Production methods of a food product / precursor and food product / precursor

Description

1 / 133 “PRODUCTION METHODS OF A FOOD PRODUCT / PRECURSOR AND FOOD PRODUCT / PRECURSOR”

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 492,918 (filed March 29, 2023), which is incorporated herein by reference in its entirety. FIELD

[0002] This disclosure is situated in the field of polysaccharides and food products. For example, the disclosure relates to the in situ production of alpha-glucan in food products and food precursors. REFERENCE TO THE LIST OF SEQUENCES SUBMITTED ELECTRONICALLY

[0003] The official copy of the sequence listing is submitted electronically via EFS-Web as a file named NB42205WOPCT_SequenceListing.xml, created on March 25, 2024, and which is approximately 36 kilobytes in size and is deposited simultaneously with the descriptive report. The sequence listing contained in this file forms part of the descriptive report and is incorporated herein by reference in its entirety. BACKGROUND

[0004] Texture is a key parameter of quality and value for a variety of food products. Currently, stabilizers such as starch, carboxymethylcellulose (CMC), cellulose, guar gum, pectin, xanthan gum, and several others are common additives in food products to improve texture. Texture is related to the consumer's eating sensation and has a strong impact on consumer perception.

[0005] One disadvantage of added stabilizers is that they can increase the total carbohydrate level and calorie content of foods and often require special handling during processing. Petition 870250087473, dated 09 / 26 / 2025, pp. 143 / 302 2 / 133 Plant-based or non-dairy protein food alternatives, such as soy, almond, pea, bean, rice, or oat milks or fresh fermented products, are one of the fastest-growing segments across all food product categories worldwide (2016, Mäkinen et al., Crit. Rev. Food Sci. Nutr. 56: 339-349; Sethi et al., 2016, J. Food Science Technol.). However, most plant-based proteins lack functional balance (Sethi et al., ibid.). Consequently, most plant-based and dairy-free products contain additives such as emulsifiers, thickening agents, and other stabilizers, which contradict the pursuit of a clean label (2017, Asioli et al., Food Res. Int. 99: 58-71; Mäkinen et al., ibid.).

[0006] As an example of issues that can be raised by the addition of stabilizers, fresh fermented products containing added starch may require special handling during processing so as not to lose the texture created by the starch through shear forces. Furthermore, it is well known that added starch can negatively impact products in several ways. First, starch diminishes the “brightness” of yogurt or plant-based fresh fermented products, negatively impacting the consumer's visual perception. Additionally, added starch often leads to undesirable sensory dryness in yogurt. SUMMARY

[0007] In one embodiment, the present disclosure relates to a method of producing a food product / precursor, wherein the method comprises: (a) providing a food product / precursor comprising at least water and sucrose, and (b) bringing the food product / precursor into contact with at least one glucosyltransferase enzyme and a second enzyme that uses sucrose as a substrate, wherein the glucosyltransferase enzyme is: (i) a glucosyltransferase enzyme that synthesizes alpha-1,6-glucan, wherein Petition 870250087473, dated 09 / 26 / 2025, pp. 144 / 302 3 / 133 less about 50% of the glycosidic linkages of 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 alpha-1,3-glucan are alpha-1,3 linkages, typically wherein at least one alpha-glucan is produced in the food product / precursor, wherein the food product / precursor, after step (b), optionally possesses one or more of the following characteristics compared with the food product / precursor before step (b): (I) increased texture, but wherein the increased texture is less than it would be if the second enzyme were not present in the food product / precursor, (II) reduced sugar content, but wherein the reduction in sugar content is less than it would be if the second enzyme were not present in the food product / precursor food, typically where the sugar content comprises monosaccharides and disaccharides from the food product / precursor,and / or (III) reduced sweetness, but where the reduced sweetness is less than it would be if the second enzyme were not present in the food product / precursor. In some alternative embodiments in this document, the provision of the second enzyme that uses sucrose as a substrate is optional.

[0008] In another embodiment, the present disclosure relates to a method of producing a food product / precursor, wherein the method comprises: (a) providing a food product / precursor comprising at least water, sucrose and at least one carbohydrate-containing ingredient, and (b) bringing the food product / precursor into contact with at least one glucosyltransferase enzyme, wherein the glucosyltransferase enzyme is: (i) a glucosyltransferase enzyme that synthesizes alpha-1,6-glucan, wherein at least about 50% of the glycosidic linkages of 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 alpha-1,3-glucan are alpha-1,3 linkages, typically wherein at least one alpha-glucan is produced Petition 870250087473, dated 09 / 26 / 2025, page 145 / 302 4 / 133 in the food product / precursor, wherein the carbohydrate-containing ingredient comprises at least one acceptor molecule for at least one glucosyltransferase enzyme, whereby the food product / precursor, after step (b), optionally has one or more of the following characteristics compared with the food product / precursor before step (b): (I) increased texture, but wherein the increased texture is less than it would be if the carbohydrate-containing ingredient were not present in the food product / precursor, (II) reduced sugar content, but wherein the reduction in sugar content is less than it would be if the carbohydrate-containing ingredient were not present in the food product / precursor, typically wherein the sugar content comprises monosaccharides and disaccharides of the food product / precursor, and / or (III) reduced sweetness,but where the reduced sweetness is less than it would be if the carbohydrate-containing ingredient were not present in the food product / precursor.

[0009] In another embodiment, the present disclosure relates to a food product or food precursor produced by a method in this document. BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES

[0010] FIG. 1: Sugar content in samples of YO-MIX 410 yogurt. See Example 1.

[0011] FIG. 2: The apparent viscosity of YOMIX 410 yogurt samples extracted at a shear rate of 11.7 Hz, which provides a measurement of the product's consistency. See Example 1.

[0012] FIG. 3: The apparent viscosity of YOMIX 410 yogurt samples extracted at a shear rate of 249 Hz, which provides a measurement of the product's mouthfeel. See Example 1.

[0013] FIG. 4: Flow curves resulting from rotational rheological tests are shown. See Example 2. Petition 870250087473, dated 09 / 26 / 2025, pp. 146 / 302 5 / 133

[0014] FIG. 5: Sugar content of YO-MIX PRIME 900, YO-MIX 410, YO-MIX T42, YO-MIX M01 and YO-MIX 863 yogurt samples treated with various GTF enzyme regimes. See Example 2.

[0015] FIG. 6A: Radar chart of sensory data measured for YO-MIX 863 yogurt samples with 3% starch or treatment with vGTFJ only (0:100% (GTF 0768:vGTFJ)) or both vGTFJ and GTF 0768 (90:10% (GTF 0768:vGTFJ)). See Example 2.

[0016] FIG. 6B: Radar chart of sensory data measured for samples of YO-MIX PRIME 900 yogurt with 3% starch or treatment with vGTFJ only (0:100% (GTF 0768:vGTFJ)) or both vGTFJ and GTF 0768 (90:10% (GTF 0768:vGTFJ)). See Example 2.

[0017] FIG. 6C: Radar chart of sensory data measured for YO-MIX 410 yogurt samples with 3% starch or treatment with vGTFJ only (0:100% (GTF 0768:vGTFJ)) or both vGTFJ and GTF 0768 (90:10% (GTF 0768:vGTFJ)). See Example 2.

[0018] FIG. 6D: Radar plot of sensory data measured for YO-MIX T42 yogurt samples with 3% starch or treatment with vGTFJ only (0:100% (GTF 0768:vGTFJ)) or both vGTFJ and GTF 0768 (90:10% (GTF 0768:vGTFJ)). See Example 2.

[0019] FIG. 6E: Radar chart of sensory data measured for YO-MIX M01 yogurt samples with 3% starch or treatment with vGTFJ only (0:100% (GTF 0768:vGTFJ)) or both vGTFJ and GTF 0768 (90:10% (GTF 0768:vGTFJ)). See Example 2.

[0020] FIG. 7: The apparent sweetness of YO-MIX 863, YO-MIX PRIME 900, YO-MIX 410, YO-MIX T42 and YO-MIX M01 yogurt samples treated with vGTFJ only (0:100% (GTF 0768:vGTFJ)) or both vGTFJ and GTF 0768 (90:10% (GTF 0768:vGTFJ)), relative to the 3% starch samples (not treated with enzyme). See Example 2. Petition 870250087473, dated 09 / 26 / 2025, pp. 147 / 302 6 / 133

[0021] FIG 8.: Sugar content in YO-MIX 410 yogurts treated with glucosyltransferase enzymes (vGTFJ, GTF 0768) and / or lactase / transgalactosidase enzymes. See Example 3.

[0022] FIG. 9: The apparent viscosity of YOMIX 410 yogurt samples treated with glucosyltransferase enzymes (vGTFJ, GTF 0768) and / or lactase / transgalactosidase enzymes, as measured by extraction at a shear rate of 11.7 Hz, which provides a measurement of the product consistency. See Example 3.

[0023] FIG. 10: The apparent viscosity of YOMIX 410 yogurt samples treated with glucosyltransferase enzymes (vGTFJ, GTF 0768) and / or lactase / transgalactosidase enzymes, as measured by extraction at a shear rate of 249 Hz, which provides a mouthfeel measurement. See Example 3.

[0024] FIG. 11: Sugar content of AE substrates with or without treatment with GTF 0974, GTF 0768 or v2GTFJ. See Example 4.

[0025] FIG. 12: For each of the AE substrates with or without treatment with GTF 0974, GTF 0768, or v2GTFJ, the height of the bar indicates the percentage reduction in sucrose compared to the reference substrate as initially prepared (e.g., before incubation with the NURICA enzyme to form substrate C, and before incubation with the TGO enzyme to form substrate E). The interior of each bar shows the relative content of oligosaccharides and polysaccharides (based on dry weight) as a percentage of the total oligosaccharide and polysaccharide content. See Example 4.

[0026] FIG. 13: The apparent viscosity of AE substrates with or without treatment with GTF 0974, GTF 0768 or v2GTFJ, as measured by extraction at a shear rate of 11.7 Hz, which provides a measurement of the product consistency. See Example 4. Petition 870250087473, dated 09 / 26 / 2025, pages 148 / 302 7 / 133

[0027] FIG. 14: The apparent viscosity of AE substrates with or without treatment with GTF 0974, GTF 0768 or v2GTFJ, as measured by extraction at a shear rate of 249 Hz, which provides a mouthfeel measurement. See Example 4.

[0028] FIG. 15A: Neutral beverages initially prepared without maltose and then treated with GTF 0768, vGTFJ or combinations thereof. For each resulting product, the height of the bar indicates the percentage reduction in sucrose compared to the reference (without GTF treatment) (the reference is at 0%, therefore no bar). The interior of each bar shows the relative content of oligosaccharides and polysaccharides (based on dry weight) as a percentage of the total content of oligosaccharides and polysaccharides formed in each GTF-treated product. See Example 5.

[0029] FIG. 15B: Neutral beverages initially prepared with maltose and then treated with GTF 0768, vGTFJ or combinations thereof. For each resulting product, the height of the bar indicates the percentage reduction in sucrose compared to the reference (without GTF treatment) (the reference is at 0%, therefore no bar). The interior of each bar shows the relative content of oligosaccharides and polysaccharides (based on dry weight) as a percentage of the total content of oligosaccharides and polysaccharides formed in each GTF-treated product. See Example 5.

[0030] FIG. 16: The apparent viscosity of neutral beverages, initially with or without maltose before treatment with GTF, resulting from treatment with GTF 0768, vGTFJ or a combination thereof, as measured by extraction at a shear rate of 11.7 Hz, which provides a measurement of the product consistency. See Example 5.

[0031] FIG. 17: The apparent viscosity of neutral beverages, initially with or without maltose before treatment with GTF, resulting from treatment with GTF 0768, vGTFJ or a combination thereof, according to Petition 870250087473, dated 09 / 26 / 2025, pp. 149 / 302 8 / 133 measured by extraction at a shear rate of 249 Hz, which provides a measurement of mouth feel. See Example 5.

[0032] FIG. 18: Viscosity of yogurt products over time (at 1, 7, 21, 35 or 63 days; bars shown in this order for each data set). See Example 6.

[0033] FIG. 19: Sugar content of yogurt products over time (at 0, 14, 28, 42 or 56 days). See Example 6.

[0034] FIG. 20: Melting profiles of ice cream samples. See Example 7.

[0035] FIG. 21A: Texture attributes (hardness (FIG. 21A)) of ice cream samples. See Example 7.

[0036] FIG. 21B: Texture attributes (cohesiveness (FIG. 21B)) of ice cream samples. See Example 7.

[0037] FIG. 21C: Texture attributes (stickiness (FIG. 21C)) of ice cream samples. See Example 7.

[0038] FIG. 22: Extensional rheometry (Hencky strain vs. time, at a rate of 1 mm / s) of sweet condensed milk samples. See Example 8.

[0039] FIG. 23: Treatment of skim milk with invertase alone (top row) or invertase plus vGTFJ (bottom row). See Example 10.

[0040] FIG. 24: Treatment of yogurt with invertase alone (bottom row) or invertase plus vGTFJ (top row). See Example 10.

[0041] FIG. 25: Samples of ketchup prepared, in part, using a mixture of GTF 0768 and vGTFJ enzymes. See Example 12.

[0042] FIG. 26: Samples of ketchup prepared, in part, using mixtures of GTF 0768 and vGTFJ enzymes. See Example 13. Petition 870250087473, dated 09 / 26 / 2025, pp. 150 / 302 9 / 133 Table 1. Summary of protein SEQ ID numbers Description SEQ ID NO. of GTF 0768 protein. Mature form of the Leuconostoc pseudomesenteroides protein, but with two additional N-terminal amino acids. 1 (1449 aa) GTF 0768. Mature wild-type form of the L. pseudomesenteroides protein (US2016 / 0122445). 2 (1447 aa) vGTFJ. Variant of GTF 6855 (SEQ ID NO: 5 below). 3 (1341 aa) Variant of GTF 6855 (SEQ ID NO: 5 below). 4 (1341 aa) GTF 6855, derivable from Streptococcus salivarius SK126. The first 178 amino acids of the protein are deleted compared to the accession no. in GENBANK ZP_04061500.1; an initial methionine is included. 5 (1341 aa) GTF 7527, derivable from Streptococcus salivarius. The first 178 amino acids of the protein are deleted compared to the GENBANK accession number CAA77900.1; an initial methionine is included. 6 (1341 aa) GTF 2678, derivable from Streptococcus salivarius K12. The first 188 amino acids of the protein are deleted compared to the GENBANK accession number EJO16940.1; an initial methionine is included. 7 (1341 aa) GTF 2919, derivable from Streptococcus salivarius PS4. The first 92 amino acids of the protein are deleted compared to the GENBANK accession number EIC80898.1; an initial methionine is included. 8 (1340 aa) GTF 2765, derivable from Streptococcus sp. C150 unknown. The first 193 amino acids of the protein are deleted compared to the GENBANK accession number ZP_08047301.1; an initial methionine is included. 9 (1340 aa) Wild-type GTF corresponding to GTF 6855, Streptococcus salivarius SK126 (GENBANK accession number ZP 04061500.1). 10 (1518 aa) GTF 0768. N-terminus truncated form A of the mature form of the L. pseudomesenteroides protein (SEQ ID NO: 2). The first 24 amino acids of the protein are deleted compared to SEQ ID NO: 2. 11 (1423 aa) GTF 0768. N-terminus truncated form B of the mature form of the L. pseudomesenteroides protein (SEQ ID NO: 2).The first 72 amino acids of the protein are deleted compared to SEQ ID NO: 2. 12 (1375 aa) GTF 0974, derivable from Streptococcus salivarius 57.I. The first 187 amino acids of the protein are deleted compared to the accession no. in GENBANK AEJ53088.1. 13 (1392 aa) Invertase, derivable from Saccharomyces cerevisiae. Mature sequence. 14 (513 aa). DETAILED DESCRIPTION

[0043] Disclosures from all patent and non-patent literature cited are incorporated herein by reference in their entirety.

[0044] Unless otherwise stated, the terms “a”, “an”, “the” and “the”, as used in this document, are intended to encompass one or more (i.e., at least one) of the features mentioned. Petition 870250087473, dated 09 / 26 / 2025, pp. 151 / 302 10 / 133

[0045] When present, all tracks are inclusive and combinable, unless otherwise indicated. For example, when a track “1 to 5” is mentioned (i.e., 1-5), the mentioned track should be interpreted as including tracks “1 to 4”, “1 to 3”, “1-2”, “1-2 and 4-5”, “1-3 and 5” and similar tracks.

[0046] The terms “alpha-glucan”, “alpha-glucan polymer” and the like are used interchangeably in this document. An alpha-glucan is a polymer comprising glucose monomeric units linked together by alpha-glycosidic linkages. In typical embodiments, an alpha-glucan in this document 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 in this document include graft copolymers as currently disclosed, as well as alpha-1,3-glucan and alpha-1,6-glucan.

[0047] The terms “alpha-1,3-glucan”, “poly-alpha-1,3-glucan”, “alpha-1,3-glucan polymer” and the like are used interchangeably in this document. 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 other linkages in alpha-1,3-glucan in this document are typically alpha-1,6, although some linkages may also be alpha-1,2 and / or alpha-1,4. Alpha-1,3-glucan, as presently disclosed, may feature an alpha-1,3-glucan side chain in this document. In some respects, alpha-1,3-glucan can be characterized as an alpha-1,3-glucan "homopolymer," which is alpha-1,3-glucan that is not part of a dextran-alpha-1,3-glucan copolymer.

[0048] The terms “dextran”, “dextran polymer”, “molecule of Petition 870250087473, dated 09 / 26 / 2025, pp. 152 / 302 The terms “11 / 133 dextran”, “alpha-1,6-glucan” and similar terms refer in this document to a water-soluble alpha-glucan comprising at least 50%, 60%, 70%, 80% or 90% alpha-1,6-glycosidic linkages (the remainder of the linkages typically being alpha-1,3). Enzymes capable of synthesizing dextran from sucrose may be described as “dextranassacarases” (EC 2.4.1.5). A “substantially linear” dextran (“primarily linear” and similar terms) has 5% or less branching before being modified in this document to have alpha-1,3-glucan side chains. A “linear” dextran has no branching before being modified in this document to have alpha-1,3-glucan side chains. Branching, if present before the modification of the Dextran with alpha-1,3-glucan side chains can be short, having one (pendulous) monomer to three glucose monomers in length.However, in some respects, dextran can be “dendritic,” which is a branched structure emanating from a core in which there are chains (containing mainly or only alpha-1,6 linkages) that iteratively branch off from one another (e.g., one chain can be a branch of another chain, which in turn is a branch of another chain, and so on). However, in still some respects, dextran is not dendritic but has a branching-on-branching structure that does not emanate from a core. Dextran, as used in a glucosyltransferase reaction in the present document for the synthesis of alpha-1,3-glucan (to produce a dextran-alpha-1,3-glucan copolymer), can optionally be characterized as an “initiator” or an “acceptor.” In some respects, dextran can be characterized as a "homopolymer" of dextran, which is dextran that is not part of a dextran-alpha-1,3-glucan copolymer.

[0049] The term “copolymer” refers in this document to a polymer comprising at least two different types of alpha-glucan, such as dextran and alpha-1,3-glucan. Petition 870250087473, dated 09 / 26 / 2025, pp. 153 / 302 12 / 133

[0050] The terms “graft copolymer”, “branched copolymer” and the like generally refer in this document 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.

[0051] Examples of graft copolymers in this document are “dextran-alpha-1,3-glucan graft copolymers” (and similar terms) comprising a backbone comprising dextran and one or more alpha-1,3-glucan side chains. The backbone itself, in some respects, may be a branched dextran as disclosed in this document; the addition of alpha-1,3-glucan side chains to such a backbone (thus forming a graft copolymer in this document) may be done, for example, by enzymatic extension from non-reducing ends presented by short branches (alpha-1,2, -1,3 or 1,4 branching, each typically consisting of a single glucose monomer, i.e., pendant glucose).Short branches (which may be enzymatically extended to an alpha-1,3-glucan side chain) may be present in a dextran that would otherwise be linear or mostly linear, or they may be present in a branching dextran. In some respects, alpha-1,3-glucan may also be synthesized from non-reducing ends of dextran backbones, such as in embodiments where the dextran backbone is linear or mostly linear, or in embodiments where the dextran backbone is branched (e.g., dendritic or non-dendritic (the branches do not emanate from a core), but with branching-to-branching structure); such alpha-1,3-glucan is not, technically speaking, a dextran side chain, but rather an extension of the dextran backbone(s).

[0052] The percentage of branching in an alpha-glucan in Petition 870250087473, dated 09 / 26 / 2025, pp. 154 / 302 13 / 133 in this document refers to that percentage of all linkages in the alpha-glucan that represent branching points. For example, the alpha-1,3 branching percentage in an alpha-glucan in this document refers to that percentage of all linkages in the glucan that represent alpha-1,3 branching points. Unless otherwise indicated, the linkage percentages disclosed in this document are based on the total linkages of a glucan or the portion of a glucan to which a disclosure specifically refers.

[0053] The terms “linkage”, “glycosidic linkage” and the like refer to the covalent bonds that connect the sugar monomers in a saccharide compound (oligosaccharides and / or polysaccharides). Examples of glycosidic linkages include 1,6-alpha-D-glycosidic linkages (also referred to herein as “alpha-1,6” linkages), 1,3-alpha-D-glycosidic linkages (also referred to herein as “alpha-1,3” linkages), 1,4-alpha-D-glycosidic linkages (also referred to herein as “alpha-1,4” linkages), and 1,2-alpha-D-glycosidic linkages (also referred to herein as “alpha-1,2” linkages). The glycosidic linkages of a glucan polymer in this document may also be referred to as “glycosidic linkages”. In this document, "alpha-D-glucose" is referred to as "glucose".

[0054] The glycosidic linkage profile of an alpha-glucan in this document can be determined using any method known in the art. For example, a linkage profile can be determined using nuclear magnetic resonance (NMR) spectroscopy methods (e.g., 13C NMR or 1H NMR). These and other methods that can be used are disclosed, for example, in Food Carbohydrates: Chemistry, Physical Properties, and Applications (SW Cui, Ed., Chapter 3, SW Cui, “Structural Analysis of Polysaccharides”, Taylor & Francis Group LLC, Boca Petition 870250087473, dated 09 / 26 / 2025, pages 155 / 302 14 / 133 Raton, FL, 2005), which is incorporated herein by reference.

[0055] The “molecular weight” of an alpha-glucan in this document may be represented as weight-average molecular weight (Mw) or number-average molecular weight (Mn), whose units are Daltons (Da) or grams / mol. In some respects, molecular weight may 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 a monomeric unit Mi. In the case of the glucan polymer, Mi = 162.14. In some respects, molecular weight may sometimes be given as “DP” (degree of polymerization), which simply refers to the number of glucoses comprised in the alpha-glucan on an individual molecule basis.Several methods are known in the art for calculating these various molecular weight determinations, such as high-performance liquid chromatography (HPLC), size exclusion chromatography (SEC), or gel permeation chromatography (GPC).

[0056] The term “sucrose” refers in this document 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 commonly known as table sugar. Sucrose may alternatively be referred to as “alpha-D-glucopyranosyl-(1^2)-beta-D-fructofuranoside”. “Alpha-D-glucopyranosyl” and “glucosyl” are used interchangeably in this document.

[0057] The terms “sugar” or “sugars”, unless used to refer specifically 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 gluco-oligosaccharide) and / or optionally any oligosaccharide (e.g., Petition 870250087473, dated 09 / 26 / 2025, pages 156 / 302 15 / 133 example, 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 in this document. The sugars in this document are typically water-soluble.

[0058] The terms “glucosyltransferase”, “glucosyltransferase enzyme”, “GTF”, “glucansucrase” and the like are used interchangeably in this document. The activity of a glucosyltransferase in this document catalyzes the reaction of the substrate sucrose to produce the products alpha-glucan and fructose. Other products (byproducts) of a GTF reaction may include glucose, various soluble glycooligosaccharides, 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 in this document is classified in the family 70 of glycoside hydrolases (GH70) according to the CAZy (“Carbohydrate-Active EnZymes”) database (Cantarel et al., Nucleic Acids Res. 37: D233238, 2009).The term "dextranassacrase" (and similar terms) may optionally be used to characterize a glucosyltransferase enzyme that produces dextran.

[0059] The term “glucosyltransferase catalytic domain” refers in this document 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.

[0060] The terms “enzymatic reaction”, “glucosyltransferase reaction”, “glucan synthesis reaction”, “reaction composition”, “reaction formulation” and the like are used interchangeably herein. Petition 870250087473, dated 09 / 26 / 2025, pp. 157 / 302 16 / 133 document and generally refer to a reaction that initially comprises water, sucrose, at least one active glucosyltransferase enzyme and optionally other components. Components that may additionally be present in a glucosyltransferase reaction typically after it has begun include fructose, glucose, leucrose, soluble glycooligosaccharides (e.g., DP2-DP7) (these may be considered as products or byproducts depending on the glucosyltransferase used) and / or insoluble alpha-glucan product(s) of DP8 or higher. It is understood that certain glucan products, such as alpha-1,3-glucan with a degree of polymerization (DP) of at least 8 or 9, are insoluble in water and therefore do not dissolve in a glucan synthesis reaction.The term “under suitable reaction conditions,” as used in this document, refers to reaction conditions that support the conversion of sucrose into 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 the synthesis of alpha-glucan polymer; the glucosyl groups involved in this process may thus optionally be referred to as the glucosyl component or fraction (or similar terms) of a glucosyltransferase reaction.

[0061] The “yield” of an alpha-glucan product in a glucosyltransferase reaction, in some respects, in this document represents the molar yield based on the sucrose converted. The molar yield of an alpha-glucan product can be calculated based on the moles of alpha-glucan product divided by the moles of sucrose converted. The moles of sucrose converted can be calculated as follows: (initial mass of sucrose - final mass of sucrose) / molecular weight of sucrose (342 g / mol). This molar yield calculation can be considered as a Petition 870250087473, dated 09 / 26 / 2025, pages 158 / 302 17 / 133 measure of the reaction selectivity for alpha-glucan. In some respects, the “yield” of an alpha-glucan product in a glucosyltransferase reaction can be based on the glucosyl component of the reaction. Such a yield (glucosyl-based yield) can be measured using the following formula: Alpha-glucan yield = ((IS / 2 - (FS / 2 + LE / 2 + GL + SO)) / (IS / 2 - FS / 2)) x 100%.

[0062] The fructose estimate from a glucosyltransferase reaction can be calculated to ensure that HPLC data, if applicable, are not out of range (90-110% is considered acceptable). The fructose estimate can be calculated using the following formula: Estimated fructose content = ((180 / 342 x (FS + LE) + FR) / (180 / 342 x IS)) x 100%.

[0063] In the two formulas above, IS is [Initial Sucrose], FS is [Final Sucrose], LE is [Leucrose], GL is [Glucose], SO is [Soluble Oligomers] (gluco-oligosaccharides) and FR is [Fructose]; the concentrations of each substrate / product above given in double parentheses are in units of grams / L and are as measured by HPLC, for example.

[0064] The terms “invertase”, “beta-D-fructofuranoside fructohydrolase”, “beta-fructosidase”, “beta-fructofuranosidase”, “glucosaccharase” and the like refer to an enzyme that can irreversibly catalyze the hydrolysis of sucrose into glucose and fructose (i.e., an enzyme that can use sucrose to produce invert sugar). An invertase in the present document is from Enzyme Commission (EC) No. 3.2.1.26, and classified under the glycoside hydrolase family 32 (GH32; Henrissat, Biochem. J. 280: 309-316). An invertase is an example of a second enzyme in the present disclosure that uses sucrose as a substrate.

