WO2021252814A1 - Production et utilisations d'oligosaccharides et de glycosides de stéviol - Google Patents

Production et utilisations d'oligosaccharides et de glycosides de stéviol Download PDF

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WO2021252814A1
WO2021252814A1 PCT/US2021/036889 US2021036889W WO2021252814A1 WO 2021252814 A1 WO2021252814 A1 WO 2021252814A1 US 2021036889 W US2021036889 W US 2021036889W WO 2021252814 A1 WO2021252814 A1 WO 2021252814A1
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reb
derivative
disaccharide
glucopyranoside
trisaccharide
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Christopher Bellovin
Nikola Nikolic
Shawn YOON
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Corn Products Development, Inc.
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A23L2/60—Sweeteners
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • A23L27/33—Artificial sweetening agents containing sugars or derivatives
    • A23L27/36—Terpene glycosides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/602—Glycosides, e.g. rutin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01031—Laminaribiose phosphorylase (2.4.1.31)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use

Definitions

  • Steviol glycosides are a class of compounds that are capable of imparting a sweet taste to food and beverage products. These compounds are found in the leaves of Stevia rebaudiana (Bertoni), a perennial shrub that is native to certain regions of South America, e.g., Brazil and Paraguay.
  • Steviol glycosides are characterized structurally by a common single base, steviol. However, different steviol glycosides are present in varying amounts in naturally occurring stevia plants.
  • Reb A is prevalent in large amounts in the wild.
  • steviol glycosides such as Reb M and Reb J are present in much smaller amounts in naturally occurring stevia plants.
  • the prevalence of certain rebaudiosides in nature is not correlated with the desirability of their traits. Therefore, many steviol glycosides that are present in nature in small amounts relative to other steviol glycosides have desirable properties.
  • Reb M is a desirable, natural, high-intensity sweetener that possesses attributes that are pleasant to the human sensory system.
  • plants synthesize Reb M through as many as six steps.
  • stevia plants may use four different types of UDP-glycosyl transferases along the way of producing Reb M from steviol. Because of the number of steps and the number of different enzymes that are required, the final yield of Reb M in nature is very low.
  • Currently known approaches for generating Reb M synthetically rely on bioconversion pathways that mimic the natural step by step process for synthesizing Reb M. Therefore, they suffer from similar drawbacks of being time-consuming and cumbersome.
  • One strategy for synthesizing steviol glycoside compounds that occur in nature in small amounts is to start with other compounds that differ at only the C13 and/or C19 positions.
  • a first embodiment is directed to a method for producing a steviol glycoside comprising conjugating a stevia compound to an oligosaccharide to form the steviol glycoside, wherein said conjugating is via either enzymatic synthesis (i.e., bioconversion) in the presence of a transglycosylation enzyme or chemical conversion.
  • the stevia compound may, for example, be steviol or a partially deglycosylated steviol glycoside relative to the desired final steviol glycoside.
  • a second embodiment is directed to a method for synthesizing a disaccharide or a disaccharide derivative comprising exposing glucose or a glucose derivative to glucose-1-phosphate (Glc-1-P) in the presence of a phosphorylase, e.g., either laminaribiose phosphorylase or sophorose phosphorylase, to form Pi (inorganic phosphate) and the disaccharide or disaccharide derivative.
  • a phosphorylase e.g., either laminaribiose phosphorylase or sophorose phosphorylase
  • UDP-Glc UDP-glucose
  • a naturally occurring compound such as, e.g., naringin, neohesperidin or rutin to an oxidizing agent to form the free neohesperidoside or rutin and oxidized aglycone.
  • a third embodiment provides a method for synthesizing a trisaccharide or a trisaccharide derivative comprising exposing a disaccharide or a disaccharide derivative to UDP-Glc in the presence of a UDP glycosyltransferase, wherein the UDP glycosyltransferase is capable of ⁇ -1,2-transglycosylation or ⁇ -1,3- transglycosylation to form UDP and the trisaccharide or trisaccharide derivative.
  • the disaccharide or disaccharide derivative may be synthesized according to any methods described herein or according to other methods known by persons of ordinary skill in the art.
  • a fourth embodiment provides a method for synthesizing a trisaccharide or a trisaccharide derivative comprising exposing glucose or a glucose derivative to UDP- Glc in the presence of a UDP glycosyltransferase, wherein the UDP glycosyltransferase is capable of ⁇ -1,2-transglycosylation or ⁇ -1,3-transglycosylation to form UDP and the trisaccharide or trisaccharide derivative.
  • a fifth embodiment provides a method for synthesizing a steviol glycoside comprising obtaining an oligosaccharide such as a disaccharide, disaccharide derivative, trisaccharide, or a trisaccharide derivative according to any embodiments described herein and conjugating the oligosaccharide to a stevia compound. If the disaccharide or disaccharide derivative is conjugated to the stevia compound, optionally another sugar moiety can be added to the conjugated disaccharide or disaccharide derivative to form a conjugated trisaccharide, or a trisaccharide derivative.
  • a sixth embodiment provides compositions and methods for making compositions containing an oligosaccharide such as a disaccharide, disaccharide derivative, trisaccharide, or a trisaccharide derivative according to any embodiments described herein for use in cosmetic, health and beauty, pharmaceutical and medical applications.
  • an oligosaccharide such as a disaccharide, disaccharide derivative, trisaccharide, or a trisaccharide derivative according to any embodiments described herein for use in cosmetic, health and beauty, pharmaceutical and medical applications.
  • Various embodiments described herein are particularly advantageous for synthesizing steviol glycosides that have an oligosaccharide such as a disaccharide and/or trisaccharide (or derivatives thereof) at the location of C19 (at the location of the C19-COOH group) or at C13 (at the location of the C13-OH group).
  • the oligosaccharide (or its derivative) can be attached through an O moiety at C13 to form an ether-like bond or through an O moiety at C19 to form an ester-like bond. If there is a moiety other than the -COOH at the C19 site and/or the -OH at the C13 site of the steviol base of the stevia compound, then either moiety or both moieties can first be detached by a chemical method or an enzymatic method.
  • stevia compounds such as Tetrasaccharide or trisaccharide derivative at that position
  • sensory-pleasing e.g., pleasing to taste and/or or smell
  • steviol glycosides such as Reb J or Reb M or other targets.
  • Various embodiments described herein are particularly advantageous for synthesizing oligosaccharides and using oligosaccharides in diverse applications, including, but not limited to, personal care products, beauty care products, pharmaceuticals, and food and beverage products.
  • Figure 1 shows a flow chart that represents a method of an embodiment of the present invention.
  • Figure 2 shows results of TLC that confirm that use of UGTSL2 led to the production of disaccharide derivatives that were produced from phenyl- ⁇ -D- glucopyranoside, salicin, methyl arbutin, and ⁇ -arbutin.
  • Figure 3 shows results of TLC that confirm that use of EUGT11 led to the production of disaccharide derivatives from methyl ⁇ - D-glucopyranoside, phenyl ⁇ - D-glucopyranoside, salicin, ⁇ -arbutin, methyl arbutin and hexyl ⁇ -D- glucopyranoside.
  • Figure 4 shows results of TLC that confirm that use of UGT76G1 led to the production of disaccharide derivatives from phenyl ⁇ -D-glucopyranoside, salicin, arbutin, and methyl arbutin.
  • FIG. 5 shows results of TLC that confirms that use of UDP-glucose dependent ⁇ -1,2-transglycosyltransferase (UGTSi or EUGT11) led to the production of disaccharide from corresponding glucose derivatives and use of ⁇ -1,3- transglycosyltransferase (UGTSr or UGT76G1) led to the production of trisaccharide derivatives from corresponding disaccharide derivatives with high yield.
  • Figure 6 shows a gel that confirms that pure neohesperidose was obtained from naringin with high yield.
  • Steviol Glycosides [0026] In accordance with figure 1, one or more embodiments described herein may be used to synthesize steviol glycosides 900 through the conjugation of stevia compounds 700 to oligosaccharides 800 such as disaccharides, disaccharide derivatives, trisaccharides, and trisaccharide derivatives.
  • oligosaccharides 800 such as disaccharides, disaccharide derivatives, trisaccharides, and trisaccharide derivatives.
  • the steviol glycosides that may be produced according to the present invention are any steviol glycosides that have an oligosaccharide at position C13 and/or position C19 of the steviol base.
  • the steviol glycoside has a steviol base conjugated to a disaccharide, a disaccharide derivative, a trisaccharide, or a trisaccharide derivative at one or both of C13 and C19.
  • the steviol glycosides that may be synthesized according to the present invention include, but are not limited to, Reb B, Reb I, Reb J, Reb M, Reb O, Reb D, Reb D2, Reb D4, Reb W, Reb WB1, Reb WB2, Reb N, Reb C, Dulcoside A, Dulcoside A1, and Reb K.
  • conjugation of the stevia compounds to oligosaccharides is by enzymatic synthesis, which refers to conjugation in the presence of an enzyme catalyst.
  • the enzyme is a transglycosylation enzyme.
  • Transglycosylation enzymes are capable of catalyzing the conjugation of a sugar moiety such as an oligosaccharide to another moiety such as a stevia compound.
  • the enzyme is capable of conjugating the oligosaccharide through an oxygen moiety at the C19-COOH site or at the C13-OH site of the steviol base in a single step.
  • an oligosaccharide or a derivative of an oligosaccharide may replace a moiety (e.g., an -H, an -OH moiety) at a C13 or C19 position of a stevia compound.
  • transglycosylation enzymes at a temperature of about 10 °C to about 100 °C for a period of about 1 to about 72 hours, under mixing conditions of about 10 rpm to about 1000 rpm, and at a pH of about 2 to about 11.
  • transglycosylation enzymes include but are not limited to disaccharide glycosyltransferases, trisaccharide glycosyltransferases, and ⁇ - glucosidases that are capable of catalyzing the formation of glyco-ether or glyco-ester bonds.
  • Other enzymes that may be of use in the enzymatic synthesis steviol glycosides include but are not limited to esterases, proteases, and lipases.
  • the enzyme is a trisaccharide glycosyltransferase, which is an enzyme that can transfer a trisaccharide unit such as 2- ⁇ -D- glucopyranosyl-[3- ⁇ -D-glucopyranosyl]-D-glucose from its derivatives or conjugate its free form to a stevia compound at the C13 or C19 such as steviol or a steviol glycoside intermediate to produce the desired steviol glycoside or desired steviol glycosides.
