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

Abstract

The application describes a large scale process for the in vitro production of an olive cell culture. The application further describes a composition in a form of a powder comprising olive fruit / leaf cells grown in vitro. The cell culture of olive cells manufactured in vitro in a large scale according to the process of the invention comprises high level of verbascoside with undetectable levels of tyrosol, hydroxytyrosol, and oleuropein.

Description

OLIVE DERIVED CELL CULTURE AND METHODS FOR PREPARING AND USING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. ProvisionalPatent Application Nos. 63 / 707,284, filed on October 15, 2024 and 63 / 712,822, filed on October 28, 2024, titled “OLIVE DERIVED CELL CULTURE AND METHODS FOR PREPARING AND USING THE SAME", the contents of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION

[0002] The invention is directed to olive derived cell cultures, a process for the large scale production of such cell cultures, as well as methods of using the sameBACKGROUND OF THE INVENTION

[0003] Large scale processes are known in the art and are necessary for the industrial production of various materials. Since large scale processes cannot be performed by the same means as small scale processes, specific processes for the large scale production of materials must be designed, even if small scale processes exist.

[0004] Nutraceuticals are sometimes prepared using synthetic processes that provide the desired active ingredients, e.g., polyphenols, which are naturally found in fruit cells. However, the use of synthetic processes does not provide the natural ingredients along with the active ingredients, which sometimes contribute to the efficiency of the formulation.

[0005] Other types of nutraceuticals are prepared from the natural plants; however, all known large scale processes for preparing nutraceuticals from plants include the extraction of the prepared plant cells in order to obtain the desired active ingredient. However, when plants containing polyphenols, for example, are extracted, the final product may be bitter. Also, only certain parts of the plant may be successfully extracted since only they contain the desired amounts of the active ingredients.

[0006] Small scale processes for the preparation of fruit cells are known in the art; however, large scale processes are more difficult to design since they tend to amplify the production of the primary metabolites, while minimizing the productionsof the secondary metabolites. Since active ingredients, such as polyphenols, are secondary metabolites their production in large-scale processes is complex.

[0007] Nutraceuticals derived from polyphenol-containing fruit extracts are known for their beneficial effects. However, it has been shown that the therapeutic effect of fruit extracts is dependent on species, location, year (annual climate), processing etc. and therefore reliance on natural fruits as a source of these regulatory compounds does not lead to a homogeneous or consistent supply of material. Furthermore, fruits are often contaminated by residual fungicides, pathogens, pesticides and pollutants.

[0008] Thus, there is a need in the art for a large scale process for preparing plant cells from natural ingredients, which includes the control of production of the primary and the secondary metabolites of the plant cells. There is need for natural (phyto) compositions that may be prepared in a large scale process in which the amount of the active ingredient is consistent and recurrent (e.g., clonal preparations), is highly bioavailable and easily administered for the treatment and prevention of various diseases and disorders.

[0009] Olive products, Olive oil, table olives and fruits extracts, as well as plant parts, such as leaves, exhibit potent biological properties attributable to the presence of polyphenols. Polyphenols content of olive fruits contain primarily phenolics, terpenes and sterols, all of which are present in various plant parts, such as, bark, leaf and fruit. The major phenolic compounds are oleuropein, demethyloleuropein, 3-4 DHPEA-EDA, ligstroside, tyrosol, hydroxytyrosol, verbascoside and lignans (Alagna et al. BMC Plant Biology 2012, 12:162).

[0010] Due to the extensive knowledge about the olive’s health attributes and increasing public awareness about functional food, the demand for olive byproduct has increased tremendously in the western world. As a result of this trend, the extent of olive growth was increased significantly in many regions throughout the world, and industries that produced olive products have been developed.

[0011] Thus, there is a need in the art for a large scale process for preparing olive plant cells, in which there is a control of the amount of certain primary and the secondary metabolites of olive plant cells. Further, compositions prepared in a large scale process would prove to be and be consistent and recurrent (and therefore, would be advantageous over olive secondary metabolites obtained by other methods.SUMMARY OF THE INVEN TION

[0012] Embodiments of the invention are directed to 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.