[0065] A “second enzyme that uses sucrose as a substrate”, “second enzyme that uses sucrose”, “second enzyme” and terms Petition 870250087473, dated 09 / 26 / 2025, pp. 159 / 302 18 / 133 similar terms in this disclosure refer to an enzyme, other than a glucosyltransferase of this disclosure, that can catalyze a reaction that converts sucrose into one or more different compounds, thereby reducing the amount of sucrose present. Such a second enzyme is typically not intended to refer to a glucosyltransferase (GH70) in this document. Examples of a suitable second enzyme in this disclosure include invertase, fructosyltransferase enzyme (FTF, EC 2.4.1.99) (e.g., inulosaccharase, levansucrase), sucrose synthase (EC 2.4.1.13), sucrose phosphorylase (EC 2.4.1.7), and sucrose alpha-glucosidase (EC 3.2.1.48).

[0066] The term “in situ”, as used in this document, characterizes a glucosyltransferase reaction(s) that occur(s) within a food product or precursor thereof and thereby produce(s) alpha-glucan within the food product (or precursor) itself. Such alpha-glucan produced (e.g., graft copolymer, alpha-1,3-glucan and / or alpha-1,6-glucan) may 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 may be soluble or insoluble in a food product / precursor as described in this document. The in situ production of alpha-glucan in a food product / precursor typically replaces the addition of alpha-glucan as an ingredient in food described herein, although such addition may be carried out if desired (e.g., to supplement the alpha-glucan produced in situ).The term “in situ”, in aspects relevant to this document, also characterizes a reaction(s) of a second enzyme (e.g., invertase) that occurs(s) within a food product or its precursor and thus produces(s) fructose and glucose within the food product (or precursor) itself. In general, the in situ reactions of glucosyltransferase and the second enzyme of this disclosure occur during the same time period and thus compete for the sucrose substrate. Petition 870250087473, dated 09 / 26 / 2025, pp. 160 / 302 19 / 133

[0067] The terms “volume percentage”, “percentage of volume”, “% by volume”, “% v / v” and the like are used interchangeably in this document. The volume percentage of a solute in a solution can be determined using the formula: [(volume of solute) / (volume of solution)] x 100%.

[0068] The terms “weight percentage”, “weight percentage (% by weight)”, “weight-weight percentage (% w / w)” and similar terms are used interchangeably in this document. Weight percentage refers to the percentage of a material on a mass basis as comprised in a composition, mixture or solution.

[0069] The terms “weight / volume percentage”, “% w / v” and similar terms are used interchangeably in this document. The weight / volume percentage 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 may be insoluble in the liquid (i.e., be a solid phase in a liquid phase, such as in the case of a dispersion) or soluble in the liquid (i.e., be a solute dissolved in the liquid).

[0070] The terms “ingestable product” and “ingestable composition” are used interchangeably in this document and refer to any substance that, alone or in combination with another substance, can be taken orally (i.e., by mouth), whether intended for consumption or not. Thus, an ingestible product includes food products / beverages. “Food products / beverages” refers to any edible product intended for consumption (e.g., for nutritional purposes) by humans or animals, including solids, semi-solids, or liquids. A “food” in this document may optionally be referred to as a “food product,” “foodie,” or another similar term, for example. In this document, unless otherwise stated, a beverage or other ingestible liquid Petition 870250087473, dated 09 / 26 / 2025, pp. 161 / 302 20 / 133 is an example of a food product. Although this disclosure generally refers to foods and food precursors that are, by definition, intended for ingestion or eventual ingestion (food precursor first transformed into food before being consumed), the disclosure also refers to other ingestible products (e.g., supplement, nutraceutical, pharmaceutical product) comprising alpha-glucan produced in situ. A food precursor in this document may be (i) a food as it exists before one or more processing steps (e.g., fermentation, maturation, cooling / freezing, heating, baking, mixing) that make it a food product intended for direct consumption, and / or (ii) an ingredient for use in the preparation of a food product, for example.In some respects, a food precursor may characterize a food product or ingredient as it exists prior to treatment with one or more GTF enzymes and second enzymes in a method described in this document.

[0071] The term “texture”, as used in this document in reference to a food product / precursor, means in this document the consistency of the food product / precursor and / or the sensory perception of the food product / precursor, for example, by visual, tactile or oral / gustatory processing. An “improvement” in texture means an increase in consistency and / or an increase in sensory perception. Unless otherwise indicated, as used in this document, the “consistency” of a food product / precursor means the apparent viscosity extracted at a 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 consistency. The apparent viscosity extracted at a shear rate of approximately 230-270 Hz (e.g., ~249 Hz) during a rheological analysis is correlated with the “mouthfeel”; an increase Petition 870250087473, dated 09 / 26 / 2025, pp. 162 / 302 21 / 133 of the apparent viscosity at that shear rate indicates an increase in mouthfeel.

[0072] “Dairy product / precursor” and similar terms in this document refer to a food product / precursor that contains milk and / or is made from milk. In some respects, a dairy product / precursor contains at least about 2.5, 5 or 10% by weight of milk or milk solids.

[0073] “Lactase-treated milk / dairy products” and similar terms in this document refer to milk / dairy products or precursors treated with one or more lactase enzymes to reduce the amount of lactose sugar they contain.

[0074] “Reduced-lactose milk / dairy products” and similar terms in this document refer to milk / dairy products or precursors in which the percentage by weight of lactose is about 2% or less, for example. “Lactose-free milk / dairy products” and similar terms in this document refer to milk / dairy products or precursors in which the percentage by weight of lactose is about 0.5% by weight or less, for example.

[0075] “Yogurt”, “milk-based yogurt”, “fermented dairy product” and similar terms in this document generally refer to a dairy food / beverage produced by the acidification of lactic fermentation of a dairy substrate, such as milk. Such a product may optionally contain secondary ingredients such as fruits, vegetables, sugars, flavors, etc.

[0076] “Flour” and similar terms in this document refer to the powder produced by grinding (crushing) grains / cereals, roots / tubers, beans / legumes, or nuts / seeds, for example. Typically, the material that is ground to form flour is entered into the grinding process in its raw, dry form. A flour in this document made from grains may optionally be called a “grain flour”. “Semolina” and Petition 870250087473, dated 09 / 26 / 2025, pp. 163 / 302 22 / 133 Other similar terms in this document refer to a substance that is similar to flour, but with a larger / coarser grain or particle size. Semolina is not ground / crushed as finely as flour. Semolina in this document made from grains may optionally be called "grain semolina". Flour and semolina are commonly used as ingredients in various food products. Flour and semolina produced from a grain may optionally be characterized as grain derivatives in this document.

[0077] “Dough”, “pasta” and similar terms herein refer to a mixture comprising at least (i) flour and / or semolina and (ii) a liquid (e.g., water or milk), and typically in a form suitable (hard / firm) for kneading or rolling. Dough may optionally be referred to with reference to the grain, grain derivative or other material from which it is derived (e.g., wheat dough, wheat flour dough, corn flour dough, cornmeal dough). Since dough is typically not consumed as food before further processing (e.g., baking), dough may optionally be characterized as a “food precursor”.

[0078] A “baked food” (and similar terms) in this document refers to a food that has been baked during its preparation. Cooking in this document refers to a process of applying dry heat to a food / food precursor over a period of time during food preparation. Cooking is generally carried out in an enclosed (typically confined) space, such as inside an oven. Bread is an example of a food whose preparation process consists of cooking.

[0079] An “extruded food” (and similar terms) in this document refers to a food that has been extruded during its preparation. Food extrusion is a process by which a mixture of ingredients is extruded. Petition 870250087473, dated 09 / 26 / 2025, pp. 164 / 302 23 / 133 (e.g., dough) is forced through an opening in a perforated device (e.g., plate or die), which is typically designed specifically for the food being extruded. After this step, the extruded food is typically then cut to a specific size.

[0080] The terms “dietary fiber”, “glucan fiber” and the like in this document refer to an alpha-glucan that is indigestible and / or does not raise blood glucose levels when administered enterally to a mammal. In general, a dietary fiber in this document is not significantly hydrolyzed by endogenous enzymes in the upper gastrointestinal tract of mammals, such as humans.

[0081] “Fermentation” and similar terms in this document as applied to the 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, yeasts).

[0082] A composition in this document that is “dry” or “dried” typically has less than 5, 4, 3, 2, 1, 0.5 or 0.1 percent by weight of water included therein.

[0083] The terms “aqueous liquid,” “aqueous fluid,” “aqueous conditions,” “aqueous environment,” “aqueous system,” and the like, as used herein, may refer to water or an aqueous solution. An “aqueous solution” herein may comprise one or more dissolved salts, wherein the maximum total salt concentration may be about 3.5% by weight in some embodiments. Although aqueous liquids herein typically comprise water as the sole solvent in the liquid, an aqueous liquid may optionally comprise one or more other solvents (e.g., polar organic solvent) that are miscible in water. Thus, an aqueous solution may comprise a solvent with at least Petition 870250087473, dated 09 / 26 / 2025, pp. 165 / 302 24 / 133 approximately 10% by weight of water.

[0084] An “aqueous composition” in this document has a liquid component comprising about, or at least about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99 or 100 percent by weight of water, for example. Examples of aqueous compositions include mixtures, solutions, dispersions (e.g., suspensions, colloidal dispersions) and emulsions, for example.

[0085] Alpha-glucan in some aspects of this disclosure may 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 in this document is, for example, the ability of dispersed particles of a dispersion, or liquid droplets dispersed in another liquid (emulsion), to remain dispersed (for example, about, or at least about, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% by weight of the dispersion particles or liquid droplets of the emulsion being in a dispersed state) for a period of about, or at least about, 2, 4, 6, 9, 12, 18, 24, 30 or 36 months after the initial preparation of the dispersion or emulsion. A stable dispersion or emulsion can, for example, resist separation, sedimentation, flocculation, and / or total coalescence of dispersed / emulsified material.

[0086] An alpha-glucan in this document that is “insoluble,” “insoluble in aqueous medium,” or “insoluble in water” (and similar terms) does not dissolve (or does not dissolve significantly) in this document in water or other aqueous conditions, optionally where the aqueous conditions are at a pH of 4–9 (e.g., pH 6–8) and / or at a temperature of about 1 to 130 °C (e.g., 20–25 °C). In some respects, less than 1.0 gram (e.g., no detectable amount) of an aqueous-insoluble alpha-glucan dissolves in 1000 milliliters of such aqueous conditions (e.g., water at 23 °C). In contrast, an alpha-glucan that is “soluble,” “soluble Petition 870250087473, dated 09 / 26 / 2025, pp. 166 / 302 25 / 133 in aqueous medium”, “soluble in water” and similar terms dissolve significantly under the above aqueous conditions.

[0087] The term “viscosity,” as used in this document, refers to the resistance of a food product / precursor to deformation at a given rate. Viscosity can also be defined as a measure of the extent to which a fluid (aqueous or non-aqueous) resists a force that tends 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 encompassed by the term viscosity, since both parameters are directly correlated through the density of a food product / precursor. Several units of viscosity that may be used in this document include centipoise (cP, cps) and Pascal-second (Pa-s), for example. One centipoise is one hundredth of a poise; one poise is equal to 0.100 kg-m-1-s-1.

[0088] As used in this document, the term “polypeptide” is defined as a chain of amino acid residues, usually with a defined sequence. As used in this document, the term “polypeptides” is interchangeable with the terms “peptides” and “proteins”. Typical amino acids contained in the polypeptides in this document include (respective three-letter codes shown in parentheses): alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), valine (Val, V).

[0089] The terms “sequence identity”, “identity” and the like, as used in this document in relation to polypeptide sequences, refer to amino acid residues in two sequences Petition 870250087473, dated 09 / 26 / 2025, pp. 167 / 302 26 / 133 which are equal when aligned for maximum match along a specified comparison window. Thus, “sequence identity percentage”, “identity percentage” and similar terms refer to the value determined by comparing two optimally aligned sequences along a comparison window, where the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) 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 where the identical amino acid residue occurs in both sequences to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the results by 100 to generate the sequence identity percentage.

[0090] The percentage of identity can be readily determined by any known method, including, but not limited to, those described in: 1) Computational Molecular Biology (Lesk, AM, Ed.) Oxford University: NY (1988); 2) Biocomputing: Informatics and Genome Projects (Smith, DW, Ed.) Academic: NY (1993); 3) Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, Eds.) Human: 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.

[0091] The preferred methods for determining the percentage of identity are designed to provide the best match between the tested sequences. The methods for determining identity and similarity are coded in publicly available computer programs, for example. Sequence alignments and percentage of identity calculations can be performed using the MEGALIGN program from the suite of Petition 870250087473, dated 09 / 26 / 2025, pages 168 / 302 27 / 133 LASERGENE bioinformatics computing (DNASTAR Inc., Madison, WI), for example. Multiple sequence alignment can be performed, for example, using the Clustal alignment method which encompasses several varieties of the algorithm, including the Clustal V alignment method (described by Higgins and Sharp, CABIOS. 5: 151-153 (1989); Higgins, DG 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. The default parameters for pairwise alignments and calculation of the percentage of identity of protein sequences using the Clustal method can be KTUPLE = 1, GAP PENALTY = 3, WINDOW = 5 and SAVED DIAGONALS = 5.For nucleic acids, these parameters can be K-TUPLE = 2, GAP PENALTY = 5, WINDOW = 4 and SAVED DIAGONALS = 4. Additionally, the Clustal W alignment method can be used (described by Higgins and Sharp, CABIOS. 5: 151-153 (1989); Higgins, DG et al., Comput. Appl. Biosci. 8: 189-191 (1992); Thompson, JD 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.). The standard parameters for multiple alignment (protein / nucleic acid) can be: GAP PENALTY = 10 / 15, GAP LENGTH PENALTY = 0.2 / 6.66, divergent sequence alignment delay (%) = 30 / 30, DNA transition weight = 0.5, protein weight matrix = Gonnet series and DNA weight matrix = IUB.

[0092] Several polypeptide amino acid sequences are disclosed in this document as characteristic of certain embodiments. Variants of these sequences that are at least about 70-85%, 85-90%, or 90%-95% identical to the sequences disclosed in this document. Petition 870250087473, dated 09 / 26 / 2025, pp. 169 / 302 28 / 133 may be used or used as a reference. Alternatively, a variant amino acid sequence may have at least 70%, 71%, 72%, 73%, 74%, %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, % or 99.5% identity with a sequence disclosed in this document. A variant amino acid sequence in this document has the same function / activity as 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 in this document that does not begin with a methionine or valine may typically still comprise at least an initial methionine or an initial valine at the N-terminus of the amino acid sequence.In contrast, any polypeptide amino acid sequence disclosed in this document beginning with a methionine or valine may optionally lack such a methionine or valine residue. In some respects, any polypeptide amino acid sequence disclosed in this document that begins with a methionine or valine may instead have, respectively, a valine or methionine as the first amino acid residue.

[0093] The terms “aligns with”, “corresponds to” and the like may be used interchangeably in this document. Some aspects of this document refer to a glucosyltransferase comprising at least one amino acid substitution at a position corresponding to at least one particular amino acid residue of SEQ ID NO: 10. An amino acid position of a glucosyltransferase or subsequence thereof (e.g., catalytic domain or catalytic domain plus glucan-binding domains) (we may refer to such an amino acid position or sequence as a “query” position or sequence) may be characterized to Petition 870250087473, dated 09 / 26 / 2025, pp. 170 / 302 29 / 133 correspond to a particular amino acid residue of SEQ ID NO: 10 (we may refer to such an amino acid position or sequence as a “reference” position or sequence) if (1) the lookup sequence can be aligned with the reference sequence (for example, where an alignment indicates that the lookup sequence and the reference sequence (or a subsequence of the reference sequence) are at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90% identical) and (2) the lookup amino acid position aligns directly with (aligns directly against) the reference amino acid position in the alignment of (1).In general, one can align a query amino acid sequence with a reference sequence (SEQ ID NO: 10 or a subsequence of SEQ ID NO: 10) using any alignment algorithm, tool, and / or program described and disclosed in this document (e.g., BLASTP, ClustalW, ClustalV, Clustal-Omega, EMBOSS) to determine the percentage of identity. Just to give one more example, one can align a query sequence with a reference sequence in this document using the Needleman-Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol. 48: 443-453, 1970) as implemented in the Needle program of the “European Molecular Biology Open Software Suite” (EMBOSS (e.g., version 5.0.0 or later), Rice et al., Trends Genet. 16: 276-277, 2000). The parameters of such an EMBOSS alignment may include, for example: a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 replacement matrix (EMBOSS version of BLOSUM62).

[0094] The numbering of particular amino acid residues of SEQ ID NO: 10 in this document (e.g., Tyr-185, Val-186, Leu513, Gln-588, Phe-607, Ile-608, Lys-625, Arg-741, Val-1188, Lys-1327, Glu1332, Asp-1418, Ala-1419, Ser-1420, Thr-1421, Arg-1424, Leu-1425, Thr-1431, Glu-1450) is relative to the total length amino acid sequence of Petition 870250087473, dated 09 / 26 / 2025, pp. 171 / 302 30 / 133 SEQ ID NO: 10. The first amino acid (i.e., position 1, Met-1) of SEQ ID NO: 10 is at the beginning of the signal peptide. Unless otherwise disclosed, substitutions in this document are relative to the full-length amino acid sequence of SEQ ID NO: 10 as the reference sequence.

[0095] A “non-native glucosyltransferase” in this document (“mutant”, “variant”, “modified” and similar terms may also be used to describe such a glucosyltransferase) has at least one amino acid substitution at a position corresponding to a particular amino acid residue of SEQ ID NO: 10 (SEQ ID NOs: 3 and 4 are examples of a non-native GTF).Such at least one amino acid substitution is typically in the place of the amino acid residue(s) that normally (natively) occur(s) at the same position in the native (original) counterpart of the non-native glucosyltransferase (i.e., although SEQ ID NO: 10 is used as a reference for the position, an amino acid substitution in the present document is relative to the native counterpart of a non-native glucosyltransferase) (considered another way, when aligning the sequence of a non-native glucosyltransferase with SEQ ID NO: 10, determining whether a substitution exists at a specific position does not itself depend on the respective amino acid residue in SEQ ID NO: 10, but depends on which amino acid exists at the position in question in the native counterpart of the non-native glucosyltransferase).The amino acid that normally occurs at the relevant site in the native counterpart of the glycosyltransferase is often (but not always) the same as (or conserved with) the particular amino acid residue of SEQ ID NO: 10 for which the alignment is made. A non-native glucosyltransferase may optionally have other amino acid alterations (mutations, deletions, and / or insertions) relative to the sequence of its native counterpart.

[0096] The term “isolated” designates a substance (or a process) Petition 870250087473, dated 09 / 26 / 2025, pp. 172 / 302 31 / 133 in a form or environment that does not occur in nature. A non-limiting example of an isolated substance includes any substance that does not occur naturally, such as a food product, food precursor, or graft copolymer described herein (as well as enzymatic reactions used to prepare these materials). The embodiments disclosed herein are believed to be synthetic / artificial (could not have been made without human intervention / involvement) and / or have properties that do not occur naturally.

[0097] The term “augmented,” as used in this document, may refer to a quantity or activity that is at least approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 50%, 100%, or 200% higher than the quantity or activity with which the augmented quantity or activity is being compared. The terms “augmented,” “high,” “enhanced,” “greater than,” “improved,” and the like are used interchangeably in this document.

[0098] Some aspects of this disclosure relate to a method of producing a food product / precursor. Such a method may include: (a) providing a food product or food precursor (“food product / precursor”) comprising at least water and sucrose, and (b) bringing the food product / precursor into contact with at least one glucosyltransferase enzyme and a second enzyme that uses sucrose as a substrate, wherein the glucosyltransferase enzyme is one or more of: (i) a glucosyltransferase enzyme that synthesizes alpha-1,6-glucan, in which at least about 50% of the glycosidic linkages of alpha-1,6-glucan Petition 870250087473, dated 09 / 26 / 2025, pp. 173 / 302 32 / 133 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 alpha-1,3-glucan are alpha-1,3 linkages, typically wherein at least one alpha-glucan is produced in the food product / precursor, wherein the food product / precursor, after step (b), optionally has one or more of the following characteristics compared with the food product / precursor before step (b): (I) increased texture, but wherein said increased texture is less than it would be if the second enzyme were not present in the food product / precursor, (II) reduced sugar content, but wherein said reduction in sugar content is less than it would be if the second enzyme were not present in the food product / precursor, optionally wherein said sugar content comprises monosaccharides and disaccharides of the food product / precursor and / or (III) reduced sweetness, but wherein said reduced sweetness is less than it would be if the second enzyme were not present in the food product / precursor. [009 9] In some alternative aspects of this document, the provision of the second enzyme that uses sucrose as a substrate is optional. For example, some embodiments of this disclosure relate to a method of producing a food product / precursor, where such method may comprise: (a) provide a food product or food precursor (“food product / precursor”) comprising at least water, sucrose and at least one carbohydrate-containing ingredient (optionally in addition to the source) Petition 870250087473, dated 09 / 26 / 2025, pp. 174 / 302 33 / 133 of sucrose), and (b) bring the food product / precursor into contact with at least one glucosyltransferase enzyme, wherein the glucosyltransferase enzyme is one or more of: (i) a glucosyltransferase enzyme that synthesizes alpha-1,6-glucan, wherein at least about 50% of the glycosidic linkages of 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 alpha-1,3-glucan are alpha-1,3 linkages, wherein the carbohydrate-containing ingredient comprises at least one acceptor molecule for at least one glucosyltransferase enzyme, typically wherein at least one alpha-glucan is produced in the food product / precursor, wherein the food product / precursor, after step (b), optionally has one or more of the following characteristics compared with the food product / precursor before step (b): (I) increased texture, but wherein said increased texture is less than it would be if the carbohydrate-containing ingredient were not present in the food product / precursor, (II) reduced sugar content, but wherein said reduced sugar content is less than it would be if the carbohydrate-containing ingredient were not present in the food product / precursor, optionally wherein said sugar content comprises monosaccharides and disaccharides of the food product / precursor and / or (III) reduced sweetness, but wherein said reduced sweetness is less than it would be if the carbohydrate-containing ingredient were not present. Petition 870250087473, dated 09 / 26 / 2025, pages 175 / 302 34 / 133 present in the food product / precursor.

[0100] Stage (b) of production of a food product / precursor may involve contact of a food product / precursor with at least: (i) a glucosyltransferase (GTF) enzyme that synthesizes alpha-1,6-glucan, wherein at least about 50% of the glycosidic linkages of alpha-1,6-glucan are alpha-1,6 linkages, and / or (ii) a GTF enzyme that synthesizes alpha-1,3-glucan, wherein at least about 50% of the glycosidic linkages of alpha-1,3-glucan are alpha-1,3 linkages.

[0101] In some respects, a GTF (dextranassacrase) enzyme that synthesizes alpha-1,6-glucan in the present document may comprise an amino acid sequence that is about 100% identical, or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% identical, to SEQ ID NO: 1, 2, 11 or 12 (GTF 0768) and has GTF activity. However, in some respects, a GTF enzyme that synthesizes alpha-1,6-glucan may be as disclosed in any of the U.S. patent application publications Nos. 2017 / 0218093, 2018 / 0282385, 2018 / 0291311, or 2016 / 0122445, each of which is 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 may 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 may be used, or a GTF comprising an amino acid sequence that is approximately 100% identical, or at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, If the amino acid sequence of any of these GTF enzymes is 98%, 98.5%, 99%, or 99.5% identical (and has GTF activity), it can be used. Petition 870250087473, dated 09 / 26 / 2025, pp. 176 / 302 35 / 133

[0102] A dextranesaccharase in the present document is capable of producing dextran comprising about, or at least about, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, %, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% of alpha-1,6-glycosidic linkages, for example. Such a percentage profile of alpha-1,6 linkages takes into account the total of all linkages in dextran (alpha-1,6-glucan main chains and, if present, branched portions thereof). Dextran as disclosed elsewhere in this document, such as in a homopolymer or graft copolymer, may have any of the above linkage profiles, for example.

[0103] A dextranesaccharase in the present document is capable of producing dextran with a weight-average 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, 100001000000 or 100000-1000000 Daltons, for example. In some respects, Mw is approximately at least approximately or less than approximately 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 in this document, as in a grafted homopolymer or copolymer, may have any of the above molecular weight profiles, for example. Petition 870250087473, dated 09 / 26 / 2025, pp. 177 / 302 36 / 133

[0104] In some respects, a GTF enzyme synthesizing alpha1,3-glucan in the present document may 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 has GTF activity; these amino acid sequences are disclosed in U.S. Patent Application Publication No. 2019 / 0078063, which is incorporated herein by reference.It is observed that such GTF enzyme comprising SEQ ID NO: 2, 4, 8, 10, 14, 20, 26, 28, 30 or 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%) of alpha-1,3 linkages. A GTF enzyme that synthesizes alpha-1,3-glucan, in some respects, may be that identified as GTF 0974 (SEQ ID NO: 13 in the present document, SEQ ID NO: 110 of US2018 / 0291311) or a GTF comprising an amino acid sequence that is about 100% identical, or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% identical, to the preceding amino acid sequence of GTF 0974 (and has GTF activity).Any of the above GTF enzyme amino acid sequences can be modified as described in this document to increase product yield, modify product molecular weight, and / or improve GTF performance and / or stability.

[0105] A GTF enzyme for producing alpha-1,3-glucan in the present document can, in some respects, synthesize alpha-1,3-glucan with a yield of at least about 40%, 45%, 50%, 55%, 60%, 65%, Petition 870250087473, dated 09 / 26 / 2025, pp. 178 / 302 37 / 133%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%. The yield, in some respects, can be calculated based on the glucosyl component of the reaction and / or calculated using HPLC or NIR spectroscopy. The 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 that have a modified amino acid sequence so that the enzyme produces more products (alpha-1,3-glucan and fructose) and fewer byproducts (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 in the present document 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 Application Publication No.° 2019 / 0078063. A modified GTF enzyme, for example, may comprise one or more amino acid substitutions corresponding to those in Tables 3-7 (ibid.) and which are associated with an alpha-1,3-glucan yield of at least 40% (the position numbering of this at least one substitution corresponds to the position numbering of SEQ ID NO: 62, as disclosed in U.S. Patent Application Publication No. 2019 / 0078063). A set of amino acid modifications as presented in Tables 6 or 7 (ibid.) may be used, for example.

[0106] The amino acid sequence of a GTF enzyme for the synthesis of alpha-1,3-glucan has been modified in some respects so that the enzyme produces alpha-1,3-glucan with a molecular weight (DPw) that is less than the molecular weight of the alpha-1,3-glucan produced by its original GTF counterpart. Examples of a suitable modified GTF enzyme are disclosed in Tables 3 and 4 of U.S. Patent Application Publication No. Petition 870250087473, dated 09 / 26 / 2025, pp. 179 / 302 38 / 133 2019 / 0276806, which is incorporated herein by reference. A modified GTF enzyme, for example, may comprise one or more amino acid substitutions corresponding to those in Tables 3 and / or 4 (ibid.) and which are associated with a molecular weight of the alpha-1,3-glucan product that is at least 5% lower than the molecular weight of the alpha-1,3-glucan produced by the original enzyme (the position numbering of this at least one substitution corresponds to the position numbering of SEQ ID NO: 62, as disclosed in U.S. Patent Application Publication No. 2019 / 0276806). A set of amino acid modifications as presented in Table 4 (ibid.) may be used, for example.