  • a trisaccharide unit such as 2- ⁇ -D- glucopyranosyl-[3- ⁇ -D-glucopyranosyl]-D-glucose from its derivatives or conjugate its free form
  • a stevia compound at the C13 or C19 such as steviol or a steviol glycoside intermediate to produce the desired steviol glycoside or desired steviol glycosides.
  • the pH is from about 2 to about 10
  • the temperature is from about 10 °C to 100 °C
  • the time is from 1 to 72 hours
  • conjugation of the stevia compounds to oligosaccharides is by chemical conversion.
  • chemical conversion may be in the presence sulfuric acid-silica for coupling the oligosaccharide and the stevia compound, or under other acidic conditions or under alkali conditions.
  • Controlling the conditions may be accomplished by controlling pH, temperature, moisture conditions, and time, as well as by protecting and de-protecting other locations on the stevia compound such as C13.
  • Stevia compounds include steviol, as well as steviol glycosides and derivatives thereof.
  • the stevia compounds are selected such that the moiety located at or attached to the C19 position and/or the moiety located at or attached to the C13 position can be replaced with an oligosaccharide under thermodynamically friendly conditions, i.e., conditions under which the activation energy is reasonable under experimental or industrial conditions, to generate the desired steviol glycosides.
  • the stevia compounds of one or more embodiment may be obtained from naturally occurring sources 600 or from other steviol glycosides 500.
  • the stevia compound may first be separated from other compounds and purified. Methods for obtaining stevia compounds from naturally occurring sources are well-known to persons of ordinary skill in the art.
  • a steviol glycoside may be exposed to caustic conditions such as those created by the introduction of at least one of potassium hydroxide, sodium hydroxide, sodium carbonate, calcium oxide, calcium hydroxide, or lithium hydroxide, or acidic conditions such as those created by the introduction of HCl, sulfuric acid, phosphoric acid or nitric acid.
  • caustic conditions such as those created by the introduction of at least one of potassium hydroxide, sodium hydroxide, sodium carbonate, calcium oxide, calcium hydroxide, or lithium hydroxide
  • acidic conditions such as those created by the introduction of HCl, sulfuric acid, phosphoric acid or nitric acid.
  • the stevia compound is formed by combining another stevia compound and one or more of water, an acid, a base, or a sugar, in the presence of a caustic agent in a mixture of methanol or ethanol in water.
  • the stevia compound is formed by conjugation of a stevia compound and one or more of water, an acid, a base, or a sugar in the presence of a caustic agent in a mixture of hydrogen peroxide and potassium permanganate. In some embodiments, the stevia compound is formed by the conjugation of a stevia compound and one or more of water, an acid, a base, or a sugar in the presence of a caustic agent in a mixture of hydrogen peroxide and potassium permanganate. [0040] In one embodiment, the stevia compound is Reb B.
  • the Reb B used in the present invention may be obtained from naturally occurring sources or formed by cleavage through chemical hydrolysis or enzymatic hydrolysis of a moiety at one or both of C13 and C19.
  • the Reb B may be formed by the chemical hydrolysis of at least one of Reb A, Reb D, Reb D2, Reb I, Reb J, Reb M, Reb M2, Reb N, Reb T, and Reb O in the presence of a caustic agent such as potassium hydroxide, sodium hydroxide, sodium carbonate, calcium oxide, calcium hydroxide, or lithium hydroxide.
  • a caustic agent such as potassium hydroxide, sodium hydroxide, sodium carbonate, calcium oxide, calcium hydroxide, or lithium hydroxide.
  • Reb B may also be formed by the use of a biocatalyst capable of hydrolyzing ⁇ -glucosyl ester bonds.
  • the Reb B can itself be combined with an oligosaccharide or it can first be converted into another stevia compound such as steviol, which can be done under either acidic or caustic conditions or by enzymatic hydrolysis.
  • the stevia compound is Reb WB2.
  • the Reb WB2 may be obtained from naturally occurring sources or formed by cleavage through chemical hydrolysis under acidic conditions such as exposure of Reb WB1, Reb W, or Reb D4 to HCl at one or both of position C13 and 19.
  • the Reb WB2 may be formed by enzymatic synthesis or cleavage of another stevia compound.
  • the Reb WB2 can itself be combined with an oligosaccharide or it can first be converted into another stevia compound such as steviol, which can be done under either acidic or caustic conditions or by enzymatic hydrolysis.
  • the stevia compound e.g., steviol, Reb B, or Reb WB2 is formed by deglycosylation under alkali or acidic conditions, in methanol, in ethanol, in a methanol/water solution, or in an ethanol/water solution.
  • the stevia compound e.g., steviol, Reb B, or Reb WB2 is formed by deglycosylation by hydrogen peroxide and potassium permanganate at a pH of from about 7 to about 13.
  • One may, for example, keep these environments at a temperature of from about 10 °C to about 60 °C for about 0.1 hours to about 72 hours, while mixing at from about 10 rpm to about 1000 rpm.
  • the oligosaccharides are molecules that can be conjugated to stevia compounds at, for example, the C19 or C13 position through glyco-ester or glyco-ether bonds.
  • the oligosaccharides are molecules that can be conjugated to stevia compounds at, for example, the C19 or C13 position through glyco-ester or glyco-ether bonds.
  • one can avoid the requirement of multiple steps that accompany the addition of individual monomer sugar moieties.
  • the oligosaccharide is selected from the group consisting of sophorose, a derivative of sophorose, laminaribiose, a derivative of laminaribiose, 2- ⁇ -D-glucopyranosyl-(3- ⁇ - D-glucopyranosyl)-D-glucose, and a derivative of 2- ⁇ -D-glucopyranosyl-(3- ⁇ -D- glucopyranosyl)-D-glucose.
  • the oligosaccharide is selected from the group consisting of neohesperidose, a derivative of neohesperidose, rutinose, a derivative of rutinose, 2- ⁇ -L-rhamnosyl-(3- ⁇ -D-glucopyranosyl)-D-glucose, and a derivative of 2- ⁇ -L-rhamnosyl-(3- ⁇ -D-glucopyranosyl)-D-glucose.
  • the oligosaccharides and their derivatives may be obtained from many different sources, including naturally occurring sources or through known synthesis techniques.
  • Some embodiments provide methods for synthesizing oligosaccharides.
  • oligosaccharides may be used for any purpose, or they may be used in combination with other embodiments described herein for conjugation with stevia compounds to form one or more steviol glycoside.
  • some embodiments are directed to forming oligosaccharides 800 from glucose and/or glucose derivatives 300 in the presence of one or more phosphorylase, a glycosyltransferase or a chemical catalyst.
  • Glucose derivatives that may be of use in accordance with one or more embodiment described herein include but are not limited to alkyl and aryl derivatives of glucose.
  • the alkyl moieties are 1 to 12 carbons or 2 to 8 carbons or 3 to 6 carbons.
  • the alkyl moieties may be branched or linear and substituted or unsubstituted.
  • the aryl compounds may, for example, have 3 to 12 carbons or 4 to 8 carbons or 5 to 6 carbons. Further, theses derivatives may be substituted or unsubstituted. [0050] In some embodiments, one exposes glucose or a glucose derivative to a phosphorylase to form a disaccharide or disaccharide derivative such as sophorose, a derivative of sophorose, laminaribiose, or a derivative of laminaribiose.
  • the glucose derivative is selected from Me- ⁇ -D-Glc, Me- ⁇ -D-Glc, salicin, ⁇ -salicin, arbutin, methyl arbutin, ⁇ -arbutin, phenyl ⁇ -D-glucopyranoside, ⁇ -D- glucopyranoside, hexyl- ⁇ -D-glucopyranoside, coniferin, aesculin, sinigrin, amygdalin, and ruberythic acid.
  • glucosides that may be used in various embodiments of the present invention include but are not limited to ethyl-, propyl-, butyl-, pentyl-, heptyl-, octyl-, nonyl-, allyl-, and vinyl- beta-D-glucopyranoside and derivatives thereof.
  • the phosphorylase is capable of ⁇ -(1,2)- transglycosylation or ⁇ -(1,3)-transglycosylation.
  • the phosphorylase may be laminaribiose (1,3- ⁇ -oligoglucan) phosphorylase or sophorose (1,2- ⁇ -oligoglucan) phosphorylase, and a by-product is inorganic phosphate (Pi).
  • the method further comprises exposing the disaccharide or disaccharide derivative and UDP-glucose to a UDP- glycosyltransferase to form a trisaccharide or trisaccharide derivative and UDP.
  • UDP-glycosyltransferases include but are not limited to: beta-1,2-UDP glycosyltransferases such as UGT91D1, UGT91D2, UGT91D2e, UGTSL2, EUGT11, UGT91C1 from Oryza sativa, UGT91C1 from Arabidopsis thaliana, UGT2, UGT-B, and HV1; beta-1,3-UDP glycosyltransferases such as UGT76G1, CP1, CR1, and UGT-A; C13-OH UDP glycosyltransferases such as UGT85C2 and UGT1; and C19- COOH UDP glycosyl transferases such as UGT74G1 and UGT3.
  • beta-1,2-UDP glycosyltransferases such as UGT91D1, UGT91D2, UGT91D2e, UGTSL2, EUGT11, UGT91C1 from Oryza sativa, UGT91C1 from Arabidops
  • enzymes that may be advantageous for use when forming disaccharides and trisaccharides from glucose and glucose derivatives in the presence of glucose 1 phosphate include but are not limited to cellobiose phosphorylase, gentiobiose phosphorylase, cellodextrin phosphorylase, maltose phosphorylase, maltodextrin phosphorylase, starch phosphorylase, glycogen phosphorylase, sucrose phosphorylase, kojibiose phosphorylase, trehalose phosphorylase and nigerose phosphorylase.
  • enzymes recited herein may use an enzyme that differs from one of those enzymes by one or more amino acids but retains the same or similar functionality and catalyzes the same or similar reactions at the same if not higher efficiency levels.
  • these enzymes differ from the recited enzymes at an active site.
  • these enzymes differ from the recited enzymes at a site other than an active site.