[0013] According to some embodiments, the size of each bioreactor used in the process is larger than the one in which the olive cells were previously grown.

[0014] According to some embodiments, 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.

[0015] According to some embodiments, the large scale process of the invention further includes additional steps of inoculating the olive cells from the second bioreactor into any number of sequential intermediate bioreactors.

[0016] In some embodiments, there is provided a process for the in vitro production of an olive cell culture of olive leaf, or olive fruit cells grown in vitro in a large scale 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 the size of each bioreactor used in the process is larger than the one in which the olive cells were previously grown; wherein the olive cells harvested from the last bioreactor are dried and wherein the olive cells are grown in bioreactors in a growth medium comprising between about 2-5% sucrose, 9-20 mg / 1, ZnSO4.7H2O, 7-20 mg / 1 K ;BCh 0.1-1 mg / 1, CuSO45H2O, 400-900 mg / 1 MgSO4, 100-500 mg / 1 Ca(NO3)2mg / 1 and 1 SO- SOO mg / 1 KH2PO4, 350-650 mg / 1 KC1, 700-1800mg / l KNO3or 200-1500mg / l NH4NO3between about 0.2-lmg / l folic acid, between about 2-8 mg / 1 nicotinic acid, between about 0.2-lmg / l thiamine, between about 0.02-0.08 mg / 1 biotin and pea pepton, wherein the olive fruit / leaf cells grown in vitro comprises verbascoside in the amount of at least between 40 -200 g / kg dry weight and wherein the olive fruit / leaf cells grown in vitro do not comprise detectable levels of tyrosol, hydroxytyrosol, and oleuropein.

[0017] In some embodiments, 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.

[0018] In some embodiments, the process further including additional steps of inoculating the olive cells from the second bioreactor into any number of sequential intermediate bioreactors.

[0019] In some embodiments, any one of the bioreactors is a 3— 9-liter bioreactor.

[0020] In some embodiments, any one of the bioreactors is a 30-65-liter bioreactor. In some embodiments, any one of the bioreactors is a 30 -200-liter bioreactor. In some embodiments, any one of the bioreactors is a 200-400-liter bioreactor. In some embodiments, any one of the bioreactors is a 200-1100-liter bioreactor. In some embodiments, any one of the bioreactors is a 1000-2000-liter bioreactor. In some embodiments, any one of the bioreactors is a 2000-5000-liter bioreactor. In some embodiments, any one of the bioreactors is a 2000-10000-liter bioreactor. In some embodiments, at least one bioreactor is a disposable bioreactor having air flow of more than 800 liter / h, 1000 liter / h, 1200 liter / h, 1500 liter / h or more.

[0021] In some embodiments, the disposable bioreactor is made from one or more layers of polyethylene. In some embodiments, the disposable bioreactor includes an inner and an outer layer prepared from polyethylene and a middle layer prepared from nylon. In some embodiments, the growth medium further comprises a pea pepton.

[0022] In some embodiments, the pea pepton is in amount of between 50 to500g / l. In some embodiments, the pea pepton is in amount of between 100 to 400g / l. In some embodiments, wherein the pea pepton is in amount of between 200 to 300g / l. In some embodiments, the growth medium is enriched with one or more of vitamin, cytokine, auxin, glycine, myo inositol, nicotinic acid, pyridoxine, biotin, thiamine, folic acid, 2,4-D, or 2iP. In some embodiments, the amount of 2, 4-D is between 0.3-0.6 mg / 1. In some embodiments, the amount of 2iP is between 0.07-0.2 mg / 1. In some embodiments, the amount of glycine is between 0.5-6 mg / 1. In some embodiments, theamount of myo inositol is between 50-200 mg / 1. In some embodiments, the amount of nicotinic acid is between 2-8 mg / 1. In some embodiments, the amount of pyridoxine HC1 is between 0.1-lmg / l. In some embodiments, the amount of thiamine HC1 is between 0.2-0.8 mg / 1. In some embodiments, the amount of biotin is between 0.01-0.1 mg / 1. In some embodiments, the amount of folic acid is between 0.2-0.8 mg / 1.