[0107] The amino acid sequence of a GTF enzyme for the synthesis of alpha-1,3-glucan has been modified in some respects so that the enzyme produces alpha-1,3-glucan with a molecular weight (DPw) that is greater than the molecular weight of the alpha-1,3-glucan produced by its original GTF counterpart. Examples of a suitable modified GTF enzyme are disclosed in Tables 3, 4 and 5 of U.S. Patent Application Publication No. 2019 / 0078062, which is incorporated herein by reference. A modified GTF enzyme, for example, may comprise one or more amino acid substitutions corresponding to those in Tables 3, 4 and / or 5 (ibid.) and which are associated with a molecular weight of the alpha-1,3-glucan product that is at least 5% higher than the molecular weight of the alpha-1,3-glucan produced by the original enzyme (the position numbering of this at least one substitution corresponds to the position numbering of SEQ ID NO: 62, as disclosed in U.S. Patent Application Publication No.° 2019 / 0078062). For example, a set of amino acid modifications as presented in Table 5 (ibid.) can be used.

[0108] In some respects, a modified GTF for alpha-1,3-glucan synthesis (i) comprises at least one amino acid substitution or Petition 870250087473, dated 09 / 26 / 2025, pp. 180 / 302 39 / 133 a set of amino acid substitutions (as described above with respect to 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 55960 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 Application Publication No. 2019 / 0078063, which is incorporated into this document by reference). Each of these subsequences is the approximate catalytic domain of each respective reference sequence and produces alpha-1,3glucan comprising at least about 50% (e.g., > 90% or > 95%) of alpha-1,3 linkages.In some respects, 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 to a subsequence thereof, such as SEQ ID NO: 4 (without the initial methionine thereof), or to positions 55-960 of SEQ ID NO: 4 (approximate catalytic domain) (each of these sequences as disclosed in U.S. Patent Application Publication No. 2019 / 0078063).

[0109] In the present disclosure, SEQ ID NOs: 5, 6, 7, 8, 9, and 10 (Table 1) are the same amino acid sequences, respectively, as SEQ ID NOs: 4, 65, 30, 28, 20, and 62 as disclosed in U.S. Patent Application Publication No. 2019 / 0078063. Thus, each of the SEQ ID NOs: 5, 6, 7, 8, 9, and 10 presently disclosed may be used in any of the disclosed aspects, as appropriate. For example, a GTF enzyme synthesizing alpha-1,3-glucan in the present document may comprise a Petition 870250087473, dated 09 / 26 / 2025, pp. 181 / 302 40 / 133 amino acid sequence that is approximately 100% identical, or at least approximately 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 to 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 may be modified as described herein to affect, for example, yield and / or molecular weight and / or stability. of alpha-1,3-glucan.

[0110] In some respects, a GTF enzyme for alpha-1,3-glucan synthesis has been modified in such a way that the enzyme has improved performance and / or stability benefit(s). Such a modification may consist, for example, of having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions compared with a corresponding original GTF enzyme (e.g., a mature wild-type GTF or an active subsequence thereof, such as a catalytic domain).Performance and / or stability benefits exemplified in this document include one or more of the following: increased thermal stability, increased storage stability, increased solubility, improved 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 yield of glucan product (and / or decreased yield of byproduct (e.g., leucrose)). In some respects, a performance benefit is obtained 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 above characteristics may be of about, or at least about, 5%, 10%, 15%, 20%, 25%, or 30%, for example, compared to activity. Petition 870250087473, dated 09 / 26 / 2025, pp. 182 / 302 41 / 133 respective of an original GTF enzyme that has not been modified.

[0111] Some examples of GTF enzymes producing alpha-1,3glucan modified in this document with improved performance and / or stability benefit(s) comprise or consist of SEQ ID NO: 3 (vGTFJ) or 4. Note 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 skeleton to make substitutions in order to provide SEQ ID NOs: 3 and 4).

[0112] It is observed that, in comparison with SEQ ID NO: 5, SEQ ID NO: 3 has the following amino acid substitutions: Tyr-8-Asn (i.e., position 8 is replaced by Asn, instead of Tyr, compared with SEQ ID NO: 5), Val-9-Ala, Leu-336-Tyr, Gln-411-Leu, Phe-430-Tyr, Lys-448-Ala, Arg-564-Ser, Thr-1254-Gln and Glu-1273-Phe (in addition to having a valine at position 1). The positions of each of these substitutions correspond, respectively, to the Tyr-185, Val-186, Leu-513, Gln-588, Phe-607, Lys-625, Arg-741, Thr-1431, and Glu-1450 positions of SEQ ID NO: 10, which is used in this document as the reference sequence.

[0113] It is observed that, in comparison with SEQ ID NO: 5, SEQ ID NO: 4 has the following amino acid substitutions: Leu-336-Tyr, Phe-430-Tyr, Ile-431-Val, Lys-448-Ala, Arg-564-Ser, Val-1011-Glu, Lys-1150-His, Glu-1155-Ala, Asp-1241-Lys, Ala-1242-Glu, Ser-1243-Gly, Thr-1244-Ser, Arg-1247-Leu and Leu-1248-Val (in addition to having a valine in position 1). The positions of each of these substitutions correspond, respectively, to the positions Leu-513, Phe-607, Ile-608, Lys-625, Arg-741, Val-1188, Lys-1327, Glu-1332, Asp-1418, Ala-1419, Ser-1420, Thr-1421, Arg-1424 and Leu-1425 of SEQ ID NO: 10, which is used in this document as a reference sequence.

[0114] In some aspects of the present disclosure, a modified GTF enzyme may comprise or consist of a sequence of Petition 870250087473, dated 09 / 26 / 2025, pp. 183 / 302 42 / 133 amino acids that are 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 (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 above respects may optionally be, alternatively, a methionine or may be deleted.

[0115] In some aspects of the present disclosure, a modified GTF enzyme may comprise or consist of an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 4 and has one or more (or all) of the following amino acid residues: 336-Tyr, 430-Tyr, 431-Val, 448-Ala, 564-Ser, 1011-Glu, 1150-His, 1155-Ala, 1241-Lys, 1242-Glu, 1243-Gly, 1244-Ser, 1247-Leu and / or 1248-Val. The valine at position 1 of SEQ ID NO: 4 in any of the above aspects may optionally be, alternatively, a methionine or may be deleted.

[0116] Any of the amino acids mentioned above in respect of SEQ ID NOs: 3 and 4 may optionally be, alternatively, another amino acid selected from Table 2 based on amino acid conservation. Table 2. Conservation of amino acids For the amino acid Code Substitute with any of: Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, DMet, D-Ile, Orn, D-O, D-Asp, D-Asp, Asparagine D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine ​​C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine, D-Gln, D-Gln, Asn, D-Asn D-Glu, Asp, D-Asp Glutamic acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, b-Ala, Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, D-Leu, D-Leucine, D-Leucine, Value D-Val, Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Petition 870250087473, of 26 / 09 / 2025, p. 184 / 302 43 / 133 For the amino acid code, replace with any one of the following: Methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4 or 5-phenylproline, cis-3,4 or 5-phenylproline Proline P D-Pro, LI-thioazolidino-4-carboxylic acid, D- or L1-oxazolidino-4-carboxylic acid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), DMet(O), L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), DMet(O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

[0117] In some respects, a GTF enzyme that produces alpha- A modified 1,3-glucan with improved performance and / or stability benefit(s) comprises one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions at position(s) corresponding to the amino acid residue(s) Tyr-185, Val-186, Leu-513, Gln-588, Phe-607, Ile-608, Lys-625, Arg-741, Val-1188, Lys-1327, Glu-1332, Asp-1418, Ala-1419, Ser-1420, Thr-1421, Arg-1424, Leu-1425, Thr-1431 and / or Glu-1450 of SEQ ID NO: 10. For example, a GTF enzyme producing modified alpha-1,3-glucan may Understanding amino acid substitutions at positions corresponding to the following amino acid residues of SEQ ID NO: 10: (i) Tyr-185, Val-186, Leu-513, Gln-588, Phe-607, Lys-625, Arg-741, Thr-1431 and / or Glu-1450 (these positions correspond to those in SEQ ID NO GTF: 3); (ii) Leu-513, Phe-607, Ile-608, Lys-625, Arg-741, Val-1188, Lys1327, Glu-1332, Asp-1418, Ala-1419, Ser-1420, Thr-1421, Arg-1424 and / or corresponding positions (corresponding Leu-1424 those in GTF to SEQ ID NO: 4); (iii) Tyr-185, Val-186, Lys-625, Thr-1431 and / or Glu-1450; (iv) Ile-608, Lys-625, Lys-1327, Glu-1332, Asp-1418, Ala-1419, Ser-1420, Thr-1421, Arg-1424 and / or Leu-1425; Petition 870250087473, of 26 / 09 / 2025, p. 185 / 302 44 / 133 (v) Leu-513, Gln-588, Phe-607, Lys-625 and / or Arg-741; (vi) Leu-513, Phe-607, Ile-608, Lys-625 and / or Arg-741; and / or (vii) Leu-513, Phe-607, Lys-625 and / or Arg-741.

[0118] In some respects relating to a modified alpha-1,3-glucan-producing GTF enzyme with improved performance and / or stability benefit(s), (a) the amino acid substitution at a position corresponding to the Tyr-185 amino acid residue of SEQ ID NO: 10 may be with an Asn residue or any residue that is conserved with Asn (e.g., Table 2); (b) the amino acid substitution at a position corresponding to amino acid residue Val-186 of SEQ ID NO: 10 may be with an Ala residue or any residue that is conserved with Ala (e.g., Table 2); (c) amino acid substitution at a position corresponding to amino acid residue Leu-513 of SEQ ID NO: 10 may be with a Tyr, Phe or Trp residue, or any residue that is conserved with Tyr, Phe or Trp (e.g., Table 2); (d) amino acid substitution at a position corresponding to amino acid residue Gln-588 of SEQ ID NO: 10 may be with a Leu residue or any residue that is conserved with Leu (e.g., Table 2); (e) amino acid substitution at a position corresponding to amino acid residue Ile-608 of SEQ ID NO: 10 may be with a Val or Tyr residue, or any residue that is conserved with Val or Tyr (e.g., Table 2); (f) amino acid substitution at a position corresponding to the amino acid residue Lys-625 of SEQ ID NO: 10 may be Petition 870250087473, dated 09 / 26 / 2025, pp. 186 / 302 45 / 133 with an Ala residue or any residue that is preserved with Ala (e.g., Table 2); (g) the amino acid substitution at a position corresponding to the amino acid residue Arg-741 of SEQ ID NO: 10 may be with a Ser residue or any residue that is conserved with Ser (e.g., Table 2); (h) amino acid substitution at a position corresponding to amino acid residue Val-1188 of SEQ ID NO: 10 may be with a Glu residue or any residue that is conserved with Glu (e.g., Table 2); (i) the amino acid substitution at a position corresponding to the amino acid residue Lys-1327 of SEQ ID NO: 10 may be with a His residue or any residue that is conserved with His (e.g., Table 2); (j) amino acid substitution at a position corresponding to amino acid residue Glu-1332 of SEQ ID NO: 10 may be with an Ala residue or any residue that is conserved with Ala (e.g., Table 2); (k) amino acid substitution at a position corresponding to the amino acid residue Asp-1418 of SEQ ID NO: 10 may be with a Lys residue or any residue that is conserved with Lys (e.g., Table 2); (l) the amino acid substitution at a position corresponding to amino acid residue Ala-1419 of SEQ ID NO: 10 may be with a Glu residue or any residue that is conserved with Glu (e.g., Table 2); (m) amino acid substitution at a position corresponding to amino acid residue Ser-1420 of SEQ ID NO: 10 may be Petition 870250087473, dated 09 / 26 / 2025, pp. 187 / 302 46 / 133 with a Gly residue or any residue that is preserved with Gly (e.g., Table 2); (n) the amino acid substitution at a position corresponding to the amino acid residue Thr-1421 of SEQ ID NO: 10 may be with a Ser residue or any residue that is conserved with Ser (e.g., Table 2); (o) amino acid substitution at a position corresponding to amino acid residue Arg-1424 of SEQ ID NO: 10 may be with a Leu residue or any residue that is conserved with Leu (e.g., Table 2); (p) amino acid substitution at a position corresponding to amino acid residue Leu-1425 of SEQ ID NO: 10 may be with a Val residue or any residue that is conserved with Val (e.g., Table 2); (q) amino acid substitution at a position corresponding to amino acid residue Thr-1431 of SEQ ID NO: 10 may be with a Gln residue or any residue that is conserved with Gln (e.g., Table 2); and / or (r) amino acid substitution at a position corresponding to amino acid residue Glu-1450 of SEQ ID NO: 10 may be with a Phe residue or any residue that is conserved with Phe (e.g., Table 2).

[0119] Although it is believed that a modified alpha1,3-glucan-producing GTF enzyme, in some respects, only needs to have a catalytic domain, the modified GTF may be comprised of 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. Petition 870250087473, dated 09 / 26 / 2025, pages 188 / 302 47 / 133

[0120] Although amino acid substitutions in a modified alpha-1,3-glucan-producing GTF enzyme are generally disclosed, in some respects, with respect to the corresponding positions in SEQ ID NO: 10, such substitutions may alternatively be stated simply with respect to their position number in the amino acid sequence used to produce the modified GTF itself (e.g., SEQ ID NO: 5 (optionally without initial methionine therein) or positions 55-960 of SEQ ID NO: 5 (approximate catalytic domain)), as convenience may dictate. This 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 their direct alignment with the corresponding position(s) in SEQ ID NO: 10.

[0121] A GTF producing alpha-1,3-glucan in the present document 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%, %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, %, 96%, 97%, 98%, 99% or 100% alpha-1,3-glycosidic linkages, for example. Alpha-1,3-glucan, as disclosed elsewhere in this document, such as in a grafted homopolymer or copolymer, may have any of the above linkage profiles, for example.

[0122] A GTF producing alpha-1,3-glucan in the present document is capable of producing alpha-1,3-glucan with a DPw, DPn or DP of 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 Petition 870250087473, dated 09 / 26 / 2025, pp. 189 / 302 48 / 133 of these values. Just as examples, DPw, DPn, or DP can be approximately 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 in this document, as in a grafted homopolymer or copolymer, may have any of the above molecular weight profiles, for example.

[0123] In some respects, a GTF enzyme may be any as disclosed in this document and include 1–300 (or any integer between these (e.g., 10, 15, 20, 25, 30, 35, 40, 45, or 50)) residues at the N-terminus and / or C-terminus. Such additional residues may be from a corresponding wild-type sequence from which the GTF enzyme is derivable, or they may be a heterologous sequence, such as an epitope marker (at the N-terminus or C-terminus) or a heterologous signal peptide (at the N-terminus), for example. A GTF enzyme in this document typically lacks an N-terminal signal peptide; such an enzyme may optionally be characterized as mature if its signal peptide has been removed during a secretion process.

[0124] A GTF enzyme in the present paper can typically be derived from bacteria. Examples of bacterial GTF enzymes are those derived from 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. Petition 870250087473, of 26 / 09 / 2025, p. 190 / 302 49 / 133 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.

[0125] A GTF enzyme in this document can be prepared by fermentation of a suitably modified microbial strain, for example. The production of recombinant enzymes 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 appropriately. Such an expression cassette may be incorporated into a suitable plasmid or integrated into the microbial host chromosome. The expression cassette may include a transcription terminator nucleotide sequence following the amino acid-coding sequence.The expression cassette may also include, between the promoter sequence and the amino acid coding sequence of GTF, a nucleotide sequence encoding a signal peptide (e.g., heterologous signal peptide) designed for direct secretion of the GTF enzyme. At the end of fermentation, the cells can be suitably disrupted (usually 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... Petition 870250087473, dated 09 / 26 / 2025, pp. 191 / 302 50 / 133 GTF can be used without further isolation. If GTF has been secreted (i.e., is present in the fermentation broth), it can optionally be used as an isolate from, or as comprised within, 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.

[0126] The alpha-glucan produced in step (b) of the production of a food product / precursor in some respects (e.g., typically when using both a GTF that synthesizes alpha-1,6-glucan and a GTF that synthesizes alpha-1,3-glucan) comprises a graft copolymer comprising: (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 (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 side chain are alpha-1,3 linkages.

[0127] Such a graft copolymer may be aqueous soluble or aqueous insoluble. The dextran backbone of an alpha-glucan graft copolymer in the present document may be dextran, as presently disclosed, for example, or may be as disclosed (e.g., molecular weight, linkage / branching profile, production method) in U.S. patent application publications Nos. 2016 / 0122445, 2017 / 0218093, 2018 / 0282385, 2020 / 0165360 or 2019 / 0185893, each of which is incorporated herein by reference. In some respects, a dextran backbone (before being integrated into a graft copolymer) has been branched into alpha-1,2 and / or alpha-1,3; The percentage of alpha-1,2 and / or alpha-1,3 branching of a graft copolymer backbone in this document may be approximately, at least approximately, or less than approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, Petition 870250087473, dated 09 / 26 / 2025, pages 192 / 302 51 / 133%, 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. The alpha-1,3-glucan side chain(s) of an alpha-glucan graft copolymer in this document may be alpha-1,3-glucan as presently disclosed, for example, or may be as disclosed (e.g., molecular weight, linkage profile) in U.S. Patents Nos. 7000000, 8871474, 10301604 or 10260053, or in U.S. Patent Application Publications Nos. 2019 / 0112456, 2019 / 0078062, 2019 / 0078063, 2018 / 0340199, 2018 / 0021238, 2018 / 0273731, 2017 / 0002335, 2015 / 0232819, 2015 / 0064748, 2020 / 0165360, 2020 / 0131281 or 2019 / 0185893, each of which is incorporated herein by reference.

[0128] One, two, three or more different GTF enzymes that synthesize alpha-1,6-glucan in the present document may be used, for example, in step (b) of the production of a food product / precursor. Similarly, one, two, three or more different GTF enzymes that synthesize alpha-1,3-glucan in the present document may be used. In some respects, alpha-1,6-glucan-producing GTF(s) may be added to (brought into contact with) a food product / precursor before adding alpha-1,3-glucan-producing GTF(s), while in some respects, these two types of GTF enzymes may be added at approximately the same time (simultaneously). However, in some respects, alpha-1,3-glucan-producing GTF(s) may be added to a food product / precursor before adding alpha-1,6-glucan-producing GTF(s).However, in some respects, a dextran as disclosed in this document, but produced exogenously to the food product / precursor, can be added as an ingredient to a food product / precursor that already contains an alpha-1,3-glucan-producing GTF. Petition 870250087473, dated 09 / 26 / 2025, pages 193 / 302 52 / 133 has been added or will be added. While not bound to any specific theory, it is believed that the addition of at least one alpha-1,6-glucan producing GTF (and / or exogenously produced dextran) and at least one alpha-1,3-glucan producing GTF in step (b) of the production of a food product / precursor allows the production of a dextran-alpha-1,3-glucan graft copolymer as currently disclosed, possibly in conjunction with the production of dextran and / or alpha-1,3-glucan homopolymer(s) (i.e., alpha-1,6-glucan and / or alpha-1,3-glucan produced independently of the production of the graft copolymer).However, it is believed to be possible that, in some aspects of use of both an alpha-1,6-glucan producing GTF and an alpha-1,3-glucan producing GTF, only dextran and / or alpha-1,3-glucan homopolymer(s) will be produced, with little (e.g., < 5% by weight of all glucan products) or no graft copolymer production.

[0129] The molecular weight and / or binding profile of alpha-glucan produced by a GTF enzyme (dextranassacrase or alpha-1,3-glucan-producing GTF), as generally disclosed above, can be 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 respects, the molecular weight and / or binding profile of alpha-glucan produced by one or both of these types of GTF enzyme in a food product / precursor in this document may differ from those produced in the previous isolated reaction.

[0130] In some aspects where both an alpha-1,6-glucan-producing GTF and an alpha-1,3-glucan-producing GTF are used, the ratio between the alpha-1,6-glucan-synthesizing GTF enzyme and the alpha-1,3-glucan-synthesizing GTF enzyme in step (b) is about 85:15 to about 95:5. However, in some aspects, the ratio between the alpha-1,6-glucan-producing GTF Petition 870250087473, dated 09 / 26 / 2025, pp. 194 / 302 53 / 133 and the alpha-1,3-glucan-producing GTF can be approximately 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 vary between any two of these ratios (for example, approximately 82.5:17.5 to 97.5:2.5, 87.5:12.5 to 92.5:7.5, 89:11 to 91:9, 17.5:82.5 to 2.5:97.5, 12.5:87.5 to 7.5:92.5, 11:89 to 9:91). The quantity of each enzyme (active enzyme) for the purpose of determining a ratio between them in this document may be based on a molar, weight, or GTF activity basis, for example. The activity of a GTF enzyme for the preparation of a ratio in this document may optionally be determined as disclosed in U.S. Patent Application Publication No. 2014 / 0087431, which is incorporated herein by reference, and / or as disclosed in the examples below.For example, a full complement (e.g., “100%”) of a GTF enzyme for establishing a ratio in this document may be that amount of enzyme capable of converting most (e.g., >95%, >98%, >99%), or all, of the sucrose in a GTF reaction comprising or consisting of water, sucrose (e.g., 50 or 100 g / L), to GTF and optionally buffer / salt in a given period of time (e.g., 6, 12, 18, 24, 30 or 36 hours); such a measured amount may optionally be characterized as a standardized amount of GTF.

[0131] A GTF enzyme (or any other enzyme as presently disclosed, such as a second enzyme that utilizes sucrose) for use in a method in this document is typically in purified form. A purified enzyme may be essentially free of insoluble and / or soluble components of an organism / a cell used to produce the enzyme, and / or any medium that has been used for cellular fermentation of the enzyme. In some respects, a purified enzyme denotes an enzyme preparation containing less than 10%, 8%, 6%, 5%, 4%, 3%, 2 Petition 870250087473, dated 09 / 26 / 2025, pages 195 / 302 54 / 133%, 1%, 0.5% or 0.1% by weight of another material (e.g., polypeptide material) with which the enzyme is natively or recombinantly associated. In some respects, a GTF and / or any other enzyme in this document is not comprised of or otherwise associated with (e.g., expressed by) a microbial cell (e.g., bacterial, yeast, fungal, algal) that may be present (e.g., endogenously or purposefully added) in a food product / precursor in this document; however, in some respects, a GTF and / or any other enzyme in this document is comprised of or otherwise associated with (e.g., expressed by) a microbial cell (e.g., bacterial, yeast, fungal, algal), such as one that heterologously expresses the enzyme(s) (i.e., recombinant cells).The contact of a food product / precursor with GTF enzyme(s) in this document is not typically performed in an oral cavity or other environment where unpurified / unisolated GTF enzymes may be present.

[0132] A GTF enzyme (or any other enzyme as presently disclosed, such as a second enzyme that uses sucrose) for use in a method in this document may be comprised in a filtration-sterilized preparation, for example. In some respects, an enzyme may be sterilized by in-line filtration while the enzyme is applied to a food product / precursor during step (b) in this document. In some respects, an enzyme may be added to a food product / precursor that has been pasteurized (after pasteurization) or, alternatively, an enzyme may be added before pasteurization of the food product / precursor. In some respects, an enzyme may be added to a food product / precursor that has been fermented (after fermentation) or, alternatively, an enzyme may be Petition 870250087473, dated 09 / 26 / 2025, pp. 196 / 302 55 / 133 added during or before the fermentation of the food product / precursor. A GTF enzyme (or any other enzyme as presently disclosed, such as a second enzyme that utilizes sucrose), in some respects, for use in a method in this document may be comprised in a preparation (for addition in step (b) in this document) that is substantially free of (e.g., < 0.5, < 0.1, < 0.05 % by weight) 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 of such other enzyme(s).

[0133] A food product / precursor in this document may be brought into contact, in step (b), with one or more GTF enzymes and / or one or more second enzymes that use sucrose in this document by mixing / agitation / combination, for example. The incubation of GTF enzyme(s) and / or second enzyme(s) in the food product / precursor may be for a sufficient time for the GTF(s) to produce alpha-glucan in the food product / precursor, such as for approximately, or at least approximately, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 24, 30, 36, 42, 48, 72, 96, 0.53, 0.5-2.5, 0.5-2, 0.5-1.5, 1-3, 1-2.5, 1-2, 1-1.5, 1.5-3 or 1.5-2 hours, or for approximately, or at least approximately, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 days (or a range between any two of these hours and / or days).The temperature for incubating one or more GTF and / or secondary enzymes in a food product / precursor in this document may be approximately 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, 550, 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. Petition 870250087473, dated 09 / 26 / 2025, pp. 197 / 302 56 / 133 °C, for example.

[0134] Typically, a food product / precursor brought into contact with a GTF enzyme and / or second enzyme in this document contains water (i.e., is an aqueous composition), and / or water is introduced into the food product / precursor before or during contact with one or more of these enzymes. GTF and / or second enzyme(s) may be added to a food product / precursor in dry form (e.g., powder, flakes, freeze-dried enzyme preparation) (typically to an aqueous food product / precursor) or in wet form. In some respects, a food product / precursor may be combined with a GTF and / or second enzyme(s) under dry conditions (the resulting combination is dry), after which water or an aqueous solution is added, which in turn allows the production of alpha-glucan by GTF to proceed.Thus, depending on how ingredients and GTF enzyme(s) are introduced to each other, steps (a) and (b) can optionally be considered as being carried out simultaneously or separately. The water content of a food product / precursor as provided in step (a) or step (b) after the addition of GTF and / or second enzyme(s) can be about, or at least about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99% by weight, for example. The pH of a food product / precursor in this document and / or the pH for the incubation of one or more GTFs and / or secondary enzymes in a food product / precursor in this document may be approximately 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 4.0-10.0, 4.0-9.0, 4.0-8.0, 4.5-10.0, 4.5-9.0, 4.5-8.0, 5.0-10.0, 5.0-9.0, 5.0-8.0, 5.5-10.0, 5.5-9.0, 5.5-8.0, 6.0-10.0, 6.0-9.0 or 6.0-8.0, for example.A food product / precursor, in some respects, can be acidic (e.g., pH < 3.0, 3.2, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 or 6.5), neutral (e.g., pH 6.5-7.5) or basic / alkaline (e.g., pH > 7.5, 8.0, 8.5, 9.0, 9.5). Petition 870250087473, dated 09 / 26 / 2025, pages 198 / 302 57 / 133

[0135] A GTF enzyme and / or second enzyme in this document may optionally be provided in step (b) of a method by introducing a recombinantly modified cell (e.g., a microbial cell, such as a bacterial or fungal / yeast cell) into the food product / precursor provided in step (a), wherein the cell recombinantly (heterologously) expresses and secretes the GTF enzyme and / or second enzyme into and / or around the food product / precursor. Such a cell may be that of a microbe that is recombinantly modified and useful in food processing (e.g., fermentation), such as a microbial cell disclosed in this document (as applicable).In some respects, a recombinantly modified cell that is supplied to the food product / precursor in step (a) may be inactive and / or nonviable in some way, such as by being killed (but preferably in a way that retains the cell shape / structure). For example, a cell may be rendered inactive and / or nonviable by irradiation or treatment with a sterilizing chemical agent / product (e.g., ethylene oxide). Typically, the means for cell inactivation and / or death preserve at least part of the three-dimensional shape / structure of the cell and / or ensure that a GTF and / or second enzyme(s) that was / were expressed by the cell remain active and typically remain associated with the inactive / nonviable cell (e.g., by association with a cell membrane through an optional transmembrane domain or membrane-binding domain of the GTF and / or second enzyme (e.g., fusion to the GTF and / or second enzyme)).An inactive / non-viable cell is typically porous and can optionally be immobilized on a support (e.g., an inert, water-insoluble material, such as a particle or surface).