  • the amino acid sequences of the enzymes disclosed herein are available through the scientific and patent literature as well as through publicly available databases such at NCBI (National Center for Biotechnology Information), which is within the United States National Institute of Health. The NCBI database is incorporated by reference.
  • these enzymes have an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence as it appears in the NCBI database.
  • the method comprises exposing a glucose derivative and UDP-Glc in e.g., a ratio of at least two UDP-Glc to one glucose derivative, to a UDP-glycosyltransferase.
  • the glucose derivative may be selected from the group consisting of methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, allyl-, and vinyl- ⁇ -D-glucopyranoside, methyl- ⁇ -D-Glc, salicin, ⁇ -salicin, arbutin, methyl arbutin, ⁇ -arbutin, phenyl ⁇ -D-glucopyranoside, ⁇ -D-glucopyranoside, coniferin, aesculin, sinigrin, amygdalin, and ruberythic acid.
  • the UDP glycosyltransferase may be capable of conducting ⁇ -1,2-transglycosylation. e.g., it may be EUGT11, UGT91C1, UGT91D2, UGT91D2e, HV1, UGT2, UGT-B or UGTSL2, to form a trisaccharide derivative and UDP.
  • the pH is from about 2 to about 10
  • the temperature is from about 10 °C to 100 °C
  • the time is from 1 to 72 hours
  • other embodiments are directed to forming oligosaccharides 800 by hydrolyzing another molecule from a naturally occurring source 400 where one of the products is the oligosaccharide.
  • the method of hydrolysis is initiated enzymatically and comprises contacting a naturally occurring compound, such as naringin or neohesperidin, with a beta-glucosidase to initiate enzymatic hydrolysis of the glycoside from the aglycone, to yield the free neohesperidoside.
  • the method of hydrolysis is initiated chemically and comprises contacting a naturally occurring compound, such as naringin or neohesperidin, with an oxidizing agent to initiate oxidative cleavage of the aryl-ether bond joining the aglycone and the glycoside, to yield the free neohesperidoside and oxidized aglycone.
  • a naturally occurring compound such as naringin or neohesperidin
  • an oxidizing agent to initiate oxidative cleavage of the aryl-ether bond joining the aglycone and the glycoside, to yield the free neohesperidoside and oxidized aglycone.
  • Suitable oxidizing agents for the oxidative cleavage of flavonoid glycosides include but are not limited to potassium permanganate and hydrogen peroxide.
  • Examples of processes in which steviol glycosides are produced according to one or more embodiment described herein include but are not limited to: [0057] Reb A ⁇ Reb B ⁇ Reb M; [0058] stevioside ⁇ steviol-1,2-bioside ⁇ Reb D4; [0059] steviol ⁇ Reb B or Reb WB2 ⁇ Reb M; [0060] Reb C ⁇ Dulcoside B ⁇ 3 C19-Glc -O-D-glucopyranosyl-Reb K; [0061] Steviol ⁇ Reb B ⁇ Reb M; [0062] Reb A ⁇ Reb B ⁇ Reb M; [0063] Reb A ⁇ Reb B ⁇ Reb N; [0064] Reb A ⁇ Reb B ⁇ Reb T; [0065] Reb WB1 ⁇ Reb WB2 ⁇ Reb M; [0066] steviol ⁇ Dulcoside B; and [0067] steviol ⁇ Dulcoside A1.
  • steviol glycoside After producing a steviol glycoside, one may purify the steviol glycoside to form a purified product.
  • Methods for purifying steviol glycosides are well-known to persons of ordinary skill in the art and include, but are not limited to, filtration and passing through an HPLC column.
  • An example of a method for purification is: (1) place the material in a centrifuge reaction medium for removing a precipitate; (2) load that supernatant on a macroporous resin; (3) elute Reb D and M from the resin with different fractions of ethanol/water mixture; (4) remove the solvent to obtain a powder form of the product; (5) dissolve the powder in an ethanol/water mixture; (6) crystallize the substance at a lower temperature; and (7) isolate crystals by filtration, washing and drying.
  • Uses of Steviol Glycosides [0070] The steviol glycosides made in accordance with one or more embodiment described herein may be used in diverse applications, including but not limited to being incorporated into food products and beverage products.
  • Examples of food products include, but are not limited to, confections, condiments, chewing gum, frozen foods, canned foods, soy-based products, salad dressings, mayonnaise, vinegar, ice cream, cereal compositions, baked goods, dairy products such as yogurts, soups, sauces, ketchup, fruit prep, jams and jellies, gluten-free applications, alternative dairy (almond, oat, coconut, soy), and tabletop sweetener compositions.
  • beverages include, but are not limited to, ready-to-drink products that are carbonated (e.g., colas or other soft drinks, sparkling beverages, and malts) or non-carbonated (e.g., fruit juices, nectars, vegetable juices, sports drinks, energy drinks, enhanced water, coconut waters teas, coffees, cocoa drinks, beverages containing milk, beverages containing cereal extracts, smoothies, and alcoholic beverages), as well as powdered beverage products that are to be combined with a liquid base such as water, milk, or club soda and beverage concentrates such as throw syrups, e.g., 5 + 1 and 9 + 1 throw syrups.
  • carbonated e.g., colas or other soft drinks, sparkling beverages, and malts
  • non-carbonated e.g., fruit juices, nectars, vegetable juices, sports drinks, energy drinks, enhanced water, coconut waters teas, coffees, cocoa drinks, beverages containing milk, beverages containing cereal extracts, smoothies, and alcoholic beverages
  • the steviol glycosides made in accordance with one or more embodiment described herein may also be used in dental compositions and pharmaceutical compositions.
  • the steviol glycosides made in accordance with one or more embodiment described herein may be the sole sweetening component or other sweeteners may also be incorporated into the product.
  • the steviol glycosides may be combined with at least one additional sweetener, such as a carbohydrate sweetener.
  • carbohydrate sweeteners include, but are not limited to, sucrose, fructose, glucose, erythritol, maltitol, lactitol, sorbitol, mannitol, xylitol, D-psicose, D-tagatose, leucrose, trehalose, galactose, rhamnose, cyclodextrin (e.g., ⁇ -cyclodextrin, ⁇ -cyclodextrin, and ⁇ -cyclodextrin), ribulose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, palatinose or isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythr
  • additional sweeteners include but are not limited to high potency sweeteners such as mogroside IV, mogroside V, mogroside VI, iso-mogroside V, grosmomoside, neomogroside, siamenoside, monatin and its salts (monatin SS, RR, RS, SR), curculin, glycyrrhizic acid and its salts, thaumatin, monellin, mabinlin, brazzein, hernandulcin, phyllodulcin, glycyphyllin, phloridzin, trilobatin, baiyunoside, osladin, polypodoside A, pterocaryoside A, pterocaryoside ⁇ , mukurozioside, phlomisoside I, periandrin I, abrusoside A, and cyclocarioside I.
  • high potency sweeteners such as mogroside IV, mogroside V,
  • Steviol glycosides made in accordance with one or more embodiment described herein may also be combined with one or more additives that may or may not also be additional sweeteners.
  • additives include but are not limited to carbohydrates, polyols, amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts, including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavorants and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, emulsifiers, weighing agents, gums, colorants, flavonoids, alcohols, polymers, essential oils, anti-fungal agents and combinations thereof.
  • Steviol glycosides made in accordance with one or more embodiment described herein may also be combined with one or more bulking agents that may also qualify as additives or additional sweeteners.
  • Bulking agents may, for example, be used to facilitate a direct substitution of the steviol glycosides of the present invention for sugar in applications such as baking, cooking, and tabletop uses.
  • bulking agents include but are not limited to: a bulk sweetener such as sucrose, dextrose, invert sugar maltose, dextrin, maltodextrin, fructose, levulose, and galactose; a low glycemic carbohydrate such as fructo-oligosaccharides, galacto- oligosaccharides, mannitol, xylitol, lactitol, erythritol, and malitol; a fiber, such as polydextrose, resistant maltodextrin, resistant starch, soluble corn fiber, and cellulose; and a hydrocolloid, such as pectin, guar gum, carboxymethylcellulose, locust bean gum, cassia gum, and alginate.
  • a bulk sweetener such as sucrose, dextrose, invert sugar maltose, dextrin, maltodextrin, fructose, levulose, and gal
  • oligosaccharides such as the disaccharides and trisaccharides of the present invention may also be used in applications and products apart from in conjunction with stevia compounds.
  • These oligosaccharides include but are not limited to sophorose, a derivative of sophorose, laminaribiose, a derivative of laminaribiose, 2- ⁇ -D-glucopyranosyl-(3- ⁇ -D-glucopyranosyl)-D-glucose, a derivative of 2- ⁇ -D-glucopyranosyl-(3- ⁇ -D-glucopyranosyl)-D-glucose, neohesperidose, a derivative of neohesperidose, rutinose, a derivative of rutinose, 2- ⁇ -L-rhamnosyl-(3- ⁇ -D-glucopyranosyl)-D-glucose, and a derivative of 2- ⁇
  • oligosaccharides of the invention are one more the following: increased solubility, increased selectivity, such as membrane transport and enzyme inhibition, increased stability, and modification of physio-chemical properties such as surface tension, which may, for example be advantageous for drug delivery. Additional benefits may include being digestion resistant or non-caloric saccharides.
  • Examples of starting glucose derivatives that may be used separately or in combination to form oligosaccharides such as disaccharides, trisaccharides, and derivatives thereof, optionally for use apart from in conjunction with stevia compounds, include but are not limited to: Methyl ⁇ -D-glucopyranoside; Phenyl ⁇ -D-glucopyranoside; Hydroxyphenyl ⁇ - or ⁇ -D-glucopyranoside ( ⁇ - or ⁇ -Arbutin); Methylarbutin; 2-(Hydroxymethyl)phenyl ⁇ -D-glucopyranoside (Salicin); and Hexyl ⁇ -D-glucopyranoside.
  • embodiments of the invention may be used to create transglycosylated products from salicin that are anti-inflammatory, from arbutin and/or ⁇ -arbutin that are inhibitors of tyrosinase, and from hexyl ⁇ -D- glucospyranoside that are surfactants that may, for example, be used in pharmaceutical and personal care products.
  • one may use methods of the invention to produce an oligosaccharide or an oligosaccharide derivative from ⁇ - or ⁇ -arbutin (hydroxyphenyl ⁇ - or ⁇ -D-glucopyranoside) or methyl arbutin or combinations thereof.
