactose, maltose, isomaltose, MOS, IMO, GOS). One or more glycosidase enzymes (glycosidic-active enzyme) can be used, for example, in a food product / precursor to reduce or eliminate the presence of disaccharide(s) and / or oligosaccharide(s), and can be added, for example, in the same or similar manner in which a GTF is added (e.g., time, temperature, pH), and can be added before, during, or after the addition of GTF. A glycosidase herein can be, for example, a beta-galactosidase (EC 3.2.1.23; e.g., lactase [EC 3.2.1 .108]) or alpha-glucosidase (EC 3.2.1.20). Suitable beta-galactosidases herein include Bonlacta™ lactase (IFF) and lactases disclosed in U.S. Patent No. 10531672, which is incorporated herein by reference. A lactase herein is a type of betagalactosidase enzyme that catalyzes hydrolysis of lactose to glucose and galactose. Suitable alpha-glucosidases herein include those disclosed in U.S. Patent Appl. Publ. No. 2015 / 0240278, which is incorporated herein by reference. In some aspects, an alpha-glucosidase that is used in the disclosed method is able to hydrolyze an alpha-1 ,4 or alpha-1 ,6 glucosidic linkage, or and / or is unable to hydrolyze an alpha-1 ,3 glucosidic linkage. A glycosidase herein can be dosed into a food product / precursor herein at about 0.1-1.5, 0.1-1.25, 0.1-1.0, 0.1-0.75, 0.1-0.5, 0.2-1.5, 0.2-1.25, 0.2-1.0, 0.2-0.75, 0.2-0.5, 0.5-1.5, 0.5-1.25, 0.5-1.0, 0.5-0.75, 0.75-1.5, 0.75-1.25, or 0.75-1.0 % (v / w), for example.

[0115] A food product / precursor in some aspects can be a dairy product / precursor, such as a dairy beverage or food. Suitable examples of a dairy product / precursor herein include milk, cheese, yogurt, dessert, cream, and butter. Milk herein can be whole milk (e.g., ~3% fat), ~2% fat milk, ~1% fat milk (“low-fat”), or fat-free (non-fat) milk, for example. Milk, whether used directly as a beverage or as a precursor for preparing a dairy product / precursor herein, can be from a cow, goat, sheep, buffalo, yak, llama, camel, or horse, for example. Milk can optionally be pasteurized before, or after, contacting it with one or more GTF enzymes herein. A cheese herein can be, for example, hard or semi-hard cheese (e.g., Cheddar, mozzarella, Swiss, parmesan, provolone), soft or semi-soft cheese (e.g., ricotta, cottage cheese, feta, American, brie), processed, or non-processed. A yogurt herein can be, for example, whole milk yogurt, low-fat yogurt, fat-free yogurt, or Greek yogurt (e.g., plain, low-fat, non-fat). A yogurt herein can optionally contain fruit and / or be flavored. A yogurt herein can optionally be drinkable (i.e. , yogurt beverage). A dairy dessert in some aspects can be a pudding (e.g., whole milk, 2% milk), frozen yogurt (e.g., low-fat), ice cream (e.g., low-fat), sherbet, milk shake, gelato, or custard; thus, in some aspects a dairy dessert can be a frozen dairy dessert (e.g., ice cream, sherbet, milk shake, frozen yogurt, gelato). Ice cream can be hard ice cream or soft (soft-serve) ice cream, for example. A dairy beverage in some aspects can be milk, chocolate milk, coffee milk, flavored milk, yogurt beverage, kumis, ryazhenka, ayran, lassi, cholado, licuado, or kefir. A dairy cream can be clotted cream (e.g., >55% milkfat), heavy cream (e.g., >36% milkfat), whipping cream (e.g., 30%-36% milkfat), light cream (e.g., 18%-30% milkfat), sour cream (>18% milkfat), half-and-half (e.g., 10.5%-18% milkfat), or ice cream (e.g., >10% milkfat). A dairy product / precursor herein can be lactose-free or have a reduced lactose content, for example. A dairy product / precursor herein can be a fermented dairy product / precursor (e.g., yogurt, buttermilk, creme fraiche, quark, fromage frais, soured milk, vinegar), for example. Some dairy products / precursors herein include dairy confections such as milk chocolate, white chocolate, caramel, and toffee. A dairy product / precursor in some aspects can be any as disclosed in U.S. Patent Appl. Publ. Nos. 2021 / 0282422, 2013 / 0230623, 2005 / 0244541 , 2017 / 0135360, 2009 / 0304864, 2017 / 0094987, or 2003 / 0152685, or U.S. Patent Nos. 5482728 or 6352734, all of which are incorporated herein by reference.

[0116] A food product / precursor in some aspects can be a syrup or beverage, for example, such as any of those disclosed in U.S. Patent Appl. Publ. Nos. 2010 / 0040728, 2017 / 0006902, 2017 / 0218093, 2013 / 0216652, 20180146699, 2009 / 0123603, 2021 / 0076724, or 2017 / 0332670, all of which are incorporated herein by reference. A beverage in some aspects can be a juice (e.g., fruit juice such as orange juice, apple juice, mango juice, peach juice, banana juice, date juice, apricot juice, grapefruit juice, papaya juice, pineapple juice, raspberry juice, strawberry juice, pear juice, tangerine juice, or cherry juice; vegetable juice such as carrot juice, tomato juice, or mixed- vegetable juice), sweetened beverage (soda / soft drink, sweetened tea of coffee), ready- to-drink (RTD), or any other beverage having natural and / or added sugar (sucrose). A food product / precursor in some aspects 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), Hollandaise sauce, hummus, guacamole, pesto sauce, miso, Worcestershire sauce, salad dressing, salsa verde, sesame oil, sour cream, tartar sauce, or teriyaki sauce.