[0136] In some respects (for example, with or without the provision of a second enzyme in the present document), the activity of Petition 870250087473, dated 09 / 26 / 2025, pages 199 / 302 58 / 133 one or more GTF enzymes in step (b) may be modulated by the inclusion of a carbohydrate-containing ingredient (together with water and sucrose) in the food product / precursor provided in step (a). A carbohydrate-containing ingredient may comprise, for example, a monosaccharide (e.g., any in this document, such as glucose or fructose), a disaccharide (e.g., any in this document, such as lactose or maltose) and / or an oligosaccharide (e.g., any in this document, such as a gluco-oligosaccharide (e.g., DP3-15, MOS, IMOS, GOS)) (e.g., the carbohydrate-containing ingredient comprises a monosaccharide and / or disaccharide). In some respects, a carbohydrate-containing ingredient comprises at least fructose and / or glucose.In some respects, a carbohydrate-containing ingredient may be in the form of a food ingredient, such as any in this document that comprises at least one acceptor molecule for at least one glucosyltransferase enzyme of this disclosure. For example, a carbohydrate-containing ingredient may comprise a syrup, such as corn syrup or high fructose corn syrup (HFCS). As another example, a carbohydrate-containing ingredient may comprise a tomato-based ingredient, such as tomato paste, tomato concentrate, tomato puree, or tomato sauce.

[0137] A “food product / precursor” (i.e., a food product or precursor) as provided in step (a) of a method in some aspects of the method of this disclosure may comprise sucrose that is endogenous to the food product / precursor (e.g., its sucrose is native) and / or may comprise sucrose that has been added to the food product / precursor (during or after its preparation as an ingredient). Step (a) may thus optionally comprise the addition of sucrose to the food product / precursor. The sucrose content of a product / precursor Petition 870250087473, dated 09 / 26 / 2025, pages 200 / 302 59 / 133 The food product finally supplied in step (a) in this document, regardless of the original source of the sucrose, may 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 percent by weight, for example. In some respects, the sucrose may be supplied as refined white sucrose or in an unrefined form, as disclosed in U.S. Patent No. 9719121, for example, which is incorporated herein by reference. Sucrose may optionally be added to a food product / precursor by adding GTF and / or second enzyme(s) to the food product / precursor.

[0138] In some respects, a food product / precursor as provided in step (a) of a method in this document further comprises at least one disaccharide in addition to sucrose and / or at least one oligosaccharide. An oligosaccharide may 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 respects, a polysaccharide in this document has more than 15 or 20 monomeric units. A disaccharide and / or oligosaccharide in this document may 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 in this document (besides sucrose) include maltose, isomaltose, lactose, lactosaccharose, nigerose, leucrose, trehalulose, maltulose, isomaltulose, and turanose.Examples of oligosaccharides in this document include glucooligosaccharides (gluco-oligomers), such as malto-oligosaccharides (MOS), isomalto-oligosaccharides (IMO), and galacto-oligosaccharides (GOS).

[0139] A disaccharide and / or oligosaccharide may be added to a food product / precursor during or after the preparation of the food product / precursor. Such addition may originate from a source Petition 870250087473, dated 09 / 26 / 2025, pages 201 / 302 60 / 133 physically outside the food product / precursor (i.e., as an ingredient) and / or can be obtained through in situ production in the food / precursor, such as by means of 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) to produce a disaccharide and / or oligosaccharide may be added, for example, in the same or similar manner to that in which a GTF enzyme is added in this document (e.g., time, temperature, pH) and may be added before, during or after the addition of the GTF enzyme. Such an enzyme may be a transglucosidase (EC (enzyme code) 2.4.1.24) or a transgalactosylated beta-galactosidase. Suitable transglucosidases in this document include TGO FoodPro® and those disclosed in U.S. patent application publications nos. 2008 / 0229514 or 2015 / 0240279, or in U.S. patent nos.° 4689296, all of which are incorporated herein by reference. A transglucosidase EC 2.4.1.24 (also referred to as “1,4-alpha-glucan 6-alpha-glucosyltransferase”) can transfer an alpha-D-glucosyl residue from an alpha-1,4-glucan, -oligosaccharide (i.e., MOS) or -disaccharide (i.e., maltose) to the primary hydroxyl group of free glucose or glucose in an alpha-1,4-glucan, -oligosaccharide (i.e., MOS) or -disaccharide. Thus, a transglucosidase EC 2.4.1.24 produces isomalto-oligosaccharides (IMO) (e.g., DP3-DP5 or DP3-DP6) in some respects. Suitable transgalactosylation beta-galactosidases in this document are disclosed in U.S. Patent Application Publication No. 2013 / 0189746 or in U.S. Patents Nos. 10531672 or 10683523, for example, which are incorporated herein by reference.A transgalactosylated beta-galactosidase is an enzyme that degrades lactose by transferring galactose from lactose to galactose, glucose, or another acceptor, thus producing galacto-oligosaccharides (GOS) (for example, GOS can also be an acceptor to form a longer GOS). Petition 870250087473, dated 09 / 26 / 2025, pp. 202 / 302 61 / 133 A particular example of such an enzyme is Nurica™(IFF). A transgalactosylated transglucosidase or beta-galactosidase in the present document can be measured in a food product / precursor in the present document at approximately 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.

[0140] In some respects, a food product / precursor as provided in step (a) of a method in this document has, in addition to sucrose, little (e.g., less than 0.5, 0.25, 0.1, 0.05, 0.025 or 0.01% by weight, or undetectable) or no disaccharide and / or oligosaccharide (or little or no specific disaccharide or oligosaccharide). A food product / precursor as produced in step (b) of a method in this document may also have, for example, little or no disaccharide and / or oligosaccharide (or little or no specific disaccharide or oligosaccharide) and also have little (e.g., as above) or no sucrose. A disaccharide or an oligosaccharide in such respects may be any as disclosed in this document (e.g., lactose, maltose, isomaltose, MOS, IMO, GOS).One or more glycosidase enzymes (active glycosidic enzymes) may be used, for example, in a food product / precursor to reduce or eliminate the presence of disaccharide(s) and / or oligosaccharide(s), and may be added, for example, in the same or similar manner to that in which a GTF and / or second enzyme in this document is added (e.g., time, temperature, pH), and may be added before, during, or after the addition of the GTF and / or second enzyme. A glycosidase in this document may 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 in this document include Bonlacta™(IFF) lactase and the lactases disclosed in [reference to relevant documentation]. Petition 870250087473, dated 09 / 26 / 2025, pages 203 / 302 U.S. Patent No. 10531672, which is incorporated herein by reference. A lactase in this document is a type of beta-galactosidase enzyme that catalyzes the hydrolysis of lactose into glucose and galactose. Appropriate alpha-glucosidases in this document include those disclosed in U.S. Patent Application Publication No. 2015 / 0240278, which is incorporated herein by reference. In some respects, an alpha-glucosidase used in the disclosed method is capable of hydrolyzing an alpha-1,4 or alpha-1,6 glycosidic bond, and / or is incapable of hydrolyzing an alpha-1,3 glycosidic bond. A glucosidase in the present document can be measured in a food product / precursor in the present document at approximately 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.

[0141] A second enzyme in the present disclosure that uses sucrose as a substrate may be an invertase, for example. An invertase may in some respects be (derivable from) a bacterial, fungal (yeast), plant or mammalian invertase, for example. An invertase may in some respects be (derivable from) a microbial invertase. An invertase in some respects may be (derivable) from a species of Aspergillus (e.g., A. niger, A. aculeatus, A. oryzae, A. fumigatus), Fusarium (e.g., F. graminearum, F. oxysporum), Kluveromyces (e.g., K. lactis), Penicillium (e.g., P. chrysogenum, P. hirsutum, P. italicum), Saccharomyces (e.g., S. cerevisiae), Talaromyces (e.g., T. minoluteus), or Thielavia (e.g., T. terrestris). Examples of suitable invertases may be as disclosed in any of U.S. Patent No. 6337201, U.S. Patent Application Publications No.os2012 / 0122192, 20180007918 and 2022 / 0073893, the publication of international patent application no. WO2022243311 and the accession numbers in GENBANK. Petition 870250087473, dated 09 / 26 / 2025, pages 204 / 302 63 / 133 CAB95010, NP_012104 and CAA06839, all of which are incorporated herein by reference. An invertase in some respects may comprise, or consist of, 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:14 or an invertase amino acid sequence disclosed in any of the aforementioned incorporated references and has invertase activity. A second enzyme in this document, such as an invertase, may optionally be produced and / or have any modifications (e.g., be in a mature form (e.g., secreted form) and / or contain heterologous amino acid residues at its N and / or C termini), as disclosed in this document for a GTF enzyme and / or as disclosed in any of the previously mentioned incorporated references.

[0142] One, two, three or more second enzymes may be used in a disclosure method, for example. Typically, if two or more second enzymes are used, they are of the same enzyme type (e.g., two or more different invertases); however, two or more different types of second enzymes may optionally be used (e.g., at least one invertase and at least one fructosyltransferase). In some respects, the concentration of one or more secondary enzymes (e.g., of active secondary enzyme(s)) in a food product / precursor may be approximately, or at least approximately, 5, 10, 15, 20, 25, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, 5-1000, 5-500, 5-300, 5-200, 5-100, 5-80, 5-50, 20-1000, 20-500, 20-300, 20-200, 20-100, 20-80, 20-50, 50-1000, 50-500, 50-300, 50-200, 50-100, 50-80, 100-1000, 100-500, 100-300 or 100-200 ppm (parts per million).Although any concentration of GTF from this disclosure may be used in conjunction with a second enzyme as described herein, in some cases... Petition 870250087473, dated 09 / 26 / 2025, pp. 205 / 302 64 / 133 aspects, the concentration of GTF (active GTF) in a food product / precursor may be approximately, or at least approximately, 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.5, 1.0, 0.01-1.0, 0.01-0.5, 0.01-0.25, 0.01-0.1, 0.05-1.0, 0.05-0.5, 0.05-0.25, 0.05-0.1, 0.1-1.0, 0.1-0.5 or 0.1-0.25 % by weight. The conditions (e.g., time, temperature, pH, order of addition, mixing / contact medium, mixing order) for treating a food product / precursor with a second enzyme can typically be as disclosed in this document for a GTF enzyme. A second enzyme can typically be added to a food product / precursor at approximately the same time that a GTF is added to the food product / precursor; however, in some respects, a second enzyme can be added before (e.g., 1, 2.5, 5, 10, 15, 20, or 30 minutes before) the addition of GTF, or after (e.g., 1, 2.5, 5, 10, 15, 20, or 30 minutes after) the addition of GTF.

[0143] A food product / precursor may, in some respects, be a dairy product / precursor, such as a dairy beverage or food. Suitable examples of a dairy product / precursor in this document include milk, cheese, yogurt, dessert, cream, and butter. The milk in this document may be whole milk (e.g., ~3% fat), milk with ~2% fat, milk with ~1% fat (“skimmed”), or fat-free milk, for example. The milk, whether used directly as a beverage or as a precursor for the preparation of a dairy product / precursor in this document, may be from cow, goat, sheep, buffalo, yak, llama, camel, or horse, for example. The milk may optionally be pasteurized before or after coming into contact with one or more GTF and second enzymes in this document.A cheese in this document may be, for example, a hard or semi-hard cheese (e.g., cheddar, mozzarella, Swiss, parmesan, provolone), a soft or semi-soft cheese (e.g., ricotta, cottage cheese, feta, American, brie), processed or unprocessed. A yogurt no. Petition 870250087473, dated 09 / 26 / 2025, pages 206 / 302 65 / 133 of this document may be, for example, whole milk yogurt, low-fat yogurt, fat-free yogurt, or Greek yogurt (e.g., plain, low-fat, fat-free). A yogurt described in this document may optionally contain fruit and / or be flavored. A yogurt in this document may optionally be drinkable (i.e., yogurt drink). A dairy dessert, in some respects, may be a pudding (e.g., whole milk, 2% milk), frozen yogurt (e.g., low-fat), ice cream (e.g., low-fat), fruit ice cream, milkshake, sorbet, or cream; thus, in some respects, a dairy dessert may be a frozen dairy dessert (e.g., ice cream, fruit ice cream, milkshake, frozen yogurt, gelato). Ice cream may be hard ice cream or soft-serve ice cream (soft-serve ice cream), for example.A dairy beverage, in some respects, may be milk, chocolate milk, coffee milk, flavored milk, yogurt drink, kumis, ryazhenka, ayran, lassi, cholado, licuado, or kefir. A dairy cream may be coagulated cream (e.g., > 55% milk fat), cream (e.g., > 36% milk fat), whipping cream (e.g., 30%-36% milk fat), light cream (e.g., 18%-30% milk fat), sour cream (> 18% milk fat), half-and-half (e.g., 10.5%-18% milk fat), or ice cream (e.g., > 10% milk fat). A dairy product / precursor in this document may be lactose-free or have a reduced lactose content, for example. A dairy product / precursor in this document may be a fermented dairy product / precursor (e.g., yogurt, buttermilk, cream, quark, fromage frais, sour milk, vinegar), for example.Some dairy products / precursors in this document include dairy candies such as milk chocolate, white chocolate, caramel, and toffee. A dairy product / precursor, in some respects, may be any as disclosed in WO2020 / 010176, in U.S. Patent Application Publications Nos. 2013 / 0230623, 2005 / 0244541. Petition 870250087473, dated 09 / 26 / 2025, pp. 207 / 302 66 / 133 2017 / 0135360, 2009 / 0304864, 2017 / 0094987 or 2003 / 0152685, or in U.S. patents nos. 5482728 or 6352734, all of which are incorporated herein by reference.

[0144] A food product / precursor may, in some respects, be a flour-based or semolina-based dough, a baked product (bakery product) or an extruded product, such as any of those disclosed in WO2021 / 034561 or in U.S. Patent Application Publications Nos. 2017 / 0218093 or 2022 / 0322685, which are incorporated herein by reference.Examples of baked goods, or dough (precursor) thereof, include bread (e.g., rolls, sourdough, rye, whole wheat, pita, flatbread, tortilla, cornbread, brioche, white bread, baguette, bagels, banana bread, ciabatta, brown bread, challah, focaccia, multigrain bread, breadsticks, Irish bread, pumpernickel, potato bread, biscuits, English muffins, whole grain bread, unleavened bread, lavash, croutons, pizza crust) (with or without yeast), cake (e.g., carrot cake, red velvet cake, angel food cake, pound cake, chocolate cake, white cake, Black Forest cake, tiramisu, coffee cake, cheesecake, devil's cake, upside-down cake, Boston cream pie, Swiss roll, lemon cake, shortcake, chiffon cake, butter cake, spice cake, rum cake, sponge cake, marble cake, coconut cake, pandan cake), muffins, brownies, Scones, biscuits, bars, creams, pies, crackers, pretzels, candies, puddings and tarts.Examples of an extruded product include pasta (e.g., spaghetti, rotini, fusilli, penne, bucatini, macaroni / macaroni, rigatoni, fettuccine, linguine, vermicelli, ziti, farfalle, gomiti / elbows, rotelle), cereals (e.g., directly expanded cereal, filled cereals, flakes, breakfast cereals), some bakery products (e.g., croutons, breadsticks, flatbreads), pre-made biscuit dough, dry and semi-moist pet food (e.g., kibble) and snacks (e.g., cheese curls, filled pastries, potato chips (e.g., chips). Petition 870250087473, dated 09 / 26 / 2025, pp. 208 / 302 67 / 133 of corn, pita bread chips, processed potato chips, tortilla chips), savory snacks (e.g., vegetable sticks), puffed molded products such as curls (e.g., cheese curls), balls, tubes, bananas, cups, bowls, discs, puffed baby food). The dough in this document may be extruded (e.g., see above) and / or flattened / rolled (e.g., lasagna), fresh or dried, long or short, small / soup pasta (pastina), filled (e.g., tortellini, ravioli, agnolotti, tortelli), stretched (e.g., cencioni, corzetti, foglie d'ulivo, orecchiette) and / or egg pasta, e.g.

[0145] A food product / precursor, in some respects, may be a syrup or a beverage, for example, such as any of those disclosed in U.S. patent application publications 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 respects may be a juice (for example, 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 (carbonated beverage / soda, sweetened tea or coffee), ready-to-drink (RTD) beverage, or any other beverage containing natural and / or added sugar (sucrose).

[0146] A food product / precursor can in some respects be a condiment (table condiment) or any other preparation (of liquid or solid consistency) that is added to a food (typically cooked food) to impart a flavor and / or to intensify a flavor. Examples of condiments in this document Petition 870250087473, dated 09 / 26 / 2025, pages 209 / 302 68 / 133 includes / comprises any tomato-based condiment (e.g., ketchup, tomato sauce, salsa, marinara sauce), mustard (e.g., yellow mustard, Dijon mustard), seasoning, horseradish, wasabi, hot sauce, chili sauce / oil, mayonnaise, aioli, barbecue sauce, soy sauce, Alfredo sauce, au jus, Béarnaise sauce, cranberry sauce, chutney (mango chutney, onion chutney, tamarind chutney), cocktail sauce, fish sauce, oyster sauce, hoisin sauce, sriracha sauce, marmalade, fruit preserves (compote / jelly), Hollandaise sauce, hummus, guacamole, pesto sauce, miso, Worcestershire sauce, salad dressing, salsa verde, sesame oil, sour cream, tartar sauce, or teriyaki sauce.

[0147] A ketchup (or catsup) in this document typically comprises at least tomatoes (e.g., in the form of a puree or in another disintegrated form, such as a paste, before or after the addition of one or more other ketchup ingredients), water, sugar / sweetener and vinegar (e.g., distilled vinegar) and optionally additionally comprises salt (e.g., NaCl) and / or spices / seasonings (e.g., onion powder, garlic powder, mustard powder, cumin, allspice, coriander, cinnamon). A tomato ingredient for preparing a ketchup may be in the form of tomato paste, tomato sauce or tomato puree, for example. In some respects, a ketchup comprises about, or at least about, 30, 35, 40, 45, 50, 30-50, 30-45, 30-40, 30-35, 35-50, 35-45 or 35-40% by weight of tomato paste, tomato concentrate or other form / ingredient of tomato solids (typically comprising some amount of water, i.e., typically not dry solids).In some respects, a ketchup comprises approximately, or at least approximately, 20, 25, 30, 35, 40, 45, 50, 55, 60, 20-60, 20-50, 20-40, 20-30, 30-60, 30-50, or 30-40 percent by weight of water used as the initial ingredient (i.e., the preceding amount of water does not include water introduced by other ingredients, such as tomato paste). A ketchup is... Petition 870250087473, dated 09 / 26 / 2025, pp. 210 / 302 69 / 133 typically prepared by cooking the tomato ingredient(s) (optionally along with one or more other ingredients) and blending them to form a sauce or other liquid consistency. An example of a ketchup in this document comprises tomato concentrate (e.g., from ripe red tomatoes), vinegar, high fructose corn syrup (this ingredient is optional), corn syrup, salt, spices, onion powder, and natural flavoring. Other examples of ketchup in this document have a formulation (and methodological setup) as described for Table 16 (formulation 5, 6 or 7), Table 18 (formulation 3, 5, 6, 7 or 8), Table 20 (formulation 4, 5 or 6) or Table 23 (formulation 2 or 3) below, where the quantity / content of each ingredient, and / or the setup parameters (incubation time and temperature), is / are within 5% or 10% of the respective values ​​presented for a given formulation.Each of the preceding formulations refers to its non-GTF ingredients, since such a formulation may be the basis on which one or more GTF enzymes as presently disclosed may be included as an ingredient(s) (i.e., the GTF component does not necessarily have to be the same as presented in the preceding tables). In some respects, a ketchup comprises a vegetable or fruit ingredient (e.g., banana, guava, tamarind, mushroom, or any fruit / vegetable disclosed in this document) instead of (or in addition to) a tomato ingredient.

[0148] A food precursor, in some respects, may be a fermented food precursor, for example, such as any of those disclosed in WO2002 / 034061. In some respects, a food precursor provided in step (a) of a method in this document is fermented, while in some respects a food precursor is fermented (or further fermented) during or after step (b). Thus, step (a) of a method in Petition 870250087473, dated 09 / 26 / 2025, pages 211 / 302 70 / 133 This document may optionally comprise a fermentation step of a food product / precursor (e.g., before or after the addition of sucrose, if applicable). Thus, step (b) of a method in this document may optionally comprise the fermentation of the food product / precursor while it is brought into contact with a GTF and a second enzyme. Thus, a method in this document may optionally comprise, after step (b), a fermentation step of the food product / precursor. One or more bacterial and / or yeast cultures may be used for the fermentation of a food product / precursor in this document. Suitable bacteria for food fermentation in this document include lactic acid bacteria, for example, species of the family Lactobacillaceae, such as those of the genus Pediococcus (e.g., P. acidilactici, P. pentosaceus), genus Lactobacillus (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, as subsp.L. bulgaricus), genus Lactococcus (e.g., L. lactis, as subsp. L. cremoris), genus Leuconostoc (e.g., L. citreum, L. mesenteroides), and genus Streptococcus (e.g., S. thermophilus). Bacteria suitable for food fermentation may in some respects be species of the genus Bifidobacterium (e.g., B. bifidum, B. longum, B. animalis, B. breve, B. infantis) or the genus Propionibacterium (e.g., P. freudenreichii, such as subsp. P. shermanii) (propionic acid bacteria).In some respects, a bacterium for food fermentation in this document can be characterized as Gram-positive, spherical, rod-shaped, anaerobic, aerobic, acid-tolerant, non-spore-forming, GRAS (generally considered safe), and / or probiotic. A bacterium for food fermentation (by... Petition 870250087473, dated 09 / 26 / 2025, pages 212 / 302 71 / 133 example, yogurt or other lactic fermentation) in some respects may be an acidic culture / strain (or blend) (e.g., (YO-MIX 863, YO-MIX 410 or YO-MIX T42, available from IFF) that produces foods with a pH of approximately, for example, 2.5-4.5, 3.0-4.5, 4.2-4.4 or 4.3, or it may be a mild culture / strain (or blend) (e.g., YO-MIX PRIME 900 or YO-MIX M01, available from IFF) that produces foods with a pH of approximately, for example, 4.6-5.5, 4.6-6.0, 4.5-4.7 or 4.6. 20 to 25 °C (room temperature)). A mixture of bacteria in a culture for food fermentation may comprise one, two, three, four, five, six or more different species and / or subspecies of bacteria, for example.The genus *Cerevisiae*, *S. pastorianus*, *S. boulardii*, *S. kluyveri*), the genus *Pichia* (e.g., *P. kluyveri*, *P. fermentans*) and the genus *Candida* (e.g., *C. humilis*, *C. famata*). A yeast, in some respects, can be characterized as baker's yeast, brewer's yeast, winemaking yeast, probiotic yeast, budding / fission yeast, or GRAS. A yeast mixture in a culture for food fermentation may comprise one, two, three, four, five, six, or more different species and / or subspecies of yeast, for example. A food product / precursor that is fermented or will be fermented may be a dairy product as defined in this document (for example, as above, such as yogurt), beer, beer wort, wine, pomace, cider, miso, kimchi, sauerkraut, pickles / pickle juice, soy curd, tofu, kombucha, soy sauce, bread, fermented dough or meat, for example.

[0149] A food product / precursor, in some respects, can be a sweet, for example. Examples of sweets in this document. Petition 870250087473, dated 09 / 26 / 2025, pp. 213 / 302 72 / 133 include boiled sugars (cooked hard candies (i.e., hard candies)), dragees, jelly candies, gums, licorice, chewy candies, caramels, toffee, dulce de leche, chewing gum, chewing gum, nougat, chewy pastes, halawa, tablets, lozenges, icing, meringue, pudding, gels (e.g., fruit gels, gelatin dessert), aerated candies, marshmallows, baked candies.

[0150] A food precursor product may, in some respects, be a non-dairy food precursor product. For example, a non-dairy food precursor product may be a plant-based milk (milk substitute) or comprise a plant-based milk (and have little or no (e.g., < 0.5% by weight) dairy ingredient(s) such as lactose, whey, casein and / or milk fat). In some respects, a non-dairy food precursor product is fermented (e.g., a non-dairy yogurt precursor product, such as a plant-based yogurt precursor product).The plant-based ingredient(s) that form the basis for a non-dairy food product / precursor in this document may be nuts / seeds (e.g., almonds, cashews, macadamia nuts, hemp seeds, quinoa, flax seeds), grains / cereals (e.g., oats, rice), fruits (e.g., coconut, banana), or vegetables (e.g., vegetables such as beans (e.g., soybeans, mung beans) and peas), for example. In some respects, a non-dairy food product / precursor is a milk from any of the nuts / seeds, grains / cereals, fruits, or vegetables mentioned above; a fermented form of any of these milks may be a yogurt, for example.

[0151] A food product / precursor, in some respects, may be a creamy soup, meat broth, sauce (e.g., tomato sauce), salad dressing, mayonnaise, compote, jam, marmalade, syrup, pie filling, batter for frying, pancake / waffle batter, glaze and Petition 870250087473, dated 09 / 26 / 2025, pages 214 / 302 73 / 133 cake frosting, whipped frosting, pet food or animal / livestock feed.

[0152] A food product / precursor may, in some respects, 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, fruits / vegetables (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 melon, Inodorus melon, kiwi, lemon, lime, carrot, tomato), or fruit / vegetable juice (concentrated juice), 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.These one or more additional ingredients may be as disclosed in U.S. Patent Application Publications Nos. 2016 / 0122445 or 2017 / 0218093 (both incorporated herein by reference), for example, and / or may be natural or artificial.Examples of suitable ingredients as sweeteners (or for any other purpose, such as flavoring) include acesulfame potassium, advantame, agave syrup, alitame, aspartame, barley malt syrup, birch syrup, brazein, brown rice syrup, cane juice, caramel, coconut sugar, corn syrup, curculin, cyclamate, dextrose, erythritol, fructooligosaccharide, fructose (levulose), galactose, glucose (dextrose), glycerol (glycerin), glycyrrhizin, golden syrup, high fructose corn syrup (e.g., HFCS-42, -55, -90), high maltose corn syrup (HMCS), honey, hydrogenated starch hydrolysate (HSH), isomaltooligosaccharide (IMO), inulin, invert sugar, isomalt, lactitol, lactose. Petition 870250087473, dated 09 / 26 / 2025, pages 215 / 302 74 / 133 maltitol, maltodextrin, maltose, mannitol, maple syrup, miraculin, molasses (e.g., black molasses), monatin, monellin, monk fruit, neohesperidin dihydrochalcone, neotame, palm sugar, pentadine, polydextrose, unrefined cane sugar, refiners' syrup, saccharin, sorbitol (glucitol), sorghum syrup, stevia / steviol glycoside (e.g., a rebaudioside such as rebaudioside A, rebaudioside D or rebaudioside M), sucralose, sugar alcohol, tagatose, thaumatin, trehalose, xylitol and yacon syrup.