  • Arbutin may for example, be obtained in nature, and ⁇ -arbutin and ⁇ -arbutin may, for example, be chemically synthesized. These resulting oligosaccharide or oligosaccharide derivatives may, for example, be used in creams such as skin- whitening creams. Among the benefits or these compounds are that they may be used as tyrosine inhibitors because they have anti-melanogenic activity. [0079] By way of another non-limiting example, one may use methods of the present invention to produce an oligosaccharide or an oligosaccharide derivative from salicin (2-(hydroxymethyl)phenyl ⁇ -D-glucopyranoside) and/or derivatives of salicin.
  • the salicin and/or its derivatives may, for example be obtained as extracts from willow (Salix) bark and Populus species.
  • the resulting compounds may, for example, be used in creams such as anti-inflammatory creams e.g., acne creams, and in analgesics that are applied topically, or administered orally, subcutaneously, intranasally or intravenously.
  • creams such as anti-inflammatory creams e.g., acne creams, and in analgesics that are applied topically, or administered orally, subcutaneously, intranasally or intravenously.
  • one may use methods of the present invention to produce an oligosaccharide or an oligosaccharide derivative from hexyl ⁇ -D-glucopyranoside and/or derivatives of hexyl ⁇ -D-glucopyranoside.
  • oligosaccharides examples include but are not limited to hexyl laminaribiose, hexyl sophorose, and hexyl tri-saccharides.
  • the resulting compounds may, for example, be used as surfactants in cosmetics, personal care products, pharmaceuticals, and foods.
  • the oligosaccharides or oligosaccharide derivatives may have one or more the following desirable properties: antimicrobial activity, being biodegradable, and having low toxicity.
  • Glycosylated hexyl ⁇ -D- glucopyranoside may be particularly advantageous as a pharmaceutical non-ionic surfactant for solubilizing insoluble medicines and delivering medicines.
  • the oligosaccharides may be used as active or inactive ingredients in pharmaceutical compositions or formulations or in personal or beauty care compositions, formulations, or products.
  • a beauty care product is a product such as creams, lotions, color applications, concealers, moisturizers or other products applied to a person’s skin, hair or face to improve one’s appearance.
  • a personal care product is a product such as soaps, shampoos, conditioners, body washes, and face washes that are designed to clean or to aid in hygienic care of one’s body.
  • the oligosaccharides may be used as medicaments for the treatment, prevention, or reduction of symptoms of a disease, disorder or a condition.
  • oligosaccharides may be administered in a therapeutically effect amount to a subject, e.g., a human, in need thereof. Thus, they may be used to treat, prevent or ameliorate the symptoms of a disease, disorder or condition.
  • the disease, disorder, or condition may for, example, be a skin condition such as, a rash, acne, or psoriasis.
  • the oligosaccharides of the invention may also be used in combination with one or more other active ingredients in a pharmaceutical compositions or formulations or in personal or beauty care compositions or formulations.
  • the oligosaccharides When used in combination with one or more other active ingredients, i.e., ingredients that are intended to or will create a physiological change or response, the oligosaccharides may themselves be active and thus capable of creating create a physiological change or response or inactive and used as, for example a surfactant or carrier or intended to impart a benefit with respect to dispersion, flow, or other parameter.
  • Examples of cosmetics, beauty, body and hair care products, compositions and formulations that may comprise the oligosaccharides of and/or produced by the present invention include but are not limited to creams, lotions, washes, rinses, pomades, gels, powders, colorants, sprays, shampoos, conditioners, lipsticks, rouges, foundation, lip liner, masks, dyes, bleaching agents, eyeliner, mascara, tanning agents, sunscreens, and nail polishes.
  • oligosaccharides of the present invention When one or more oligosaccharides of the present invention is used in a cosmetics, beauty, body or hair care product, other ingredients that are now known or that come to be known for inclusion in those products may also be included, for example, a bioactive agent that is an oil soluble and/or water soluble component and/or an anti-wrinkle or other agent that may be effective for skin health, such as whitening, antioxidant, and elasticity enhancement.
  • a bioactive agent that is an oil soluble and/or water soluble component and/or an anti-wrinkle or other agent that may be effective for skin health, such as whitening, antioxidant, and elasticity enhancement.
  • compositions or formulations that comprise the oligosaccharides are beauty or personal care products, they may also comprise one or more of cleansing agents, solvents, thickeners, and neutralizers.
  • Examples of cleansing agents include, but are not limited to, lipids and soaps commonly found in shampoos and body washes, e.g., ammonium lauryl sulfate, cocamidopropyl betaine, cocobetaine, and lauroamphoacetate.
  • Examples of solvents include, but are not limited to, alcohols, water, and mixtures thereof.
  • thickeners include but are not limited to carbomers, xanthan gum, acrylate/CCCC10 - 230 alkyl acrylate crossovers, ammonium acryloyl dimethyltaurate/vinylpyrrolidine copolymers, hydroxypropyl starch glycolate, tapioca starch, gelangum, hydroxyethylcellulose sodium polyacrylate, polyacrylate cross polymer -11, and ammonium acryloyldimethtaurate/behex -25 methacrylate crossovers.
  • Examples of neutralizers include but are not limited to amine-based neutralizer such as (arginine), tromethamine (Tromethamine), triethylamine (Triethylamine) with the neutralizing agent, water, and mixtures thereof may be used.
  • amine-based neutralizer such as (arginine), tromethamine (Tromethamine), triethylamine (Triethylamine) with the neutralizing agent, water, and mixtures thereof may be used.
  • Examples of food products and beverage products in which the oligosaccharides may be included even when not part of steviol glycosides are those that are identified in this specification for use with steviol glycosides. In these products, the oligosaccharides may, for example, be mixed with one or more food ingredients or beverage ingredients, such as other sugars, proteins, fats, vitamins, and minerals.
  • Examples of medicaments and pharmaceuticals in which the oligosaccharides may be used include, but are not limited to, oral formulations, such as tablets, capsules, caplets, elixirs, intranasal formulations, intravenous formulations, and topical formulations.
  • oral formulations such as tablets, capsules, caplets, elixirs, intranasal formulations, intravenous formulations, and topical formulations.
  • those other active ingredients may, for example, be analgesics such as acetaminophen or NSAIDS, or compounds that treat, prevent or ameliorate one or more of the following conditions: neurologic conditions, cancers, psychological conditions, musculo-skeletal conditions, pulmonary conditions, and cardiac conditions.
  • a method for producing a steviol glycoside comprising conjugating a stevia compound to an oligosaccharide to form the steviol glycoside, wherein said conjugating is via either enzymatic synthesis in the presence of a transglycosylation enzyme or chemical conversion.
  • the stevia compound is selected from the group consisting of steviol, Reb B, and Reb WB2 and the oligosaccharide is a disaccharide, disaccharide derivative, trisaccharide or trisaccharide derivative.
  • the steviol glycoside is selected from the group consisting of Reb I, Reb B, Reb J, Reb M, Reb D, Reb D2, Reb D4, Reb W, Reb WB1, Reb WB2, Reb N, Reb C, Dulcoside A, Dulcoside A1, and Reb K. 4.
  • the method of any of embodiments 1 to 4, wherein the conjugating is at position C13 of the stevia compound. 7.
  • the conjugating is via enzymatic synthesis in the presence of the transglycosylation enzyme. 8.
  • the transglycosylation enzyme is capable of forming a glyco-ester bond or a glyco-ether bond.
  • the transglycosylation enzyme is a ⁇ - glucosidase.
  • said conjugating is via chemical conversion. 12.
  • the method of embodiment 11, wherein the chemical conversion comprises exposing the stevia compound and the oligosaccharide to at least one of sulfuric acid silica, alkali, and acid under conditions that selectively conjugate the oligosaccharide to the stevia compound at a desired location.
  • the method of embodiment 13 further comprising chemical hydrolysis of at least one of Reb A, Reb D, Reb I, Reb D2, Reb M, Reb M2, Reb J, Reb N, Reb T or Reb O under caustic conditions created by the introduction of at least one of potassium hydroxide, sodium hydroxide, sodium carbonate, calcium oxide, calcium hydroxide and lithium hydroxide to form Reb B. 15.
  • the method of embodiment 13 further comprising enzymatic hydrolysis of at least one of Reb A, Reb D, Reb I, Reb D2, Reb M, Reb M2, Reb J, Reb N, Reb T or Reb O to form Reb B.
  • the method of embodiment 16 further comprising hydrolysis via enzymatic hydrolysis or chemical treatment of at least one of Reb WB1, Reb W, or Reb D4 to form Reb WB2.
  • the method of embodiment 17, wherein the hydrolysis is via chemical treatment.
  • the method of embodiment 18, wherein the chemical treatment is under caustic hydrolysis conditions that cause cleavage at C19.
  • 20. The method of embodiment 18, wherein the chemical treatment is under oxidative conditions that cause cleavage at C19.
  • the UDP-glycosyltransferase is selected from the group consisting of EUGT11, UGT76G1, UGT91C1, UGTSL2, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGT4, UGT-A, UGT-B, HV1, CP1, CR1, UGT85C2, and UGT74G1. 29.
  • UDP-glycosyltransferase is selected from the group consisting of EUGT11, UGT76G1, UGT91C1, UGTSL2, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGT4, UGT-A, UGT-B, HV1, CP1, CR1, UGT85C2, and UGT74G1. 31.
  • glucose derivative is selected from the group consisting of methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, allyl-, and vinyl- ⁇ -D-glucopyranoside, methyl- ⁇ -D-glucopyranoside, salicin, ⁇ -salicin, arbutin, methyl arbutin, ⁇ -arbutin, phenyl ⁇ - D-glucopyranoside, and ⁇ -D-glucopyranoside. 32.
  • oligosaccharide is a trisaccharide or trisaccharide derivative and the method further comprises exposing a disaccharide to UDP-Glc in the presence of a UDP glycosyltransferase to form the oligosaccharide.
  • UDP-glycosyltransferase is selected from the group consisting of EUGT11, UGT76G1, UGT91C1, UGTSL2 , UGT91D1, UGT91D2, UGT91D2e, UGT2, UGT4, UGT-A, UGT-B, HV1, CP1, CR1, UGT85C2, and UGT74G1. 34.
