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(12) Patent Application Publication (10) Pub. No.: US 2016/0213039 A1 KUMAR Et Al

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US 20160213039A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0213039 A1 KUMAR et al. (43) Pub. Date: Jul. 28, 2016 (54) STEVIOL GLYCOSIDES A23G 9/34 (2006.01) A618/24 (2006.01) (71) Applicant: DSM IPASSETS B.V., Heerlen (NL) A68/73 (2006.01) A618/34 (2006.01) (72) Inventors: Manoj KUMAR, Echt (NL); Marieke A618/86 (2006.01) NIJMEIJER, Echt (NL) A61K 8/44 (2006.01) A68/60 (2006.01) (73) Assignee: DSM IPASSETS B.V., Heerlen (NL) A2.3L 2/60 (2006.01) A61O 11/00 (2006.01) (21) Appl. No.: 14/908,146 (52) U.S. Cl. 22) PCT Fled:1-1. Jul. 31, 2014 CPC ................. A23L I/2363 (2013.01);s A23L 2/60 (22) 1. l. 3, (2013.01); A23L 2/02 (2013.01); A2ID 13/062 (86). PCT No.: PCT/EP2014/066SSS (2013.01); A2ID 2/36 (2013.01); A23G 1/40 (2013.01); A23G 9/34 (2013.01); A23L I/238 S371 (c)(1), (2013.01); A61O II/00 (2013.01); A61K 8/24 (2) Date: Jan. 28, 2016 (2013.01); A61K 8/73 (2013.01); A61K 8/731 O O (2013.01); A61K 8/345 (2013.01); A61K 8/738 Related U.S. Application Data (2013.01); A61K 8/86 (2013.01); A61K 8/442 (63) Continuation of application No. 13/956,226, filed on (2013.01); A61K 8/602 (2013.01); A23V Jul. 31, 2013, now abandoned. 2002/00 (2013.01); A61 K 2800/59 (2013.01) Publication Classification (57) ABSTRACT (51) Int. Cl. The present invention relates to a product which is a foodstuff, A2.3L I/236 (2006.01) beverage, pharmaceutical composition, tobacco, nutraceuti A2.3L 2/02 (2006.01) cal, oral hygiene composition or cosmetic comprising a A2ID I3/06 (2006.01) Sweetener composition, wherein the Sweetener composition A2ID 2/36 (2006.01) comprises one or more fermentatively-produced Steviol gly A23G L/40 (2006.01) cosides. Patent Application Publication Jul. 28, 2016 Sheet 1 of 11 US 2016/0213039 A1 Not Hind Pst Sai Nde ECOR pUG7-EcoRV kanMX 3982 bps ind Fig. 1 Patent Application Publication Jul. 28, 2016 Sheet 2 of 11 US 2016/0213039 A1 ONA2.0: T thMG1 PCR: Transformation -5-YPRcTau3r- ERG20,or- orthmG1 > s:------------------------------------------------------------g. KAN is Bisis 3'-YPRcTau3 x Cre recombinase r-r-r-r Fig. 2 Patent Application Publication Jul. 28, 2016 Sheet 3 of 11 US 2016/0213039 A1 A g SO bp i -- - ERG9 . < CTF8 recy TRP1 > rap - a CTF8 ----- 98 bp Transformation B - ERG9 > pe TRP repH < CTF8 - Fig. 3 Patent Application Publication Jul. 28, 2016 Sheet 4 of 11 US 2016/0213039 A1 RA28 UGT2.1a s s-chro5.0 3 NAT-T cra 3'-ch:89.01. - s-chroea usta ass NATs cRE - is 3-chro90 x Cre reconnairase w 5'-Chr09,81 ug2.1a :- 2 3'-C39, Fig. 4 Patent Application Publication Jul. 28, 2016 Sheet 5 of 11 US 2016/0213039 A1 GGPP - RebA A NA2.0: KOs. UGTGT1 . momen B Fig. 5 Patent Application Publication Jul. 28, 2016 Sheet 6 of 11 US 2016/0213039 A1 3**************38 :***SCO Fig. 6 Patent Application Publication Jul. 28, 2016 Sheet 7 of 11 US 2016/0213039 A1 Fig. 7 Patent Application Publication Jul. 28, 2016 Sheet 8 of 11 US 2016/0213039 A1 Aviii.38: i:8: N83: 3:38: 33ice {{}}: 8-8383tw8 Rei. A 8 at 88ses: 388 & Fig. 8 Patent Application Publication Jul. 28, 2016 Sheet 9 of 11 US 2016/0213039 A1 38&c & dia ... Sweet arosa:3:. .xii;tex Si38:ver: - ". .8-3. ... ." ... ... ::g::::g Cooking : ...: Citrus : 8:35:icfway: is is 38 WYY: eitasy R&A weeks as&c: Ra: A Fig. 9 Patent Application Publication Jul. 28, 2016 Sheet 10 of 11 US 2016/0213039 A1 &authdrying... -- ... Sweet artisatis origi:x Coci:8: 3:38: tag &strigant ... E8ty; Riatto: Bitter sites 35t S$88.3. *:::::::::::8:338 888. A «838 &S::::::: , Fig. 10 Patent Application Publication Jul. 28, 2016 Sheet 11 of 11 US 2016/0213039 A1 $3:3: $33.t 3:3: & S$838c:s 38:a::it::tic crgies 8tte site tasks : Redirit ... - 8:ow it::t MSc::::::::::: 8:8; & M888; 8.3888 &y ix Fig. 11 US 2016/0213039 A1 Jul. 28, 2016 STEVOL, GLYCOSDES may be further processed to