[0117] A food product / precursor in some aspects can be a fermented food product / precursor, for example, such as any of those disclosed in W02002 / 034061. In some aspects, a food product / precursor provided for use in a method herein is fermented, while in some aspects a food product / precursor is fermented (or further fermented) during or after performing a step of contacting with a GTF(s). Fermentation can be a means of acidifying a food product / precursor (e.g., to any acidic pH herein), for example. One or more bacterial and / or yeast cultures can be used for fermentation of a food product / precursor herein. Suitable bacteria for food fermentation herein include lactic acid bacteria, for example, such as Lactobacillaceae family species such as those of the Pediococcus genus (e.g., P. acidilactici, P. pentosaceus), Lactobacillus genus (e.g., L sakei, L fermentum [formerly L cellobiosus], L rhamnosus, L plantarum, L brevus, L kefir, L casei, L paracasei, L acidophilus, L salivarius, L buchneri, L helveticus, L reuteri, L johnsonii, L crispatus, L gasseri, L delbruecki such as subsp. L bulgaricus), Lactococcus genus (e.g., L lactis such as subsp. L cremoris), Leuconostoc genus (e.g., L citreum, L mesenteroides), and Streptococcus genus (e.g., S. thermophilus). Suitable bacteria for food fermentation in some aspects can be species from the Bifidobacterium genus (e.g., B. bifidum, B. longum, B. animalis, B. breve, B. infantis) or Propionibacterium genus (e.g., P. freudenreichii such as subsp. P. shermanif) (propionic acid bacteria). In some aspects, a bacteria for food fermentation herein can be characterized as Gram-positive, sphere-shaped, rod-shaped, anaerobic, aerobic, acid-tolerant, non-sporulating, GRAS (generally regarded as safe), and / or probiotic. A bacteria for food fermentation (e.g., yogurt or other dairy fermentation) in some aspects can be an acidic culture / strain (or mix) (e.g., (YO-MIX 863, YO-MIX 410, or YO-MIX T42, available from IFF) that produces food with a pH of about, for example, 2.5-4.5, 3.0-4.5, 4.2-4.4, or 4.3, or it can be a mild culture / strain (or mix) (e.g., YO-MIX PRIME 900 or YO-MIX M01 , available from IFF) that produces food with a pH of about, for example, 4.6-5.5, 4.6-6.0, 4.5-4.7, or 4.6. A bacteria herein (e.g., mild of acidic) can optionally be mesophilic (temperature for optimal growth typically at 20 to 25 °C [room temperature]). A mix of bacteria in a culture for food fermentation can comprise one, two, three, four, five, six or more different species and / or sub-species of bacteria, for example. Suitable yeast for food fermentation in some aspects include species from the Saccharomyces genus (e.g., S. cerevisiae, S. pastorianus, S. boulardii, S. kluyveri), Pichia genus (e.g., P. kluyveri, P. fermentans), and Candida genus (e.g., C. humilis, C. famata). A yeast in some aspects can be characterized as baker’s (baking) yeast, brewing yeast, wine-making yeast, probiotic yeast, budding / fission yeast, or GRAS. A mix of yeast in a culture for food fermentation can comprise one, two, three, four, five, six or more different species and / or sub-species of yeast, for example. A food product / precursor that is fermented, or will be fermented, can be a 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.

[0118] A food product / precursor in some aspects can be a non-dairy food product / precursor. For example, a non-dairy food product / precursor can be a plantbased milk (milk substitute) or comprise a plant-based milk (and lack, or have little of [e.g., < 0.5 wt%], any dairy ingredients] such as lactose, whey, casein, and / or milk fat). In some aspects, a non-dairy food product / precursor is fermented (e.g., a non-dairy yogurt product / precursor such as a plant-based yogurt product / precursor). Plant-based ingredient(s) forming the basis for a non-dairy food product / precursor herein can be from nuts / seeds (e.g., almonds, cashews, macadamias, hemp seed, quinoa, flax seed), grains / cereal (e.g., oats, rice), fruit (e.g., coconut, banana), or vegetables (e.g., legumes such as beans [e.g., soybeans, mug beans] and peas), for example. In some aspects, a non-dairy food product / precursor is a milk of any of the foregoing nuts / seeds, grains / cereal, fruit, or vegetables; a fermented form of any of these milks can be a yogurt, for example.

[0119] A food product / precursor in some aspects can be a cream soup, gravy, sauce (e.g., tomato sauce), salad dressing, mayonnaise, jam, jelly, marmalade, syrup, pie filling, batter for fried foods, batter for pancakes / waffles, cake icing and glazes, whipped topping, pet food, or animal / livestock feed. A food product / precursor in some aspects can comprise one or more additional ingredients such as a vegetable component (e.g., vegetable oil, vegetable protein, vegetable carbohydrates), enzyme, fat, oil, flavoring agent, microbial culture (e.g., probiotic culture), salt, sweetener, acid (e.g., acetic acid), vinegar, fruit / vegetable (e.g., 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, com syrup, curculin, cyclamate, dextrose, erythritol, fructo-oligosaccharide, fructose (levulose), galactose, glucose (dextrose), glycerol (glycerin), glycyrrhizin, golden syrup, high fructose com syrup (e.g., HFCS-42, - 55, -90), high maltose com syrup (HMCS), honey, hydrogenated starch hydrolysate (HSH), isomalto-oligosaccharide (IMO), inulin, inverted sugar, isomalt, lactitol, lactose, maltitol, maltodextrin, maltose, mannitol, maple syrup, miraculin, molasses (e.g., blackstrap molasses), monatin, monellin, monk fruit, neohesperidin dihydrochalcone, neotame, palm sugar, pentadin, polydextrose, rapadura, refiners syrup, saccharin, sorbitol (glucitol), sorghum syrup, stevia I steviol glycoside (e.g., a rebaudioside such as rebaudioside A, rebaudioside D, or rebaudioside M), sucralose, sugar alcohol, tagatose, thaumatin, trehalose, xylitol, and yacon syrup.