  • oligosaccharide is a trisaccharide or trisaccharide derivative and the method further comprises exposing a disaccharide derivative to UDP-Glc in the presence of a UDP glycosyltransferase to form the oligosaccharide.
  • UDP-glycosyltransferase is selected from the group consisting of EUGT11, UGT76G1, UGT91C1 and UGTSL2, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGT4, UGT-A, UGT-B, HV1, CP1, CR1, UGT85C2, and UGT74G1.
  • disaccharide derivative is selected from the group consisting of sophorose, a derivative of sophorose, laminaribiose, a derivative of laminaribiose, 2- ⁇ -D-glucopyranosyl-(3- ⁇ - D-glucopyranosyl)-D-glucose, and a derivative of 2- ⁇ -D-glucopyranosyl-(3- ⁇ -D- glucopyranosyl)-D-glucose. 37.
  • the method according to embodiment 36 further comprising forming the disaccharide derivative by exposing glucose or a glucose derivative to Glc-1-P in the presence of laminaribiose phosphorylase, sophorose phosphorylase, gentiobiose phosphorylase, cellobiose phosphorylase, or cellodextrin phosphorylase to form inorganic phosphate and the disaccharide derivative. 38.
  • glucose derivative is selected the group consisting of methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, allyl-, and vinyl- ⁇ -D-glucopyranoside, methyl- ⁇ -D-glucopyranoside, salicin, ⁇ -salicin, arbutin, methyl arbutin, ⁇ -arbutin, phenyl ⁇ -D-glucopyranoside, and ⁇ -D- glucopyranoside. 39.
  • a method for synthesizing a disaccharide or a disaccharide derivative comprising exposing glucose or a glucose derivative to Glc-1-P in the presence of either or both of laminaribiose phosphorylase or sophorose phosphorylase to form inorganic phosphate and the disaccharide or the disaccharide derivative. 40.
  • a method for synthesizing a trisaccharide or a trisaccharide derivative comprising the method of embodiment 39 and exposing the disaccharide or the disaccharide derivative to UDP-Glc in the presence of a UDP glycosyltransferase, wherein the UDP glycosyltransferase is capable of ⁇ -1,2-transglycosylation or ⁇ -1,3- transglycosylation to form UDP and the trisaccharide or trisaccharide derivative.
  • the UDP glycosyltransferase is selected from the group consisting of UGTSL2, UGT91D1, UGT91D2, UGT91D2e.
  • the UDP glycosyltransferase is UGT76G1.
  • a method for synthesizing a trisaccharide or a trisaccharide derivative comprising exposing glucose or a glucose derivative to UDP-Glc in the presence UDP glycosyltransferase, wherein the UDP glycosyltransferase is capable of ⁇ -1,2-transglycosylation or ⁇ -1,3-transglycosylation to form UDP and the trisaccharide or trisaccharide derivative.
  • UDP glycosyltransferase is selected from the group consisting of UGTSL2, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGT4, UGT-A, UGT-B, HV1, CP1, CR1, UGT85C2, and UGT74G1.
  • UGTSL2, UGT91D1, UGT91D2, UGT91D2e is selected from the group consisting of UGTSL2, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGT4, UGT-A, UGT-B, HV1, CP1, CR1, UGT85C2, and UGT74G1.
  • UGT76G is selected from the group consisting of UGTSL2, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGT4, UGT-A, UGT-B, HV1, CP1, CR1, UGT85C2, and UGT74G1.
  • glucose derivative is selected from the group consisting of methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, allyl-, and vinyl- ⁇ -D-glucopyranoside, methyl- ⁇ -D- glucopyranoside, salicin, ⁇ -salicin, arbutin, methyl arbutin, ⁇ -arbutin, phenyl ⁇ -D- glucopyranoside, and ⁇ -D-glucopyranoside. 47.
  • UDP-glycosyltransferase is selected from the group consisting of EUGT11, UGT76G1, UGT91C1, UGTSL2, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGT4, UGT-A, UGT-B, HV1, CP1, CR1, UGT85C2, and UGT74G1.
  • the oligosaccharide is a disaccharide or disaccharide derivative and the method further comprises exposing a glucose derivative to an UDP-Glc in the presence of a UDP glycosyltransferase to form the oligosaccharide. 51.
  • UDP-glycosyltransferase is selected from the group consisting of EUGT11, UGT76G1, UGT91C1, UGTSL2, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGT4, UGT-A, UGT-B, HV1, CP1, CR1, UGT85C2, and UGT74G1. 52.
  • glucose derivative is selected from the group consisting of methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, allyl-, and vinyl- ⁇ -D-glucopyranoside, methyl- ⁇ -D-glucopyranoside, salicin, ⁇ -salicin, arbutin, methyl arbutin, ⁇ -arbutin, phenyl ⁇ -D-glucopyranoside, and ⁇ -D-glucopyranoside. 53.
  • oligosaccharide is a disaccharide or disaccharide derivative and the method further comprises exposing glucose to an UDP-L-Rhamnose in the presence of a UDP rhamnosyltransferase to form the oligosaccharide.
  • the UDP-rhamnosyltransferase is selected from the group consisting of UGT-B, EUGT11, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGTSL2, UGT76G1, UGT4, UGT-A, HV1, CP1, CR1, UGT85C2, and UGT74G1. 55.
  • oligosaccharide is a disaccharide or disaccharide derivative and the method further comprises exposing a glucose derivative to an UDP-L-Rhamnose in the presence of a UDP rhamnosyltransferase to form the oligosaccharide.
  • UDP-rhamnosyltransferase is selected from the group consisting of UGT-B, EUGT11, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGTSL2, UGT76G1, UGT4, UGT-A HV1, CP1, CR1, UGT85C2, and UGT74G1.
  • glucose derivative is selected from the group consisting of methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, allyl-, and vinyl- ⁇ -D-glucopyranoside, methyl- ⁇ -D-glucopyranoside, salicin, ⁇ -salicin, arbutin, methyl arbutin, ⁇ -arbutin, phenyl ⁇ -D-glucopyranoside, and ⁇ -D-glucopyranoside. 58.
  • oligosaccharide is a trisaccharide or trisaccharide derivative and the method further comprises exposing glucose to an UDP-Glc and an UDP-L-Rhamnose in the presence of a UDP glycosyltransferase and UDP-rhamnosyltransferase to form the oligosaccharide. 59.
  • UDP-glycosyltransferase is selected from the group consisting of EUGT11, UGT76G1, UGT91C1, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGT4, UGT-A, UGT-B, HV1, CP1, CR1, UGT85C2, UGT74G1 and UGTSL2 and the UDP-rhamnosyltransferase is selected from the group consisting of UGT-A, EUGT11, UGT91D1, UGT91D2, UGT91D2e, UGTSL2, UGT76G1, UGT2, UGT4, UGT-B, HV1, CP1, CR1, UGT85C2, and UGT74G1. 60.
  • oligosaccharide is a trisaccharide or trisaccharide derivative and the method further comprises exposing a glucose derivative to an UDP-Glc and an UDP-L-Rhamnose in the presence of a UDP glycosyltransferase and UDP-rhamnosyltransferase to form the oligosaccharide. 61.
  • UDP-glycosyltransferase is selected from the group consisting of EUGT11, UGT76G1, UGT91C1, UGT91D1, UGT91D2, UGT2, UGT4, UGT-B, HV1, CP1, CR1, UGT85C2, UGT74G1 and UGTSL2 and the UDP-rhamnosyltransferase is selected from the group consisting of UGT-B, EUGT11, UGT91D1, UGT91D2, UGTSL2 and UGT76G1, UGT2, UGT4, UGT-A HV1, CP1, CR1, UGT85C2, and UGT74G1. 62.
  • glucose derivative is selected from the group consisting of methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, allyl-, and vinyl- ⁇ -D-glucopyranoside, methyl- ⁇ -D-glucopyranoside, salicin, ⁇ -salicin, arbutin, methyl arbutin, ⁇ -arbutin, phenyl ⁇ -D-glucopyranoside, and ⁇ -D-glucopyranoside.
  • the glucose derivative is selected from the group consisting of methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, allyl-, and vinyl- ⁇ -D-glucopyranoside, methyl- ⁇ -D-glucopyranoside, salicin,
  • oligosaccharide is a trisaccharide or trisaccharide derivative and the method further comprises exposing a neohesperidose to UDP-Glc in the presence of a UDP glycosyltransferase to form the oligosaccharide.
  • a neohesperidose to UDP-Glc in the presence of a UDP glycosyltransferase to form the oligosaccharide.
  • UDP-glycosyltransferase is UGT76G1.
  • oligosaccharide is a trisaccharide or trisaccharide derivative and the method further comprises exposing a neohesperidose derivative to UDP-Glc in the presence of a UDP glycosyltransferase to form the oligosaccharide.
  • UDP-glycosyltransferase is UGT76G1.
  • oligosaccharide is a disaccharide or disaccharide derivative and the method further comprises exposing a naturally-occurring compound to an oxidizing agent to obtain a disaccharide separated from an aglycone. 68.
  • disaccharide derivative is selected from the group consisting of neohesperidose, a derivative of neohesperidose, 2- ⁇ -L- rhamnosyl-(3- ⁇ -D-glucopyranosyl)-D-glucose, and a derivative of 2- ⁇ -L-rhamnosyl- (3- ⁇ -D-glucopyranosyl)-D-glucose.
  • Subject matter contemplated by the present disclosure is further set out in the following numbered embodiments: 1A.
  • a method for producing a steviol glycoside comprising conjugating a stevia compound to an oligosaccharide to form the steviol glycoside, wherein said conjugating is via either enzymatic synthesis in the presence of a transglycosylation enzyme or chemical conversion.
  • 2A The method according to embodiment 1A, wherein the stevia compound is selected from steviol, Reb B, and Reb WB2 and the oligosaccharide is a disaccharide, disaccharide derivative, trisaccharide or trisaccharide derivative.
  • steviol glycoside is selected from the group consisting of Reb I, Reb B, Reb J, Reb M, Reb D, Reb D2, Reb D4, Reb W, Reb WB1, Reb WB2, Reb N, Reb C, Dulcoside A, Dulcoside A1, and Reb K. 4A
  • the method of any of embodiments 1A to 4A, wherein the conjugating is at position C19 of the stevia compound, at position C13 of the stevia compound, or a combination thereof. 6A.
