IP Library › Granted Patent US 12,336,551
Granted Patent B2
US 12,336,551 · App. 18/456,939 · Granted Jun 24, 2025

Purified protein composition

Inventors: Carl Allen Henderson, Jr. (Brisbane, CA); Ratnayake Mudiyanselage Dunilka Nishani Ratnayake (San Mateo, CA); Ian Guiles Ronningen (San Francisco, CA); Laila Dafik (Belmont, CA)
Assignee: Impossible Foods Inc.
A23J3/20A23J1/008B01D61/145B01D61/147
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,336,551
App. No.
18/456,939
Granted
Jun 24, 2025
Kind
B2
Abstract

Described herein are methods for purifying protein, and more particularly to methods for purifying protein that minimize the development of undesirable odors and flavors in the purified protein and increase protein yield.

Claims (29)

1. A protein composition produced by a method comprising:

a) lysing an aqueous suspension of a plurality of cells to obtain a cell lysate; and

b) filtering the cell lysate to obtain a protein composition,

wherein steps a) and b) independently, are performed at a pH between about 8.5 and about 12.0; and

wherein:

(i) the protein composition forms a gel upon heating to 65° C.;

(ii) the protein composition forms a gel with a storage modulus of at least about 100 Pa when heated at or above about 85° C. for about 20 minutes;

(iii) heating a 10% (w/v) suspension of the protein composition to at least about 95° C. results in a gel with a storage modulus of at least about 100 Pa;

(iv) the protein composition forms a gel between about pH 5.5 and about pH 10.0, or

(v) the protein composition forms a gel in solution with ionic strength below about 0.5 M, wherein the ionic strength is calculated based on the concentration of non-protein solutes.

2. The protein composition of claim 1 , wherein filtering comprises microfiltration, ultrafiltration, diafiltration, or a combination thereof.

3. The protein composition of claim 1 , further comprising washing the aqueous suspension of the plurality of cells at a pH between about 8.5 and about 12.0 before step a).

4. The protein composition of claim 1 , wherein at least about 50% of the protein in the protein composition falls between about 10 kDa and about 200 kDa.

5. The protein composition of claim 1 , wherein the plurality of cells comprises microbial cells.

6. The protein composition of claim 1 , wherein the lysing is performed biochemically or chemically.

7. The protein composition of claim 1 , further comprising pasteurizing the protein composition to obtain a pasteurized protein composition.

8. The protein composition of claim 1 , wherein the protein composition forms a gel upon heating to 65° C.

9. The protein composition of claim 1 , wherein the protein composition forms a gel with a storage modulus of at least about 100 Pa when heated at or above about 85° C. for about 20 minutes.

10. The protein composition of claim 1 , wherein heating a 10% (w/v) suspension of the protein composition to at least about 95° C. results in a gel with a storage modulus of at least about 100 Pa.

11. The protein composition of claim 1 , wherein the protein composition forms a gel between about pH 5.5 and about pH 10.0.

12. The protein composition of claim 1 , wherein the protein composition forms a gel in solution with ionic strength below about 0.5 M, wherein the ionic strength is calculated based on the concentration of non-protein solutes.

13. The protein composition of claim 1 , wherein the protein composition exhibits two or more characteristics selected from the group consisting of: (i)-(v).

14. The protein composition of claim 1 , wherein the protein composition exhibits three or more characteristics selected from the group consisting of: (i)-(v).

15. The protein composition of claim 1 , wherein the plurality of cells comprises fungal cells.

16. The protein composition of claim 15 , wherein the fungal cells are selected from the group consisting of Saccharomyces, Pichia, Candida, Hansenula, Torulopsis, Kluyveromyces, Yarrowia , and Fusarium cells.

17. The protein composition of claim 1 , wherein the plurality of cells comprises bacterial cells.

18. The protein composition of claim 17 , wherein the bacterial cells are selected from the group consisting of Bacillus, Escherichia, Lactobacillus, Corynebacterium, Pseudomonas , and Methanococcus.