[0120] 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.

[0121] A food product / precursor in some aspects, such as sweetened milk herein, typically before it is contacted with at least one glucosyltransferase, can comprise at least one cellulose-based dispersion ingredient and / or a cellulose-based viscosifying ingredient. Examples of these cellulose-based ingredients can be as disclosed in EP Pat. Publ. No. EP4125399B1 , for example, which is incorporated herein by reference. A cellulose-based dispersion ingredient and / or cellulose-based viscosifying ingredient can be mixed with milk in step (a), and / or mixed with a sweetened milk of step (b), for instance to provide a sweetened milk with either or both these materials. Optionally, the sweetened milk does not comprise, or has very little (e.g., < 0.1 wt%), starch and / or pectin (and / or any other alpha-1 , 4-glucan and / or alpha-1 , 4-1 , 6-glucan).

[0122] Typically, a cellulose-based dispersion ingredient herein can be in the form of a gel, and / or can comprise a microcrystalline cellulose (MCC) dispersion. Such a dispersion typically is aqueous. MCC can be produced by any number of means, such as through partial acid hydrolysis of cellulose. MCC typically lacks amorphous regions present in unmodified cellulose, while retaining the cellulose crystal regions. MCC herein can have a weight-average Mw of about 15-100, 20-100, 15-95, or 20-95 kDa, for example. A commercial source of MCC can be of the AVICEL brand, for example.

[0123] Typically, a cellulose-based viscosifying ingredient can be in the form of an aqueous gum / structurant, and / or can comprise a water-soluble cellulose ether. Such a gum / structurant typically is aqueous. Suitable cellulose ethers herein include carboxymethyl cellulose (CMC) (or any other suitable carboxyalkyl cellulose, e.g.) and methyl cellulose (or any other suitable alkyl cellulose, e.g.), for example. A commercial source of CMC can be of the AVICEL brand, for example.

[0124] In some aspects, a preparation having both MCC and a cellulose ether (e.g., CMC) can be used for preparing sweetened milk. With such a preparation, MCC typically is dispersed in an aqueous solution of the cellulose ether. For example, a preparation can have MCC dispersed in an aqueous solution of CMC; a commercial source of such a preparation can be of the Avicel® GP 2313 brand, for example.

[0125] In some aspects, for example those in which a sweetened milk has been provided with at least one cellulose-based dispersion ingredient and / or a cellulose- based viscosifying ingredient (and / or any other suitable aspect herein, as appropriate, such as a sweetened milk that does not contain a cellulose-based ingredient), a sweetened milk can be pasteurized before it is contacted with at least one glucosyltransferase enzyme herein. Pasteurization of sweetened milk herein can be conducted using any suitable means. For example, pasteurization can be conducted at about 62-64 °C (e.g., ~63 °C) such as for a time of about 25-35 minutes (e.g., ~30 minutes) (i.e. , low-temp, long-time [LTLT] pasteurization), at about 70-75 °C (e.g., ~72 °C) such as for a time of about 10-20 seconds (e.g., -15 seconds) (i.e., high-temp, short-time [HTST] pasteurization), or at about 135-140 °C (e.g., -138 °C) such as for a time of about 1-2 seconds (i.e., ultra-high-temp. [UHT] pasteurization). Also for example, pasteurization can be conducted as disclosed in EP Pat. Publ. No.

[0126] EP4125399B1, U.S. Pat. Appl. Publ. Nos. 20080160149 or 2015 / 0064317, or Int. Pat. Appl. Publ. No. W02023 / 055902, which are each incorporated herein by reference.

[0127] In some aspects, the total level / conc. of a cellulose-based dispersion ingredient and / or cellulose-based viscosifying ingredient in a sweetened milk can be about 0.2-1.0, 0.2-0.6, 0.3-1 .0, 0.3-0.6, or 0.4 wt%. Such a content can be with respect to either of these ingredients, or a combination of these ingredients. This addition is typically done before contacting the sweetened milk with a GTF enzyme, and also typically before applying any pasteurization step ahead of GTF treatment.

[0128] A food product / precursor in some aspects after a step of contacting it with one or more GTF enzyme(s) of a method herein has increased (improved) texture as compared to the food product / precursor as it existed before such contacting. In some aspects, the texture of a food product / precursor after GTF contacting can be increased by about, or at least about, 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 750%, 1000%, 1250%, 1500%, 1750%, 2000%, 2250%, 2500%, 3000%, 3500%, 4000%, 4500%, 5000%, 5500%, 6000%, 6500%, or 7000% as compared to the texture of the food product / precursor as it existed before the GTF contacting. Texture herein can be in terms of viscosity, thickness, structure, or mouthfeel, and / or measured in units of Pascal- seconds (Pa s) or cP, for example. In some aspects, texture (thickness) can be measured by determining food product / precursor viscosity when extracted at a shear rate of about 11 to 12 Hz (e.g., 11 .7 Hz). Texture (mouthfeel) can be measured by determining food product / precursor viscosity when extracted at a shear rate of about 248-250 Hz (e.g., 249 Hz), for example. In some aspects, texture can be estimated visually (e.g., photographically or videographically).