[0153] In some respects, a food product / precursor produced by a method of the present disclosure may be concentrated, dried (e.g., into a powder), reconstituted (after concentration or drying) or processed (e.g., frozen) in any other way. Examples of such products include sweetened milk, concentrated milk, condensed milk (e.g., sweetened condensed milk), evaporated milk, milk powder, frozen dairy product (e.g., ice cream), concentrated juice or juice powder.

[0154] In some respects, a method in the present document may further comprise a freezing step of a food product / precursor (for example, a dairy food product / precursor or a plant-based food product / precursor) after step (b). This method may produce a frozen dairy product in the present document, such as frozen ice cream or yogurt, or a plant-based frozen ice cream or yogurt, for example. Freezing may be carried out at approximately -10, -15, -20, -25, -30, -35, -40, -20 to -40, -25 to -35 °C, for example.A frozen product in some respects, wherein step (b) of the method comprises the use of at least one alpha-1,3-glucan-producing GTF and a second enzyme in the present document, may have an improved melting profile (e.g., slower melting) compared with a suitable control (e.g., a frozen product that was not treated with an alpha-1,3-glucan-producing GTF and a second enzyme, but which, otherwise, was made with the same). Petition 870250087473, dated 09 / 26 / 2025, pages 216 / 302 75 / 133 ingredients and process steps). The slower melting of a frozen product, in some respects, may be a melting that is reduced by about, or at least by about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to the melting of a properly controlled product. Melting can refer to melting that occurs within approximately 45, 60, 75, 90, 100, 120, 150, or 60-120 minutes after placing a frozen product (from freezing conditions) at room temperature (e.g., approximately 20, 25, 18-25, or 20-25 °C) or an elevated temperature (e.g., approximately 25-38, 25-35, 25-32, or 25-30 °C), for example. Melting can optionally be measured according to the examples below or as disclosed in Granger et al. (2005, Int. Dairy J. 15(3): 255-262, incorporated herein by reference), for example.

[0155] A food product / precursor, in some respects, after step (b) of a method in this document, optionally has one or more of the following characteristics compared with the food product / precursor as it existed before step (b) (i.e., as it existed before being treated with one or more GTF enzymes and one or more second enzymes that use sucrose as a substrate): (I) increased texture, but wherein the increased texture is less than it would have been if the second enzyme were not present in the food product / precursor, (II) reduced sugar content, but wherein the reduction in sugar content is less than it would have been if the second enzyme were not present in the food product / precursor, typically wherein the sugar content comprises monosaccharides and disaccharides of the food product / precursor and / or (III) reduced sweetness, but wherein the reduced sweetness is less than it would have been if the second enzyme were not present in the food product / precursor. Petition 870250087473, dated 09 / 26 / 2025, pages 217 / 302 76 / 133

[0156] In some respects, the texture of a food product / precursor after step (b) may 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% compared to the texture of the food product / precursor as it existed before the step (b). However, by virtue of including at least one second enzyme in the present document during the enzymatic treatment of the food product / precursor, such increased texture is typically not as high as it would have been if the second enzyme(s) were omitted. For example, the texture obtained may be about, or less than about, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 25%-90% of the texture that would have been obtained if the second enzyme(s) were omitted.Thus, for example, this methodology can be useful for fine-tuning or modulating the texture obtained when treating a food product / precursor with GTF enzyme(s) as described in this document. For example, excessive gelation mediated by GTF enzyme(s) can be avoided to achieve a more desirable amount of gelation. Texture as described in this document can be in terms of viscosity, consistency, structure, or mouthfeel and / or measured in units of Pascal-seconds (Pa^s) or cP, for example, either of which can optionally be measured according to the examples below. In some respects, texture (consistency) can be measured by determining the viscosity of the food product / precursor when extracted at a shear rate of about 11 to 12 Hz (e.g., 11.7 Hz).The texture (mouthfeel) can be measured by determining the viscosity of the food product / precursor when extracted at a shear rate of approximately 248-250 Hz (e.g., 249 Hz). In some respects, the texture can be estimated visually (e.g.,...). Petition 870250087473, dated 09 / 26 / 2025, pp. 218 / 302 77 / 133 photographically or videographically).

[0157] In some respects, the sugar content (e.g., % by weight) in a food product / precursor after step (b) may be reduced by about, or at least about, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 20-65%, 20-60%, 25-65% or 25-60%, compared with the sugar content of the food product / precursor as it existed before step (b). However, by virtue of including at least one second enzyme in the present document during the enzymatic treatment of the food product / precursor, such reduction in sugar content is not typically as pronounced (i.e., the sugar reduction is not as high) as it would have been if the second enzyme(s) were omitted (i.e., the second enzyme attenuates the sugar reduction).For example, the magnitude of the sugar content reduction obtained may be approximately, or less than approximately, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 40%-90% of the magnitude of the sugar content reduction that would have been obtained if the second enzyme(s) were omitted. In some respects, the reduction in sugar content relates to all sugars in the food product / precursor, while in other respects, this reduction relates to a particular sugar, such as sucrose, or to the monosaccharides (e.g., including fructose and glucose, and optionally galactose) and disaccharides (e.g., including sucrose and leucrose, and optionally lactose) of the food product / precursor. The sugar content in this document can be measured by HPLC, for example, as disclosed in the examples below.

[0158] In some respects, the sweetness of a food product / precursor after step (b) may be reduced by about, or at least about, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%, compared with the sweetness of the food product / precursor as it existed before step (b). However, by virtue of including at least a second enzyme in the present Petition 870250087473, dated 09 / 26 / 2025, pages 219 / 302 78 / 133 document during the enzymatic treatment of the food product / precursor, such a reduction in sweetness is not typically as pronounced (i.e., the reduction in sweetness is not as high) as it would have been if the second enzyme(s) were omitted (i.e., the second enzyme attenuates the reduction in sweetness). For example, the magnitude of the sweetness reduction obtained may be about, or less than about, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 40%-90% of the magnitude of the sweetness reduction that would have been obtained if the second enzyme(s) were omitted. Sweetness can be measured according to the examples below, or as disclosed in U.S. Patent Application Publication No. 2009 / 0053378, for example, which is incorporated herein by reference. In some respects, sweetness can be based on theoretical sweetness.Theoretical sweetness can be calculated, for example, based on the concentration of each monosaccharide and disaccharide registered in the food product / precursor (e.g., g of sugar compound / 100 g of food product / precursor), and the relative sweetness value of each monosaccharide and disaccharide (e.g., the relative sweetness values ​​for lactose, glucose, galactose, sucrose, and fructose may be, respectively, 0.16, 0.75, 0.16, 1.0, and 1.2). Theoretical sweetness is the sum of the respective products of concentration and relative sweetness for each monosaccharide and disaccharide registered in the food product / precursor. In some respects, registered monosaccharides include fructose and glucose, and optionally galactose, and registered disaccharides include sucrose and optionally lactose.

[0159] In some respects, a food product / precursor after step (b) has an improved physical appearance compared with the physical appearance of the food product / precursor as it existed before step (b). The improved physical appearance may be increased homogeneity (e.g., visual homogeneity and / or little or no syneresis) and / or brightness. Petition 870250087473, dated 09 / 26 / 2025, pages 220 / 302 79 / 133 increased (e.g., visual brightness), for example; this increase(s) may be of about, or at least about, 5%, 10%, 20%, 25%, 30%, 40% or 50% in some respects.

[0160] In some respects, the dietary caloric content (calories that can be accessed during digestion) of a food product / precursor after step (b) may be reduced by about, or at least about, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or 75% compared with the dietary caloric content of the food product / precursor as it was before step (b).

[0161] In some respects, the dietary fiber content (e.g., percentage by weight) of a food product / precursor after step (b) may be increased by about, or at least about, 5%, 10%, 25%, 50%, 75%, 100%, 200%, 300%, 400% or 500% compared with the dietary fiber content of the food product / precursor as it existed before step (b).

[0162] Some aspects of this disclosure relate to a food product / precursor as produced by a GTF treatment method as described herein. Examples of such products / precursors are any food product / precursor as described herein. Typically, such a food product / precursor may have any characteristic as described herein (e.g., reduced sugar content, increased texture, improved physical appearance, reduced calorie content, increased dietary fiber, pH, temperature, age), as appropriate / applicable. Typically, such a food product / precursor comprises at least one GTF enzyme and at least one second enzyme as described herein and / or an alpha-glucan as described herein.

[0163] In some respects, a food product / precursor, after following a step (a) of providing a product / precursor Petition 870250087473, dated 09 / 26 / 2025, pages 221 / 302 80 / 133 food product comprising at least water, sucrose and at least one carbohydrate-containing ingredient (optionally in addition to the sucrose source), and a step (b) of bringing the food product / precursor into contact with at least one glucosyltransferase enzyme as described herein, optionally has one or more of the following characteristics compared to the food product / precursor as it existed before step (b) (i.e., as it existed before being treated with one or more GTF enzymes): (I) increased texture, but wherein the increased texture is less than it would have been if the carbohydrate-containing ingredient were not present in the food product / precursor, (II) reduced sugar content, but wherein the reduced sugar content is less than it would have been if the carbohydrate-containing ingredient were not present in the food product / precursor, typically wherein the sugar content comprises monosaccharides and disaccharides of the food product / precursor and / or (III) reduced sweetness, but wherein the reduced sweetness is less than it would have been if the carbohydrate-containing ingredient were not present in the food product / precursor.

[0164] In some respects, the texture of a food product / precursor after step (b) may 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% compared to the texture of the food product / precursor as it existed before the step (b). However, by virtue of including at least one carbohydrate-containing ingredient in this document during the enzymatic treatment of the food product / precursor, such increased texture is typically not as high as it would have been if the carbohydrate-containing ingredient(s) were omitted. Petition 870250087473, dated 09 / 26 / 2025, pages 222 / 302 81 / 133 For example, the texture obtained may be approximately, or less than approximately, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 25%-90% of the texture that would have been obtained if the carbohydrate-containing ingredient(s) were omitted. Thus, for example, this methodology may be useful for fine-tuning or modulating the texture obtained when treating a food product / precursor with GTF enzyme(s) as described in this document. For example, excessive gelation mediated by GTF enzyme(s) may be avoided to achieve a more desirable amount of gelation. The texture in this document can be in terms of viscosity, consistency, structure or mouthfeel and / or measured in units of Pascal-seconds (Pa^s) or cP, for example, and any of these can optionally be measured according to the examples below.In some respects, texture (consistency) can be measured by determining the viscosity of the food product / precursor when extracted at a shear rate of about 11 to 12 Hz (e.g., 11.7 Hz). Texture (mouthfeel) can be measured by determining the viscosity of the food product / precursor when extracted at a shear rate of about 248-250 Hz (e.g., 249 Hz). In some respects, texture can be estimated visually (e.g., photographically or videographically).

[0165] In some respects, the sugar content (e.g., % by weight) in a food product / precursor after step (b) may be reduced by about, or at least about, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 20-65%, 20-60%, 25-65% or 25-60%, compared to the sugar content of the food product / precursor as it existed before step (b). However, by virtue of including at least one carbohydrate-containing ingredient in the present document during the enzymatic treatment of the food product / precursor, such a reduction in sugar content does not Petition 870250087473, dated 09 / 26 / 2025, pages 223 / 302 82 / 133 is typically as pronounced (i.e., the sugar reduction is not as high) as it would have been if the carbohydrate-containing ingredient(s) were omitted (i.e., the carbohydrate-containing ingredient attenuates the sugar reduction). For example, the magnitude of the sugar reduction achieved may be about, or less than about, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 40%–90% of the magnitude of the sugar reduction that would have been achieved if the carbohydrate-containing ingredient(s) were omitted. In some respects, the reduction in sugar content relates to all sugars in the food product / precursor, while in other respects, this reduction relates to a particular sugar, such as sucrose, or to the monosaccharides (e.g., including fructose and glucose, and optionally galactose) and disaccharides (e.g., including sucrose and leucrose, and optionally lactose) of the food product / precursor.The sugar content in this document can be measured by HPLC, for example, as shown in the examples below.

[0166] In some respects, the sweetness of a food product / precursor after step (b) may be reduced by about, or at least about, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%, compared with the sweetness of the food product / precursor as it existed before step (b). However, by virtue of including at least one carbohydrate-containing ingredient in the present document during the enzymatic treatment of the food product / precursor, such sweetness reduction is not typically as pronounced (i.e., the sweetness reduction is not as high) as it would have been if the carbohydrate-containing ingredient(s) were omitted (i.e., the carbohydrate-containing ingredient attenuates the sweetness reduction). For example, the magnitude of the sweetness reduction obtained may be approximately, or less than approximately, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 40%–90% of the magnitude of the sweetness reduction that would have been obtained if Petition 870250087473, dated 09 / 26 / 2025, pp. 224 / 302 83 / 133 the carbohydrate-containing ingredient(s) were omitted. Sweetness can be measured according to the examples below, or as disclosed in U.S. Patent Application Publication No. 2009 / 0053378, for example, which is incorporated herein by reference. In some respects, sweetness can be based on theoretical sweetness. Theoretical sweetness can be calculated, for example, based on the concentration of each monosaccharide and disaccharide registered in the food product / precursor (e.g., g of sugar compound / 100 g of food product / precursor), and the relative sweetness value of each monosaccharide and disaccharide (e.g., the relative sweetness values ​​for lactose, glucose, galactose, sucrose, and fructose may be, respectively, 0.16, 0.75, 0.16, 1.0, and 1.2). Theoretical sweetness is the sum of the respective products of concentration and relative sweetness for each monosaccharide and disaccharide recorded in the food product / precursor.In some respects, the registered monosaccharides include fructose and glucose, and optionally galactose, and the registered disaccharides include sucrose and optionally lactose.

[0167] In some respects, a food product / precursor after step (b) has an improved physical appearance compared with the physical appearance of the food product / precursor as it existed before step (b). The improved physical appearance may be increased homogeneity (e.g., visual homogeneity and / or little or no syneresis) and / or increased brightness (e.g., visual brightness), for example; this increase(s) may be of about, or at least about, 5%, 10%, 20%, 25%, 30%, 40% or 50% in some respects.

[0168] Some aspects of the present disclosure are focused on isolated compositions / products comprising at least (I) an isolated invertase or other second isolated enzyme from the present disclosure that uses sucrose as a substrate, and (II) at least one of (i) an enzyme Petition 870250087473, dated 09 / 26 / 2025, pp. 225 / 302 84 / 133 isolated glucosyltransferase synthesizing alpha-1,6-glucan and / or (ii) an isolated glucosyltransferase enzyme synthesizing alpha-1,3-glucan. Such a composition / product may optionally further comprise at least one alpha-glucan as described herein. An isolated composition / product may be in the form of, and / or comprised within, a household care product, personal care product, industrial product, ingestible product (e.g., food precursor product, such as any disclosed herein) or pharmaceutical product, for example, as described in any of the U.S. patent application publications Nos. 2018 / 0022834, 2018 / 0237816, 2018 / 0230241, 20180079832, 2016 / 0311935, 2016 / 0304629, 2015 / 0232785, 2015 / 0368594, 2015 / 0368595, 2016 / 0122445, 2019 / 0202942 or 2019 / 0309096, or in the publication of international patent application no. WO2016 / 133734, all of which are incorporated herein by reference. In some respects, a composition / product may comprise at least one component / ingredient of a household care product, personal care product, industrial product, pharmaceutical product or ingestible product (e.g., food product / precursor), as disclosed in any of the foregoing publications and / or as herein disclosed.

[0169] Non-limiting examples of compositions and methods / processes disclosed in this document include: 1. A method (process) for producing a food product / precursor, wherein the method comprises: (a) providing a food product / precursor comprising at least water and sucrose, and (b) bringing the food product / precursor into contact with at least one glucosyltransferase enzyme and a second enzyme that uses sucrose as a substrate (wherein such second enzyme is not a glucosyltransferase in the present document), wherein the glucosyltransferase enzyme is: (i) an enzyme Petition 870250087473, dated 09 / 26 / 2025, pages 226 / 302 85 / 133 glucosyltransferase that synthesizes alpha-1,6-glucan, wherein at least about 50% of the glycosidic linkages of 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 alpha-1,3-glucan are alpha-1,3 linkages, typically wherein at least one alpha-glucan is produced in the food product / precursor, wherein the food product / precursor, after step (b), optionally possesses one or more of the following characteristics compared with the food product / precursor before step (b): (I) increased texture (or viscosity, consistency, structure, mouthfeel), but wherein the increased texture is less than it would be if the second enzyme were not present in the food product / precursor (this (i) the degree of texture formation mediated by glucosyltransferase is reduced by the second enzyme), (II) reduction of sugar content,but wherein the reduction in sugar content is less than it would be if the second enzyme were not present in the food product / precursor (i.e., the degree of sugar reduction mediated by glucosyltransferase is reduced by the second enzyme), typically wherein the sugar content comprises monosaccharides and disaccharides of the food product / precursor, and / or (III) reduced sweetness, but wherein the reduced sweetness is less than it would be if the second enzyme were not present in the food product / precursor (i.e., the degree of sweetness reduction mediated by glucosyltransferase is reduced by the second enzyme, i.e., the second enzyme helps to maintain sweetness); 2. The method of embodiment 1, wherein the second enzyme that uses sucrose as a substrate is an invertase enzyme (for example, an invertase comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 14) or fructosyltransferase enzyme; 3. The method of embodiment 1 or 2, in which the glycosyltransferase enzyme that synthesizes alpha-1,6-glucan comprises a sequence Petition 870250087473, dated 09 / 26 / 2025, pp. 227 / 302 86 / 133 amino acids that are at least 90% identical to SEQ ID NO: 1, 2, 11 or 12; 4. The method of embodiment 1, 2 or 3, in which the glucosyltransferase enzyme that synthesizes alpha-1,3-glucan comprises an amino acid sequence that is at least 90% identical to residues 55-960 of SEQ ID NO: 5, residues 54-957 of SEQ ID NO: 6, residues 55-960 of SEQ ID NO: 7, residues 55-960 of SEQ ID NO: 8, residues 55-960 of SEQ ID NO: 9, or to SEQ ID NO: 13; 5. The method of embodiment 1, 2, 3 or 4, in which both (i) the glucosyltransferase enzyme that synthesizes alpha-1,6-glucan and (ii) the glucosyltransferase enzyme that synthesizes alpha-1,3-glucan are used in step (b); 6. The method of embodiment 1, 2, 3, 4 or 5, wherein the alpha-glucan produced in step (b) comprises a graft copolymer comprising: (i) a main chain of alpha-1,6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1,6-glucan main chain are alpha-1,6 linkages, and (ii) at least one side chain of alpha-1,3-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1,3-glucan chain are alpha-1,3 linkages, wherein the alpha-glucan is aqueous soluble or aqueous insoluble; 7. The method of embodiment 1, 2, 3, 4, 5 or 6, in which step (a) comprises the addition of sucrose to the food product / precursor; 8. The method of embodiment 1, 2, 3, 4, 5, 6 or 7, in which the food product / precursor is a dairy food product / precursor; 9. The method of embodiment 1, 2, 3, 4, 5, 6 or 7, in which the food product / precursor is a non-dairy food product / precursor; 10. The method of modality 9, in which the non-dairy food product / precursor is plant-based; 11. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, in which the food product / precursor of step (a) is fermented or the method Petition 870250087473, dated 09 / 26 / 2025, pages 228 / 302 87 / 133 additionally includes, during or after step (b), the fermentation of the food product / precursor; 12. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, in which the food product / precursor is a yogurt; 13. The method of modality 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, wherein the food product / precursor is a condiment (for example, a tomato-based condiment, such as tomato ketchup), such as ketchup; 14. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, in which step (b) further comprises bringing the food product / precursor into contact with at least one isolated lactase enzyme; 15. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, in which, however, the second enzyme is not necessarily used (for example, the second enzyme, such as an invertase, is not used in the method); 16. A food product or food precursor (isolated food product / precursor) produced by the method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; 17. An isolated food product or food precursor comprising (I) an isolated invertase (e.g., according to embodiment 2) or a second isolated enzyme of the present disclosure that uses sucrose as a substrate, and (II) at least one of (i) an isolated glucosyltransferase enzyme that synthesizes alpha-1,6-glucan (e.g., according to embodiment 3) and / or (ii) an isolated glucosyltransferase enzyme that synthesizes alpha-1,3-glucan (e.g., according to embodiment 4), and optionally further comprising at least one isolated alpha-glucan and / or lactase enzyme (in some optional alternative embodiments, the Petition 870250087473, dated 09 / 26 / 2025, pp. 229 / 302 88 / 133 isolated food product or food precursor does not necessarily comprise isolated invertase or the second isolated enzyme of (I), and the isolated food product or food precursor may be any as presently disclosed, such as those cited in any of the embodiments 8, 9, 10, 11, 12 or 13); and 18. A composition / isolated product (e.g., household care product, personal care product, industrial product, ingestible product, or pharmaceutical product; or household care product, personal care product, or industrial product) comprising (I) an isolated invertase (e.g., according to embodiment 2) or a second isolated enzyme from this disclosure that uses sucrose as a substrate, and (II) at least one of (i) an isolated glucosyltransferase enzyme that synthesizes alpha-1,6-glucan (e.g., according to embodiment 3) and / or (ii) an isolated glucosyltransferase enzyme that synthesizes alpha-1,3-glucan (e.g., according to embodiment 4), and optionally further comprising at least one isolated alpha-glucan and / or lactase enzyme.

[0170] Non-limiting examples of compositions and methods / processes disclosed in this document include: 1a. A method (process) for producing a food product / precursor, wherein the method comprises: (a) providing a food product / precursor comprising at least water, sucrose and at least one carbohydrate-containing ingredient (optionally, the sucrose is contained in the carbohydrate-containing ingredient, which has one or more other carbohydrates besides sucrose; regardless of the source of the sucrose in step (a), the carbohydrate-containing ingredient has one or more other carbohydrates, as disclosed herein), and (b) bringing the food product / precursor into contact with at least one glucosyltransferase enzyme, wherein the glucosyltransferase enzyme is: (i) an enzyme Petition 870250087473, dated 09 / 26 / 2025, pages 230 / 302 89 / 133 glucosyltransferase that synthesizes alpha-1,6-glucan, wherein at least about 50% of the glycosidic linkages of 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 alpha-1,3-glucan are alpha-1,3 linkages, typically wherein at least one alpha-glucan is produced in the food product / precursor, wherein the carbohydrate-containing ingredient comprises at least one acceptor molecule for at least one glucosyltransferase enzyme, whereby the food product / precursor, after step (b), optionally possesses one or more of the following characteristics compared with the food product / precursor before step (b): (I) increased texture (or viscosity, consistency, structure, mouthfeel),but wherein the increased texture is less than it would be if the carbohydrate-containing ingredient were not present in the food product / precursor (i.e., the degree of texture formation mediated by glucosyltransferase is reduced by the carbohydrate-containing ingredient), (II) reduced sugar content, but wherein the reduction in sugar content is less than it would be if the carbohydrate-containing ingredient were not present in the food product / precursor (i.e., the degree of sugar reduction mediated by glucosyltransferase is reduced by the carbohydrate-containing ingredient), typically wherein the sugar content comprises monosaccharides and disaccharides of the food product / precursor, and / or (III) reduced sweetness, but wherein the reduced sweetness is less than it would be if the carbohydrate-containing ingredient were not present in the food product / precursor (i.e., the degree of sweetness reduction mediated by glucosyltransferase is reduced by the carbohydrate-containing ingredient, i.e.,The carbohydrate-containing ingredient helps maintain sweetness. 2a. The method of embodiment 1a, wherein step (b) comprises bringing the food product / precursor into contact with at least one glucosyltransferase enzyme and a second enzyme using Petition 870250087473, dated 09 / 26 / 2025, pp. 231 / 302 90 / 133 sucrose as a substrate (wherein such second enzyme is not a glucosyltransferase in the present document), wherein the second enzyme that uses sucrose as a substrate is an invertase enzyme (for example, an invertase comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 14) or a fructosyltransferase enzyme; 3a. The method of embodiment 1a or 2a, in which the 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 or 12; 4a. The method of embodiment 1a, 2a or 3a, in which the glucosyltransferase enzyme that synthesizes alpha-1,3-glucan comprises an amino acid sequence that is at least 90% identical to residues 55-960 of SEQ ID NO: 5, residues 54-957 of SEQ ID NO: 6, residues 55-960 of SEQ ID NO: 7, residues 55-960 of SEQ ID NO: 8, residues 55-960 of SEQ ID NO: 9, or to SEQ ID NO: 13; 5a. The method of embodiment 1a, 2a, 3a or 4a, in which both (i) the glucosyltransferase enzyme that synthesizes alpha-1,6-glucan and (ii) the glucosyltransferase enzyme that synthesizes alpha-1,3-glucan are used in step (b); 6a. The method of embodiment 1a, 2a, 3a, 4a or 5a, wherein the alpha-glucan produced in step (b) comprises a graft copolymer comprising: (i) a backbone of alpha-1,6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1,6-glucan backbone are alpha-1,6 linkages, and (ii) at least one sidebone of alpha-1,3-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1,3-glucan backbone are alpha-1,3 linkages, wherein the alpha-glucan is aqueous soluble or aqueous insoluble; 7a. The method of embodiment 1a, 2a, 3a, 4a, 5a or 6a, in which step (a) comprises the addition of sucrose to the food product / precursor; 8a. The method of modality 1a, 2a, 3a, 4a, 5a, 6a or 7a, in Petition 870250087473, dated 09 / 26 / 2025, pages 232 / 302 91 / 133 that the food product / precursor is a dairy food product / precursor; 9a. The method of embodiment 1a, 2a, 3a, 4a, 5a, 6a or 7a, in which the food product / precursor is a non-dairy food product / precursor; 10a. The method described in modality 9a, in which the non-dairy food product / precursor is plant-based; 11a. The method of embodiment 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a or 10a, in which the food product / precursor of step (a) is fermented or the method additionally comprises, during or after step (b), the fermentation of the food product / precursor; 12a. The method of embodiment 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a or 11a, in which the food product / precursor is a yogurt; 13a. The method of embodiment 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a or 12a, in which the food product / precursor is a condiment (for example, a tomato-based condiment, such as tomato ketchup), such as ketchup; 14a. The method of embodiment 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a, 12a or 13a, wherein step (b) further comprises bringing the food product / precursor into contact with at least one isolated lactase enzyme; 15a. The method of embodiment 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a, 12a, 13a or 14a, in which the carbohydrate-containing ingredient comprises a tomato-based ingredient (for example, tomato paste, tomato concentrate, tomato puree or tomato sauce); 16a. The method of embodiment 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a, 12a, 13a, 14a or 15a, wherein the carbohydrate-containing ingredient comprises syrup (for example, corn syrup or high-fructose corn syrup); Petition 870250087473, dated 09 / 26 / 2025, pages 233 / 302 92 / 133 17a. The method of embodiment 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a, 12a, 13a, 14a, 15a or 16a, wherein the carbohydrate-containing ingredient comprises a monosaccharide (for example, any one in this document), disaccharide (for example, any one in this document) and / or oligosaccharide (for example, any one in this document, such as a gluco-oligosaccharide) (for example, the carbohydrate-containing ingredient comprises a monosaccharide and / or disaccharide); 18a. The method of embodiment 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a, 12a, 13a, 14a, 15a, 16a or 17a, in which the carbohydrate-containing ingredient comprises fructose and / or glucose; 19a. The method of embodiment 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a, 12a, 13a, 14a, 15a, 16a, 17a or 18a, wherein the food product / food precursor, after step (b), has the characteristic of (I), compared with the food product / precursor before step (b), of increased texture (or viscosity, consistency, structure, mouthfeel), but wherein the increased texture is less than it would be if the carbohydrate-containing ingredient were not present in the food product / precursor (i.e., the degree of texture formation mediated by glucosyltransferase is reduced by the carbohydrate-containing ingredient; i.e., excessive gelation as mediated by glucosyltransferase is avoided to obtain a desirable amount of gelation); 20a. The method of embodiment 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a, 12a, 13a, 14a, 15a, 16a, 17a, 18a or 19a, which additionally comprises homogenizing the food product / precursor after step (b); 21a. A food product or food precursor (isolated food product / precursor) produced by the method of embodiment 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a, 12a, 13a, 14a, 15a, 16a, 17a, 18a, 19a or 20a; and Petition 870250087473, dated 09 / 26 / 2025, pages 234 / 302 93 / 133 22a. An isolated food product or food precursor comprising at least one of (i) an isolated glucosyltransferase enzyme that synthesizes alpha-1,6-glucan (for example, according to embodiment 3a) and / or (ii) an isolated glucosyltransferase enzyme that synthesizes alpha-1,3-glucan (for example, according to embodiment 4a), and optionally further comprising at least one alpha-glucan, wherein the isolated food product or food precursor may be any of those presently disclosed, such as those mentioned in any of embodiments 8a, 9a, 10a, 11a, 12a or 13a. EXAMPLES