  • oligosaccharide is a first oligosaccharide
  • the method further comprises conjugating a second oligosaccharide to the stevia compound at position C19 of the stevia compound.
  • 7A The method of any of embodiments 1A to 6A, wherein said conjugating is via enzymatic synthesis in the presence of the transglycosylation enzyme.
  • 8A The method of embodiment 7A, wherein the transglycosylation enzyme is capable of forming a glyco-ester bond or a glyco-ether bond and, optionally, is a ⁇ - glucosidase.
  • the method of embodiment 11A further comprising (i) chemical hydrolysis of at least one of Reb A, Reb D, Reb I, Reb D2, Reb M, Reb M2, Reb J, Reb N, Reb T or Reb O under caustic conditions created by the introduction of at least one of potassium hydroxide, sodium hydroxide, sodium carbonate, calcium oxide, calcium hydroxide and lithium hydroxide to form Reb B, (ii) enzymatic hydrolysis of at least one of Reb A, Reb D, Reb I, Reb D2, Reb M, Reb M2, Reb J, Reb N, Reb T or Reb O to form Reb B, or (iii) any combination thereof to form Reb B. 13A.
  • the method of any of embodiments 1A to 10A, wherein the stevia compound is Reb WB2. 14A.
  • the method of embodiment 13A further comprising hydrolysis via enzymatic hydrolysis or chemical treatment of at least one of Reb WB1, Reb W, or Reb D4 to form Reb WB2.
  • 15A The method of embodiment 14A, wherein the chemical treatment is under caustic hydrolysis conditions, oxidative conditions, or a combination thereof that causes cleavage at C19. 16A.
  • any of embodiments 1A to 10A wherein the stevia compound is Reb B or Reb WB2 and said conjugating occurs (i) in the presence of a caustic agent or an acid in an aqueous solution, a mixture of methanol or ethanol and water, or any combination thereof; (ii) in the presence of hydrogen peroxide and potassium permanganate; (iii) under conditions that cause an oxidative reaction; or (iv) any combination thereof. 18A.
  • oligosaccharide is a trisaccharide or trisaccharide derivative and the method further comprises exposing glucose, a glucose derivative, a disaccharide, a disaccharide derivative, or a combination thereof to UDP-Glc in the presence of a UDP glycosyltransferase to form the oligosaccharide. 19A.
  • UDP-glycosyltransferase is selected from EUGT11, UGT76G1, UGT91C1, UGTSL2, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGT4, UGT-A, UGT-B, HV1, CP1, CR1, UGT85C2, UGT74G1, and combinations thereof. 20A.
  • glucose derivative is selected from the group consisting of methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, allyl-, and vinyl- ⁇ -D-glucopyranoside, methyl- ⁇ -D- glucopyranoside, salicin, ⁇ -salicin, arbutin, methyl arbutin, ⁇ -arbutin, phenyl ⁇ -D- glucopyranoside, and ⁇ -D-glucopyranoside. 21A.
  • disaccharide derivative is selected from sophorose, a derivative of sophorose, laminaribiose, a derivative of laminaribiose, 2- ⁇ -D-glucopyranosyl-(3- ⁇ -D-glucopyranosyl)-D- glucose, and a derivative of 2- ⁇ -D-glucopyranosyl-(3- ⁇ -D-glucopyranosyl)-D- glucose. 22A.
  • the method according to embodiment 21A further comprising forming the disaccharide derivative by exposing glucose or a glucose derivative to Glc-1-P in the presence of laminaribiose phosphorylase, sophorose phosphorylase, gentiobiose phosphorylase, cellobiose phosphorylase, cellodextrin phosphorylase, or combinations thereof to form inorganic phosphate and the disaccharide derivative.
  • glucose derivative is selected from methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, allyl-, and vinyl- ⁇ -D-glucopyranoside, methyl- ⁇ -D-glucopyranoside, salicin, ⁇ -salicin, arbutin, methyl arbutin, ⁇ -arbutin, phenyl ⁇ -D-glucopyranoside, ⁇ -D-glucopyranoside, and combinations thereof. 24A.
  • a method for synthesizing a disaccharide or a disaccharide derivative comprising exposing glucose or a glucose derivative to Glc-1-P in the presence of either or both of laminaribiose phosphorylase or sophorose phosphorylase to form inorganic phosphate and the disaccharide or the disaccharide derivative. 25A.
  • a method for synthesizing a trisaccharide or a trisaccharide derivative comprising (i) the method of embodiment 24A and exposing the disaccharide or the disaccharide derivative to UDP-Glc in the presence of a UDP glycosyltransferase, (ii) exposing glucose or a glucose derivative to UDP-Glc in the presence UDP glycosyltransferase, or (iii) any combination thereof, wherein the UDP glycosyltransferase is capable of ⁇ -1,2-transglycosylation or ⁇ -1,3- transglycosylation to form UDP and the trisaccharide or trisaccharide derivative.
  • UDP glycosyltransferase is selected from UGTSL2, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGT4, UGT-A, UGT-B, HV1, CP1, CR1, UGT85C2, UGT74G1, UGT76G1, and combinations thereof.
  • glucose derivative is selected from methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, allyl-, and vinyl- ⁇ -D-glucopyranoside, methyl- ⁇ -D-glucopyranoside, salicin, ⁇ -salicin, arbutin, methyl arbutin, ⁇ -arbutin, phenyl ⁇ -D-glucopyranoside, and ⁇ -D-glucopyranoside. 28A.
  • UDP-glycosyltransferase is selected from EUGT11, UGT76G1, UGT91C1, UGTSL2, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGT4, UGT-A, UGT-B, HV1, CP1, CR1, UGT85C2, and UGT74G1. 31A.
  • glucose derivative is selected from the group consisting of methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, allyl-, and vinyl- ⁇ -D-glucopyranoside, methyl- ⁇ -D- glucopyranoside, salicin, ⁇ -salicin, arbutin, methyl arbutin, ⁇ -arbutin, phenyl ⁇ -D- glucopyranoside, and ⁇ -D-glucopyranoside. 32A.
  • oligosaccharide is a disaccharide or disaccharide derivative and the method further comprises exposing glucose, a glucose derivative, or a combination thereof to a UDP-L-Rhamnose in the presence of a UDP rhamnosyltransferase to form the oligosaccharide. 33A.
  • UDP-rhamnosyltransferase is selected from UGT-B, EUGT11, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGTSL2, UGT76G1, UGT4, UGT-A, HV1, CP1, CR1, UGT85C2, UGT74G1, and combinations thereof. 34A.
  • glucose derivative is selected from methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, allyl-, and vinyl- ⁇ -D-glucopyranoside, methyl- ⁇ -D-glucopyranoside, salicin, ⁇ - salicin, arbutin, methyl arbutin, ⁇ -arbutin, phenyl ⁇ -D-glucopyranoside, and ⁇ -D- glucopyranoside. 35A.
  • oligosaccharide is a trisaccharide or trisaccharide derivative and the method further comprises exposing glucose, a glucose derivative, or a combination thereof to a UDP-Glc and a UDP-L- Rhamnose in the presence of a UDP glycosyltransferase and UDP- rhamnosyltransferase to form the oligosaccharide. 36A.
  • UDP-glycosyltransferase is selected from EUGT11, UGT76G1, UGT91C1, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGT4, UGT-A, UGT-B, HV1, CP1, CR1, UGT85C2, UGT74G1, UGTSL2, and combinations thereof
  • the UDP-rhamnosyltransferase is selected from UGT- A, EUGT11, UGT91D1, UGT91D2, UGT91D2e, UGTSL2, UGT76G1, UGT2, UGT4, UGT-B, HV1, CP1, CR1, UGT85C2, UGT74G1, and combinations thereof.
  • glucose derivative is selected from methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, allyl-, and vinyl- ⁇ -D-glucopyranoside, methyl- ⁇ -D-glucopyranoside, salicin, ⁇ - salicin, arbutin, methyl arbutin, ⁇ -arbutin, phenyl ⁇ -D-glucopyranoside, and ⁇ -D- glucopyranoside. 38A.
  • oligosaccharide is a trisaccharide or trisaccharide derivative and the method further comprises exposing a neohesperidose, neohesperidose derivative, or any combination thereof to UDP-Glc in the presence of a UDP glycosyltransferase to form the oligosaccharide.
  • a neohesperidose, neohesperidose derivative, or any combination thereof to UDP-Glc in the presence of a UDP glycosyltransferase to form the oligosaccharide.
  • UDP-glycosyltransferase is UGT76G1.
  • oligosaccharide is a disaccharide or disaccharide derivative and the method further comprises exposing a naturally-occurring compound to an oxidizing agent to obtain a disaccharide separated from an aglycone. 41A.
  • the disaccharide derivative is selected from neohesperidose, a derivative of neohesperidose, 2- ⁇ -L-rhamnosyl-(3- ⁇ - D-glucopyranosyl)-D-glucose, a derivative of 2- ⁇ -L-rhamnosyl-(3- ⁇ -D- glucopyranosyl)-D-glucose, and combinations thereof.
  • 42A A method for creating a beauty or personal care composition comprising the method of embodiment 24A and mixing the disaccharide or disaccharide derivative with at least one of a cleansing agent, a solvent, a thickener, and a neutralizer.
  • the beauty or personal care composition is selected from the group consisting of shampoos, conditioners, lipsticks, rouges, foundation, lip liner, masks, dyes, colorants, bleaching agents, eyeliner, mascara, tanning agents, sunscreens, and nail polishes.
  • the beauty or personal care composition is in the form of a cream, a lotion, a wash, a rinse, a pomade, a gel, or a powder. 45A.
  • a method for creating a beauty or personal care composition comprising the method of any of embodiments 25A to 27A and mixing the trisaccharide or trisaccharide derivative with at least one of a cleansing agent, a solvent, a thickener, and a neutralizer.
  • a cleansing agent e.g., a soap, a soap, a soap, a soap, a soap, a soap, a soap, a soap, a soap, rouges, foundation, lip liner, masks, dyes, colorants, bleaching agents, eyeliner, mascara, tanning agents, sunscreens, and nail polishes. 47A.
  • the method of embodiment 46A wherein the beauty or personal care composition is in the form of a cream, a lotion, a wash, a rinse, a pomade, a gel, or a powder.