[0129] A food product / precursor in some aspects after a step of contacting it with one or more GTF enzyme(s) of a method herein has an improved physical appearance as compared to the physical appearance of the food product / precursor as it existed before the GTF contacting. Improved physical appearance can be increased homogeneity (e.g., visual homogeneity, and / or little or no syneresis) and / or increased shininess (e.g., visual shininess), for example; such increase(s) can be by about, or at least about, 5%, 10%, 20%, 25%, 30%, 40%, or 50% in some aspects. In some aspects, the dietary fiber content (e.g., weight percent) of a food product / precursor after GTF contacting can be increased by about, or at least about, 5%, 10%, 25%, 50%, 75%, 100%, 200%, 300%, 400%, or 500% as compared to the dietary fiber content of the food product / precursor as it existed before contacting it with one or more GTF enzyme(s) in a method herein.

[0130] A food product / precursor in some aspects after performing a method herein has increased (improved) sucrose level stability. For example, regarding increased sucrose level stability, the sucrose level of a food product / precursor following step (e) of GTF inactivation does not decrease, or decreases by less than about 7%, 5%, 2.5%, or 1 % by weight, as compared to the sucrose level of the product / precursor immediately prior to performing step (e), for at least 5, 10, 15, 21 , 90, or 180 days at a temperature of 15- 37 °C, 15-30°C, or 18-24 °C (e.g., ambient temperature) and typically at a pressure of 1 atm, typically wherein the at least 5, 10, 15, 21 , 90, or 180 days commences upon completion of step (e).

[0131] Some aspects of the present disclosure concern a food product / precursor as produced by a method herein. Examples of such products / precursors are any food product / precursor as disclosed herein. Typically, such a food product / precursor can have any feature as disclosed herein (e.g., increased texture, improved physical appearance, increased dietary fiber, increased sucrose level stability, pH, temperature, age), as appropriate / applicable. Typically, such a food product / precursor comprises an alpha-glucan as presently disclosed, but does not comprise any active GTF enzyme(s).

[0132] In some aspects of producing an acidified milk product, and typically in which a sweetened milk is pasteurized prior to GTF treatment, the resulting acidified milk product exhibits a low level of syneresis. For example, less than about 25%, 20%, 15%, or 10% by volume of the acidified milk product can in the form of liquid that has separated from solids. A low level of syneresis herein can characterize an acidified milk product after at least 5, 10, 15, or 20 days following completion of step (e) of inactivating the GTF, for example. In some aspects, the temperature of the acidified milk product, such as the temperature held over the foregoing time period, can be about 15-23 °C. In some aspects, such a low level of syneresis characterizes an acidified milk product that was made with sweetened milk that further contained a cellulose-based dispersion ingredient and / or cellulose-based viscosifying ingredient as presently disclosed.

[0133] In some aspects of producing an acidified milk product, and typically in which a sweetened milk is pasteurized prior to GTF treatment, and the sweetened milk further contains a cellulose-based dispersion ingredient and / or cellulose-based viscosifying ingredient as presently disclosed, the resulting acidified milk product exhibits a low level of protein instability. For example, an acidified milk product with a low level of protein instability is smooth (silky, creamy) and / or does not have any chalkiness (grittiness, powderiness), both of which oral / taste sensation features typically can be as detected by a sensory / taste evaluation. A low level of protein instability (e.g., at least 50%, 60%, 70%, 80%, 90%, or 95% less protein instability) can be as compared to the protein stability of a control acidified milk product made by the same methodology, with the only difference being that the sweetened milk (for producing the control acidified milk product) did not contain a cellulose-based dispersion ingredient and / or cellulose-based viscosifying ingredient as presently disclosed. Protein instability can occur, for example, due to heating such as from a pasteurization step and / or from inactivating GTF by heat treatment herein (step [e]). A low level of protein instability herein can characterize an acidified milk product after at least 5, 10, 15, or 20 days following completion of step (e) of inactivating the GTF, for example. In some aspects, the temperature of the acidified milk product, such as the temperature held over the foregoing time period, can be about 15-23 °C.

[0134] Non-limiting examples of compositions and methods disclosed herein include: 1. A method of producing an acidified milk product having improved texture, wherein the improved texture comprises increased / improved thickness and / or increased / improved mouthfeel, the method comprising the steps of:

[0135] (a) providing milk,

[0136] (b) adding sucrose to the milk to form sweetened milk (optionally or alternatively, steps [a] and [b] can be combined as a step of providing sweetened milk),

[0137] (c) contacting the sweetened milk with at least one glucosyltransferase to form insoluble alpha-glucan in the milk, thereby providing a milk product having the improved texture (optionally or alternatively, sucrose addition of step [b] can be conducted following adding the at least one glucosyltransferase),

[0138] (d) following step (c), or concurrently with step (c), acidifying the milk product to a pH below about 5.0, and

[0139] (e) inactivating the glucosyltransferase by submitting / subjecting the acidified milk product of step (d) to a temperature of about 70 °C to about 110 °C (or a suitable temperature above 70 °C), thereby producing an acidified milk product having a stable sucrose level and a stable level of the improved texture. 2. The method of embodiment 1 , wherein the milk is a dairy milk or plant-based milk (e.g., any non-dairy milk such as soy milk, rice milk, oat milk, almond milk).

[0140] 3. The method of embodiment 1 or 2, wherein the acidifying of step (d) is performed by microbial fermentation.

[0141] 4. The method of embodiment 3, wherein the microbial fermentation is performed by inoculation and fermentation with a starter culture.

[0142] 5. The method of embodiment 1 , 2, 3, or 4, wherein the acidifying of step (d) is performed concurrently with step (c).

[0143] 6. The method of embodiment 1 , 2, 3, 4, or 5, wherein the acidifying of step (d) is performed by directly adding a suitable acid (e.g., food-safe / food-grade acid such as citric acid) to the milk product.