19. The protein composition of claim 1 , further comprising clarifying the cell lysate to obtain a clarified lysate.

20. The protein composition of claim 19 , wherein the clarifying is performed by centrifugation to less than about 20% dry solids.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 9, 2023
From: HENDERSON, CARL ALLEN, JR.; RATNAYAKE, RATNAYAKE MUDIYANSELAGE DUNILKA NISHANI; RONNINGEN, IAN GUILES; DAFIK, LAILA
To: IMPOSSIBLE FOODS INC.
Reel/Frame 064852/0932 →
Continuity (5)
Continuation 17350508 · Jun 17, 2021
Division 16138876 · Sep 21, 2018
Provisional Application 62670478 · May 11, 2018
Provisional Application 62562298 · Sep 22, 2017
Related Publication 20240016182A1 · Jan 18, 2024
References Cited (127)
US 3043826A · Beaber et al. · 1962 [cited by applicant]
US 3821080A · Kalina et al. · 1974 [cited by applicant]
US 3959246A · Bickoff et al. · 1976 [cited by applicant]
US 4006078A · Bickoff et al. · 1977 [cited by applicant]
US 4334024A · Johal · 1982 [cited by applicant]
US 4340676A · Bourque · 1982 [cited by applicant]
US 4427580A · Kinsella et al. · 1984 [cited by applicant]
US 4588691A · Johal · 1986 [cited by applicant]
US 4683294A · Van Wijnendaele et al. · 1987 [cited by applicant]
US 5151358A · Heinsohn · 1992 [cited by applicant]
US 5328841A · Lorch et al. · 1994 [cited by applicant]
US 5407810A · Builder et al. · 1995 [cited by applicant]
US 5760189A · Vicik et al. · 1998 [cited by applicant]
US 6132795A · Holbrook et al. · 2000 [cited by applicant]
US 6174704B1 · Chu et al. · 2001 [cited by applicant]
US 7387884B2 · Suzuki et al. · 2008 [cited by applicant]
US 9011949B2 · Brown et al. · 2015 [cited by applicant]
US 9593143B2 · Colaco et al. · 2017 [cited by applicant]
US 9700067B2 · Fraser et al. · 2017 [cited by applicant]
US 9765112B2 · Oliver et al. · 2017 [cited by applicant]
US 10039306B2 · Vrljic et al. · 2018 [cited by applicant]
US 10087434B2 · Kale et al. · 2018 [cited by applicant]
US 10093913B2 · Kale et al. · 2018 [cited by applicant]
US 10287568B2 · Kale et al. · 2019 [cited by applicant]
US 11051532B2 · Henderson, Jr. et al. · 2021 [cited by applicant]
US 11771111B2 · Henderson, Jr. et al. · 2023 [cited by applicant]
US 12011016B2 · Henderson, Jr. et al. · 2024 [cited by applicant]
US 20040101947A1 · Engel et al. · 2004 [cited by applicant]
US 20040151817A1 · Fukuda et al. · 2004 [cited by applicant]
US 20040166026A1 · Bratcher et al. · 2004 [cited by applicant]
US 20040171813A1 · Garger et al. · 2004 [cited by applicant]
US 20060025579A1 · Riedl et al. · 2006 [cited by applicant]
US 20080182002A1 · Staerk et al. · 2008 [cited by applicant]
US 20080187988A1 · Warmington et al. · 2008 [cited by applicant]
US 20100304126A1 · Wu et al. · 2010 [cited by applicant]
US 20120053328A1 · Yan et al. · 2012 [cited by applicant]
US 20120164234A1 · Kwiatkowski et al. · 2012 [cited by applicant]
US 20120252065A1 · Rozenszain et al. · 2012 [cited by applicant]
US 20120292241A1 · Bertanza et al. · 2012 [cited by applicant]
US 20150087532A1 · Brown et al. · 2015 [cited by applicant]
US 20150289541A1 · Brown et al. · 2015 [cited by applicant]
US 20150305361A1 · Holz-Schietinger et al. · 2015 [cited by applicant]
US 20150335043A1 · De Jong et al. · 2015 [cited by applicant]
US 20160340411A1 · Fraser et al. · 2016 [cited by applicant]
US 20160360770A1 · Sherlock et al. · 2016 [cited by applicant]
US 20170035076A1 · Geistlinger et al. · 2017 [cited by applicant]
