Olive derived cell culture and methods for preparing and using the same

Abstract

The application further describes a composition in a form of a powder comprising olive fruit/leaf cells grown in vitro and a method of treating metabolic syndrome disorders, such as, high cholesterol level, comprising administering an effective amount of the composition. The cell line callus culture of olive cells manufactured according to the process of the invention includes high level of hydroxytyrosoll, tyrosol, oleoropein and verbascoside.

1 . A large scale process for the in vitro production of an olive cell culture of olive cells grown comprising:
 growing olive cells in a flask;   inoculating the olive cells from the flask into a first bioreactor;   inoculating the olive cells from the first bioreactor into a second bioreactor;   optionally inoculating the olive cells from the second bioreactor into a last bioreactor; and   harvesting the olive cells from the last bioreactor;   wherein the second bioreactor is a last bioreactor or an intermediate bioreactor and wherein the olive cells harvested from the last bioreactor are dried.   
     
     
         2 . The large scale process of  claim 1 , wherein the size of each bioreactor used in the process is larger than the one in which the olive cells were previously grown. 
     
     
         3 . The large scale process of  claim 1 , wherein if the second bioreactor is an intermediate bioreactor, an additional step of inoculating the olive cells to another intermediate bioreactor or to the last bioreactor is performed. 
     
     
         4 . The large scale process according to  claim 1 , further including additional steps of inoculating the olive cells from the second bioreactor into any number of sequential intermediate bioreactors. 
     
     
         5 . The large scale process of  claim 1 , wherein any one of the bioreactors is a 4-10 liter bioreactor. 
     
     
         6 . The large scale process of  claim 1 , wherein any one of the bioreactors is a 10-50 liter bioreactor. 
     
     
         7 - 8 . (canceled) 
     
     
         9 . The large scale process of  claim 1 , any one of the bioreactors is a 200-1000 liter bioreactor. 
     
     
         10 . The large scale process of  claim 1 , wherein the olive cells are grown in a modified MS growth medium. 
     
     
         11 . The large scale process of  claim 10 , wherein the growth medium is enriched with sucrose, casein hydrolysate, myo inositol, 1-naphthaleneacetic acid (NAA), kinetin, 2,4,D, [[NAA,]] BA, 2iP, or any combination thereof. 
     
     
         12 . The large scale process of  claim 1 , wherein at least one bioreactor is a disposable bioreactor. 
     
     
         13 . The large scale process of  claim 12 , wherein the disposable bioreactor is made from one or more layers of polyethylene. 
     
     
         14 . (canceled) 
     
     
         15 . The large scale process of  claim 10 , wherein the growth medium does not include plant hormones. 
     
     
         16 . (canceled) 
     
     
         17 . The large scale process of  claim 10 , wherein the growth medium is enriched with 1-6% sucrose. 
     
     
         18 . A composition in a form of a powder comprising olive fruit/leaf cells grown in vitro, whereby the olive cells are derived from one or more of section: olive pulp, olive seed, olive petiole or olive leaf. 
     
     
         19 . The composition according to  claim 18 , wherein the olive fruit cells include polyphenols selected from hydroxytyrosol, tyrosol, oleuropein, verbascoside and pinoresinol. 
     
     
         20 . The composition of  claim 18 , wherein the olive cells grown in vitro in a large scale process. 
     
     
         21 . (canceled) 
     
     
         22 . A method of treating metabolic syndrome such as affecting cholesterol level, comprising administering an effective amount of a composition comprising a powder comprising olive fruit cells grown in vitro, whereby the olive cells are derived from one or more of olive skin, olive lamellae and olive seeds. 
     
     
         23 . (canceled) 
     
     
         24 . The composition of  claim 18 , wherein the olive fruit/leaf cells grown in vitro Olive calli culture comprising comprise verbascoside in the amount of at least 4.1 mg/gr dry weight. 
     
     
         25 . (canceled) 
     
     
         26 . The large scale process of  claim 10 , wherein the modified MS growth medium for growing olive cells in vitro in bioreactor comprises:
 1.1-8.8 gr/L MS medium, 0.5 mg/L kinetin, 0.5 mg/L 2,4,D, and 40 gr/L sucrose;   1.1-8.8 gr/L MS medium, 1.3 mg/L 2,4,D, 0.06 mg/L BA, 30 gr/L and sucrose;   1.1-8.8 gr/L MS medium, 2.6 mg/L 2,4,D, 0.12 mg/L BA and 30 gr/L sucrose;   1.1-8.8 gr/L MS medium, 0.1 mg/L 2,4,D and 20 gr/L sucrose;   1.1-8.8 gr/L MS medium, 0.5 mg/L 2,4,D, 0.1 mg/L 2iP and 45 gr/L sucrose; or   1.1-8.8 gr/L MS medium, 2.0 mg/L NAA, 0.5 mg/L 2iP and 30 gr/L sucrose.   
     
     
         27 - 28 . (canceled)