[0023] In some embodiments, there is provided a process for the in vitro production of an olive cell culture of olive leaf, or olive fruit cells grown in vitro in a large scale 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 the size of each bioreactor used in the process is larger than the one in which the olive cells were previously grown; wherein the olive cells harvested from the last bioreactor are dried and wherein the olive cells are grown in bioreactors in a growth medium comprisingPea pepton; and one or more of vitamin, auxin, cytokine, glycine, sucrose, myo inositol, nicotinic acid, pyridoxine, biotin, thiamine and folic acid. In some embodiments, wherein the growth medium comprises:Pea pepton; and one or more of vitamin, cytokine, auxin s glycine, sucrose, myo inositol, nicotinic acid, pyridoxine, biotin, thiamine and folic acid.

[0024] In some embodiments, the amount of pea peptone and the one or more of vitamin, auxin, cytokine, glycine, sucrose, myo inositol, nicotinic acid, pyridoxine, biotin, thiamine and folic acid is as follows:

[0025] In some embodiments, the cells are grown in OCR medium at the stage of Erlenmeyers, wherein the medium is supplemented with pea pepton and sucrose. In some embodiments, the cells are grown in MS medium at the stage of Erlenmeyers, wherein the medium is supplemented with pea pepton and sucrose.

[0026] In some embodiments, there is provided a Olive Cell Product comprising dry cell line culture of olive cells manufactured in vitro in a large scale in accordance the process of the invention, comprising total polyphenols in an amount of 50-220 g / kg dry weight, wherein the total polyphenols comprises verbascoside in an amount of 40 -200 g / kg dry weight, 1-O-Synapoylglucose and Beta- Hydroxyverbascoside and the product does not comprise detectable levels of tyrosol, hydroxytyrosol, and oleuropein.

[0027] In some embodiments, there is provided a method of treating or preventing a fatty liver disease comprising administering the Olive Cell Product of invention to a subject in need thereof.

[0028] In some embodiments, there is provided a method of treating or preventing a steaotosis comprising administering the Olive Cell Product of invention to a subject in need thereof.

[0029] In some embodiments, there is provided a method of treating or preventing liver fibrosis comprising administering the Olive Cell Product of invention to a subject in need thereofBRIEF DESCRIPTION OF THE DRAWINGS

[0030] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanied drawings. Embodiments of the invention are illustrated by way of example and not limitation in the figures of the accompanying drawings, in which like reference numerals indicate corresponding, analogous or similar elements, and in which:

[0031] Figure 1 presents the dose-dependent effect of OCP on OA-induced lipid accumulation in HepG2 cells.

[0032] Figure 2 presents the effect of Olive Cell Product (OCP) on CollagenType I Production in LX-2 Cells in an In Vitro Fibrosis Model.DETAILED EMBODIMENTS OF THE INVENTION

[0033] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.

[0034] Embodiments of the invention are directed to a composition in a form of a powder comprising a cell culture of olive cell culture (OC) grown in vitro in large scale, whereby the cell culture of OC is derived from one or more of olive sections: olive pulp, olive seed, olive petiole or olive leaf. In an embodiment of the invention, the cell culture of OC grown in vitro in a large scale includes verbascoside in an amountof at least 40 g / kg dry weight (DW) wherein the amount of tyrosol, hydroxytyrosol and oleuropein is undetectable. In an embodiment of the invention, the cell culture of OC grown in vitro in a large scale includes verbascoside in an amount of at least 40, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 180, 190 or 200 g / kg dry weight (DW) wherein the amount of tyrosol, hydroxytyrosol and oleuropein is undetectable.

[0035] According to some embodiments, there is provided a process for the large scale in vitro production of olive cell cultures. In some embodiments of the invention, the process does not include the extraction of the olive cells. Surprisingly, the produced fruit cell cultures, manufactured in accordance with the large scale process described herein, were shown to include high amounts of verbacoside with undetectable amout of tyrosol, hydroxytyrosol and oleuropein. As used herein the term "polyphenols" refers to naturally occurring phyto organic compounds having more than one phenol group. Polyphenols may range from simple molecules, such as phenolic acid, to large, highly polymerized, compounds such as hydrolyzed tannins. The phenolic rings of polyphenols are typically conjugated to various sugar molecules, organic acids and / or lipids. Differences in this conjugated chemical structure account for the chemical classification and variation in the modes of action and health properties of the various polyphenol compounds. Examples of polyphenols include, but are not limited to, phenolics, terpenes and sterols. Typical olive polyphenols include, but are not limited to, phenolics, terpenes and sterols. The olive fruit may be of a wild or cultivated variety.