[0171] The present disclosure is further exemplified by the following examples. It should be understood that these examples, while indicating certain aspects in the present document, are provided for illustrative purposes only. From the above discussion and these examples, a person skilled in the art can determine the essential characteristics of the disclosed embodiments and, without departing from their spirit and scope, can make various alterations and modifications to adapt the disclosed embodiments to various uses and conditions. Materials / Methods Analysis of sugar content by HPLC

[0172] The sugar composition was measured by high-performance liquid chromatography (HPLC) using a Waters® 2695 separation module or a ThermoScientific Dionex™ UltraMate 3000 HPLC instrument, equipped with a Phenomonex Rezex™ RPM-Monosaccharide Pb2+ column (300 mm x 7.8 mm) and a refractive index (RI) 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 appropriate dilution in water, optionally centrifugation (10 min at 15,000 rpm) and Petition 870250087473, dated 09 / 26 / 2025, pages 235 / 302 94 / 133 sterilizing filtration. The HPLC signals were quantified relative to calibration standards of sugars eluted simultaneously. Sugar reduction was calculated by subtracting the total sum of mono- and disaccharides in the test sample from the total sum of mono- and disaccharides in the reference sample without the addition of enzymes. Yogurt preparation

[0173] Batch-mixed, pre-pasteurized (72 °C for 15 s) skimmed milk (0.1% fat) (Arla Foods, Denmark), stored at 4-6 °C, was standardized to a desired protein (% w / w), fat (% w / w) and sucrose (% w / w) content by adding skimmed milk powder (33% protein, 1.2% fat, 54% carbohydrate) from BBA Lactalis (Laval, Mayenne, France), cream (38% fat) from Arla Foods and sucrose (Granulated Sugar 500, Nordic Sugar A / S, Denmark). The standardized milk thus prepared was then pasteurized and homogenized in a plate heat exchanger pasteurizer. Homogenization was carried out at 65 °C and 200 bar, and pasteurization at 95 °C for 6 minutes; Next, the milk was cooled to 43 °C. The milk was inoculated with a thermophilic starter culture at an inoculation rate of 20 DCU / 100 L; all cultures were from IFF. Fermentation was conducted until pH 4.60, after which the product was cooled to 24 °C.The resulting yogurts were stored at 4-6 °C for viscosity measurements. Method for measuring apparent viscosity

[0174] A rotational rheological test was employed to evaluate the viscosity of the produced samples. Flow curves were obtained using an Anton Paar MCR302 rheometer (Anton Paar GmbH, Ostfildern, Germany) using an ST22-4V-40 vane geometry for aluminum cups. Samples were placed in C-CC27 aluminum cups and stored at 5 °C for at least 5 hours before analysis. The shear rate ranges applied to the samples were 0.1–350 s⁻¹, which defines the curve. Petition 870250087473, dated 09 / 26 / 2025, pages 236 / 302 95 / 133 ascending, and the reverse operation explains the descending curve (350-0.1 s-1). The measurement point duration value was selected to be at least as long as the reciprocating shear rate value, which is valid for the ascending curve. Tests were performed at a constant temperature of 10 °C, and each sample was analyzed in duplicate. A water bath was connected to the rheometer to ensure isothermal conditions.

[0175] Apparent viscosity was evaluated from flow curves, which is appropriate for fluids where the relationship between shear stress and shear rate varies with shear rate. Apparent viscosity was extracted at a shear rate of 11.7 Hz or 249 Hz. The apparent viscosity extracted at a shear rate of 11.7 Hz indicated the “consistency” of the sample. The apparent viscosity extracted at a shear rate of 249 s-1 (249 Hz) was correlated with the sensory perception of “mouthfeel”. Measuring the physical characteristics of ice cream.

[0176] The melt stability of the ice cream was analyzed according to Granger et al. (2005, Int. Dairy J. 15(3): 255-262, incorporated herein by reference), with minor modifications. A pre-weighed block of ice cream (60-70 g) was placed in a grid (mesh size 0.5 cm x 0.5 cm) at 20 °C. The grid with the ice cream was placed above a balance with a beaker. During the melting of the ice cream, the liquid flowed down the grid into the beaker placed on the balance. The liquid in the beaker was weighed every 10 seconds. Melting was defined as the percentile of the liquid in the beaker at a given time, divided by the initial weight of the ice cream in the grid at the start of the measurement. In addition, the time between placing the ice cream sample in the grid and the moment the first drop of melted ice cream reached the beaker (first drop) was recorded.

[0177] The texture of the ice cream was analyzed according to Parvar et al. Petition 870250087473, dated 09 / 26 / 2025, pp. 237 / 302 96 / 133 (2013, Food Biosci. 3: 10-18, incorporated herein by reference), with minor modifications. The analysis was performed using a TA.XTPLUS Texture Analyzer equipped with a P / 10 cylindrical measuring probe (Stable Micro System, Surrey, UK). A block of ice cream (0.24 L; 10.4 cm x 5.4 cm x 4.4 cm) was penetrated with the texture analyzer at a speed of 2 mm s⁻¹ to a depth of 15 mm. The probe was withdrawn from the ice cream at a speed of 2 mm s⁻¹. The block was measured at three points (center, 1.5 cm left of center, 1.5 cm right of center). The hardness and cohesiveness of the ice cream were defined as, respectively, the maximum force required to penetrate (maximum positive force) or extract (maximum negative force) the probe from the ice cream sample. The adhesiveness of the ice cream was defined as the negative area when plotting the force over the analysis time. All analyses were performed at a constant ice cream temperature (15 °C ± 2 °C).Since each analysis took less than a minute, external cooling of the sample was not necessary. Ultra-high performance liquid chromatography (UHPLC)

[0178] UHPLC was performed following the methodology published in Gardana et al. (2018, Journal of Chromatography A 1578: 8-14), which is incorporated herein by reference. The analysis of different steviol glycosides was performed on an Agilent 1290 UHPLC system (containing a 1290Bio multisampler, 1290 binary pump, 1290 thermostated column compartment, and a 1260 diode array detector). A Waters Acquity UPLC BEH amide column (150 x 3.0 mm internal diameter, 1.7 µm) was used for separation. The column was maintained at 35 °C and the samples at 15 °C. The mobile phases were (A) 0.05% formic acid in water and (B) 0.05% formic acid in acetonitrile. The flow rate was 0.3 mL / min and the gradient was (% of A / min): 10 / 0, 35 / 20, 50 / 20.5, 21 / 50, 10 / 21.5 or 10 / 25. Steviol glycosides were detected using a diode array detector at 200 nm. Petition 870250087473, dated 09 / 26 / 2025, pp. 238 / 302 97 / 133

[0179] Standards of stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, rebaudioside N, rubusoside and dulcoside A were dissolved in methanol (approximately 1 mg / mL) and diluted with water to be used as retention time standards.

[0180] The samples were centrifuged and the upper phase was transferred to an HPLC vial and analyzed directly by the UHPLC method. Example 1 Dextran-alpha-1,3-glucan graft copolymer produced in situ in yogurt.

[0181] In this example, a graft copolymer possessing a dextran backchain with alpha-1,3-glucan side chains was produced in yogurt in situ using a combination of a dextran saccharase and an alpha-1,3-glucan sucrase. This glucan production coincided with sugar-reducing and texturizing effects in the yogurt.

[0182] The sugar-reducing and texturizing effects of using glucosyltransferase (GTF) 0768 (SEQ ID NO: 1, also represented by SEQ ID NOs: 2, 11 and 12) and an amino acid-substituted GTF 6855 variant (SEQ ID NO: 3, “vGTFJ” in this document) alone or in combination were investigated in a yogurt production system (scale: 60 g). Both GTF enzymes use sucrose as a substrate to produce fructose and glucan (i.e., they are glucansucrases). GTF 0768 (a dextranassacrase) produces a soluble alpha-glucan with a high content of alpha-1,6 linkages (i.e., a type of dextran; see U.S. Patent Application Publication No. 20160122445, which is incorporated herein by reference), while vGTFJ stably produces in high yield an insoluble alpha-glucan that has about 100% alpha-1,3 linkages (the variant Petition 870250087473, dated 09 / 26 / 2025, pp. 239 / 302 98 / 133 GTF from SEQ ID NO: 4 can be used similarly in this document to produce a stable and high-yield insoluble alpha-glucan that has approximately 100% alpha-1,3 linkages. Fresh milk standardized to 4.0% (w / w) protein, 1.0% (w / w) fat, and 6.0% (w / w) sucrose was homogenized and pasteurized as described in Materials / Methods. GTF enzymes were then added at the inoculation step, as shown in Table 3. 100% GTF 0768 or 100% vGTFJ is the amount of each respective enzyme sample required to completely convert the sucrose within the fermentation time. YO-MIX 410 was used as a starter culture (available from IFF). After 3 days of storage at 5 °C, the sugar content (by HPLC) and texture in each yogurt sample were evaluated according to the Materials / Methods section. The resulting sugar content is shown in FIG.1 and the resulting apparent viscosity at shear rates of 11.7 Hz and 249 Hz is shown in FIGS. 2 and 3, respectively. Table 3 Sample ID GTF 0768 (SEQ ID NO: 1) (pL) vGTFJ (SEQ ID NO: 3) (pL) 100:0 % (GTF 0768:vGTFJ) 37.5 0 90:10 % (GTF 0768:vGTFJ) 33.75 24.7 75:25 % (GTF 0768:vGTFJ) 28.125 61.75 50:50 % (GTF 0768:vGTFJ) 18.75 123.5 25:75 % (GTF 0768:vGTFJ) 9.375 185.25 10:90 % (GTF 0768:vGTFJ) 3.75 222.3 0:100 % (GTF 0768:vGTFJ) 0 247 2% starch 0 0 3% starch 0 0

[0183] It was found that all samples with one or both glucosyltransferase enzymes added achieved at least a 25% reduction in sugar compared to reference samples containing starch (FIG. 1). The greatest reduction in sugar was found when only GTF 0768 was used and the least when only vGTFJ was used. This was due to the fact that GTF 0768, compared to vGTFJ, uses lactose as Petition 870250087473, dated 09 / 26 / 2025, pages 240 / 302 99 / 133 acceptor molecule to a greater extent and release less free glucose byproduct (data not shown).

[0184] Furthermore, it became clear that treatment with vGTFJ alone (0:100 % (GTF 0768:vGTFJ)) resulted in an increase in apparent viscosity at 11.7 Hz and 249 Hz compared to what was previously described in WO2020 / 010176 for another alpha-1,3-glucan producing GTF and that treatment with GTF 0768 alone (100:0 % (GTF 0768:vGTFJ)) resulted in low apparent viscosity (FIGs. 2 and 3). Furthermore, it was found (FIGs. 2 and 3) that some of the enzyme combinations resulted in only a slight increase in apparent viscosity (10:90 % (GTF 0768:vGTFJ), 25:75 % (GTF 0768:vGTFJ) and 50:50 % (GTF 0768:vGTFJ)) compared to the dosage of the GTF 0768 enzyme alone (100:0 % (GTF 0768:vGTFJ)). Surprisingly, the apparent viscosity of the 90:10% (GTF 0768:vGTFJ) samples at 11.7 Hz and 249 Hz exceeded the apparent viscosity obtained when only vGTFJ was used; this was especially the case for mouthfeel perception at a shear rate of 249 Hz (FIG. 3). The addition of 90:10% (GTF 0768:vGTFJ) had a significant impact on texture and was the only sample treated with GTF to exceed the apparent viscosity obtained when using 3% starch (FIGs. 2 and 3). Thus, the texture of the yogurt product could be adapted simply by adjusting the dosage ratio of GTF 0768 and vGTFJ.

[0185] Since vGTFJ (SEQ ID NO: 3) forms insoluble alpha-1,3-glucan and GTF 0768 (SEQ ID NO: 1) forms a soluble dextran, it was hypothesized that a specific ratio of these two enzyme types results in the formation of a dextran-soluble alpha-1,3-glucan graft copolymer that could provide a gel-like texture. This hypothesis was supported by the flow curves presented in FIG. 4. Samples with insoluble alpha-1,3-glucan had an initial shear thinning effect at a rate of Petition 870250087473, dated 09 / 26 / 2025, pages 241 / 302 100 / 133 shear below 40 Hz and then increased slightly in shear stress with an increasing shear rate. In contrast, the samples with soluble dextran and the samples with dextran-alpha-1,3-glucan graft copolymer had a shear thickening effect very similar to that of starch. Example 2 Dextran-alpha-1,3-glucan graft copolymers produced in situ in fermented yogurt with various cultures.

[0186] The sugar-reducing and texturizing effects of GTF 0768 (SEQ ID NO: 1) and vGTFJ (SEQ ID NO: 3), alone or in combination, were investigated in a yogurt production system (scale: 60 g). Fresh milk was standardized to 4.0% (w / w) protein, 1.0% (w / w) fat, 8.0% (w / w) sucrose, and 3% starch for the reference samples, which were homogenized and pasteurized as described in Materials / Methods. GTF enzymes were added at the dosage level shown in Table 4, where 100% of GTF 0768 or vGTFJ was the amount of enzyme sample required to completely convert the sucrose within the fermentation time. YO-MIX 863, YO-MIX PRIME 900, YO-MIX M01, and YO-MIX T42 were used as starter cultures (available from IFF).After 14 days of storage at 5 °C, the sugar content of each yogurt sample prepared in this way was quantified according to HPLC (Materials / Methods), and the sweetness and texturizing effect were evaluated by sensory assessment. The sensory assessment was carried out with a trained panel of ten individuals. Three training sessions and three analysis sessions were conducted with 30 mL sample portions at 10 °C. Rosehip tea and saltine crackers were used for mouthwashes between samples. The relative sugar content in each sample is shown in FIG. 5. The sensory data are presented in radar charts in FIGS. 6A-E, and the sweetness relative to the starch references is... Petition 870250087473, dated 09 / 26 / 2025, pages 242 / 302 101 / 133 shown in FIG. 7. Table 4. Enzyme dosages Sample ID GTF 0768 (SEQ ID NO: 1) vGTFJ (SEQ ID NO: 3) 100:0 % (GTF 0768:vGTFJ) 100% 0 0:100 % (GTF 0768:vGTFJ) 0 100% 90:10 % (GTF 0768:vGTFJ) 90% 10% 3% starch 0 0

[0187] It was found that all samples with added glucosyltransferase enzymes achieved at least 35% sugar reduction and up to 52% sugar reduction compared to reference samples containing starch.

[0188] As in Example 1, the yogurt treatment with only GTF 0768 (100:0% (GTF 0768:vGTFJ)) had a low impact on texture and is therefore not shown in the radar charts of FIGS. 6A-E, while the treatments with only vGTFJ (0:100% (GTF 0768:vGTFJ)) or both vGTFJ and GTF 0768 (90:10% (GTF 0768:vGTFJ)) both had a significant increase in yogurt texture. The treatment with only vGTFJ also provided a softer / shinier and less grainy / floury yogurt compared to the reference yogurt with starch. It was surprisingly discovered that more than 80% of the sweetness (relative to the starch reference) was retained in yogurt with mild cultures (pH 4.6) (YO-MIX PRIME 900 and YO-MIX M01), which contrasted with what was observed with yogurts with more acidic cultures (pH 4.3) (YO-MIX 863, YO-MIX 410, YO-MIX T42) (FIG. 7), although all the sucrose was converted in each enzyme-treated yogurt. Example 3 Combined addition of glucosyltransferase and beta-galactosidase in yogurt production.

[0189] The sugar-reducing and texturizing effects of GTF 0768 (SEQ ID NO: 1) and vGTFJ (SEQ ID NO: 3), alone or in combination, Petition 870250087473, dated 09 / 26 / 2025, pages 243 / 302 102 / 133 were investigated in conjunction with beta-galactosidase that has lactase activity or both lactase and transgalactosylation activity in a yogurt production system (scale: 60 g). The individual dosages of the enzymes GTF 0768 and vGTFJ were normalized where a dosage of 100% was the dosage required to provide total sucrose conversion when added at the inoculation step. Lactase Bonlacta™ (IFF) (i.e., a type of beta-galactosidase) was dosed at 0.9% (v / w) and Nurica™ (IFF), a lactase with transgalactosylation activity (produces galacto-oligosaccharides (GOS)), was dosed at 0.21% (v / w). Fresh milk was standardized to 4.0% (w / w) protein, 1.0% (w / w) fat, and 8.0% (w / w) sucrose and homogenized and pasteurized as described in Materials / Methods. All enzymes were added at the inoculation step according to the dosages shown in Table 5. YO-MIX 410 was used as the starter culture.After three days of storage at 5 °C, the sugar content and texture of the yogurt samples were evaluated according to the Materials / Methods section. The resulting sugar content is shown in FIG. 8 as values ​​relative to the total carbohydrates quantified in each reference sample. The resulting apparent viscosities at shear rates of 11.7 Hz and 249 Hz are shown in FIGS. 9 and 10, respectively. Table 5. Enzyme dosages Sample ID BONLACT A (pL) NURIC A (pL) GTF 0768 (SEQ ID NO: 1) (PL) vGTFJ (SEQ ID NO: 3) (PL) Ref - Reference 0 0 0 0 Ref - 100:0% (GTF 0768:vGTFJ) 0 0 32 0 Ref - 0:100% (GTF 0768:vGTFJ) 0 0 0 210 Ref - 90:10 % (GTF 0768:vGTFJ) 0 0 28 21 Bon - Reference 54 0 0 0 Bon - 100:0 % (GTF 0768:vGTFJ) 54 0 32 0 Bon - 0:100 % (GTF 0768:vGTFJ) 54 0 0 210 Bon - 90:10 % (GTF 0768:vGTFJ) 54 0 28 21 Petition 870250087473, dated 09 / 26 / 2025, pp. 244 / 302 103 / 133 ID da amostra BONLACT A (ML) NURIC A (ML) GTF 0768 (SEQ ID NO: 1) (ML) vGTFJ (SEQ ID NO: 3) (ML) Nur - Referência 0 128 0 0 Nur - 100:0 % (GTF 0768:vGTFJ) 0 128 32 0 Nur - 0:100 % (GTF 0768:vGTFJ) 0 128 0 210 Nur - 90:10 % (GTF 0768:vGTFJ) 0 128 28 21

[0190] In the reference sample with only BONLACTA, lactose was completely hydrolyzed (data not shown) and only a small amount of DP3 oligomers was formed, therefore approximately 95% of the carbohydrates in the sample were still considered sugar (i.e., DP1 (glucose, galactose) and DP2 (sucrose and lactose)) (FIG. 8). The reference sample with NURICA had only 73.7% of the carbohydrates still considered sugar, as NURICA formed galacto-oligosaccharides and lactosaccharides. The addition of GTF 0768 and / or vGTFJ resulted in a 37-46% reduction in sugar (resulting in 5463% residual sugar) in the samples treated without beta-galactosidase or with BONLACTA, leaving only 44% of the carbohydrates as sugar in the samples (FIG. 8).

[0191] The use of a beta-galactosidase together with GTF 0768 and / or vGTFJ resulted in a lower apparent viscosity at both shear rates of 11.7 Hz and 249 Hz compared to samples without beta-galactosidase (FIGs. 9 and 10). The apparent viscosity at both shear rates of 11.7 Hz and 249 Hz was, however, still significantly higher than the reference samples without the addition of glucosyltransferase enzyme(s). Again, consistent with Example 1, the samples with the addition of 90:10% (GTF 0768:vGTFJ) had the highest perceived mouthfeel at an apparent viscosity of 249 Hz, regardless of whether they were used with a beta-galactosidase or not. Thus, it was possible to combine the use of GTF 0768 and vGTFJ with a beta Petition 870250087473, dated 09 / 26 / 2025, pp. 245 / 302 104 / 133 galactosidase to obtain a significant increase in texture and, at the same time, make the yogurt product lactose-free (with BONLACTA) or with an even lower sugar content (with NURICA). Example 4 Texturizing effect and sugar reduction on various neutral substrates using glucosyltransferase enzymes in situ.

[0192] The texturizing effect of GTF 0768 (SEQ ID NO: 1), GTF 0974 (SEQ ID NO: 13) and a variant GTF 6855 from S. salivarius (“v2GTFJ”) on various milk preparations and buffered substrates was investigated. GTF 0974 (U.S. Appl. Publication No. 2018 / 0291311, incorporated herein by reference) represents a glucosyltransferase-SI enzyme from S. salivarius that produces alpha-1,3-glucan with approximately 93% alpha-1,3 linkages and approximately 7% alpha-1,6 linkages. v2GTFJ is a glucosyltransferase enzyme from S. salivarius with amino acid modification that produces alpha-1,3-glucan with approximately 100% alpha-1,3 linkages in high yield (U.S. Appl. Publication No. 2018 / 0072998, incorporated herein by reference). The AE substrates were prepared as described in Table 6.GTF 0768, GTF 0974, or v2GTFJ were then added to each substrate and incubated for 24 hours at 5 °C to completely convert the sucrose in each preparation, after which the enzymes were inactivated by heat (10 min, 95 °C). After three days of storage at 5 °C, the sugar content and texture of each preparation were evaluated according to Materials / Methods. The absolute sugar content in each preparation is shown in FIG. 11, and the percentage reduction of total sugar relative to each respective reference preparation (as initially prepared – without the addition of GTF and before incubations with other enzymes as per Table 6) is shown in FIG. 12. FIG. 12 also shows the relative content of oligosaccharides (DP3+) and polysaccharides of each preparation. The resulting apparent viscosities are shown in... Petition 870250087473, dated 09 / 26 / 2025, pages 246 / 302 105 / 133 shear rates of 11.7 Hz and 249 Hz are shown in FIGS. 13 and 14, respectively. Table 6. Preparation of AE substrates Substrate Preparation Procedure A. Sucrose was dissolved in MINI MILK (4.8% lactose) (Arla Foods, Denmark) to a final concentration of 7.5% (w / w). B. 1.7 g / L of BONLACTA was added to MINI MILK (4.8% lactose) and the milk was incubated at 5°C for 24 hours. The enzyme was then inactivated by heating the milk to 95°C for 10 min. The milk was cooled before the addition of sucrose to a final concentration of 7.5% (w / w). C. 3.2 g / L of NURICA was added to MINI MILK (4.8% lactose) and the milk was incubated at 5°C for 24 hours. The enzyme was then inactivated by heating the milk to 95°C for 10 min. The milk was cooled before the addition of sucrose to a final concentration of 7.5% (w / w). D Maltose was dissolved in MINI MILK (4.8% lactose) to a final concentration of 5% (w / w) before adding sucrose to a final concentration of 7.5% (w / w). E Maltose was dissolved in 0.05 M sodium phosphate buffer (pH 5.5) to a concentration of 5% (w / w).The FoodPro® TGO enzyme was then added at 0.125 g / L and incubated at 60 °C for 1 hour. The enzyme was then inactivated by heating at 95 °C for 10 min. The sample was cooled to 5 °C before adding sucrose to a final concentration of 7.5% (w / w).

[0193] It was found that a sugar reduction > 35% could be achieved in all substrates treated with a GTF enzyme (FIG. 11). The highest level of sugar reduction (> 50%) observed in substrates treated with GTF was with substrates D and E, which initially contained maltose (substrate D) or isomalto-oligosaccharides (IMO) (substrate E, possibly also some residual maltose). It is observed that substrate E, before treatment with GTF, contained IMO by virtue of having been treated with the FoodPro® TGO enzyme, which is a transglucosidase that converts maltose into IMO (mainly DP3-DP5) (mainly by glucose transfer from maltose to other maltose or already extended maltose). Based on these data, it is clear that each GTF enzyme added to substrates D and E used maltose and / or IMO as acceptor molecules for the synthesis of oligo- and polysaccharides.When comparing AC substrates with DE substrates, GTF 0974 caused greater polysaccharide formation in AC substrates, while GTF 0768 and v2GTFJ caused greater oligosaccharide formation in DE substrates (FIG. 12).

