( 12 ) United States Patent

( 12 ) United States Patent

US009924728B2 (12 ) United States Patent (10 ) Patent No. : US 9 , 924 , 728 B2 Pandya et al . (45 ) Date of Patent: Mar. 27, 2018 ( 54 ) FOOD COMPOSITIONS COMPRISING ONE FOREIGN PATENT DOCUMENTS OR BOTH OF RECOMBINANT BETA -LACTOGLOBULIN PROTEIN AND WOWO 2013 / 009182 1 /2013 RECOMBINANT ALPHA - LACTALBUMIN PROTEIN OTHER PUBLICATIONS Arora et al. , “ Variations in the fat unsaponifiable matter and ( 71 ) Applicant: Perfect Day , Inc. , Berkeley , CA (US ) cholesterol contents of goat milk , ” Ind . J . Daily Sci. , Sep . 29 : 191, 1976 . (72 ) Inventors : Ryan Pandya , South San Francisco , Beare -Rogers et al. , “ Lexicon of lipid nutration ( IUPAC Technical CA (US ) ; Perumal Gandhi, Santa Report ) ,” Pure and Applied Chemistry 73 ( 4 ) :685 - 744 , 2001 . Clara , CA (US ) ; Shaowen Ji, Ann Brignon et al . , “ Preparation and amino acid sequences of human Arbor, MI ( US) ; Derek Beauchamp , kappa -casein ,” FEBS Lett. 188 ( 1 ) :48 -54 , 1985 . Dexter , MI (US ) ; Louis Hom , San Singh et al. , “ Post - translational Modification of Caseins, ” In Milk Proteins from Expression to Food , Chapter 5 , Second Edition , Carlos, CA (US ) 141 - 162 , 2014 . Choi and Jimenez - Flores , “ Expression and purification of ( 73 ) Assignee : Perfect Day, Inc ., Berkeley , CA (US ) glycosylated bovine beta -casein (L70S / P71S ) in Pichia pastoris , ” J . Agric . Food Chem . 49 ( 4 ): 1761 - 1766 , 2001 . ( * ) Notice : Subject to any disclaimer , the term of this Deshpande et al . , “ Protein Glycosylation Pathways in Filamentus patent is extended or adjusted under 35 Fungi, ” Glycobiology 18 ( 8 ) :626 -637 , 2008 . U . S . C . 154 ( b ) by 0 days . Farrell Jr. , et al. , “ Casein Micelle Structure : What Can Be Learned from Milk Synthesis and Structural Biology ? ” Curr. Opin . Colloid Interface Sci . 11 : 135 - 147 , 2006 . ( 21) Appl. No .: 15 /438 ,273 Greenberg et al. , “ Human beta - casein . Amino acid sequence and identification of phosphorylation sites , ” J. Biol. Chem . (22 ) Filed : Feb . 21, 2017 259 ( 8 ) :5132 -5138 , 1984 . Hansson et al. , “ Expression of human milk beta - casein in (65 ) Prior Publication Data Escherichia coli: comparison of recombinant protein with native isoforms, ” Protein Expr. Purif . 4 ( 5 ) :373 - 381 , 1993 . US 2017 /0164632 A1 Jun . 15 , 2017 Idiris et al . , “ Engineering of protein secretion in yeast : strategies and impact on protein production , ” Appl . Microbiol. Biotechnol . 86 ( 2 ) :403 -417 , 2010 . Related U . S . Application Data Imafidon et al ., “ Isolation , purification , and alteration of some (63 ) Continuation of application functional groups of major milk proteins: a review , ” Crit . Rev. Food . No . Sci. Nutr. 37 ( 7 ) : 663- 689 , 1997 . PCT/ US2015 / 046428 , filed on Aug . 21 , 2015 . International Preliminary Report on Patentability in International Application No. PCT/US2015 /046428 , dated Feb . 21, 2017 , 12 (60 ) Provisional application No . 62/ 040 , 393 , filed on Aug . pages . 21, 2014 International Search Report for PCT /US2015 /046428 , dated Feb . 5 , 2016 , 8 pages. (51 ) Int . CI. Jenness , “ Composition and Characteristics of Goat Milk : Review CO7K 14 /00 (2006 . 