US 20170172169A1 · Grzanich et al. · 2017 [cited by applicant]
US 20170188612A1 · Varadan et al. · 2017 [cited by applicant]
US 20170298337A1 · Kale et al. · 2017 [cited by applicant]
US 20170321203A1 · Kale et al. · 2017 [cited by applicant]
US 20170321204A1 · Kale et al. · 2017 [cited by applicant]
US 20180127764A1 · Shankar et al. · 2018 [cited by applicant]
US 20200361986A1 · Shultz et al. · 2020 [cited by applicant]
US 20200397021A1 · Henderson · 2020 [cited by applicant]
US 20210307358A1 · Henderson, Jr. et al. · 2021 [cited by applicant]
US 20220087286A1 · Henderson et al. · 2022 [cited by applicant]
US 20230210150A1 · Li et al. · 2023 [cited by applicant]
US 20240148019A1 · Li et al. · 2024 [cited by applicant]
US 20240292865A1 · Henderson, Jr. et al. · 2024 [cited by applicant]
CN 101617736B · 2011 [cited by applicant]
CN 102550795A · 2012 [cited by applicant]
CN 103732076 · 2014 [cited by applicant]
CN 103766573 · 2014 [cited by applicant]
CN 104862355A · 2015 [cited by applicant]
CN 105821104A · 2016 [cited by applicant]
CN 108409827 · 2018 [cited by applicant]
EP 1197154 · 2002 [cited by applicant]
EP 3670646 · 2020 [cited by applicant]
GB 1556297 · 1979 [cited by applicant]
GB 1597783A · 1981 [cited by applicant]
WO WO2001019969 · 2001 [cited by applicant]
WO WO2010046920 · 2010 [cited by applicant]
WO WO2011078671 · 2011 [cited by applicant]
WO WO2013013949 · 2013 [cited by applicant]
WO WO2016054375 · 2016 [cited by applicant]
WO WO2018102656 · 2018 [cited by applicant]
WO WO2018102721 · 2018 [cited by applicant]
WO WO2020127957 · 2020 [cited by applicant]
Anonymous, “Cytochrome c from equine heart”, Sigma Product Information, Aug. 28, 2008, retrieved on May 17, 2021, retrieved from URL :https://www.sigmaaldri ch.com/content/dam/sigma-aldrich/docs/Sigma/Data sheet/3/c2867… [cited by applicant]
Bansal-Mutalik et al, “Reverse micellar solutions aided permeabilization of baker's yeast,” Process Biochemistry, Jan. 1, 2006, 41(1): 133-141. [cited by applicant]
CAS No. 137397-56-9, “3-[18-(2-carboxylatoethyl)-7-ethenyl-12-[(4E,8E)-1-hydroxy-5,9,13-trimethyltetradeca-4,8,12-trienyl]-3,8,13,17-tetramethylporphyrin-21,23-diid-2-yl]propanoate;hydron;iron(2+)” retrieved on Sep. 1, … [cited by applicant]
CAS No. 17375-41-6, “Ferrous sulfate monohydrate,” PubChem, retrieved on Aug. 31, 2022, retrieved from URL<https://pubchem.ncbi.nlm.nih.gov/compound/62712>, 26 pages. [cited by applicant]
CAS No. 207399-12-0,“Ferric citrate hydrate,” PubChem, retrieved on Aug. 31, 2022, retrieved from URL<https://pubchem.ncbi.nlm.nih.gov/compound/51341966>, 19 pages. [cited by applicant]
CAS No. 22830-45-1, “Iron(II) gluconate hydrate,” PubChem, retrieved on Sep. 1, 2022, retrieved from <https://pubchem.ncbi.nlm.nih.gov/compound/443752>, 10 pages. [cited by applicant]
CAS No. 699014-53-4, “iron;(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoic acid;hydrate,” PubChem, retrieved on Sep. 1, 2022, retrieved from <https://pubchem.ncbi.nlm.nih.gov/compound/22836455>, 10 pages. [cited by applicant]
CAS No. 7782-63-0, “Ferrous sulfate heptahydrate,” PubChem, retrieved on Sep. 1, 2022, retrieved from <https://pubchem.ncbi.nlm.nih.gov/compound/62662>, 35 pages. [cited by applicant]
CAS No. 10028-22-5, “Ferric sulfate,” PubChem, retrieved on Aug. 31, 2022, retrieved from URL<https://pubchem.ncbi.nlm.nih.gov/compound/24826>, 47 pages. [cited by applicant]