[0144] 7. The method of embodiment 1 , 2, 3, 4, 5, or 6, wherein the acidifying of step (d) is to a pH of about, or below about, 4.8 (e.g., pH of about, or below about, 4.7, 4.6, 4.5, or 4.4).

[0145] 8. The method of embodiment 1 , 2, 3, 4, 5, 6, or 7, wherein the temperature of step (e) is about 75 °C (e.g., ~80, 85, or 90 °C) to about 110 °C (or a suitable temperature above 75 °C).

[0146] 9. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, or 8, wherein the temperature of step (e) is held for about, or at least about, 5 seconds (e.g., about, or at least about, 10, 15, 20, 25, or 30 seconds).

[0147] 10. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, or 9, further comprising, following step (e), storing the acidified milk product at a temperature of about 15-37 °C (e.g., 15-30 °C, 18-24 °C) (e.g., ambient temperature) and typically at a pressure of about 1 atm (e.g., the storing can be done for at least 5, 10, 15, 21 , 90, or 180 days).

[0148] 11 . The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein the stable sucrose level is stable for at least 5 days (e.g., at least 10, 15, 21 , 90, or 180 days) at a temperature of 15-37 °C (e.g., 15-30°C, 18-24 °C) (e.g., ambient temperature) and typically at a pressure of 1 atm, typically wherein the at least 5 days commences upon completion of step (e).

[0149] 12. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 , wherein the stable sucrose level does not decrease, or decreases by less than about 7% by weight (e.g., less than about 5%, 2.5%, or 1 % by weight), as compared to the sucrose level of the milk product immediately prior to step (e), for at least 5 days (e.g., at least 10, 15, 21 , 90, or 180 days) at a temperature of 15-37 °C (e.g., 15-30°C, 18-24 °C) (e.g., ambient temperature) and typically at a pressure of 1 atm, typically wherein the at least 5 days commences upon completion of step (e).

[0150] 13. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12, wherein the sweetened milk of step (b) comprises about 5-60 wt% sucrose (e.g., about 5-20 or 5-15 wt% sucrose).

[0151] 14. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13, further comprising, after step (b) and before step (c), pasteurizing the sweetened milk.

[0152] 15. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14, wherein the sweetened milk of step (b) comprises a cellulose-based dispersion ingredient (i.e. , a cellulose-based aqueous gel ingredient; e.g., a microcrystalline cellulose aqueous dispersion) and / or a cellulose-based viscosifying ingredient (i.e., a cellulose-based aqueous gum / structurant ingredient; e.g., a cellulose ether [e.g., carboxymethyl cellulose ether] aqueous solution) (e.g., by virtue of the cellulose-based dispersion ingredient and / or the cellulose-based viscosifying ingredient being mixed with the milk in step [a] or being mixed with the sweetened milk of step [b]), optionally wherein the sweetened milk of step (b) has very little (e.g., < 0.1 wt%), or does not comprise, starch and / or pectin (and / or any other alpha-1 ,4-glucan and / or alpha-1 ,4- 1 ,6-glucan), optionally wherein the method further comprises, after step (b) and before step (c), pasteurizing the sweetened milk, and optionally wherein the acidified milk product of step (e) exhibits:

[0153] (i) a low level of syneresis (e.g., less than about 25%, 20%, 15%, or 10% by volume of the acidified milk product is in the form of liquid that has separated from solids) (e.g., wherein the low level of syneresis characterizes the acidified milk product after at least 5, 10, 15, or 20 days following completion of step [e], such as at a temperature of about 15-23 °C, for example),

[0154] (ii) a low level of protein instability (e.g., the acidified milk product is smooth and / or does not have any chalkiness, both of which features typically can be as detected by a sensory evaluation) (protein instability can occur due to heating such as from a pasteurization step or from step [e]) (e.g., wherein the low level of protein instability characterizes the acidified milk product after at least 5, 10, 15, or 20 days following completion of step [e], such as at a temperature of about 15-23 °C, for example).

[0155] 16. The method of embodiment 15, wherein the total level of the cellulose-based dispersion ingredient and / or the cellulose-based viscosifying ingredient in the sweetened milk is about 0.2-1.0 wt% (e.g., -0.2-0.6, -0.3-1.0, -0.3-0.6, or - 0.4 wt%). 17. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16, wherein the acidified milk product of step (e) exhibits a low level of syneresis (e.g., less than about 25%, 20%, 15%, or 10% by volume of the acidified milk product is in the form of liquid that has separated from solids) (e.g., wherein the low level of syneresis characterizes the acidified milk product after at least 5, 10, 15, or 20 days following completion of step [e], such as at a temperature of about 15-23 °C, for example), optionally wherein the sweetened milk of step (b) has very little (e.g., < 0.1 wt%), or does not comprise, starch and / or pectin (and / or any other alpha-1 ,4-glucan and / or alpha-1 ,4- 1 ,6-glucan), and optionally wherein the method further comprises, after step (b) and before step (c), pasteurizing the sweetened milk.

[0156] 18. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, or 17, wherein the at least one glucosyltransferase comprises: (i) a glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1 , 6-glucan are alpha-1 ,6 linkages, and / or (ii) a glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1 , 3-glucan are alpha-1 ,3 linkages.

[0157] 19. The method of embodiment 18, wherein: the glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan comprises an amino acid sequence that is at least 90% (e.g., >95%) identical to SEQ ID NO:1 , 2, 11 , 12, 14, or 15, and / or the glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan comprises an amino acid sequence that is at least 90% (e.g., >95%) identical to residues 55-960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55-960 of SEQ ID NO:8, residues 55-960 of SEQ ID NO:9, or SEQ ID NO:13.

[0158] 20. The method of embodiment 18 or 19, wherein: the glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan comprises an amino acid sequence that is at least 90% (e.g., >95%) identical to SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:13.