01 ) 1968 - 1979 , " J . Dairy Sci . 63 ( 10 ) : 1605 - 1630 , 1980 . ( 2006 . 01 ) Jensen , “ Bovine milk lipids, ” in Handbook of Milk Composition , A23C 11/ 08 Academic Press , pp . 543 -575 , 1995 . A23C 9 / 123 ( 2006 .01 ) Juarez et al. , “ Physico -Chemical Characteristics of Goat ' s Milk as A23C 19 /055 ( 2006 . 01 ) Distinct From Those of Cow ' s Milk , ” Int. Dairy Fed . Bulletin A23C 9 / 13 ( 2006 . 01 ) 202 :54 -67 , 1986 . A23 ) 3 / 10 ( 2006 . 01 ) Kawai et al ., “ Transformation of Saccharomyces cerevisiae and COZK 14 /47 ( 2006 .01 ) other fungi, ” Bioengineered Bugs 1 : 395 -403 , 2010 . U . S . CI. Kim et al. , “ High -Level Expression of Bovine B -Lactoglobulin in (52 ) Pichia pastoris and Characterization of Its Physicial Properties , ” CPC . .. .. .. .. A23C 11/ 08 ( 2013 .01 ) ; A23C 9 / 123 Protein Engineering 10 ( 11 ) :1339 - 1345 , 1997 . ( 2013 . 01 ) ; A23C 9 / 1315 ( 2013. 01 ) ; A23C Li et al ., “ Cell culture processes for monoclonal antibody produc 19 /055 (2013 .01 ) ; A23 ) 3 / 10 ( 2013 .01 ) ; COOK tion , ” Landes Bioscience 2 : 466 -477 , 2010 . 14 /00 ( 2013 . 01 ) ; CO7K 14 /4732 ( 2013 .01 ) Lonnerdal, “ Recombinant Human Milk Proteins — An Opportunity (58 ) Field of Classification Search and a Challenge ," Am . J . Clin . Nutr . 63 ( 4 ) :622S -6265 , 1996 , None Abstract only . See application file for complete search history. Lonnerdal, “ Recombinant Human Milk Proteins, ” Nestle Nutr. Workshop Ser . Pediatr . Program 58 : 207 - 217 , 2006 . ( 56 ) References Cited (Continued ) U . S . PATENT DOCUMENTS Primary Examiner - Anand U Desai 5 , 942, 274 A 8 / 1999 Slattery et al . ( 74 ) Attorney, Agent, or Firm — Fish & Richardson P . C . 6 , 181, 421 B11 / 2001 Aspnes et al . 6 , 270 , 827 B1 8 / 2001 Gaull et al. (57 ) ABSTRACT 9 , 591, 872 B2 * 3 / 2017 Rosado Loria . .. .. A23L 33 / 40 2010 /0119691 AL 5 / 2010 Huang et al. Disclosed herein are methods and compositions including 2014 /0065264 A1* 3 / 2014 Do .. .. A61K 38 /018 casein , and methods for making these compositions . 426 / 73 2017/ 0273328 AL 9 / 2017 Pandya et al. 30 Claims, 9 Drawing Sheets US 9, 924 , 728 B2 Page 2 References Cited Rosmaninho et al . , “ The effect of citrate on calcium phosphate ( 56 ) deposition from simulated milk ultrafiltrate ( SMUF ) , " J . Food Engineering 77 ( 3 ) :379 - 387 , 2006 . Simons et al . , " Overproduction of bovine beta -casein in Escherichia OTHER PUBLICATIONS coli and engineering of its main chymosin cleavage site ,” Protein Eng. 6 ( 7 ) : 763- 770 , 1993 . MacGibbon et al. , “ Compositions and structure of bovine milk Smouse , “ A Laboratory Continuous Deodorizer, ” Inform . 8 : 1176 lipids, ” in Advanced Dairy Chemistry , vol. 2 Lipids, pp . 1 - 42 , 2006 . 1181, 1997 . Mansson , “ Fatty acids in bovine milk fat, ” Food & Nutrition Written Opinion of the International Searching Authority for PCT/ US2015 /046428 , dated Feb . 5 , 2016 , 10 pages . Research , 2008, 52 : 1, 2008 . Yu et al. , “ The Modification and Analysis of Vegetable Oil for Park , “ Bioreactive Components in Bovine Milk ,” in Bioactive Cheese Making, ” J . Am . Oil . Chem . Soc . 