CAS No. 14875-96-8, “Protoheme,” retrieved on Sep. 1, 2022, retrieved from <https://pubchem.ncbi.nlm.nih.gov/compound/26945>, 22 pages. [cited by applicant]
CAS No. 15664-29-6, “Pheophorbide a,” retrieved on Sep. 12, 2022, retrieved from <https://pubchem.ncbi.nlm.nih.gov/compound/253193>, 26 pages. [cited by applicant]
CAS No. 17099-81-9, “Ferric-EDTA,” retrieved on Sep. 12, 2022, retrieved from <https://pubchem.ncbi.nlm.nih.gov/compound/28283>, 21 pages. [cited by applicant]
CAS No. 18535-39-2, “Heme a3,” retrieved on Sep. 12, 2022, retrieved from <https://pubchem.ncbi.nlm.nih.gov/compound/5489981>, 14 pages. [cited by applicant]
CAS No. 26598-29-8, “Heme C,” retrieved on Sep. 30, 2022, retrieved from <https://chem.nlm.nih.gov/chemidplus/rn/26598-29-8>, 2 pages. [cited by applicant]
CAS No. 69138-22-3, “Chlorophyllin ferrous-sodium complex,” retrieved on Sep. 30, 2022, retrieved from <https://www.guidechem.com/cas/69138-22-3.html>, 2 pages. [cited by applicant]
CAS Nos. 2338-05-8, “Iron citrate,” retrieved on Sep. 12, 2022, retrieved from <https://pubchem.ncbi.nlm.nih.gov/compound/4989393>, 25 pages. [cited by applicant]
CAS Nos. 299-29-6, “Ferrous gluconate hydrate,” retrieved on Sep. 12, 2022, retrieved from <https://pubchem.ncbi.nlm.nih.gov/compound/16212939>, 36 pages. [cited by applicant]
CAS Nos. 3522-50-7, “Ferric citrate,” retrieved on Sep. 12, 2022, retrieved from <https://pubchem.ncbi.nlm.nih.gov/compound/61300>, 58 pages. [cited by applicant]
CAS Nos. 7720-78-7, “Ferrous sulfate,” retrieved on Sep. 12, 2022, retrieved from <https://pubchem.nebi.nlm.nih.gov/compound/24393>, 64 pages. [cited by applicant]
Castro et al., Liquid-liquid equilibrium of water + PEG 8000 + magnesium sulfate or sodium sulfate aqueous two-phase systems at 35 oC: experimental determination and thermodynamic modeling, Braz. J. Chem. Eng., 2005, 22… [cited by applicant]
Degerli et al., “A novel concentration method for concentrating solutions of protein extracts based on dialysis techniques,” Analytical Biochemistry, Oct. 2001, 297(2):192-194. [cited by applicant]
Extended European Search Report in European Patent Application No. 15846579.9, dated Feb. 5, 2018, 6 pages. [cited by applicant]
Ferreira et al., “β-Conglycinin (7S) and glycinin (11S) exert a hypocholesterolemic effect comparable to that of fenofibrate in rats fed a high-cholesterol diet,” Journal of Functional Foods, 2(2010), pp. 275-283. [cited by applicant]
Gressent et al., “Characterization of a high-affinity binding site for the pea albumin 1b entomotoxin in the weevil Sitophilus,” Eur J Biochem. Jun. 2003;270(11):2429-35. [cited by applicant]
Guo et al., “Mini-review: In vitro Metabolic Engineering for Biomanufacturing of High-value Products”, Computational and Structural Biotechnology Journal, 2017, vol. 15, 161-167. [cited by applicant]
Hemeprotein [online] “Heme Protein Database,” retrieved on Aug. 30, 2022, retrieved from URL< http://hemeprotein.info/heme.php>, 1 page. [cited by applicant]
International Preliminary Report on Patentability in International Appln. No. PCT/US2021/020356, dated Sep. 9, 2022, 15 pages. [cited by applicant]
International Preliminary Report on Patentability in International Application No. PCT/US2015/053492, dated Apr. 4, 2017, 7 pages. [cited by applicant]
International Search Report and Written Opinion in International Application No. PCT/US2015/053492, dated Feb. 5, 2016, 10 pages. [cited by applicant]