[0036] According to some embodiments, the calli cells and / or suspension culture of olive cells is derived from one or more of olive fruit cross sections: olive fruit, or olive leaf.

[0037] In some embodiments, the term “large scale” or “large scale process” and the like, refers hereto to a process of growing in vitro plant cells, which can be olive cells, in bioreactors of 40 , 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 liters or more, i.e.2000 liters or more, 5000 liters or more, 10000 liters or more.

[0038] According to some embodiments, the relative amounts of the various polyphenols in the prepared olive fruit / leaf cells differ from the relative amounts thereof in the agricultural olive fruit. According to some embodiments, the amount of verbacoside is amplified in the prepared fruit / leaf cells, in comparison to their amount in the agricultural olive fruit.

[0039] According to some embodiments, the amount of verbascoside is between about 40-200 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0040] According to some embodiments, the amount of verbascoside is between about 40-180 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0041] According to some embodiments, the amount of verbascoside is between about 40-160 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0042] According to some embodiments, the amount of verbascoside is between about 40-140 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0043] According to some embodiments, the amount of verbascoside is between about 40-120 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0044] According to some embodiments, the amount of verbascoside is between about 40-100 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0045] According to some embodiments, the amount of verbascoside is between about 40-80 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder. In some embodiments the amount of the verbacoside is up to 200 gVkg.

[0046] According to some embodiments, the amount of verbascoside is more than 30 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0047] According to some embodiments, the amount of verbascoside is more than 40 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0048] According to some embodiments, the amount of verbascoside is more than 50 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0049] According to some embodiments, the amount of verbascoside is more than 60 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0050] According to some embodiments, the amount of verbascoside is more than 70 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0051] According to some embodiments, the amount of verbascoside is more than 80 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0052] According to some embodiments, the amount of verbascoside is more than 90 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0053] According to some embodiments, the amount of verbascoside is more than 100 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0054] According to some embodiments, the amount of verbascoside is more than 110 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0055] According to some embodiments, the amount of verbascoside is more than 120 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0056] According to some embodiments, the amount of verbascoside is more than 130 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0057] According to some embodiments, the amount of verbascoside is more than 140 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0058] According to some embodiments, the amount of verbascoside is more than 150 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0059] According to some embodiments, the amount of verbascoside is more than 160 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0060] According to some embodiments, the amount of verbascoside is more than 170 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder. According to some embodiments, the amount of verbascoside is more than 180 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0061] According to some embodiments, the amount of verbascoside is more than 190 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0062] According to some embodiments, the amount of verbascoside is less than 200 g / kg, after the olive cell cultures grown in vitro in a large scale are dried to a powder.

[0063] According to some embodiments of the invention, the amount of total olive polyphenols including 1-O-Synapoylglucose, Beta-Hydroxyverbascoside and verbascoside is more than about 100 mg / gr after the olive cell cultures are dried to a powder.

[0064] According to some embodiments, the olive cell cultures prepared according to the large scale method of the invention contain less than about 10 % w / v fat after the olive cell cultures are dried to a powder.

[0065] According to some embodiments, the olive cell cultures prepared according to the large scale method of the invention contain less than about 5 % w / v fat after the olive cell cultures are dried to a powder. According to some embodiments, the olive cell cultures prepared according to the large scale method of the invention contain less than about 4, 3, 2 or 1% w / v fat after the olive cell cultures are dried to a powder.Fat refers to any of a group of natural esters of glycerol and various fatty acids conjugated or free or any combination thereof.

[0066] As used herein, fat refers to a fat types, e.g., saturated, monounsaturated and polyunsaturated. According to some embodiments, the olive cell cultures are dried, thus concentrating the materials found therein, including the fat. 20.