77 : 911 - 915, 2000 . Components in Milk and Dairy Products , Wiley - Blackwell, pp . Zhang et al. , “ Fermentation Strategies for Recombinant Protein 15 - 42 , 2009 . Expression in the Methylotrophic Yeast Pichia pastoris , " Park , “ Rheological characteristics of goat and sheep milk , ” Small Biotechnol . Bioprocess Eng . 5 : 275 -287 , 2000 . Ruminant Res . 68 ( 1 - 2 ) : 73 - 87 , 2007 . Zhu , “ Mammalian cell protein expression for biopharmaceutical Posati et al. , “ Composition of foods: dairy and egg products : raw , production ,” Biotechnol. Adv. 30 ( 5 ) : 1158 -1170 , 2012 . processed , and prepared ,” Agricultural Handbook — U . S . Dept. of Agriculture (USA ) , No . 8 - 1 , 158 pages , 1976 . * cited by examiner U . S . Patent Mar . 27, 2018 Sheet 1 of 9 US 9 ,924 , 728 B2 Fats Glycerol ( SCFAS Proteins DNA cells Esterification HOSO Fermentation Inter -esterification 2Purification = Deodorization Carbohydrates Formulation Ash Milk (downstream processing) FIG . 1 atent Mar . 27 , 2018 Sheet 2 of 9 US 9, 924, 728 B2 FIG . 2A FIG . 2B U . S . Patent Mar . 27, 2018 Sheet 3 of 9 US 9 ,924 ,728 B2 precipitate supernatant Mr (kDa ) 250 130 8100 8 8 G ? FIG . 3 U . S . Patent Mar. 27, 2018 Sheet 4 of 9 US 9 ,924 , 728 B2 ?? 3 ? ? ? ? ?????????????????????? FIG.4 ??????????????????????????????? Alpha-actalbumin U . S . Patent Mar. 27 , 2018 Sheet 5 of 9 US 9 ,924 ,728 B2 0 .600 : : : : : : : : : : : : : : : : : : : :: : : :: : : : : : : : : : : : : 0 .500 : : : : : : :: : : : : :: : : : :: : : : : : : : : 11 : :: : : :: : : : : : : : : : : : :: : :: : : :: : : :: : : : : : : : : : : : : : : : :: : : :: : : : : :: : : : : : : : :: : : :: : : : : : :: : :: : : : : : : : : : : :: : : : : 1 : 2 : : : : :: : : ELISAsignal(OD650) : :: : :: : :: : : : .300 :: : : : : 39 : : : : : : : : : : : : :: : : : : : : : : :: : :: : : : : : :: :: : :: :: : : : : : :: : : : :: : :: : : : : :: : 0 . 200 : :: : : :: :: : : :: : :: : : : :: : : : : : :: : :: : : : : :: : :: : : : : : : : : : : : : : : :: : : : : : 01 : 2 : : 0 . 100 :: . i .. : : . : : : : : : : : : : : : : : : :: : : : : : : : : : : 1 : 1 : : : : : : : : : : : : : : :: : : : : : : : :: : : : : : : : : : : : : :: : : : : : 35331 0 .000 : : : :: : : : : : : : : 0 . 1 % ( W / V ) Yeast neg ctrl nat- LAA OST -LAA Dry Milk FIG . 5 : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :: : : :: . : : : : : : . : . : . : . : . : : :: : :: : :: :: . : :: : : :: : 1 : :: : : :: : : :: : : :: : : :: : : :: : : :: : . : :: : : :: : : : .:1. : : : : : : ELISAsignal(OD650) : : : : : : : : :: . : :: : :: : :: :: : :: : : :: : : :: : : :: : : : :: : : : : :: : : : :: : : :: : : :: : . : :: : : :: : : :: : : é : : : : : :: : : : : : :: . : : :: : : :: . : . : . : . : . : . : . : . : . : : : . : . : . : . : . : . : . : . : . : . : : . : . : . : . : . : . : . : . : . : . : : : : : : : :: : : : :: : : : : : : : : : : : : : :: . o 9999999999 999999999999 Yeast Neg Ctrl 0 .Dry 1 % Milk( w / v ) Blank LGB FIG . 6 U . S . Patent Mar . 27, 2018 Sheet 6 of 9 US 9 ,924 ,728 B2 WT Milk MW Yeast LGB 130 - 100 70 Beta - lactoglobulin FIG . 7 U . S . Patent Mar . 27, 2018 Sheet 7 of 9 US 9 ,924 ,728 B2 ? OD600 0 ZZZ ) alphaS1 beta 0 A NormalizedValue

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