[0159] 21 . The method of embodiment 18, 19, or 20, wherein the (ii) glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan is used in the contacting of step (c).

[0160] 22. The method of embodiment 18, 19, or 20, wherein both of the (i) glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan and the (ii) glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan are used in the contacting of step (c). 23. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or 22, wherein the acidified milk product of step (e) is a yogurt product (e.g., regular yogurt or drinkable yogurt).

[0161] 24. A food product produced by the method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 25.

[0162] 25. The method of embodiment 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23, however wherein a suitable food product / precursor as presently disclosed is used in place of, or in addition to, milk, thereby producing an acidified food product / precursor having a stable sucrose level and optionally a stable level of the improved texture (e.g., improved texture is optional for food products in which texture is not a discernible or sought-after feature).

[0163] EXAMPLES

[0164] The present disclosure is further exemplified in the following Examples. It should be understood that these Examples, while indicating certain aspects herein, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of the disclosed embodiments, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt the disclosed embodiments to various uses and conditions. Materials / Methods

[0165] HPLC sugar content analysis

[0166] Sugar composition was measured by high performance liquid chromatography (HPLC) with a Waters® 2695 Serrations 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 an appropriate dilution in water, optionally centrifugation (10 minutes at 15000 rpm), and a sterile filtration. Signals from the HPLC were quantified against calibration standards of sugars eluting at the same time.

[0167] Example 1

[0168] Pasteurization of Milk Base at 95 °C for Six Minutes

[0169] It was investigated whether an amino acid-substituted GTF 6855 variant (SEQ ID NO:3, “vGTFJ” herein) would be inactivated under typical milk base pasteurization conditions of 95 °C for 6 minutes applied on milk base prior to fermentation. Skimmed milk was standardized to 3.9% protein, 4.7% lactose, 1.5% fat and added 0.5% vGTFJ enzyme. The milk base was preheated to 65 °C, homogenized at 65 °C / 200 bar, and then pasteurized at 95 °C for 6 minutes at neutral pH before cooling to 5°C. Sucrose was then dissolved in the milk base at a final concentration of 8%. Two 1-mL aliquots were extracted from the base for which one was stored cold at 5 °C and the other at 30 °C for 4 hours. Ethanol was added to both samples to 30%, and then the samples were heated for 10 minutes at 83 °C for precipitation of polysaccharide and protein; the supernatants were then diluted 10-fold in water before HPLC analysis.

[0170] It was clear that the sucrose had been converted to fructose, leucrose, glucose and oligosaccharides during incubation of both samples (see Table 1). The highest conversion was observed at 30 °C. It is therefore likely not possible to inactivate the added GTF enzyme by pasteurization at 95 °C for 6 minutes at neutral pH in an effort to provide controlled conversion of sucrose.

[0171] Table 1. Soluble carbohydrate profile (% w / w)

[0172] Example 2

[0173] GTF Enzyme Used in Co-Fermentation - GTF Activity Post-Fermentation

[0174] It was confirmed in this Example that the vGTFJ enzyme (SEQ ID NO:3) would not be inactivated by the final fermentation pH of 4.6.

[0175] Skimmed milk was standardized to 1.5% fat, 3.1 % protein, and 6% sucrose, and then homogenized and pasteurized (200 bar 165-95 °C / 6 minutes). The milk base was heated to 43 °C, 20 DCU / kg YO-MIX PRIME 840 and 0.05% vGTFJ (SEQ ID NO:3) were added, and the milk base was then fermented to pH 4.6. After fermentation, the sample was cooled and stored at 5 °C. Aliquots were extracted during storage for up to 3 days. Ethanol was added to each aliquot to 30%, and then the aliquots were heated for 10 minutes at 83 °C for precipitation of polysaccharide and protein. The supernatants were further diluted 10-fold in water before HPLC analysis.

[0176] The soluble carbohydrate profile is presented in Table 2, which clearly shows that the GTF enzyme was not inactivated by the final fermentation pH of 4.6, since there was residual sucrose decrease during storage while fructose content increased. It would likely therefore not be possible to convert only part of the sucrose present in the recipe, as conversion would be expected to continue during shelf life of the product due to residual GTF enzyme activity.

[0177] Table 2. Soluble carbohydrate profile as percent relative to the total soluble carbohydrates

[0178] Example 3

[0179] GTF Inactivation at Low pH Post-Acidification

[0180] It was investigated in this Example whether applying a heat treatment following a chemical acidification would be effective in inactivating the vGTFJ enzyme (SEQ ID NO:3).

[0181] Skimmed milk was standardized to 3.0% fat, 4.3% protein and 10% sucrose. The milk was then homogenized at 62 °C with a pressure of 200 bar. Subsequently, pasteurization was done at 95 °C with a holding time of six minutes. The milk was cooled to 40 °C and split into two different bases.

[0182] For each base, pH was adjusted with citric acid. Base 1 was adjusted to pH 4.3 and base 2 was adjusted to pH 4.6. The two bases were then further split into three different batches for various heat treatments. vGTFJ (SEQ ID NO:3) was added to each batch before heat treatment. Post-acidification heat treatment was done according to Table 3.

[0183] Table 3

[0184] A sample was extracted just after addition of enzyme, before heat treatment.

[0185] Additional samples were extracted after post-acidification heat treatment and over shelf life of ambient stored product. All samples were prepared for HPLC by addition of ethanol to 30% and heating for 10 minutes at 83 °C to precipitate polysaccharide and protein. The supernatants were further diluted 10-fold in water before analyzing sucrose level relative to soluble carbohydrate (see FIG. 1).