[0067] In one embodiment of the invention, there is provided a process for the in vitro production of a cell culture of olive fruit / leaf cells grown comprising: growing olive cells in a flask; inoculating the olive cells from the flask into a first bioreactor; and harvesting the produced olive cells.

[0068] In some embodiments of the invention, there is provided a large scale process for the in vitro production of a cell culture of olive fruit / leaf 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, wherein the second bioreactor is a last bioreactor or an intermediate bioreactor, and where there may be provided some more steps with one or more intermediate bioreactors; and harvesting the olive cells from the last bioreactor; wherein the olive cells harvested from the last bioreactor are dried.

[0069] According to some embodiments, at least one of the bioreactors is a disposable bioreactor.

[0070] By a "disposable bioreactor" it is meant a bioreactor with a disposable bag, which can be for a single use bag instead of a culture vessel. The disposable bag may be prepared from three or more layers of plastic foil. In some embodiments of the invention, one layer is prepared from polyethylene, polyethylene terephthalate or LDPE to provide mechanical stability. A second layer may be prepared using nylon, PVA or PVC that acts as a gas barrier. Finally, a contact layer may be prepared from PVA or PP or another layer of polythyelene, polyethylene terephthalate or LDPE. For medical applications the single-use materials that contact the product must be certified by the European Medicines Agency or similar authorities responsible for other regions.

[0071] According to some embodiments of the invention, the disposable bioreactor is prepared from one or more layers of polyethylene. In some embodiments of the invention, the disposable bioreactor is prepared from an inner and outer layer of polyethylene and a middle nylon layer.

[0072] In general there are two different approaches for constructing singleuse bioreactors, differing in the means used to agitate the culture medium.

[0073] Some single-use bioreactors use stirrers, similarly to conventional bioreactors; however, the stirrers may be integrated into the plastic bag. The closed bag and the stirrer are pre-sterilized. In use the bag is mounted in the bioreactor and the stirrer is connected to a driver mechanically or magnetically.

[0074] Other single-use bioreactors are agitated by a rocking motion. Other single-use bioreactors are airlift bioreactor in which the reaction medium is agitated and aerated by introduction of air. This type of bioreactor does not need any mechanical agitators inside the single-use bag.

[0075] According to some embodiments, the large scale process for preparing olive cell cultures comprises a number of subsequent steps. According to some embodiments of the invention, the amount of olive cell cultures prepared in eachstep is either larger or not than that prepared in the previous step. Further, the olive cell cultures prepared in each step may be inoculated or harvested to be used as a starter for the next step of the large scale process. In the last step of the large scale process, the fruit cells are typically grown until they reach the plateau in their growth profile.

[0076] According to some embodiments, there is provided a composition comprising a complex of pholyphenols including verbascoside and 1-0- Synapoylglucose, Beta-Hydroxyverbascoside, wherein the amount of total polyphenols (including verbascoside) in respect to the amount of verbascoide is higher than 1 : 1, 1.1 : 1, 1.2: 1, 1.3: 1, 1.4: 1, 1.5: 1, 1.6: 1, 1.7:1, 1.8: 1, 1.9: 1 or 1 :2. In some embodiments, the complex of pholyphenols does not comprise detectable amounts of oleuropin, hy droxtyrosol and tyrosol.

[0077] In some embodiments, at least 99% of the total polyphenols in the product of the process of invention comprises verbascoside. In some embodiments, at least 98% of the total polyphenols in the product of the process of invention comprises verbascoside. In some embodiments, at least 97% of the total polyphenols in the product of the process of invention comprises verbascoside. In some embodiments, at least 96% of the total polyphenols in the product of the process of invention comprises verbascoside. In some embodiments, at least 95% of the total polyphenols in the product of the process of invention comprises verbascoside. In some embodiments, at least 94, 93, 92, 91 or 90% of the total polyphenols in the product of the process of invention comprises verbascoside. In some embodiments, at least 89, 88, 87, 86, 85, 84, 83, 82, 81 or 80% of the total polyphenols in the product of the process of invention comprises verbascoside. In some embodiments, at least 79, 78, 77, 76, 75, 84, 73, 72, 71 or 70% of the total polyphenols in the product of the process of invention comprises verbascoside. In some embodiments, at least 69, 68, 67, 66, 65, 64, 63, 62, 61 or 60% of the total polyphenols in the product of the process of invention comprises verbascoside. In some embodiments, at least 59, 58, 57, 56, 55, 54, 53, 52, 51 or 50% of the total polyphenols in the product of the process of invention comprises verbascoside.