remove spent fermentation medium components by down-stream purification processes. FIELD OF THE INVENTION 0010. In contrast to plant-extracted Steviol glycosides, fer 0001. The present invention relates to products compris mentatively-produced rebA may readily be produced in a ing a Sweetener composition and to a method for the prepa highly purified grade and in a form which has less residual ration of such products. The invention further relates to the bitterness and aftertaste. use of Sweetener compositions in the preparation of the prod 0011. Accordingly, the invention relates to a process of ucts and to a Steviol-glycoside containing composition. fermentative production of high purity rebA. The process is useful for producing high purity rebA with a product speci BACKGROUND TO THE INVENTION fication of at least about 95% rebA (dry weight basis). 0002. The use of artificial sweeteners such as dulcin, 0012. This product specification of fermentatively-pro Sodium cyclamate, aspartame, acylsulfame and saccharin has duced rebA is useful as a non-caloric Sweetener in various been seeing lower consumer demand for Some time. How food and beverage compositions as well as being useful in ever, non-caloric high potency Sweeteners with natural origin combination with other caloric and non-caloric Sweeteners. Such as Steviol glycosides have been receiving increasing 0013 This fermentatively-produced reb-A is useful as a demand. In addition, the Sweet Steviol glycosides have func non-caloric Sweetener in edible and chewable compositions tional and sensory properties Superior to those of many high Such as any beverages, confectioneries, bakeries, cookies, potency Sweeteners. chewing gums, and alike. 0003 Steviol glycosides (Masaya Ohta et al., J. Appl. Gly 0014. An object of the present invention is thus to provide cosci, V 57, (2010), pages 199-209) are a group of different a commercially valuable fermentative production process for Sweet diterpene glycosides, which have a single base—ste producing a highly purified Sweetener with known product viol diterpene and differ by the presence of carbohydrate specifications from a microbial production system and its use residues at positions C13 and C19. in various food products and beverages, overcoming the dis 0004. The physical and sensory properties of Rebaudio advantages of known plant extracted Steviol glycosides. side A and a number of other steviol glycosides have been 0015. In particular, fermentatively-produced rebA may be studied (Caroline Hellfritschet. al. J. Agric Food Chem, v60, (2012) pages 6782-6793). They have been tested for stability used to enhance citrus or sour attributes, total aroma impact, in carbonated beverages and found to be both heat and pH sweet aromatic complex, ethyl maltol (strawberry flavor) or stable (Chang and Cook, 1983). The Sweetness potency of brown fruit (flavor/aroma). Reb-A is between 200-400 times higher than sucrose, 0016. Herein is described a fermentation process where 0005. However, apart from its high level of sweetness, Steviol glycosides are produced via microbial fermentation. they have also intrinsic properties of post-bitter taste and Also described is a down-stream purification and recovery metallic and liquorish aftertaste. Some undesirable taste char process for recovery of fermentatively-produced steviol gly acteristics of Steviol glycosides as a result of contamination of cosides along with a highly-purified rebA of a given product other Substances such as polyphenolics and other flavor com specification. pound present in Stevia plant extracts. There are thus limita 0017. The fermentatively produced Steviol glycoside (of tions associated with the use of plant-derived Steviol glyco specific product specification) may be applied in various side extracts. foods and beverages as a Sweetener. That is to say, the present invention is based on fermentatively-produced Steviol glyco SUMMARY OF THE INVENTION sides Suitable for various food and beverage applications. 