International Search Report and Written Opinion in International Appln. No. PCT/US2021/020356, dated Aug. 16, 2021, 23 pages. [cited by applicant]
International Search Report on Patentability in International Appln. No. PCT/US2020/050774, date Jun. 9, 2021, 27 pages. [cited by applicant]
Irwin et al., “Identification of Flavor-Active Volatiles in Soy Protein Isolate via Gas Chromatography Olfactometry,” In Chemistry, Texture, and Flavor of Soy 2010, 389-400. [cited by applicant]
Kao et al. “Volatile compounds produced during deodorization of soybean oil and their flavor significance,” Journal of the American Oil Chemists' Society, Dec. 1998, 75(12):1103-1107. [cited by applicant]
Lamsal et al., “Separation of Protein Fraction in Alfalfa Juice: Effects of Some Pre-Treatment Methods,” Trans. ASAE 2003, 46(3):715-720. [cited by applicant]
Lei et al., “Compounds contributing to the odor of aqueous slurries of soy protein concentrate,” Journal of food science, Nov. 2001, 66(9):1306-1310. [cited by applicant]
Ravi et al., “Rapid profiling of soybean aromatic compounds using electronic nose,” Biosensors, May 24, 2019, 9(2): 13 pages. [cited by applicant]
Reedy et al., “Development of a heme protein structure-electrochemical function database,” Nucleic acids research, Oct. 11, 2007, 36:D307-D113. [cited by applicant]
Rickert et al., “Effect of extraction pH and temperature on isoflavone and saponin partitioning and profile during soy protein isolate production,” Journal of food science, Oct. 2004, 69(8):C623-C631. [cited by applicant]
Roland et al. “Flavor aspects of pulse ingredients,” Cereal Chemistry, Jan. 2017, 94(1):58-65. [cited by applicant]
Shi et al., “Application of Adsorption Separation Resin in Pharmaceutical Industry,” Chemical Industry Press, Sep. 2008, p. 76, 6 pages (with machine translation). [cited by applicant]
Solina et al., “Volatile aroma components of soy protein isolate and acid-hydrolysed vegetable protein,” Food chemistry, May 1, 2005, 90(4):861-873. [cited by applicant]
Srinivas et al., “Extraction and purification of a plant peroxidase by aqueous two-phase extraction coupled with gel filtration,” Process Biochemistry, Oct. 1999, 35(1/2):43-48. [cited by applicant]
Yamgata et al., “O-acetylserine and O-acetylhomoserine sulfhydrylase of yeast Further purification and characterization as a pyridoxal enzyme,” The Journal of Biochemistry, Oct. 1, 1976, 80(4): 777-785. [cited by applicant]
Yang et al., “Antihypertensive properties of spinach leaf protein digests,” J. Agric. Food Chem. 2004, 52:2223-2225. [cited by applicant]
International Preliminary Report on Patentability in International Application No. PCT/US2022/018395, dated Aug. 29, 2023, 9 pages. [cited by applicant]
International Search Report and Written Opinion in International Application No. PCT/US2022/018395, mailed on Jul. 6, 2022, 12 pages. [cited by applicant]
Schlemmer et al., “Phytate in foods and significance for humans: Food sources, intake, processing, bioavailability, protective role and analysis,” Mol. Nutr. Food Res., Sep. 2009, 53(S2):S330-S375. [cited by applicant]
Cao, “Grain and Oil Deep Processing Technology,” Northeast Forestry University Press, Jun. 2007, pp. 283-286 (with machine translation). [cited by applicant]
Zenoozian et al., “Composition and Functional Properties of Soy Protein Isolates,” Poster, Presented at International Soybean Processing and Utilization Conference, Oct. 15-20, 2000, pp. 441-442. [cited by applicant]