[0186] Surprisingly, as shown in FIG. 1 , the sucrose level was stable in each product for at least 72 days of ambient storage (shelf life). It was therefore apparent that the GTF was inactivated by the combination of a pH below 5 combined with a heat treatment (see Table 3).

[0187] Example 4

[0188] GTF Inactivation at Low pH Post-Fermentation for Drinking Yogurt

[0189] Considering the results in Example 3, it was further investigated in this Example whether applying a heat treatment post-acidification by fermentation would be effective to inactivate GTF enzyme.

[0190] A recombined milk recipe, based on water and skimmed milk powder, was standardized to 4.0% protein. Additionally, 10% sucrose was added to the milk base. Pasteurization was done at 95 °C with a holding time of 10 minutes. The base was cooled to 43 °C, which corresponded to the fermentation temperature.

[0191] YO-MIX™ 505 (20 DCU / 100 L) and vGTFJ (SEQ ID NO:3) were added simultaneously to the pasteurized milk base, which was fermented to pH 4.6. The fermented milk base (now yogurt base) was mixed 50:50 with water. Homogenization was then performed at 60 °C with a pressure of 300 bar, followed by heat treatment at 75 °C with a holding time of 25 seconds. The fermented yogurt / water recipe was cooled to 10 °C and filled into 250-mL bottles. After filling, the bottles were stored at ambient temperature. Samples were extracted over shelf life and prepared for HPLC by addition of ethanol to 30% and heating for 10 minutes at 83 °C for precipitation of polysaccharide and protein. The supernatants were further diluted 10-fold in water before being analyzed for individual carbohydrate level relative to soluble carbohydrate (see FIG. 2).

[0192] It was found that the residual sucrose (-2.4%) was stable in the product and hence there were no increase in viscosity over shelf life. This underlines that GTF enzyme can be inactivated if applying heat treatment after acidification to a pH below 5.

[0193] Example 5

[0194] GTF Inactivation for Stabilization of Ambient-Stored Long Shelf-Life Drinking Yogurt Considering the results in Example 4, it was further investigated in this Example whether applying a heat treatment post-acidification by fermentation would be effective to inactivate GTF enzyme while also providing efficient stabilization of the milk proteins. Also studied in this Example was whether GTF enzyme could replace and / or supplement the effects of using a cellulose-based stabilizer ingredient. It has been described that if applying an Avicel® stabilizer of cellulose gel and cellulose gum (Avicel® GP 2313, which is a mixture of microcrystalline cellulose [MCC] that forms a gel [dispersion], and carboxymethyl cellulose [CMC] that forms a gum [viscous solution]), then the resulting product can be unstable and show phase separation with a top watery phase and a bottom white proteinaceous phase (EP4125399B1 , incorporated herein by reference). Given this past result, it was determined herein whether treatment of milk with an alpha-1 , 3-glucan-producing GTF could serve to replace using this Avicel® stabilizer, and / or rescue the stabilizing effect of the Avicel® stabilizer. In particular, it was studied if using a GTF could eliminate phase separation brought on by Avicel® stabilizer, and / or provide protein protection / stabilization that can occur in ambient-stable yogurt (pH 4.2-4.6) made at higher thermization temperatures. Increased protein stabilization could provide products that avoid problems of chalkiness and low smoothness, which are hallmarks of protein instability (EP4125399B1).

[0195] A recombined milk recipe, standardized to 3.5 wt% protein and 1 .5 wt% fat, was prepared with the ingredients listed in Table 4. Homogenization was then performed at 65 °C at a pressure of 200 bar, followed by heat treatment at 95 °C with a holding time of 6 minutes. Fermentation of the base was done at 43 °C with YO-MIX™ 883 (40 DCU / 100 L) and vGTFJ (SEQ ID NO:3) or a blend of vGTFJ (SEQ ID NO:3) and FoodPro I invertase (IFF), that was added simultaneously to the pasteurized milk base, which was fermented to pH 4.4-4.5.

[0196] Table 4. Overview of ingredients (wt%) used in Trials 1-5 Homogenization was then performed at 42 °C at a pressure of 100 bar, followed by heat treatment at 85 °C with a holding time of 25 seconds. Each fermented yogurt was cooled to 20 °C and filled into 250-mL bottles. After filling, the bottles were stored at ambient temperature (20 °C).

[0197] Samples were extracted shortly after yogurt production and again after 19 days of yogurt storage, and prepared for HPLC by addition of ethanol to 30% and heating for 10 minutes at 83 °C for precipitation of polysaccharide and protein. The supernatants were further diluted 10-fold in water before being analyzed for individual carbohydrate level relative to soluble carbohydrate.

[0198] The resulting distribution in each Trial product of carbohydrates relative to total soluble carbohydrates is presented in Table 5. The results confirmed the enzymedependent conversion of sucrose into poly- and oligo-saccharides, nigerose, leucrose, glucose and fructose in Trials 3-5. Furthermore, the carbohydrate profile was stable over the 19 days of storage.

[0199] Table 5. Relative carbohydrate distribution profile in drinking yogurt at day 0 and day 19

[0200] A picture was taken of all Trial 1-5 products at day 19 (FIG. 3). It was apparent that Trial 2 (which only included Avicel® stabilizer, without enzyme or other polymer) had severe syneresis, whereas Trials 3-5 (included at least a GTF) were highly similar to the Trial 1 reference with only a thin top watery layer. Replacing Avicel® stabilizer with the GTF (Trial 3) showed the ability of the GTF to prevent syneresis, without the need of any polymer ingredients (starch, pectin). Including both Avicel® stabilizer and the GTF (Trials 4-5) also showed prevention of syneresis (FIG. 3).