[0078] According to some embodiments, the composition is derived from olive cell cultures grown in large scale disposable bioreactors. According to some embodiments, the composition is derived from olive cell cultures grown in large scale disposable bioreactors, according to the process described herein.

[0079] According to some embodiments, the olive cells are grown in bioreactors. According to some embodiments, the bioreactors are designed so as to allow adequate mixing and mass transfer, while minimizing the intensity of shear stress and hydrodynamic pressure. According to some embodiments of the invention, at least one of the bioreactors is a disposable bioreactor. This can be the first bioreactor or the intermediate bioreactor or the last bioreactor or any combination thereof. According to some embodiments of the invention, the disposable bioreactor is the last bioreactor, such that after growing in the last bioreactor, the cells are harvested therefrom and dried, so as to form a powder.

[0080] According to an exemplary embodiment of the invention, the first step includes the preparation of an olive cell culture in a flask, such as an Erlenmeyer or a bioreactor. According to some embodiments, the first step involves the preparation of up to 1.0L of an olive cell culture. According to further embodiments, first step involves the preparation of up to 1.5L of an olive cell culture. According to further embodiments, first step involves the preparation of up to 2.0L of an olive cell culture. According to further embodiments, first step involves the preparation of up to 4.0L of an olive cell culture.

[0081] According to some embodiments, the first step is conducted using a glass, metal or plastic flask. According to some embodiments, the flask is disposable. According to further embodiments, the flask may be reused any number of times. According to some embodiments, the flask is sterilized by any appropriate means between uses.

[0082] According to some embodiments, the first step includes the use of any appropriate medium for growing the olive cells. According to some embodiments, the medium used for growing the fruit cells includes cell growth medium, salts, vitamins, sugars, hormones or any combination thereof.

[0083] In some embodiments, the large-scale process as described herein isvitamin, cytokine, auxin, glycine, sucrose or myo inositol and sucrose 1-5%.

[0084] In some embodiments, the first step of the process for a large scale in vitro growth of olive cells is conducted in Erlenmeyer flask comprising OCR mediumas hereinafter defined wherein the OCR medium comprises sucrose 1-5% and pea pepton.

[0085] In some embodiments, the first step of the process for a large scale in vitro growth of olive cells is conducted in Erlenmeyer flask comprising MS medium as hereinafter defined for example in Table 1 of the Examples, wherein the OCR medium comprises sucrose 1-5% and pea pepton.

[0086] In some embodiments, the term “OCR growth medium” or “OCRMedium” refer to a cell growth medium comprising: CoC126H2O, CuSOd.of vitamins auxin, cytokine, glycine, biotin, nicotinic acid, folic acid, thiamine, sucrose and / or myo inositol. In some embodiments the OCR medium is as provided in Table 1 in the examples. According to further embodiments, the OCR cell growth mediumsome embodiments, pea pepton is added to the growth medium. According to further embodiments, the OCR cell growth medium includes one or more of 2-5% sucrose,In some embodiments, pea pepton, is added to the growth medium. In some embodiments, the OCR cell growth medium is further supplemented with about 2-8 mg / 1 nicotinic acid, 0.2-lmg / l thiamine, 0.02-0.08 biotin or 0.2-lmg / l folic acid. In some embodiments, pea pepton is added to the growth medium. In some embodiments, the amount of the nicotinic acid, thiamine, biotin and folic acid is as follows: 2-8 mg / 1 nicotinic acid, 0.2-lmg / l thiamine HC1, 0.02-0. lmg / 1 biotin and 0.2-lmg / l folic acid. In some embodiments, pea pepton is added to the growth medium. In some embodimewnts, the amount of 2, 4-D is between 0.3 -0.6 mg / 1, the amount of 2iP is between 0.07-0.2 mg / 1, the amount of glycine is between 0.5-6 mg / 1, the amount of myo inositol is between 50-200 mg / 1, the amount of pyridoxine HC1 is between 0.1-lmg / l.

