# US 11,028,423 B2 · Heat treatment to produce glycosides > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,028,423 B2 - Document ID: US11028423B2 - Status: Granted - Kind: B2 - Application number: 16/796,630 - Filed: 2020-02-20 - Published: 2020-09-10 - Granted: 2021-06-08 - Earliest claimed: 2015-05-29 - Assignees: Cargill, Incorporated - Applicants: Cargill, Incorporated - Inventors: James C. Anderson; Robert J. Brower, III; Ting Liu Carlson; Belit Flores; Dan S. Gaspard; Kristopher T. Mortenson; Richard Nygaard; Nicole Paulson; Maribeth Rasmussen - Web page: https://patentgrep.com/patent/US11028423B2 ## CPC - 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 - A23L 2/60: Sweeteners - A61K 31/704: attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin {(digitoxin A61K31/7048)} - C12N 15/81: for yeasts - C12N 2523/00: Culture process characterised by temperature - 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)} - A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B) - 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) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Disclosed arc methods of releasing or enriching for steviol glycosides produced by yeast. The disclosed methods enhance or improve the release or enrichment of the steviol glycosides. ## Claims ### Claim 1 A method of releasing steviol glycosides from a yeast host cell, comprising the steps of: (a) providing a composition, comprising the host cell producing one or more steviol glycosides; and (b) releasing the one or more steviol glycosides from the host cell by heating the composition to a temperature in a range from 50° C. to 95° C. for 5 minutes to 48 hours, wherein the one or more steviol glycosides are released from an intracellular portion of the host cell into an extracellular portion of the composition. ### Claim 2 (depends on 1) The method of claim 1, wherein the composition is a liquid composition comprising the host cell and a liquid media. ### Claim 3 (depends on 2) The method of claim 2, wherein the composition is obtained by separating the host cell from a fermentation medium comprising the host cell, and transferring the separated host cell into the suitable liquid media. ### Claim 4 (depends on 1) The method of claim 1, wherein the composition is a fermentation medium. ### Claim 5 (depends on 1) The method of claim 1, wherein the host cell is concentrated before the heat treatment. ### Claim 6 (depends on 1) The method of claim 1, wherein the composition is heated to the temperature in the range from 70° C. to 95° C. for 5 minutes to 2 hours. ### Claim 7 (depends on 1) The method of claim 1, wherein the composition is heated to about 50° C. for 24 hours. ### Claim 8 (depends on 1) The method of claim 1, wherein the composition is heated to about 70° C. for 5 minutes to 2 hours. ### Claim 9 (depends on 1) The method of claim 1, wherein the composition is heated to about 75° C. for 5 minutes to 2 hours. ### Claim 10 (depends on 1) The method of claim 1, wherein the composition is heated to about 95° C. for 5 minutes to 1 hour. ### Claim 11 (depends on 1) The method of claim 1, wherein an amount of the steviol glycosides released extracellularly is in a range of about 80% to about 100% of a total intracellular and extracellular amount of the steviol glycosides produced by the host cell. ### Claim 12 (depends on 1) The method of claim 1, wherein the one or more steviol glycosides is rebaudioside M (Reb M), rebaudioside D (Reb D), rebaudioside A (Reb A), rebaudioside B (Reb B), or combinations thereof. ### Claim 13 (depends on 12) The method of claim 12, wherein an amount of Reb A released extracellularly is in a range of about 30% to about 100% of a total intracellular and extracellular amount of Reb A produced by the host cell. ### Claim 14 (depends on 12) The method of claim 12, wherein an amount of Reb B released extracellularly is in a range of about 25% to about 100% of a total intracellular and extracellular amount of Reb B produced by the host cell. ### Claim 15 (depends on 12) The method of claim 12, wherein an amount of Reb D and/or rebaudioside M released extracellularly is in a range of about 30% to about 100% of a total intracellular and extracellular amount of Reb D and/or Reb M produced by the host cell. ### Claim 16 (depends on 1) The method of claim 1, wherein an extracellular nitrogen content in the composition after the heat treatment is not increased by more than 50% of a total extracellular and intracellular nitrogen content in the composition. ### Claim 17 (depends on 1) The method of claim 1, wherein an extracellular phosphorous content in the composition after the heat treatment is not increased by more than 80% of a total extracellular and intracellular phosphorous content in the composition. ### Claim 18 (depends on 1) The method of claim 1, wherein the yeast is selected from a group consisting of genus, Candida, Pichia (Hansenula), Kloeckera (Hanseniaspora), Kluyveromyces, Rhodotorula, Torulopsis, Zygosaccharomyces, Saccharomycete, Yarrowia, and Saccharomyces. ### Claim 19 (depends on 1) The method of claim 1, wherein the yeast is Saccharomyces cerevisiae. ### Claim 20 (depends on 1) The method of claim 1, wherein the released steviol glycosides are further isolated or purified from the host cell. ### Claim 21 A composition, comprising a yeast host cell producing one or more steviol glycosides and one or more steviol glycosides that are released from the host cell by heating the composition to a temperature in a range from 50° C. to 95° C. for 5 minutes to 48 hours; wherein an amount of the steviol glycosides released extracellularly is in a range of about 80% to about 100% of a total intracellular and extracellular amount of the steviol glycosides produced by the host cell.