[0201] To evaluate protein protection in the various trials, a sensory evaluation of the smoothness / chalkiness of each product was performed at day 19. It was clear that the Trial 3 sample had a very high level of chalkiness and would not be characterized as smooth. However, the products of T rials 4 and 5 were very similar to the T rial 1 reference, having high smoothness and low to no chalkiness. It was therefore surprisingly found that that the combination of Avicel® stabilizer of cellulose gel and cellulose gum and vGTFJ provides excellent protein protection and stabilization in ambient-stored yogurt made under the disclosed conditions.

[0202] To verify the complete inactivation of enzymes (Trials 3-5), additional sucrose was mixed into samples of each of Trials 1-5 products (day 19) in duplicate to a final concentration of 2% (w / w). One set was then immediately heat-treated at 95 °C for 10 minutes, while the other set was incubated at 30 °C for 3 hours before being heat- treated at 95 °C for 10 minutes. All samples were prepared for HPLC analysis to evaluate whether any residual enzyme activity resided that could convert the added sucrose during the 3-hour incubation. The results of this study are presented in FIG. 4 and confirm that no residual enzyme activity was present in any of the samples, as there was no decline in sucrose over the 3 hour incubation at 30 °C.

Claims

CLAIMSWhat is claimed is:

1. A method of producing an acidified milk product having improved texture, wherein said improved texture comprises increased thickness and / or improved mouthfeel, said method comprising the steps of:(a) providing milk,(b) adding sucrose to the milk to form sweetened milk,(c) contacting said sweetened milk with at least one glucosyltransferase to form insoluble alpha-glucan in the milk, thereby providing a milk product having said improved texture,(d) following step (c), or concurrently with step (c), acidifying the milk product to a pH below about 5.0, and(e) inactivating the glucosyltransferase by subjecting the acidified milk product of step (d) to a temperature of about 70 °C to about 110 °C, thereby producing an acidified milk product having a stable sucrose level and a stable level of said improved texture.

2. The method of claim 1 , wherein the milk is a dairy milk or plant-based milk.

3. The method of claim 1 , wherein the acidifying of step (d) is performed by microbial fermentation.

4. The method of claim 3, wherein the microbial fermentation is performed by inoculation and fermentation with a starter culture.

5. The method of claim 3, wherein the acidifying of step (d) is performed concurrently with step (c).

6. The method of claim 1 , wherein the acidifying of step (d) is performed by directly adding a suitable acid to the milk product.

7. The method of claim 1 , wherein the acidifying of step (d) is to a pH of about, or below about, 4.8.

8. The method of claim 1 , wherein said temperature of step (e) is about 75 °C to about 110 °C.

9. The method of claim 1 , wherein said temperature of step (e) is held for about, or at least about, 5 seconds.

10. The method of claim 1 , further comprising, following step (e), storing the acidified milk product at a temperature of about 15-37 °C and typically at a pressure of about 1 atm.11 . The method of claim 1 , wherein said stable sucrose level is stable for at least 5 days at a temperature of 15-37 °C and typically at a pressure of 1 atm.

12. The method of claim 1 , wherein said stable sucrose level does not decrease, or decreases by less than about 7% by weight, as compared to the sucrose level of the milk product immediately prior to step (e), for at least 5 days at a temperature of 15-37 °C and typically at a pressure of 1 atm.

13. The method of claim 1 , wherein the sweetened milk of step (b) comprises about 5-60 wt% sucrose.

14. The method of claim 1 , further comprising, after step (b) and before step (c), pasteurizing the sweetened milk.

15. The method of claim 1 , wherein the sweetened milk of step (b) comprises a cellulose-based dispersion ingredient and / or a cellulose-based viscosifying ingredient, optionally wherein the sweetened milk of step (b) has very little, or does not comprise, starch and / or pectin, optionally wherein the method further comprises, after step (b) and before step (c), pasteurizing the sweetened milk, and optionally wherein the acidified milk product of step (e) exhibits:(i) a low level of syneresis,(ii) a low level of protein instability.

16. The method of claim 15, wherein the total level of the cellulose-based dispersion ingredient and / or the cellulose-based viscosifying ingredient in the sweetened milk is about 0.2-1.0 wt%.

17. The method of claim 1 , wherein the acidified milk product of step (e) exhibits a low level of syneresis, optionally wherein the sweetened milk of step (b) has very little, or does not comprise, starch and / or pectin, and optionally wherein the method further comprises, after step (b) and before step (c), pasteurizing the sweetened milk.

18. The method of claim 1 , wherein said at least one glucosyltransferase comprises:(i) a glucosyltransferase enzyme that synthesizes alpha-1 ,6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1 ,6-glucan are alpha- 1 ,6 linkages, and / or(ii) a glucosyltransferase enzyme that synthesizes alpha-1 ,3-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1 ,3-glucan are alpha- 1 ,3 linkages.

19. The method of claim 18, wherein: said glucosyltransferase enzyme that synthesizes alpha-1 , 6-glucan comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1 , 2, 11 , 12, 14, or 15, and / or said glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan comprises an amino acid sequence that is at least 90% identical to residues 55- 960 of SEQ ID NO:5, residues 54-957 of SEQ ID NO:6, residues 55-960 of SEQ ID NO:7, residues 55-960 of SEQ ID NO:8, residues 55-960 of SEQ ID NO:9, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:13.

20. The method of claim 18, wherein said (ii) glucosyltransferase enzyme that synthesizes alpha-1 , 3-glucan is used in said contacting of step (c).21 . The method of claim 18, wherein both of said (i) glucosyltransferase enzyme that synthesizes alpha-1 ,6-glucan and said (ii) glucosyltransferase enzyme that synthesizes alpha-1 ,3-glucan are used in said contacting of step (c).

22. The method of claim 1 , wherein the acidified milk product of step (e) is a yogurt product.

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

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