[0194] Surprisingly, just the addition of GTF 0768 or Petition 870250087473, dated 09 / 26 / 2025, pp. 247 / 302 The addition of 106 / 133 v2GTFJ resulted in increases in apparent viscosity at both shear rates of 11.7 Hz and 249 Hz on substrates AC (FIGs. 13-14). In contrast to the fresh fermented yogurt samples (see above), GTF 0768 had the greatest impact on apparent viscosity at the 249 Hz shear rate, most likely due to the fact that it produces soluble polysaccharide. The change in reaction product characteristics, primarily towards oligosaccharides on substrates D and E, resulted in samples with little or no increase in texture. It was therefore clear that by adding a GTF enzyme such as GTF 0768 or v2GTFJ, a high reduction in sugar (> 50%) could be achieved in food products containing both sucrose and maltose (and / or IMO) without significantly altering the texture. Example 5 Dextran-alpha-1,3-glucan graft copolymers produced in situ in neutral beverages

[0195] The texturizing effect of GTF 0768 (SEQ ID NO: 1) and vGTFJ (SEQ ID NO: 3) added alone or in combination was investigated in two neutral beverage recipes. The beverage base was 10% (w / w) skimmed milk powder and 5% (w / w) sucrose with (B) or without (A) 4% (w / w) maltose dissolved in tap water. GTF enzymes were added to the cold beverage base as shown in Table 7 and incubated for 24 hours at 5 °C before thermal inactivation of the GTF enzymes at 95 °C for 10 minutes. The individual enzymatic dosages of the GTF enzymes were normalized where the 100% dosage is the dosage required to provide total sucrose conversion during the 24 hours at 5 °C. After three days of storage at 5 °C, the sugar content and texture of each preparation were evaluated according to the Materials / Methods section. FIGS. 15A (without maltose) and 15B (with maltose) show the percentage reduction in total sugar compared to the reference preparation. FIGS.Tables 15A-B also show the relative content of oligosaccharides. Petition 870250087473, dated 09 / 26 / 2025, pages 248 / 302 107 / 133 (DP3+) and polysaccharides from each preparation. The resulting apparent viscosities at shear rates of 11.7 Hz and 249 Hz are shown in FIGS. 16 and 17, respectively. Table 7. Enzyme dosages Sample ID GTF 0768 (SEQ ID NO: 1) vGTFJ (SEQ ID NO: 3) 100:0 % (GTF 0768:vGTFJ) 100% 0% 90:10 % (GTF 0768:vGTFJ) 90% 10% 75:25 % (GTF 0768:vGTFJ) 75% 25% 50:50 % (GTF 0768:vGTFJ) 50% 50% 25:75 % (GTF 0768:vGTFJ) 25% 75% 10:90 % (GTF 0768:vGTFJ) 10% 90% 0:100 % (GTF 0768:vGTFJ) 0% 100% reference 0% 0%

[0196] Although vGTFJ had a high texturizing effect on fresh fermented products (see above), it was found in this example to have almost no impact on texture (FIGs. 16-17) (similar to v2GTFJ, see above) due to the production of insoluble polysaccharides. Surprisingly, the combined addition of GTF 0768 and vGTFJ, which resulted in the formation of soluble dextran-alpha1,3-glucan graft copolymer, was found to have an impressive effect on texture readings of consistency and mouthfeel (FIGs. 16-17). Furthermore, the texture could be tailored by adjusting the mixing ratio of GTF 0768 and vGTFJ in the maltose-free neutral beverage. The use of GTF 0768 alone also increased the texture; However, the polysaccharides formed were considered too sticky / viscous, which would typically not be preferred for a smooth-drinking beverage.Consistent with the results of Example 4, it was generally possible, with GTF treatment, to obtain a higher percentage of sugar reduction in neutral beverages containing maltose (compared to neutral beverages without maltose) without significantly altering the texture, which is due to a greater preference for the formation of oligosaccharides (compare FIGS. 15A and 15B). Petition 870250087473, dated 09 / 26 / 2025, pages 249 / 302 108 / 133 Example 6: Thickening of plant-based yogurt using glucosyltransferase enzymes in situ.

[0197] vGTFJ (SEQ ID NO: 3) or a combination of GTF 0768 (SEQ ID NO: 1) and vGTFJ (90:10% (GTF 0768:vGTFJ)) were evaluated in coconut yogurt. Starch-stabilized yogurt, made with 6% sucrose, 30% coconut cream (iTi Tropicals, Lawrence Township, New Jersey, USA), 6% tapioca starch, and 58% water, was used as a reference. The experimental yogurt base contained 6% sucrose, 30% coconut cream, and 64% tap water. The ingredients for each of the two bases were mixed under high agitation before pasteurization, which was performed by short-duration pasteurization at high temperature at 195 °F (90.56 °C) for 30 seconds. Homogenization occurred downstream of the heating stage at a total pressure of 3,000 psi in a two-stage homogenizer. After homogenization, each mixture was cooled to 110 °F (43.3 °C), after which fermentation was carried out with Danisco® VEGE 022 culture.Fermentation was considered complete when the yogurt pH reached 4.65 or less. Two different yogurt samples were made by adding GTF enzyme(s) to the experimental base during the fermentation step: (i) vGTFJ alone (0:100% (GTF 0768:vGTFJ)) and (ii) a combination of GTF 0768 and vGTFJ (90:10% (GTF 0768:vGTFJ)). A third yogurt was prepared in which no GTF enzyme was added to the experimental base during the fermentation step. A yogurt was also prepared using the reference yogurt base (base with tapioca starch) without the addition of GTF enzyme.

[0198] It became clear that the addition of GTF enzyme(s) provided a color benefit to the yogurt products. The reference yogurt with starch showed a yellowish color. However, the two experimental yogurts with one or both GTF enzymes did not exhibit a significant color change. Petition 870250087473, dated 09 / 26 / 2025, pages 250 / 302 109 / 133 (data not shown) compared to yogurt produced (white color) using the experimental base without the addition of GTF enzyme.

[0199] The viscosity of each yogurt was measured over a period of time representing an example of product shelf life (63 days) (FIG. 18). Measurements were taken 1, 7, 21, 35, or 63 days after yogurt production using a Brookfield DV3T™ viscometer (Brookfield Engineering Laboratories, Middleboro, MA, USA) with an RV 6 spindle after 30 s at a rotational speed of 20 rpm at 40 °F (4.4 °C). The pH values ​​of the yogurt measured over this period are presented in Table 8. Table 8. pH of yogurt Sample ID Day 1 Day 21 Day 35 Day 65 Reference (6% starch) 4.48 4.50 4.49 4.46 90:10% (GTF 0768:vGTFJ) 4.53 4.58 4.59 4.61 0:100% (GTF 0768:vGTFJ) 4.41 4.46 4.42 4.40

[0200] The sugar composition of the yogurt was also measured throughout this period on days 0, 14, 28, 42 and 56 by HPLC (Materials / Methods). The % (w / w) of total sugar in each sample is shown in FIG. 19.

[0201] The control yogurt without added GTF enzyme or starch showed low viscosity throughout its shelf life (FIG. 18). In contrast, the reference yogurt with starch and both experimental yogurts with added GTF enzyme(s) showed high viscosity. All yogurts had a similar pH from production to shelf life. In the samples containing added GTF enzyme(s), the sugar was reduced by approximately 40%, which was consistent throughout the tested shelf life. Furthermore, it was found that almost no oligosaccharides formed (< 0.6% w / w) in the GTF-treated yogurts, unlike what was observed in the dairy-based yogurts above (e.g., Examples 4-5) which initially contained endogenous or added disaccharides such as lactose or maltose. This lack of oligosaccharide production allowed for a higher proportion of polysaccharide formation, which in turn led to textural formation. Petition 870250087473, dated 09 / 26 / 2025, pages 251 / 302 110 / 133 pronounced (consistency) (FIG. 18). Example 7 Improved physical properties of ice cream produced using glucosyltransferase enzymes in situ

[0202] The ice creams were prepared according to Table 9 with or without various combinations of glucose syrup, hydrocolloid, NURICA at 0.32%, BONLACTA at 0.17%, GTF 0768 (SEQ ID NO: 1) and vGTFJ (SEQ ID NO: 3). The ice cream base for samples 2102-3-(2-7) was prepared by mixing water, skimmed milk powder (9%), sucrose (12%), whey powder (2.5%) and emulsifier (0.35%, CREMODAN SUPER MB) at room temperature before adding refined coconut oil (4%, KRISTAL) and heating to 70 °C. The ice cream bases were then homogenized at 78 °C and pasteurized at 84 °C for 30 seconds. After cooling to 5 °C, the various enzymes were added according to Table 9 during the ice cream maturation, and the mixtures were incubated for 24 hours before freezing (light extrusion with 100% overshoot) in a hardening operation at 30 °C.The individual enzymatic dosages of GTF 0768 and vGTFJ were standardized where a dosage of 100% is the dosage required to provide total sucrose conversion during 24 hours at 5 °C. A reference ice cream (2021-3-1) was also prepared containing additional glucose syrup (4%) and hydrocolloid (0.2%, CREMODAN DC 100).

[0203] The melting profile and texture attributes (hardness, cohesiveness, adhesiveness) of the ice cream samples were measured according to the Materials / Methods. Table 9 Sample ID Sample Description 2102-3-1 + hydrocolloid, + glucose syrup, without enzyme 2102-3-2 without enzyme 2102-3-3 100:0 % (GTF 0768:vGTFJ) 2102-3-4 0:100 % (GTF 0768:vGTFJ) Petition 870250087473, dated 09 / 26 / 2025, pages 252 / 302 111 / 133 Sample ID Sample Description 2102-3-5 10:90% (GTF 0768:vGTFJ) 2102-3-6 0:100% (GTF 0768:vGTFJ) and NURICA 2102-3-7 0:100% (GTF 0768:vGTFJ) and BONLACTA

[0204] All samples 2102-3-(3-7) had between 47-60% reduction in sugar compared to sample 2102-3-2. Surprisingly, all samples with added enzyme that included the vGTFJ enzyme had an improved melting profile (slower melting) compared to the plain ice cream base (sample 2102-3-2) (FIG. 20). This melting profile was at least equal to or better than the melting profile of the reference sample containing hydrocolloid and glucose syrup (sample 2102-3-1). Furthermore, it was found that when the vGTFJ enzyme was combined with GTF 0768 or a beta-galactosidase (NURICA or BONLACTA), the ice cream samples performed equivalently to the reference sample containing hydrocolloid and glucose syrup (sample 2102-3-1) in both cohesiveness and adhesiveness (FIG. 21B and 21C).Only the hardness increased in all cases using GTF 0768 or vGTFJ, but it was clear that the hardness could be improved (decreased) by the combined addition of GTF 0768 and vGTFJ (2102-3-5 vs. 2102-3-3 and 2102-3-4) or by a combined addition of vGTFJ and BONLACTA (2102-3-7) (FIG. 21A). Example 8 Improved physical properties of sweetened condensed milk produced using glucosyltransferase enzymes in situ

[0205] The in situ effect of glucosyltransferase enzymes was investigated in a condensed milk application.

[0206] The recipe for each experimental sample of condensed milk (trial nos. 1-8) is presented in Table 10. To produce each sample, 800 g of skimmed milk powder (Arla, Aarhus, Denmark), 1523 g of sucrose (Nordic Sugar, Copenhagen, Denmark) and approximately 800 g of water were mixed at 50 °C until no lumps were visible. The mixture was Petition 870250087473, dated 09 / 26 / 2025, pages 253 / 302112 / 133 additionally stirred and cooled to 5 °C. For assays 3-8, GTF 0768 (SEQ ID NO: 1) and / or vGTFJ (SEQ ID NO: 3) were added to individual mixtures, which were then further stirred for 10 minutes and left for 24 hours at 5 °C until further processing. For the GTF enzyme assay, 100% of GTF 0768 or vGTFJ was the amount of enzyme sample required to completely convert sucrose within 24 hours of incubation at 5 °C. Experimental samples 1 and 2 acted as references / controls and did not contain GTF enzyme, but otherwise were treated the same as experimental samples 3-8. Next, milk fat anhydrase (AMF, 315 g per assay, Coreman, Goe, Belgium) and an emulsifier (7 g of RECODANTMRS 100, not included in assays 2 and 3) were melted together at 70 °C, after which 7 g of lecithin (SOLEC SF-D, IFF, Copenhagen, Denmark) were added to the melted materials.The different melted materials and corresponding skim milk / sugar pastes were combined and mixed at 50 °C. The mixtures were subsequently pasteurized at 90 °C for 3 minutes, homogenized (35 bar, 80 °C), and cooled to 30 °C under vigorous stirring. Except for trial 3, lactose seeds (1.75 g of crystalline lactose, Variolac® 992, Arla, Viby, Denmark) were added during this mixing. All mixtures were then cooled to 15 °C and stored for 18 hours in a tank to allow crystallization. Subsequently, all condensed milk samples were placed in cups and cooled to 5 °C. Table 10. Sweetened condensed milk recipes Experimental sample number 1 2 3 4 5 6 7 8 Sample ID Ref Ref ()Est ab (-)Estab ()Seed 50:50 % (GTF 0768:vGT 100:0 % (GTF 0768:vGT FJ) 90:10 % (GTF 0768:vGT FJ) 50:50 % (GTF 0768:vGT FJ) 10:90 % (GTF 0768:vGT FJ) 0:100 % (GTF 0768:vGT FJ) Petition 870250087473, dated 09 / 26 / 2025, pp. 254 / 302 113 / 133 Experimental sample number 1 2 3 4 5 6 7 8 FJ) Butter oil (AMF) 9% 9% 9% 9% 9% 9% 9% 9% SOLEC SF-D 0.2% 0.2% 0.2% 0.2% 0.2% 0.2% 0.2% 0.2% Skimmed milk powder 22.85% 22.85% 22.85% 22.85% 22.85% 22.85% 22.85% 22.85% Sucrose 43.5% 43.5% 43.5% 43.5% 43.5% 43.5% 43.5% 43.5% Lactose (Variolac® 992 BG100) 0.05% 0.05% - 0.05% 0.05% 0.05% 0.05% 0.05% RECODA N RS 100 0.2% - - 0.2% 0.2% 0.2% 0.2% 0.2% Water 24.2% 24.4% 22.83% 23.80% 23.55% 22.58% 21.60% 21.36% All percentage values ​​for ingredients are given by weight.

[0207] Extensional rheometry was performed on experimental samples 1-8 (S1-S8, FIG. 22) using a VADER 1000 filament stretching device (Rheo Filament ApS, Copenhagen, Denmark). This device measures the dimensional change and stresses acting on a fluid bridge (filament) that forms between two plates during vertical separation of the plates. In this specific experiment, a constant vertical separation rate of 1 mm / s was used. From the change in filament radius, the so-called Hencky strain is calculated. The degree of vertical separation that a filament can sustain (i.e., how “fibrous” the sample is) in mm is numerically equal to the time the filament remains stable, which results from using a separation rate of 1 mm / s.

[0208] FIG. 22 shows the Hencky strain versus time for experimental samples 1-8 (S1-S8); in all samples, significant differences in strain values ​​were observed at different times. Petition 870250087473, dated 09 / 26 / 2025, pages 255 / 302 114 / 133 greater than 5 seconds. From this point onward, the samples varied significantly in their extensibility, or “fibrousness,” as can be seen by the increase in the time a filament can be sustained under deformation. Based on the data in FIG. 22, the samples can be classified in terms of their fibrousness / extensibility as follows: S5 > S7 > S8 « S6 > S4 > S3 > S1 > S2. It is noteworthy that, while treatments with GTF 0768 (S4) or vGTFJ (S8) alone, or with a 50%:50% combination of both GTF enzymes (S6), increased fibrousness / extensibility compared to the corresponding reference / control sample that received no GTF treatment (S1), treatments with a combination of 90% GTF 0768: 10% vGTFJ (S5) or a combination of 10% GTF 0768: 90% vGTFJ (S7) resulted in greater increases in fibrousness / extensibility (FIG. 22).

[0209] When the above analysis was performed at 50 mm / s, filament lengths of 40 mm to 65 mm were observed at the breaking point (data not shown). Example 9 Controlled texture formation in plant-based compositions using glucosyltransferase enzymes in situ.

[0210] Five 102.6 g sample solutions (Samples 1-5) were prepared, each composed of tap water and sucrose (40% by weight). The sample solutions were brought to 35 °C and a high-texturizing mixture of GTF 0768 (SEQ ID NO: 1) and vGTFJ (SEQ ID NO: 3) was added to four of the solutions (Samples 2-5) at time zero. At specific time points for each sample, strawberry puree (30 g), strawberry pieces (70 g), and sucrose (6.4 g) were added to Samples 2 (15 minutes), 3 (20 minutes), 4 (25 minutes), and 5 (30 minutes). These same ingredients / quantities were also added to the reference sample. Petition 870250087473, dated 09 / 26 / 2025, pages 256 / 302 115 / 133 without GTF enzymes (Sample 1) at the 30-minute time point. After this addition, all Samples 1-5 were incubated for a total of 60 minutes (e.g., Sample 4 was incubated for 35 minutes after the addition) at 35 °C and then heat-treated for 5 minutes at 95 °C. The sugar composition of each final product sample was measured by HPLC (Materials / Methods) and the samples were subjected to visual / manual evaluation.

[0211] The results of the visual / manual evaluation are presented in Table 11. Notably, based on these data, it became evident that the texture / viscosity resulting from GTF enzyme activity could be modified by varying the time at which the fruit ingredients were added. Although the early addition of fruit ingredients reduced the texturizing effects of GTF activity on the final product (e.g., Sample 2, Table 11), the later addition allowed for GTF activity that was sufficient to provide texturization to the final product (e.g., Samples 4 and 5, Table 11). Probably consistent with these visual data, the early addition of fruit ingredients resulted in a higher oligosaccharide to polysaccharide formation ratio, while the later addition of fruit ingredients resulted in a lower oligosaccharide to polysaccharide formation ratio (data not shown). Table 11. Effect of adding fruit ingredients on the GTF-based texturization of a food product. Sample No. Time without fruit ingredients Total incubation time at 35°C Visual assessment of product samples 1 without GTF 30 min 60 min Fine, liquid texture. Pieces of fruit floating. 2 15 min 60 min Fine, liquid texture, but with more texture than Sample 1. Pieces of fruit floating. 3 20 min 60 min Still liquid texture, but with more texture than Sample 2. Pieces of fruit distributed. 4 25 min 60 min Soft gel with good marmalade texture. Pieces of fruit evenly distributed. 5 30 min 60 min Strong gel. Pieces of fruit evenly distributed. More mixing force is needed to produce the product without gel pieces. Petition 870250087473, dated 09 / 26 / 2025, pages 257 / 302 116 / 133 Example 10: Modification of glucosyltransferase-mediated texturization of dairy products using invertase. Milk

[0212] A skimmed milk base was prepared by having 9% by weight of skimmed milk powder (33% protein, 1.2% fat, 54% carbohydrate; BBA Lactalis, Laval, Mayenne, France) and 8% by weight of sucrose (Granulated Sugar 500; Nordic Sugar A / S, Denmark) dissolved in tap water. This base was tempered at 40 °C.

[0213] A yeast invertase (SEQ ID NO: 14) was added to aliquots of skim milk base at 5, 20, 80, or 200 ppm, alone or in combination with vGTFJ (SEQ ID NO: 3, added at 0.1 wt%). These aliquots were incubated at 40 °C; samples were removed from each aliquot after 10, 30, 60, 120, or 150 minutes of incubation and inactivated by heating at 95 °C for 5 minutes. An image was taken of the resulting texture of each 150-minute sample (FIG. 23), and the sugar / oligosaccharide composition of each sample was measured according to Materials / Methods.

[0214] As shown in FIG. 23 (bottom row), a significant amount of texture was formed as a result of the vGTFJ conversion of sucrose to alpha-glucan, and this texture was reduced / adjusted by the inclusion of invertase. This texture-tempering effect of invertase was enzyme dose-dependent. Incubation of invertase alone in the samples, without the addition of vGTFJ, did not result in any texture formation (FIG. 23, top row).

[0215] The sugar / oligosaccharide composition of each sample after 150 minutes of incubation confirmed that there was a dose-dependent increase in the hydrolysis of sucrose by invertase (Table 12). Similarly, this analysis confirmed that invertase competed with vGTFJ for the use of the sucrose substrate. This competition was Petition 870250087473, dated 09 / 26 / 2025, pages 258 / 302 117 / 133 evidenced by the fact that more sucrose was consumed, but less free glucose was formed, in the presence of vGTFJ, since instead, glucose was incorporated into poly- and oligosaccharides through transglycosylation. Table 12. Oligosaccharide and sugar composition (% w / w) of skim milk samples treated with invertase and optionally with glucosyltransferase (150-minute incubation) vGTFJ Invertases and Oligosaccharides of Sucrose and Lactose and Glucose and Leucrose and Fruits and - 5 ppm 0.25 7.23 4.92 0.32 0.07 0.16 20 ppm 0.27 6.29 4.74 0.64 0.10 0.52 80 ppm 0.53 3.99 4.56 1.67 0.11 1.53 200 ppm 0.38 1.27 4.37 2.82 0.07 2.61 0.1 % by weight 5 ppm 1.00 2.23 4.01 0.42 0.21 2.67 20 ppm 1.15 1.37 3.76 0.57 0.21 2.72 80 ppm 1.22 0.36 3.52 1.03 0.26 3.06 200 ppm 0.99 0.12 3.60 1.66 0.22 3.01 Yogurt

[0216] A skimmed milk base was prepared by having 9% by weight of skimmed milk powder (33% protein, 1.2% fat, 54% carbohydrate; BBA Lactalis, Laval, Mayenne, France) and 8% by weight of sucrose (Granulated Sugar 500; Nordic Sugar A / S, Denmark) dissolved in tap water. This base was tempered at 43 °C. The skim milk base was inoculated with a thermophilic starter culture (YO-MIX PRIME 900) at an inoculation rate of 20 DCU / 100 L. The above yeast invertase (SEQ ID NO: 14) was added to individual aliquots of the culture-inoculated skim milk base at 50, 100, 200, or 300 ppm, alone or in combination with vGTFJ (SEQ ID NO: 3, added at 0.1% by weight). Fermentation of each aliquot was conducted until pH 4.60 was reached, after which the product was cooled to 4 °C. The aliquot products were stored at 4 °C overnight before being shaken.An image was taken of the resulting texture of each aliquot product (FIG. 24), and the composition of. Petition 870250087473, dated 09 / 26 / 2025, pages 259 / 302 118 / 133 of the sugar / oligosaccharide content of each product was measured according to the Materials / Methods.

[0217] As shown in FIG. 24 (top row), a significant amount of texture was formed during fermentation as a result of the vGTFJ conversion of sucrose to alpha-glucan, and this texture was reduced / adjusted by the inclusion of invertase. This texture-tempering effect of invertase was enzyme dose-dependent. Incubation of invertase alone in the samples, without the addition of vGTFJ, did not result in any texture formation (FIG. 24, bottom row).

[0218] The sugar composition (monosaccharides and disaccharides) of each sample was analyzed. Based on this analysis, the percentage of total sugar reduction and theoretical sweetness were calculated for each sample relative to a reference sample that did not receive any enzyme (Table 13). This confirmed that sugar reduction was mediated by vGTFJ activity and that invertase alone did not result in any significant sugar reduction. Furthermore, invertase treatment generally maintained theoretical sweetness, while the use of vGTFJ alone maintained only 67% of the theoretical sweetness (Table 13). Thus, the inclusion of invertase with vGTFJ not only reduces vGTFJ-mediated texture formation but also reduces the degree of vGTFJ-mediated sugar reduction while increasing theoretical sweetness as a result of increased free glucose formation. Table 13. Percentage reduction in total sugar and theoretical sweetness calculated relative to a reference sample with no added enzyme. vGTFJ Invertase % reduction of total sugarb % of theoretical sweetnessc relative to the reference 0.1% - 31.6 67 0.1% 50 ppm 28.4 70 0.1% 100 ppm 23.9 74 0.1% 200 ppm 19.6 79 Petition 870250087473, dated 09 / 26 / 2025, pages 260 / 302 119 / 133 vGTFJ Invertase % reduction of total sugarb % of theoretical sweetnessc relative to the reference 0.1% 300 ppm 13.7 85 a - a - 0.0 100 - 50 ppm 2.7 94 - 100 ppm 0.8 99 - 200 ppm 3.3 96 - 300 ppm 1.6 98 aReference sample bThe sugars measured were all monosaccharides and disaccharides in the sample cThe theoretical sweetness for each sample was calculated as a sum of the relative sweetness of individual sugars in the sample multiplied by their respective concentrations (g of sugar / 100 g of sample). In particular, given that the relative sweetness values ​​of lactose, glucose, galactose, sucrose, and fructose are, respectively, 0.16, 0.75, 0.16, 1.0, and 1.2, the theoretical sweetness of each sample was calculated as: 0.16 x (g of lactose / 100 g) + 0.75 x (g of glucose / 100 g) + 0.16 x (g of galactose / 100 g) + 1.0 x (g of sucrose / 100 g) + 1.2 x (g of fructose / 100 g). Example 11 Optimization of glucosyltransferase dosage

[0219] The individual enzymatic dosages of GTF 0768 (SEQ ID NO: 1) and vGTFJ (SEQ ID NO: 3) were standardized so that a dosage of 100% was the dosage required for GTF to provide total sucrose conversion during a 24-hour incubation at 5 °C. A 10:90% mixture (GTF 0768:VGTFJ) was prepared.

[0220] Two milk bases (A and B), which can be used for the production of ice cream, for example, were prepared by (A) dissolving skimmed milk powder (10%), sucrose (14.5%) and glucose (2.5%) in demineralized water, or (B) dissolving skimmed milk powder (10%) and sucrose (18%) in demineralized water. The 10:90% mixture (GTF 0768:vGTFJ) was Petition 870250087473, dated 09 / 26 / 2025, pages 261 / 302 120 / 133 added to each milk base that had been tempered to 5 °C with or without invertase (IFF, FoodPro® I) and / or lactase (IFF, BONLACTA) according to Table 14. When invertase was included, it was to replace part of the 10:90% mixture (GTF 0768:vGTFJ) until a point where total sucrose depletion should still be achieved within the incubation time. Table 14. Enzyme dosage in milk bases Sample No. Milk base Dosages in g / L 10:90% (GTF 0768:vGTFJ) Invertase and Lacta and Total dosage of 10:90% (GTF 0768:vGTF J) + invertase 1 A 8.34 - - 8.34 2 A 8.34 - 1.70 8.34 3 A 7.51 0.08 - 7.59 4 A 6.67 0.16 - 6.84 5 A 5.84 0.24 - 6.08 7 A 5.01 0.32 1.70 5.33 8 A 4.17 0.40 - 4.57 9 A 4.17 0.40 1.70 4.57 10 A 2.09 0.60 1.70 2.69 11 A - - - 12 B 10.36 - - 10.36 13 B 10.36 - 1.70 10.36 14 B 7.25 0.30 1.70 7.55 15 B 6.21 0.40 1.70 6 B 5.18 0.50 - 5.68 17 B 5.18 0.50 1.70 5.68 18 B 2.59 0.75 1.70 3.34 19 B - - -

[0221] Since invertase showed significantly higher specific activity against sucrose than the 10:90% mixture (GTF 0768:vGTFJ), it was found that the total dosage volume of 10:90% (GTF 0768:vGTFJ) plus invertase decreased with increasing invertase dosage. A benefit of this was improved cost-in-use and reduced transfer of formulation ingredients, such as glycerol. High glycerol transfer could negatively affect application performance, for example, Petition 870250087473, dated 09 / 26 / 2025, pages 262 / 302 121 / 133 in milk-based ice cream, causing a reduction in the freezing point.

[0222] The milk bases with added enzyme(s) (Table 14) were incubated for 24 hours at 5 °C before heat inactivation at 95 °C for 10 minutes. The sugar / oligosaccharide composition of each sample was measured according to the Materials / Methods to verify if a sugar reduction > 30% was achieved.

[0223] The results of the carbohydrate analysis are presented in Table 15. Surprisingly, it was found in milk base A (samples 1-11) that a sugar reduction > 30% was achieved for all samples that included invertase, except for sample 10. In sample 10, the invertase ratio was too high, causing most of the sucrose to be hydrolyzed into glucose and fructose, thus providing less sucrose available to form oligosaccharides by the 10:90% mixture (GTF 0768:vGTFJ). This enzymatic regime resulted in a sugar reduction of only 20.82% in sample 10. Surprisingly, the total dosage volume of 10:90% (GTF 0768:vGTFJ) plus invertase can be reduced by more than 45% while still achieving a sugar reduction > 30% (Table 15, comparing sample 8 with sample 1). It was also found that the inclusion of a lactase could improve sugar reduction (Table 15, comparing sample 2 with sample 1, and sample 9 with sample 8), potentially since the monosaccharides produced by lactase could, in turn, serve as acceptor molecules for glucosyltransferases.

