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 leaf, seed or olive fruit cells grown in vitro, wherein the olive fruit/seed/leaf cells grown in vitro comprises verbascoside in the amount of at least 4.1 mg/gr dry weight 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 and wherein the olive cells are grown in a modified MS growth medium enriched with sucrose, casein hydrolysate, myo inositol, 1-naphthaleneacetic acid (NAA), kinetin, 2,4,D, NAA, BA, 2iP, or any combination thereof.   
     
     
         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 . The large scale process of  claim 1 , wherein any one of the bioreactors is a 50-200 liter bioreactor. 
     
     
         8 . The large scale process of  claim 1 , wherein any one of the bioreactors is a 200-500 liter bioreactor. 
     
     
         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 at least one bioreactor is a disposable bioreactor. 
     
     
         11 . The large scale process of  claim 10 , wherein the disposable bioreactor is made from one or more layers of polyethylene. 
     
     
         12 . The large scale process of  claim 11 , wherein the disposable bioreactor includes an inner and an outer layer prepared from polyethylene and a middle layer prepared from nylon. 
     
     
         13 . The large scale process of  claim 11 , wherein the growth medium does not include plant hormones. 
     
     
         14 . The large scale process of  claim 11 , wherein the growth medium includes plant hormones. 
     
     
         15 . The large scale process of  claim 1 , wherein the growth medium is enriched with 1-6% sucrose. 
     
     
         16 . The large scale process of  claim 1 , wherein the modified MS growth medium for growing olive calli comprises:
 a) 1.1-8.8 gr/L MS medium, 0.5 mg/L kinetin, 0.5 mg/L 2,4,D, 40 gr/L sucrose and 6 gr/L agar;   b) 1.1-8.8 gr/L MS medium, 1.3 mg/L 2,4,D, 0.06 mg/L BA, 30 gr/L sucrose and 6 gr/L agar;   c) 1.1-8.8 gr/L MS medium, 2.6 mg/L 2,4,D, 0.12 mg/L BA, 30 gr/L sucrose, 0.25 gr/L charcoal, and 6 gr/L agar;   d) 1.1-8.8 gr/L MS medium, 0.1 mg/L 2,4,D, 20 gr/L sucrose, and 6 gr/L agar;   e) 1.1-8.8gr/L MS medium, 0.5 mg/L 2,4,D, 0.1 mg/L 2iP, 45 gr/L sucrose, and 6 gr/L agar; or   f) 1.1-8.8 gr/L MS medium, 2.0 mg/L NAA, 0.5 mg/L 2iP, 30 gr/L sucrose, and 6 gr/L agar.   
     
     
         17 . The large scale process of  claim 1 , 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.