  • 48A A method for creating a food or beverage product comprising the method of embodiment 24A and mixing the disaccharide or disaccharide derivative with a food or beverage ingredient.
  • 49A A method for creating a food or beverage product comprising the method of any of embodiment 25A to 27A and mixing the trisaccharide or trisaccharide derivative with a food or beverage ingredient.
  • 50A A method for creating a pharmaceutical composition comprising the method of embodiment 24A and mixing the disaccharide or a disaccharide derivative with at least one active ingredient. 51A.
  • composition is in the form of a tablet, a capsule, a caplet, an elixir, an intranasal formulation, an intravenous formulation, or a topical formulation.
  • 52A A method for creating a pharmaceutical composition comprising the method of any of embodiments 25A to 27A and mixing the trisaccharide or a trisaccharide derivative with at least one active ingredient.
  • Subject matter contemplated by the present disclosure is further set out in the following numbered embodiments: 1B.
  • a disaccharide or disaccharide derivative comprising at least glucose derivative selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D- glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside in a cosmetic product. 2B.
  • a disaccharide or disaccharide derivative comprising at least one glucose derivative selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside in a beauty care product or a personal care product.
  • a disaccharide or disaccharide derivative comprising at least one glucose derivate selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside in a food product or beverage product. 4B.
  • a disaccharide or disaccharide derivative comprising at least one glucose derivative selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside in a pharmaceutical product. 5B.
  • disaccharide or disaccharide derivative comprises two glucose derivatives selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside.
  • each of the two glucose derivatives of the disaccharide or disaccharide derivative are derived from the same glucose derivative selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ --D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside. 7B.
  • a trisaccharide or trisaccharide derivative comprising at least one glucose derivative selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ - D-glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside in a cosmetic product. 8B.
  • a trisaccharide or trisaccharide derivative comprising at least glucose derivative selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D- glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside in a beauty care product or a personal care product.
  • a trisaccharide or trisaccharide derivative comprising at least one glucose derivative selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside in a food product or beverage product. 10B.
  • a trisaccharide or trisaccharide derivative comprising at least one glucose derivative selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside in a pharmaceutical product. 11B.
  • trisaccharide or trisaccharide derivative comprises three glucose derivatives selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D- glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D- glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside. 12B.
  • each of the three units of the trisaccharide or trisaccharide derivative is derived from the same glucose derivative selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D- glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D- glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside. 13B.
  • a cosmetic composition comprising an oligosaccharide, wherein the oligosaccharide is a disaccharide, a disaccharide derivative, a trisaccharide, or a trisaccharide derivative and the oligosaccharide comprises at least glucose derivative selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D- glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D- glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside. 14B.
  • the oligosaccharide comprises two glucose derivatives selected from the group consisting of methyl ⁇ -D- glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D- glucopyranoside. 15B.
  • oligosaccharide comprises three glucose derivatives selected from the group consisting of methyl ⁇ -D- glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D- glucopyranoside. 16B.
  • a beauty care or personal care product comprising an oligosaccharide, wherein the oligosaccharide is a disaccharide, a disaccharide derivative, a trisaccharide, or a trisaccharide derivative and the oligosaccharide comprises at least one glucose derivative selected from the group consisting of methyl ⁇ -D- glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D- glucopyranoside. 17B.
  • oligosaccharide comprises two glucose derivatives selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D- glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside. 18B.
  • oligosaccharide comprises three glucose derivatives selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside. 19B.
  • a food product or beverage product comprising an oligosaccharide, wherein the oligosaccharide is a disaccharide, a disaccharide derivative, a trisaccharide, or a trisaccharide derivative and the oligosaccharide comprises at least one glucose derivative selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D- glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside. 20B.
  • oligosaccharide comprises two glucose derivatives selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D- glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D--glucopyranoside. 21B.
  • oligosaccharide comprises three glucose derivatives selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D- glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside. 22B.
  • a pharmaceutical product comprising an oligosaccharide, wherein the oligosaccharide is a disaccharide, a disaccharide derivative, a trisaccharide, or a trisaccharide derivative and the oligosaccharide comprises at least one glucose derivative selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D- glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside. 23B.
  • oligosaccharide comprises two glucose derivatives selected from the group consisting of methyl ⁇ -D- glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D- glucopyranoside, hydroxyphenyl ⁇ -D- glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D- glucopyranoside. 24B.
  • oligosaccharide comprises three glucose derivatives selected from the group consisting of methyl ⁇ -D- glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D- glucopyranoside.
  • Subject matter contemplated by the present disclosure is further set out in the following numbered embodiments: 1C.
  • a method for producing a steviol glycoside comprising conjugating a stevia compound to an oligosaccharide to form the steviol glycoside, wherein said conjugating is via either enzymatic synthesis in the presence of a transglycosylation enzyme or chemical conversion.
  • 2C The method of embodiment 1C, wherein the stevia compound is selected from steviol, Reb B, and Reb WB2 and the oligosaccharide is a disaccharide, disaccharide derivative, trisaccharide or trisaccharide derivative.
  • steviol glycoside is selected from the group consisting of Reb I, Reb B, Reb J, Reb M, Reb D, Reb D2, Reb D4, Reb W, Reb WB1, Reb WB2, Reb N, Reb C, Dulcoside A, Dulcoside A1, and Reb K, and the conjugating is at position C19 of the stevia compound, at position C13 of the stevia compound, or a combination thereof. 4C.
  • the method of any of embodiments 1C to 5C, wherein the stevia compound is Reb B. 7C.
  • the method of embodiment 6C further comprising (i) chemical hydrolysis of at least one of Reb A, Reb D, Reb I, Reb D2, Reb M, Reb M2, Reb J, Reb N, Reb T or Reb O under caustic conditions created by the introduction of at least one of potassium hydroxide, sodium hydroxide, sodium carbonate, calcium oxide, calcium hydroxide and lithium hydroxide to form Reb B, (ii) enzymatic hydrolysis of at least one of Reb A, Reb D, Reb I, Reb D2, Reb M, Reb M2, Reb J, Reb N, Reb T or Reb O to form Reb B, or (iii) any combination thereof to form Reb B. 8C.
  • 9C The method of embodiment 8C, wherein the chemical treatment is under caustic hydrolysis conditions, oxidative conditions, or a combination thereof that causes cleavage at C19. 10C.
  • the method of embodiment 8C, wherein the hydrolysis is via enzymatic hydrolysis, and, optionally, the enzymatic hydrolysis occurs via a ⁇ -glucosidase. 11C.
  • the oligosaccharide is a trisaccharide or trisaccharide derivative and the method further comprises exposing glucose, a glucose derivative, a disaccharide, a disaccharide derivative, or a combination thereof to UDP-Glc in the presence of a UDP glycosyltransferase to form the oligosaccharide and wherein the UDP-glycosyltransferase is selected from EUGT11, UGT76G1, UGT91C1, UGTSL2, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGT4, UGT-A, UGT-B, HV1, CP1, CR1, UGT85C2, UGT74G1, and combinations thereof.
  • a method for synthesizing a disaccharide or a disaccharide derivative comprising exposing glucose or a glucose derivative to Glc-1-P in the presence of either or both of laminaribiose phosphorylase or sophorose phosphorylase to form inorganic phosphate and the disaccharide or the disaccharide derivative. 13C.
  • a method for synthesizing a trisaccharide or a trisaccharide derivative comprising (i) the method of embodiment 12C and exposing the disaccharide or the disaccharide derivative to UDP-Glc in the presence of a UDP glycosyltransferase, (ii) exposing glucose or a glucose derivative to UDP-Glc in the presence UDP glycosyltransferase, or (iii) any combination thereof, wherein the UDP glycosyltransferase is capable of ⁇ -1,2-transglycosylation or ⁇ -1,3-transglycosylation to form UDP and the trisaccharide or trisaccharide derivative, and the UDP glycosyltransferase is selected from UGTSL2, UGT91D1, UGT91D2, UGT91D2e, UGT2, UGT4, UGT-A, UGT-B, HV1, CP1, CR1, UGT85C2, UGT74G1, UGT76G
  • a method for creating a beauty or personal care composition comprising the method of embodiment 12C and mixing the disaccharide or disaccharide derivative with at least one of a cleansing agent, a solvent, a thickener, and a neutralizer, wherein the beauty or personal care composition is selected from the group consisting of shampoos, conditioners, lipsticks, rouges, foundation, lip liner, masks, dyes, colorants, bleaching agents, eyeliner, mascara, tanning agents, sunscreens, and nail polishes. 15C.
  • a method for creating a beauty or personal care composition comprising the method of embodiment 13C and mixing the trisaccharide or trisaccharide derivative with at least one of a cleansing agent, a solvent, a thickener, and a neutralizer, wherein the beauty or personal care composition is selected from the group consisting of shampoos, conditioners, lipsticks, rouges, foundation, lip liner, masks, dyes, colorants, bleaching agents, eyeliner, mascara, tanning agents, sunscreens, and nail polishes.
  • a method for creating a food or beverage product comprising the method of embodiment 12C and mixing the disaccharide or disaccharide derivative with a food or beverage ingredient. 17C.
  • a method for creating a food or beverage product comprising the method of embodiment 13C and mixing the trisaccharide or trisaccharide derivative with a food or beverage ingredient.
  • 18C A method for creating a pharmaceutical composition comprising the method of embodiment 12C and mixing the disaccharide or disaccharide derivative with at least one active ingredient, wherein the pharmaceutical composition is in the form of a tablet, a capsule, a caplet, an elixir, an intranasal formulation, an intravenous formulation, or a topical formulation. 19C.
  • a method for creating a pharmaceutical composition comprising the method of embodiment 13C and mixing the trisaccharide or trisaccharide derivative with at least one active ingredient, wherein the pharmaceutical composition is in the form of a tablet, a capsule, a caplet, an elixir, an intranasal formulation, an intravenous formulation, or a topical formulation. 20C.
  • a product comprising an oligosaccharide, wherein the oligosaccharide is a disaccharide, a disaccharide derivative, a trisaccharide, or a trisaccharide derivative and the oligosaccharide comprises at least one glucose derivative selected from the group consisting of methyl ⁇ -D-glucopyranoside, phenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D-glucopyranoside, hydroxyphenyl ⁇ -D- glucopyranoside, methylarbutin, salicin and hexyl ⁇ -D-glucopyranoside, wherein the product is a beauty care product, a personal care product, a food or beverage product, or a pharmaceutical.