0006. One of the main ways to improve the taste quality is 0018. According to the invention, there is thus provided a the fermentative production of steviol glycosides. Another fermentatively derived product which is a foodstuff, bever way to produce highly purified individual glycosides with age, pharmaceutical composition, tobacco, nutraceutical, desired clean taste characteristics and minimal content of oral hygiene composition or cosmetic comprising a Sweet accompanying compounds is to tailor make specific Steviol ener composition, wherein the Sweetener composition com glycosides via designed biosynthesis in a fermentation pro prises one or more fermentatively-produced Steviol glyco duction host and fermentation thereof. sides. 0007 Accordingly, the invention is related to processes 0019. The invention also provides a method for the prepa for microbial production of steviol glycosides by fermenta ration of a product which is a foodstuff, beverage, pharma tion and to fermentatively-produced Steviol glycosides, such ceutical composition, tobacco, nutraceutical, oral hygiene as rebaudioside A (rebA), of certain product specification and composition or cosmetic comprising a Sweetener composi use thereof. That is to say, the present invention relates to a tion, which method comprises preparing a said product and microbial fermentation process for producing a highly puri incorporating a Sweetener composition comprising one or fied rebA and to its use thereof in various food products and more fermentatively-produced Steviol glycosides. Such a beverages. method may also comprise fermenting a microorganism as 0008. A process for the fermentative production and herein described and recovering rebaudioside A from the recovery of diterpene glycosides, from microbial fermenta microorganism and/or extracellular medium.
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  • CHTF18 Monoclonal Antibody (M01), Clone 1F5

    CHTF18 Monoclonal Antibody (M01), Clone 1F5

    CHTF18 monoclonal antibody (M01), clone 1F5 Catalog # : H00063922-M01 規格 : [ 100 ug ] List All Specification Application Image Product Mouse monoclonal antibody raised against a partial recombinant Western Blot (Cell lysate) Description: CHTF18. Immunogen: CHTF18 (AAH18184, 886 a.a. ~ 975 a.a) partial recombinant protein with GST tag. MW of the GST tag alone is 26 KDa. Sequence: GVHRPAPRNHEQRLEHIMRRAAREEQPEKDFFGRVVVRSTAVPSAGDT APEQDSVERRMGTAVGRSEVWFRFNEGVSNAVRRSLYIRDLL enlarge Western Blot (Transfected Host: Mouse lysate) Reactivity: Human Isotype: IgG2a Kappa Quality Control Antibody Reactive Against Recombinant Protein. Testing: enlarge Western Blot (Recombinant protein) Sandwich ELISA (Recombinant protein) enlarge Western Blot detection against Immunogen (35.53 KDa) . ELISA Storage Buffer: In 1x PBS, pH 7.4 Storage Store at -20°C or lower. Aliquot to avoid repeated freezing and thawing. Instruction: MSDS: Download Datasheet: Download Publication Reference 1. Two Different Replication Factor C Proteins, Ctf18 and RFC1, Separately Control PCNA-CRL4Cdt2-Mediated Cdt1 Proteolysis during S Phase and following UV Irradiation. Shiomi Y, Hayashi A, Ishii T, Shinmyozu K, Nakayama J, Sugasawa K, Nishitani H.Mol Cell Biol. 2012 Jun;32(12):2279-88. Epub 2012 Apr 9. 2. Stable interaction between the human PCNA loader complex Ctf18-RFC and DNA polymerase {epsilon} is mediated by the cohesion specific subunits, Ctf18, Dcc1 and Ctf8. Murakami T, Takano R, Takeo S, Taniguchi R, Ogawa K, Ohashi E, Tsurimoto T.J Biol Chem. 2010 Sep 7. [Epub ahead of print] Page 1 of 3 2016/12/7 Applications Western Blot (Cell lysate) CHTF18 monoclonal antibody (M01), clone 1F5 Western Blot analysis of CHTF18 expression in HeLa ( Cat # L013V1 ). Protocol Download Western Blot (Transfected lysate) Western Blot analysis of CHTF18 expression in transfected 293T cell line by CHTF18 monoclonal antibody (M01), clone 1F5.