[0224] Similar data were obtained for milk base B (samples 12-19) (Table 15). Again, the total dosage volume of 10:90% (GTF 0768:vGTFJ) plus invertase can be reduced by more than 45% while still achieving a sugar reduction > 30% (Table 15, comparing sample 16 with sample 12). However, in milk base B, there was a greater impact on sugar reduction if lactase was included. Surprisingly, in Petition 870250087473, dated 09 / 26 / 2025, pages 263 / 302 122 / 133 presence of lactase, the total dosage volume of 10:90% (GTF 0768:vGTFJ) plus invertase can be reduced by more than 67% while still achieving a sugar reduction > 42% (Table 15, comparing sample 18 with sample 12). Table 15. Oligosaccharide and sugar composition (% w / w) of milk bases treated with enzymes Sample No. DP3+ Sucrose Nitrogen Lactose Glucose Leucrose Galactose Fructose Total Soluble Carbohydrate Sugar % Sugar Reduction 1 6.80 0.00 1.17 3.95 0.84 1.24 0.38 5.19 19.57 12.77 42.69 2 7.18 0.00 2.37 0.00 2.52 1.10 0.84 5.09 19.09 11.92 46.51 3 6.56 0.00 1.11 3.93 0.94 1.14 0.00 5.12 18.80 12.24 45.06 4 6.91 0.00 1.25 4.28 1.13 1.18 0.35 5.55 20.65 13.74 38.34 5 6.35 0.00 1.28 4.06 1.16 0.96 0.39 5.40 19.60 13.26 40.50 7 6.22 0.00 2.86 0.00 3.15 0.35 0.95 6.64 20.17 13.95 34.70 8 6.35 0.00 1.95 4.70 1.96 0.99 0.00 5.27 21.22 14.87 30.46 9 6.21 0.00 3.19 0.00 3.61 0.34 0.98 6.43 20.77 14.55 31.88 10 4.69 0.00 3.66 0.00 5.05 0.23 0.94 7.04 21.60 16.92 20.82 11 0.45 13.05 0.00 5.11 2.63 0.00 0.57 0.00 21.81 21.37 0.00 12 5.70 1.22 0.00 2.65 0.34 1.37 0.00 6.24 17.51 ​​11.82 47.07 13 7.12 0.00 1.51 0.00 1.20 0.86 0.78 6.46 17.92 10.80 51.60 14 6.48 0.00 1.64 0.00 1.35 0.65 0.78 6.18 17.08 10.60 52.52 15 6.51 0.00 1.76 0.00 1.50 0.57 0.80 6.65 17.78 11.27 49.50 16 7.07 0.00 1,48 4.05 0.94 2.02 0.00 6.21 21.77 14.70 31.54 17 6.44 0.00 2.14 0.00 1.81 0.48 0.76 6.80 18.43 11.99 46.27 18 4.78 0.00 2.56 0.00 2.60 0.34 0.76 6.47 17.51 ​​12.73 42.98 19 0.45 16.59 0.00 5.51 0.00 0.00 0.00 0.23 22.77 22.32 0.00 Total soluble carbohydrate = Sugar + DP3 + B Sugar = sucrose + nitrogen + lactose + glucose + leucrose + galactose + fructose.

[0225] Based on these results, it would be logical to expect the same dosage reduction benefit trend for sugar from including invertase, regardless of whether an incubation is performed at different temperatures or pH. For example, the same reduction benefit trend would be expected. Petition 870250087473, dated 09 / 26 / 2025, pages 264 / 302 Dosage of 123 / 133 sugar to include invertase during fermentation with a culture to produce a fresh fermented dairy product. Example 12 Impact of glucose syrup on glucan formation during condiment production.

[0226] The impact of corn syrup (glucose syrup) on alpha-glucan production by GTF in a ketchup formulation and any resulting texture imparted by such alpha-glucan to the formulation were investigated. In doing so, the effects of various glucose syrup concentrations were tested. A second enzyme in this document that uses sucrose as a substrate (e.g., invertase) was not used in this of the following examples, although it would be possible to use such an additional enzyme as desired or appropriate.

[0227] The corn syrup used in this example contained 17-21% dextrose, 11-16% maltose, and 9-16% trisaccharides (e.g., maltotriose) on a dry weight basis, all of which saccharide components could possibly act as acceptor molecules for GTF enzymes and thus reduce the molecular weight of alpha-glucan produced in the ketchup formulation. Therefore, different amounts of corn syrup were tested. The 10:90% enzyme mixture (GTF 0768:vGTFJ) from Example 11 was used in the preparation of these formulations. Ketchup samples (1-9) were prepared in 15 g portions based on the ingredients / formulations shown in Table 16. Table 16. Ketchup formulations and processing regimes Formulation (each ingredient in % by weight) 1 2 3 4 5 6 7 8 9 Water 21.85 23.35 24.35 24.35 34.35 39.35 41.85 42.85 44.35 Corn syrup at 77.5% SS, DE 42 20 20 20 20 10 5 2.5 1.5 0 90:10% (GTF 0768:vGTFJ) - - - 0.23 0.23 0.23 0.23 0.23 0.23 Sugar 10 10 10 10 10 10 10 10 10 Petition 870250087473, dated 09 / 26 / 2025, pages 265 / 302 124 / 133 Formulation (each ingredient in % by weight) 1 2 3 4 5 6 7 8 9 Salt 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 Starch (waxy corn)* 2.5 1 - - - - - - - GRINDSTED® FF 5128 stabilizing system - 0.4 - - - - - - - Onion powder 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Potassium sorbate 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Garlic powder 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Ketchup spices qs* qs* qs* qs* qs* qs* qs* qs* qs* Additions after incubation and enzymatic termination Tomato paste 34 34 34 34 34 34 34 34 34 10% vinegar 9 9 9 9 9 9 9 9 9 % total 100 100 100 100 100 100 100 100 100

[0228] To prepare each ketchup sample (Table 16), the ingredients water and corn syrup were mixed before the addition of the enzyme mixture. The dry materials (sugar, salt, preservatives, spices, and stabilizer) were mixed and added to the wet phase under stirring. After incubation for 20 hours at 5 °C, the ingredients tomato paste and vinegar were added and mixed well into each sample. All prepared samples were then heated to 90 °C for 10 minutes to gelatinize the starch or to inactivate the GTF enzymes. Small aliquot samples were extracted and stored frozen until analyzed for carbohydrates by HPLC.

[0229] The resulting ketchup samples had significant visual differences (FIG. 25). Samples 1 and 2 represented low and high texture references, respectively, where acceptable ketchup textures should generally fall between them. Sample 3 (without stabilizer) and sample 4 (GTF + 20% corn syrup mixture) had less texture than sample 1 and were therefore unacceptable. Samples 5-7 (GTF + 2.5-10% corn syrup mixture) had acceptable textures, although further homogenization might be desirable to improve smoothness. Samples 8 and 9 contained large, gelled lumps and could not be Petition 870250087473, dated 09 / 26 / 2025, pages 266 / 302 125 / 133 additionally analyzed.

[0230] The results of the carbohydrate analysis are shown in Table 17. Some of the components (including maltose) were not well separated from sucrose, which is why sucrose is slightly overestimated and maltose is missing from the overall view. The data fully support the visual conclusion of FIG. 25 that glucan formation increased with decreasing corn syrup levels. However, a significant impact on sweetness is expected if the corn syrup content is reduced in the recipe to allow for GTF-based texturizing. Table 17. Carbohydrate levels in % w / w Sample DP3+ Sucrose Glucose Leucrose Fructose Total soluble saccharides Polysaccharides Insoluble 1 10.37 13.18 4.89 0.00 0.90 29.33 2.83 2 10.94 14.03 5.27 0.00 0.96 31.21 0.96 3 11.32 14.39 5.36 0.00 0.99 32.06 0.10 4 18.24 3.92 3.95 0.73 2.58 29.43 2.77 5 13.16 1.07 2.81 0.80 2.71 20.54 3.96 6 10.04 0.51 2.64 0.95 2.96 17.10 3.55 7 7.18 0.38 2.24 1.05 2.54 13.39 5.33

[0231] It became very clear that the texture decreased with increasing levels of corn syrup due to reduced polysaccharide formation. It was therefore found that the inclusion of a sugar syrup in recipes would be a valuable tool for GTF-based texture modulation in the production of condiments (e.g., ketchup) and other food products / precursors. The sugar syrup can be any corn syrup or high-fructose corn syrup containing acceptor molecules for glucosyltransferase. If such a syrup is not included in the recipe, then the inclusion of an invertase in the incubation step would be relevant for texture modulation. Example 13 Order of ingredient addition and impact of homogenization.

[0232] For the following ketchup formulation trial, the Petition 870250087473, dated 09 / 26 / 2025, pages 267 / 302 126 / 133 batch size was increased to 100 g. The ingredients are shown in Table 18. These formulations were subjected to incubation steps (20 hours at 5 °C) and GTF enzyme termination (90 °C for 10 minutes).

[0233] Two different mixtures of GTF 0768 and vGTFJ were tested, of which a 90:10% mixture (GTF 0768:vGTFJ) was highly texturizing (trials 3, 5, 7 and 8) and a 60:40% mixture (GTF 0768:vGTFJ) was less texturizing (trials 4 and 6). As it was demonstrated that corn syrup levels have an impact on texture (Example 12), it was decided to include 2.5% by weight of corn syrup (trials 3, 4 and 7, Table 18), or no corn syrup (trials 5, 6 and 8, Table 18), during incubation with GTF enzymes, and then add the rest of the corn syrup (up to 20% by weight) after incubation with GTF. It is therefore expected that the resulting formulations will have a sweetness similar to the references. It was also tested whether the presence of tomato paste during incubation with GTF enzyme could interrupt gelation and thus produce a smoother product (tests 7 and 8, Table 18).For assays 5 and 6, which did not include corn syrup during enzyme incubation, these formulations were expected to form gel lumps / clumps, and thus these samples were homogenized with a TURRAX homogenizer after termination of GTF enzyme activity. Small aliquot samples were extracted and stored frozen until analyzed for carbohydrates by HPLC. Table 18. Ketchup formulations and processing regimes Formulation (each ingredient in % by weight) 1 2 3 4 5 6 7 8 Water 23 24 24.35 24.35 24.35 24.35 58.35 58.35 Corn syrup at 77.5% SS, DE 42 20 20 2.5 2.5 - - 2.5 - 90:10% (GTF 0768:vGTFJ) - - 0.23 - 0.23 - 0.23 0.23 60:40% (GTF 0768:vGTFJ) - - - 0.38 - 0.38 - - Tomato paste - - - - - - 34 34 Sugar 10 10 10 10 10 10 10 10 Petition 870250087473, dated 09 / 26 / 2025, pages 268 / 302 127 / 133 Formulation (each ingredient in % by weight) 1 2 3 4 5 6 7 8 Salt 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 Starch (waxy corn)* 1 - - - - - - - GRINDSTED® FF 5128 stabilizing system 0.4 - - - - - - - Onion powder 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Potassium sorbate 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Garlic powder 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Ketchup spices qs* qs* qs* qs* qs* qs* qs* qs* Additions after incubation and enzymatic termination Tomato paste 34 34 34 34 34 34 - - Vinegar at 10% 9 9 9 9 9 9 9 9 Corn syrup at 77.5% SS, DE 42 - - 17.5 17.5 20 20 17.5 20 % total 100 100 100 100 100 100 100 100

[0234] The resulting ketchup samples were visually inspected (FIG. 26). Samples 2 (without stabilizer) and 4 (60:40% (GTF 0768:vGTFJ) with 2.5% corn syrup during enzymatic incubation) were found to exhibit some syneresis, which is unacceptable. All samples 3, 5, 6, 7, and 8 showed acceptable texture, although samples 3 and 7 were not as thick as reference sample 1, which was due to the presence of 2.5% corn syrup during incubation with GTF enzyme. It was further found that if corn syrup were not included during enzymatic incubation, then a uniform product could be obtained by applying homogenization after production or by including tomato paste during incubation with GTF enzyme.

[0235] The results of the carbohydrate analysis are shown in Table 19. As in the previous experiment (Example 12), some of the components (including maltose) were not well separated from the sucrose, thus explaining why sucrose was slightly overestimated and maltose was absent in the analysis. It was also because of this poor separation that sucrose did not appear to be fully reduced by enzymatic action in these Petition 870250087473, dated 09 / 26 / 2025, pages 269 / 302 128 / 133 data, although it was probably fully reduced. The data further confirmed that samples 3-8 were more similar to reference samples 1 and 2 in terms of total soluble carbohydrates, as the total levels of the corn syrup ingredient were maintained, although the order of addition was altered between the samples. Table 19. Carbohydrate levels in % w / w Sample DP3+ Sucrose Glucose Leucrose Fructose Total soluble saccharides Polysaccharides Insoluble 1 13.58 15.00 5.34 0.00 2.22 36.13 0.00 2 12.10 14.89 4.81 0.00 2.15 33.95 0.00 3 13.33 6.70 4.11 0.77 4.64 29.57 4.39 4 13.27 7.96 4.15 1.14 4.42 30.94 3.02 5 14.23 6.55 4.17 1.74 4.37 31.07 2.89 6 13.58 7.98 4.89 1.48 4.43 32.36 1.59 7 13.89 6.88 3.72 1.11 4.49 30.09 3.86 8 12.37 6.70 3.66 1.22 4.29 28.24 5.72 Example 14 Impact of available sucrose on the texture of 30% tomato concentrate ketchup

[0236] The ketchup samples prepared in Examples 12 and 13 without corn syrup or other added carbohydrate acceptor molecules present during incubation with GTF enzyme exhibited excessive gelation. The tomato paste added during incubation with GTF enzyme (Example 13) prevented excessive GTF-induced gelation and interrupted network formation, presumably by providing carbohydrate acceptor molecules due to its natural carbohydrate content. Example 13 thus demonstrated that it was possible to adjust the texture generated by GTF, not only by adding pure carbohydrate mixtures, but also by adding carbohydrate-containing food ingredients such as tomato paste. This result indicated that food ingredients naturally containing carbohydrates can likely be used to adjust the texture provided by GTF activity in a food product / precursor in situ. Petition 870250087473, dated 09 / 26 / 2025, pp. 270 / 302 129 / 133

[0237] In the present example, 1.8 kg ketchup formulations were prepared according to the ingredient list in Table 20. These formulations were subjected to incubation steps (20 hours at 5 °C) and GTF enzyme termination (90 °C for 10 minutes). Dry materials (sugar, salt, preservatives, stabilizer, spices) were mixed and dissolved in the aqueous phase with (tests 4, 5 and 6), or without (tests 1 and 2), GTF enzyme mixtures under stirring for 2 minutes. Tomato paste and vinegar were added for tests 1 and 2, and the material was heated to 90 °C for 2 minutes under stirring (before incubation at 5 °C for 20 hours). For tests 4, 5 and 6, only tomato paste was added and mixed before incubation at 5 °C for 20 hours. After incubation, the vinegar and the remaining sugar were added for tests 4 and 5. Small aliquot samples were extracted and stored frozen until analyzed for carbohydrates by HPLC. Table 20. Ketchup formulations and processing regimes Formulation (each ingredient in % by weight) 1 2 4 5 6 Water 39.65 41.65 41.471 41.383 41.294 90:10 % (GTF 0768:vGTFJ) - - 0.179 0.267 0.356 Tomato paste 30 30 30 30 30 12% vinegar 6 6 Sugar 20 20 10 15 20 Salt 2 2 2 2 2 Onion powder 0.1 0.1 0.1 0.1 0.1 Potassium sorbate 0.1 0.1 0.1 0.1 0.1 FF 4109 2 Garlic powder 0.05 0.05 0.05 0.05 0.05 Ketchup spices 0.1 0.1 0.1 0.1 0.1 Additions after incubation and enzymatic termination Vinegar at 12% 6 6 6 Sugar 10 5% total 100 100 100 100 100

[0238] The results of the carbohydrate analysis are shown in Table 21. The results clearly demonstrate how the levels of the GTF enzyme reaction products (DP3+ oligosaccharides, fructose) and byproduct Petition 870250087473, dated 09 / 26 / 2025, pages 271 / 302 130 / 133 (leukoresin) increased for samples 4, 5, and 6 due to more substrate (sucrose) being available during the 20-hour incubation at 5°C. This logically resulted in increased sugar reduction. Table 21. Carbohydrate levels in % w / w Sample ID DP3+ Sucrose Glucose Leucrose Galactose Fructose Carbohydrate Total soluble sugar % of sugar % of sugar reduction 1 2.63 20.60 3.17 0.00 0.00 3.17 29.57 26.94 0.00 2 2.47 18.64 2.64 0.00 0.00 2.76 26.51 24.04 0.00 4 3.93 12.65 1.62 0.97 0.00 4.83 23.99 20.07 16.54 5 5.88 7.33 1.31 2.00 0.00 6.55 23.08 17.20 28.46 6 7.85 2.02 0.88 3.19 0.00 7.38 21.31 13.46 44.00

[0239] The viscosity (Pas) of each ketchup sample was measured at 20 °C using an Anton Paar 301 rheometer with a rotor / cup measuring system (CC27). Each sample was allowed to equilibrate for 10 minutes after sample transfer to the rheometer. The shear rate was then increased from 0.1 s-1 to 100.0 s-1 in a logarithmic sweep measuring 10 points per decade over a time period of 200 seconds. The apparent viscosity at 0.1, 1, 10 and 100 Hz for each sample is presented in Table 22. The increased sucrose conversion clearly resulted in increased viscosity (compare sample 2 with samples 4, 5 and 6). Although the apparent viscosity at low shear rates of the samples with GTF enzymes was not as high as the reference sample with stabilizer, they were found to be comparable in terms of mouthfeel (apparent viscosity at high shear rate).Furthermore, no excessive gelation was observed in the samples. Table 22. Apparent viscosity at 0.1, 1.10 and 100 Hz Sample ID Viscosity (Pa s) at a shear rate of 0.1 s-1 Viscosity (Pa s) at a shear rate of 1.0 s-1 Viscosity (Pa s) at a shear rate of 10.0 s-1 Viscosity (Pa s) at a shear rate of 100.0 s-1 1 119.7 27.4 4.1 0.7 2 26.8 4.9 1.0 0.2 Petition 870250087473, dated 09 / 26 / 2025, pages 272 / 302 131 / 133 Sample ID Viscosity (Pas) at a shear rate of 0.1 s-1 Viscosity (Pa s) at a shear rate of 1.0 s-1 Viscosity (Pa s) at a shear rate of 10.0 s-1 Viscosity (Pa s) at a shear rate of 100.0 s-1 4 61.6 15.3 3.0 0.7 5 72.3 16.4 3.3 0.8 6 80.7 17.6 3.5 0.8 Example 15 GTF-based texture formation in ketchup to replace the level of tomato concentrate used for texturizing.

[0240] It was investigated whether it would be possible to produce a ketchup with 30% by weight of tomato concentrate and use GTF activity in it to produce a texture comparable to the texture of a ketchup that has 65% by weight of tomato concentrate. 1.8 kg batches of ketchup were prepared according to the ingredient list in Table 23. These formulations were subjected to incubation steps (20 hours at 5 °C) and GTF enzyme termination (90 °C for 10 minutes). Dry materials (sugar, salt, preservatives, spices) were mixed and dissolved in the aqueous phase with (tests 2 and 3) or without (tests 1 and 4) GTF enzymes under stirring for 2 minutes. Tomato paste and vinegar were then added for tests 1 and 4 and the material was heated to 90 °C for 2 minutes under stirring (before incubation at 5 °C for 20 hours). For trials 2 and 3, only tomato paste was added and mixed before incubation at 5 °C for 20 hours. Table 23. Ketchup formulations and processing regimes Formulation (each ingredient in % by weight) 1 2 3 4 Water 41.65 41.747 41.294 16.65 90:10 % (GTF 0768:vGTFJ) - 0.179 0.356 - Tomato paste 30 30 30 65 12% vinegar 6 6 Sugar 20 10 20 10 Salt 2 2 2 2 Onion powder 0.1 0.1 0.1 0.1 Potassium sorbate 0.1 0.1 0.1 0.1 FF4109 0.05 0.05 0.05 0.05 Garlic powder 0.1 0.1 0.1 0.1 Petition 870250087473, dated 09 / 26 / 2025, pages 273 / 302 132 / 133 Formulation (each ingredient in % by weight) 1 2 3 4 Ketchup spices 41.65 41.747 41.294 16.65 Additions after incubation and enzymatic termination Vinegar at 12% 6 6 Sugar 10% total 100 100 100 100

[0241] The viscosity (Pa s) of each ketchup sample was measured at 20 °C using an Anton Paar 301 rheometer with a rotor / cup measuring system (CC27). Each sample was allowed to equilibrate for 10 minutes after sample transfer to the rheometer. The shear rate was then increased from 0.1 s-1 to 100.0 s-1 in a logarithmic sweep measuring 10 points per decade over a time period of 200 seconds. The apparent viscosities at 0.1, 1, 10 and 100 Hz are presented in Table 24. The increased sucrose conversion clearly resulted in increased viscosity of the 30% tomato concentrate ketchups (compare sample 1 with samples 2 and 3). Although the apparent viscosity of the samples with GTF enzymes (samples 2 and 3) was not as high as the reference sample with 65% tomato concentrate (sample 4), they presented an acceptable ketchup texture. Table 24. Apparent viscosity at 0.1, 1.10 and 100 Hz Sample ID Viscosity (Pas) at a shear rate of 0.1 s-1 Viscosity (Pa s) at a shear rate of 1.0 s-1 Viscosity (Pa s) at a shear rate of 10.0 s-1 Viscosity (Pa s) at a shear rate of 100.0 s-1 1 24.3 4.3 0.8 0.2 2 67.7 15.7 3.0 0.7 3 85.0 17.8 3.5 0.8 4 194.2 33.3 6.2 1.0

[0242] The GTF enzyme incubations in the ketchup formulations in this example and in Examples 12-14 were carried out at 5 °C. However, these incubations could have been carried out at a temperature between 0 °C and 50 °C, for example, by adjusting the GTF enzyme dosages (generally, less enzyme would be needed at higher temperatures). Furthermore Petition 870250087473, dated 09 / 26 / 2025, pp. 274 / 302 133 / 133 of this, incubations with GTF enzyme could have been carried out at any pH between 3.7 and 8, at which the applied enzymes are typically both active. Petition 870250087473, dated 09 / 26 / 2025, pp. 275 / 302

Claims

1 / 5 CLAIMS 1. METHOD OF PRODUCING A FOOD PRODUCT / PRECURSOR, said method being characterized in that it comprises: (a) providing a food product / precursor comprising at least water and sucrose, and (b) bringing the food product / precursor into contact with at least one glucosyltransferase enzyme and a second enzyme that uses sucrose as a substrate, wherein the glucosyltransferase enzyme is: (i) a glucosyltransferase enzyme that synthesizes alpha-1,6-glucan, wherein at least about 50% of the glycosidic linkages of 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 alpha-1,3-glucan are alpha-1,6 linkages alpha-1,3, wherein at least one alpha-glucan is produced in the food product / precursor, wherein the food product / precursor, after step (b),optionally has one or more of the following characteristics compared to the food product / precursor before step (b): (I) increased texture, but wherein said increased texture is less than it would be if the second enzyme were not present in the food product / precursor, (II) reduced sugar content, but wherein said reduced sugar content is less than it would be if the second enzyme were not present in the food product / precursor, typically wherein said sugar content comprises monosaccharides and disaccharides of the food product / precursor, Petition 870250087473, dated 09 / 26 / 2025, p. 276 / 302 2 / 5 and / or (III) reduced sweetness, but wherein said reduced sweetness is less than it would be if the second enzyme were not present in the food product / precursor.

2. METHOD, according to claim 1, characterized in that said second enzyme which uses sucrose as a substrate is an invertase enzyme or a fructosyltransferase enzyme.

3. METHOD, according to claim 1, characterized in that 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 or 12.

4. METHOD, according to claim 1, characterized in that said glucosyltransferase enzyme that synthesizes alpha-1,3-glucan comprises an amino acid sequence that is at least 90% identical to residues 55-960 of SEQ ID NO: 5, residues 54-957 of SEQ ID NO: 6, residues 55-960 of SEQ ID NO: 7, residues 55-960 of SEQ ID NO: 8, residues 55-960 of SEQ ID NO: 9, or to SEQ ID NO:

13.

5. METHOD, according to claim 1, characterized in that both said (i) glucosyltransferase enzyme that synthesizes alpha1,6-glucan and said (ii) glucosyltransferase enzyme that synthesizes alpha1,3-glucan are used in step (b).

6. METHOD, according to claim 5, characterized in that said alpha-glucan produced in step (b) comprises a graft copolymer comprising: (i) a main chain of alpha-1,6-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1,6-glucan main chain are alpha-1,6 linkages, and (ii) at least one side chain of alpha-1,3-glucan, wherein at least about 50% of the glycosidic linkages of the alpha-1,3-glucan chain are alpha-1,3 linkages, wherein said alpha-glucan is aqueous soluble or aqueous insoluble.

7. METHOD according to claim 1, characterized in that step (a) comprises adding sucrose to the food product / precursor.

8. METHOD according to claim 1, characterized in that the food product / precursor is a dairy food product / precursor.

9. METHOD according to claim 1, characterized in that the food product / precursor is a non-dairy food product / precursor.

10. METHOD, according to claim 9, characterized in that the non-dairy food product / precursor is plant-based.

11. METHOD, according to claim 1, characterized in that the food product / precursor of step (a) is fermented, or the method further comprises, during or after step (b), the fermentation of the food product / precursor.

12. METHOD, according to claim 1, characterized in that the food product / precursor is a yogurt.

13. METHOD, according to claim 1, characterized in that step (b) further comprises bringing the food product / precursor into contact with a lactase enzyme.

14. FOOD PRODUCT / PRECURSOR characterized in that it is produced by the method as defined in claim 1.

15. METHOD OF PRODUCING A FOOD PRODUCT / PRECURSOR, the method being characterized by Petition 870250087473, dated 09 / 26 / 2025, page. 278 / 302 4 / 5 fact comprising: (a) providing a food product / precursor comprising at least water, sucrose and at least one carbohydrate-containing ingredient, and (b) bringing the food product / precursor into contact with at least one glucosyltransferase enzyme, wherein the glucosyltransferase enzyme is: (i) a glucosyltransferase enzyme that synthesizes alpha-1,6-glucan, wherein at least about 50% of the glycosidic linkages of 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 alpha-1,3-glucan are alpha-1,3 linkages, wherein at least one alpha-glucan is produced in the food product / precursor,wherein the carbohydrate-containing ingredient comprises at least one acceptor molecule for at least one glucosyltransferase enzyme, wherein the food product / food precursor, after step (b), optionally has one or more of the following characteristics compared with the food product / food precursor before step (b): (I) increased texture, but wherein the increased texture is less than it would be if the carbohydrate-containing ingredient were not present in the food product / food precursor, (II) reduced sugar content, but wherein the reduction in sugar content is less than it would be if the carbohydrate-containing ingredient were not present in the food product / food precursor, typically wherein the sugar content comprises monosaccharides and disaccharides of the food product / food precursor, Petition 870250087473, dated 09 / 26 / 2025, p. 279 / 302 5 / 5 and / or (III) reduced sweetness,but where the reduced sweetness is less than it would be if the carbohydrate-containing ingredient were not present in the food product / precursor. Petition 870250087473, dated 09 / 26 / 2025, pp. 280 / 302.

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