  • Oxidative Cleavage using Potassium Permanganate [0094] 100 mL acetone and a stir-bar were added to a 250 mL Erlenmeyer flask. The flask was placed in an ice bath on top of a stir-plate and stirred until the solution temperature was less than 5 °C. 8 mL of 0.5 M ammonium hydroxide was added slowly to the acetone solution under stirring. 1000 mg naringin was then dissolved in the ammonia-acetone solution under stirring. The solution (2 °C) was then taken out of the ice bath and placed back on the stir-plate.
  • Reaction digest (0.2 mL) was mixed with 0.05 mL ethyl acetate and 0.75 mL acetonitrile, and then loaded to a 30 mL glass column packed with silica gel 60.
  • Neohesperidose was eluted and isolated using acetonitrile, ethyl acetate, water ratio of 80:5:20 v/v, respectively, as a mobile phase.
  • Under gravity flow separation ⁇ 1 mL/min
  • neohesperidose collected from approximately 1-hour of sample addition until 1.5-hour mark. Samples were then freeze-dried via standard SOP procedure to remove solvent and obtain purified white powder as end product.
  • TLC analysis was performed using TLC silica gel 60 F254 (EMD Millipore) and solvent (acetonitrile, ethyl acetate, water ratio of 80:5:20 v/v, respectively).
  • the plate was developed by immersing it in a developing solution, followed by heat treatment (130 ⁇ C; 7 min.) as explained in Robyt, J.F., Thin-Layer Chromatography of Carbohydrates in Biochemical Techniques, Theory and Practice, Robyt, J.F and White, B.J., eds, Books/Cole Publishing Co., Monterey, CA., pp 107-109, 1987.
  • IPTG isopropyl ⁇ -D-1- thiogalactopyranoside
  • the culture was further grown at 18 ⁇ C for 18-22 hours.
  • Cells were collected by centrifugation (8000 RPM; 20 min; 25 ⁇ C), cell pellets were used within an hour or stored at -18 ⁇ C for up to 3 days.
  • the cell pellets were resuspended using BugBuster ® Protein Extraction Reagent (EMD Millipore) plus Benzonase ® Endonuclease (EMD Millipore); 5 mL of lysing agent is needed per gram of wet cell and 25 units nuclease per mL of lysing agent.
  • Resuspended cells were incubated at 25 ⁇ C for 10 ⁇ 30 minutes with gentle stirring (110 rpm). Alternately, cell pellets were resuspended in cold (4 ⁇ C) lysis buffer (300 mM potassium chloride, 50 mM potassium dihydrogen phosphate, 5 mM imidazole, pH 8), 15 mL lysis buffer per gram wet cells (pellet) is needed, followed by cell disruption by sonication at 4 ⁇ C (35% amplitude, pulses on/off for15 seconds each over 30 minutes). Cell debris was clarified by centrifugation (16,000 x g; 60 min; 4 ⁇ C).
  • Supernatant was loaded to an equilibrated Ni-NTA (Qiagen) affinity column using equilibration buffer (300 mM potassium chloride, 50 mM potassium dihydrogen phosphate, 5 mM imidazole, pH 8). After eluting of the protein sample, the column was washed 2 times with 20 mL equilibration buffer to remove unbound contaminant proteins. The His-tagged UGT recombinant polypeptides were eluted using an elution buffer (equilibration buffer with 250 mM imidazole).
  • equilibration buffer 300 mM potassium chloride, 50 mM potassium dihydrogen phosphate, 5 mM imidazole, pH 8.
  • Disaccharide derivatives were produced by a coupling reaction system.
  • the reaction system contained 50 mM phosphate buffer (pH 7.0) with 1 mM magnesium chloride, 5 mM glucose derivative.
  • Glucose derivatives that were used were glucose, methyl ⁇ -D-glucopyranoside, methyl ⁇ -D-glucopyranoside, phenyl ⁇ - D-glucopyranoside, salicin, arbutin, methyl arbutin, ⁇ -arbutin, hexyl- ⁇ -D- glucopyranoside or heptyl- ⁇ -D-glucopyranoside, 5 mM UDP-Glucose and UDP- glycotransferase as prepared in Example 3. The reaction was incubated at 37 ⁇ C for 18-20 hours. Disaccharide reaction products were immediately identified using TLC (thin layer chromatography).
  • reaction could be terminated by heat treatment (85 ⁇ C; 5 min.) and preserved at -18 ⁇ C for long term storage.
  • TLC analysis was performed using TLC silica gel 60 F254 (EMD Millipore) and solvent (ethyl acetate, methanol, acetic acid ratio of 60:20:20 v/v, respectively).
  • a developing solution was prepared by dissolving 3.0 g of N-(1- naphthyl)ethylene diamine in 950 mL of methanol, stirring at room temperature. The dissolved solution was cooled in an ice water bath, to which was added 50 mL concentrated sulfuric acid.
  • Trisaccharide derivatives were produced by a coupling reaction system. A disaccharide derivative was produced as in Example 4. The reaction system contained disaccharide reaction products (from example 4) and UDP- glycotransferase as prepared in Example 3. The reaction was performed at 37 ⁇ C for 18-20 hours. Trisaccharide reaction products were identified using TLC. The same TLC analysis method that was used in Example 4 was used here, except here there was detection of the trisaccharide.
  • EXAMPLE 6 Preparing Phosphorylases [00117] Phosphorylases were prepared using same method as example 3 except the experiment used expression constructs for the phosphorylases (sophorose phosphorylase and laminaribiose phosphorylase).
  • EXAMPLE 7 Production of Disaccharides by Phosphorylases -Prophetic Example [00118] Disaccharides derivatives may be produced by a coupling reaction system.
  • the system may contain 0.1M MES buffer (pH 7) with 1 mM magnesium chloride, 5 mM glucose-1-phosphate, 5 mM glucose and be repeated with glucose derivatives (methyl- ⁇ -D-glucopyranoside, methyl- ⁇ -D-glucopyranoside, phenyl- ⁇ -D- glucopyranoside, salicin, arbutin, ⁇ -arbutin, methyl arbutin, hexyl- ⁇ -D- glucopyranoside, or n-heptyl ⁇ -D-glucopyranoside) and purified phosphorylase (sophorose phosphorylase or laminaribiose phosphorylase, prepared in example 6).
  • glucose derivatives methyl- ⁇ -D-glucopyranoside, methyl- ⁇ -D-glucopyranoside, phenyl- ⁇ -D- glucopyranoside, salicin, arbutin, ⁇ -arbutin, methyl arbutin, hexyl- ⁇ -
  • Disaccharide reaction products may next be identified using TLC (thin layer chromatography). Alternately, the reaction could be terminated by heat treatment (85 ⁇ C; 5 min.) and preserved at -18 ⁇ C for long term storage. The same TLC analysis method that was used in Example 4 may be used in this example.
  • EXAMPLE 8 Production of Disaccharide Derivatives by UDP-glycosyltransferases at large scale [00119] Disaccharide derivatives were produced by transglycosylation reaction using UDP-glycosyltransferase at 40 mL of reaction.
  • the reaction system contained 50 mM phosphate buffer (pH 7.0) with 1 mM magnesium chloride, 5 mM glucose derivative, 5 mM UDP-glucose and 9-10% (v/v) UDP-glycotransferase ( ⁇ -1,2- glycosyltransferase such as UGTSi or EUGT11).
  • Glucose derivatives that were used are as follows: salicin, arbutin, ⁇ -arbutin and hexyl- ⁇ -D-glucopyranoside.
  • the reaction was incubated at 37 ⁇ C for 18-20 hours, followed by termination via heat treatment (>85 ⁇ C; 10 min.) Disaccharide reaction products were immediately identified using TLC (thin layer chromatography).
  • reaction products could be preserved at -18 ⁇ C for long term storage.
  • TLC analysis was performed using TLC silica gel 60 F254 (EMD Millipore) and irrigation solvent mixture (ethyl acetate, methanol, acetic acid ratio of 60:20:20 v/v, respectively).
  • the plate was developed by immersing it in a developing solution, followed by heat treatment (130 ⁇ C; 7 min.) as explained in Robyt, J.F., Thin-Layer Chromatography of Carbohydrates in Biochemical Techniques, Theory and Practice, Robyt, J.F and White, B.J., eds, Books/Cole Publishing Co., Monterey, CA., pp 107-109, 1987.
  • a developing solution was prepared by dissolving 3.0 g of N-(1- naphthyl)ethylene diamine in 950 mL of methanol, stirring at room temperature. The dissolved solution was cooled in an ice water bath, to which was added 50 mL concentrated sulfuric acid.
  • EXAMPLE 9 Production of Trisaccharide Derivatives by UDP-glycosyltransferases at large scale [00121] Trisaccharide derivatives were produced from disaccharide derivatives by subsequent transglycosylation reaction using UDP-glycosyltransferase at 40 mL reaction. A disaccharide derivative was produced as in Example 8.
  • the reaction system contained disaccharide reaction products (from example 8), additional UDP- glucose (5 mM) and 9-10% (v/v) UDP-glycotransferase ( ⁇ -1,3- glycosyltransferase such as UGTSr or UGT76G1).
  • the reaction was performed at 37 ⁇ C for 18-20 hours.
  • Trisaccharide reaction products were identified using TLC. The same TLC analysis method that was used in Example 1 was used in this example, except that here there was detection of the trisaccharide. Alternatively, the reaction products could be preserved at -18 ⁇ C for long term storage.
  • UDP-glucose dependent ⁇ -1,3-transglycosyltransferase (UGTSr or UGT76G1) produced trisaccharide derivatives from corresponding disaccharide derivatives with high yield (lane 3, 6, 9 and 12 in figure 5).

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Abstract

L'invention concerne des procédés de synthèse de glucosides et de glycosides de stéviol. L'invention concerne plus spécifiquement, des procédés de synthèse de glycosides de stéviol d'ordre supérieur tels que Reb M, Reb J et Reb N. Par conjugaison d'oligosaccharides à des composés de stévia, il est possible de synthétiser efficacement et de manière efficiente ces glycosides de stéviol. Des procédés, des compositions et des utilisations sont également fournis pour des oligosaccharides.
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