# US 2018/0020709 A1 · Rebaudioside m biosynthetic production and recovery methods

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- Number: US 2018/0020709 A1
- Document ID: US20180020709A1
- Status: Published application
- Kind: A1
- Application number: 15/550,075
- Filed: 2016-02-10
- Published: 2018-01-25
- Earliest claimed: 2014-04-16
- Applicants: PureCircle USA Inc.
- Inventors: Avetik MARKOSYAN
- Web page: https://patentgrep.com/patent/US20180020709A1

## CPC

- A23L 27/36: {Terpene glycosides}
- A21D 2/18: Carbohydrates
- A21D 2/36: Vegetable material
- A21D 13/062: with modified sugar content; Sugar-free products
- A21D 13/40: Products characterised by the type, form or use
- A23C 9/1307: {Milk products or derivatives; Fruit or vegetable juices; Sugars, sugar alcohols, sweeteners; Oligosaccharides; Organic acids or salts thereof or acidifying agents; Flavours, dyes or pigments; Inert or aerosol gases; Carbonation methods}
- A23L 2/02: containing fruit or vegetable juices
- A23L 2/54: Mixing with gases
- A23L 2/60: Sweeteners
- A61K 31/704: attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin {(digitoxin A61K31/7048)}
- A61K 36/28: Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
- C12G 3/04: by mixing, e.g. for preparation of liqueurs
- C12P 19/18: produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
- C12P 19/22: produced by the action of a beta-amylase, e.g. maltose
- C12P 19/56: having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
- C12P 33/00: Preparation of steroids
- C12Y 204/01019: Cyclomaltodextrin glucanotransferase (2.4.1.19)
- A23V 2002/00: Food compositions, function of food ingredients or processes for food or foodstuffs
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- A21D: TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS (preservation of flour or dough before baking A23B40/00; preservation of bakery products A23B45/00)
- A23C: DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF (preservation thereof A23B11/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12G: WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES (beer C12C); PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES
- A23V: INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION

## Abstract

Various recovery processes are provided for the complete recovery of low soluble steviol glycosides obtained in recombinant microorganisms. Soluble α-glycosyl steviol glycosides were fully recovered in downstream processing and then converted to steviol glycosides by hydrolases. The obtained steviol glycosides were purified and used as sweeteners, sweetness enhancers, flavor enhancers, and flavor modifiers in foods, beverages, cosmetics and pharmaceuticals.

## Claims

### Claim 1

A process for producing terpenoid glycoside comprising the steps of:
a. producing α-glycosyl derivative of terpenoid glycoside having at least one α-glycosyl residue;
b. hydrolyzing the produced α-glycosyl derivative of terpenoid glycoside to obtain the terpenoid glycoside.

### Claim 2 (depends on 1)

The process of claim 1 further comprising the step of:
c. purifying the obtained terpenoid glycoside.

### Claim 3 (depends on 1)

The process of claim 1 wherein step (a) the terpenoid glycoside is synthesized by a recombinant microorganism.

### Claim 4 (depends on 1)

The process of claim 1 wherein step (a) the terpenoid glycoside is synthesized by at least one biocatalyst.

### Claim 5 (depends on 1)

The process of claim 1 wherein step (a) the terpenoid glycoside is synthesized by at least one enzyme.

### Claim 6 (depends on 1)

The process of claim 1 wherein step (a) occurs in recombinant microorganism.

### Claim 7 (depends on 1)

The process of claim 1 wherein step (a) occurs outside of recombinant microorganism.

### Claim 8 (depends on 1)

The process of claim 1 wherein step (a) occurs on the surface of recombinant microorganism.

### Claim 9 (depends on 1)

The process of claim 1 wherein step (b) occurs outside of recombinant microorganism.

### Claim 10 (depends on 1)

The process of claim 1 wherein step (b) occurs by contacting α-glycosyl derivative of terpenoid glycoside with at least one biocatalyst.

### Claim 11 (depends on 1)

The process of claim 1 wherein step (b) occurs by contacting α-glycosyl derivative of terpenoid glycoside with at least one enzyme.

### Claim 12 (depends on 1)

The process of claim 1 wherein the terpenoid glycoside is selected from the group consisting of stevia extract, steviol glycosides, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside L, rebaudioside M, rebaudioside N, rebaudioside O, dulcoside A, steviolbioside, rubusoside, glycosylated steviol glycosides, glucosylated steviol glycosides as well as any other steviol glycoside(s) found in Stevia rebaudiana plant and mixtures thereof.

### Claim 13 (depends on 1)

The process of claim 1 wherein the terpenoid glycoside is selected from the group consisting of Luo Han Guo extract, mogrol glycosides, mogrosides, mogroside mogroside II, mogroside II B, mogroside II E, mogroside III, mogroside III A2, mogroside IV, mogroside V, mogroside VI, neomogroside, grosmomoside siamenoside I, 7-oxo-mogroside II E, 11-oxo-mogroside A1, 11-deoxy-mogroside III, -oxomogroside IV A, 7-oxo-mogroside V, 11-oxo-mogroside V, as well as any other mogrol glycoside(s) found in Siraitia grosvenorii plant and mixtures thereof.

### Claim 14 (depends on 1)

A flavor composition comprising terpenoid glycoside of claim 1 and at least one additional flavoring agent selected from the group consisting of, lemon, berry, orange, fruit, banana, grape, pear, pineapple, mango, bitter almond, cola, cinnamon, sugar, cotton candy, vanilla, terpenoid glycosides, NSF01, NSF02, NSF03, NSF04 and combination thereof.

### Claim 15 (depends on 1)

A food ingredient comprising terpenoid glycoside of claim 1 and at least one additional food ingredient selected from the group consisting of acidulants, organic and amino acids, coloring agents, bulking agents, modified starches, gums, texturizers, preservatives, antioxidants, emulsifiers, stabilisers, thickeners, gelling agents, and combination thereof.

### Claim 16 (depends on 1)

A food, beverage, cosmetic, pharmaceutical product or other consumable comprising terpenoid glycoside of claim 1.