[0087] In some embodiments, the amount of the pea pepton is 50-700 mg / 1.In some embodiments, the amount of the pea pepton is 100-500 mg / 1. In some embodiments, the amount of the pea pepton is 200-300 mg / 1.

[0088] According to further embodiments, the cell growth medium includesacid, 0.2-lmg / l thiamine, 0.02-0.08 mg / 1 biotin and 0.2-lmg / l folic acid. In some embodiments, pea pepton is added to the growth medium.

[0089] In some embodiments, the amount of the nicotinic acid, thiamine, biotin and folic acid is as follows: 2-8 mg / 1 nicotinic acid, 0.2-lmg / l thiamine HC1, 0.02-0. lmg / 1 biotin and 0.2-lmg / l folic acid. In some embodiments, pea pepton is added to the growth medium. In some embodimewnts, the amount of 2, 4-D is between 0.3- 0.6 mg / 1 , the amount of 2iP is between 0.07-0.2 mg / 1, the amount of glycine is between 0.5-6 mg / 1, the amount of myo inositol is between 50-200 mg / 1, the amount of pyridoxine HC1 is between 0.1-lmg / l.

[0090] In some embodiments, the amount of the pea pepton is 50-700 mg / 1. In some embodiments, the amount of the pea pepton is 100-500 mg / 1. In some embodiments, the amount of the pea pepton is 200-300 mg / 1.

[0091] According to further embodiments, the cell growth medium includes one or more of between about 1-5% sucrose, 9-20mg / l ZnSO4.7H2O, 7-20 mg / 1 H3BCh. 0.1-1 mg / 1 CuSO45H2O, 400-900 mg / 1 MgSO4, 100-500 mg / 1 Ca(NO3)2, 150-500 mg / 1 KH2PO4, 350-650 mg / 1 KC1, 700-1800mg / l KNCh or 200-1500mg / l NH4NCh 2-8 mg / 1 nicotinic acid, 0.2-lmg / l thiamine, 0.02-0.08 biotin and 0.2-lmg / l folic acid. In some embodiments, pea pepton is added to the growth medium.

[0092] According to some embodiments, the cell growth medium includes one or more of about 1, 2, 3, 4, or 5% sucrose, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20mg / l ZnSO4.7H2O, about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg / 1 H3BO3about 0.1, 0.2, O.3.O.4. 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mg / 1 CuSO45H2O, about 400, 500, 600, 700, 800 or 900 mg / 1 MgSO4, about 100, 200, 300, 400 or 500 mg / 1 Ca(NO3)2, about 150, 200, 250, 300, 350, 400, 450 or 500 mg / 1 KH2PO4, about 350, 400, 450, 500, 550, 600 or 650 mg / 1 KC1, about 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or 1800mg / l KNCh or about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500mg / l NH4NO3;about 2, 3, 4, 5, 6, 7 or 8 mg / 1 nicotinic acid, about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or lmg / 1 thiamine, about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, or 0.08 mg / 1 biotin and about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or lmg / 1 folic acid.

[0093] In some embodiments, pea pepton is added to the growth medium.

[0094] In some embodiments, the growth medium comprises the followingbetween about 150-500 mg / 1 KH2PO4, between about 800-1500 mg / 1 KNO3, between about 100-500 mg / 1 Ca(NO3)2 and between about 350-650 mg / 1 KC1.

[0095] In some embodiments, the medium comprises the following salts:

[0096] In some embodiments, the growth medium comprises the followingbetween about 250-500 mg / 1 KH2PO4, between about 900-1260 mg / 1 KNO3, between about 100-500 mg / 1 Ca(NCO3)2 and between about 350-650 mg / 1 KC1.

[0097] In some embodiments, pea peptone and sucrose are added. In some embodiments, the amount of the pea pepton is between 50-700 mg / 1. In some embodiments, the amount of the pea pepton is between 100-600 mg / 1. In some embodiments, the amount of the pea pepton is between 150-550 mg / 1. In some embodiments, the amount of the pea pepton is between 200-400 mg / 1. In some embodiments, the amount of the pea pepton is between 200-300 mg / 1. In some embodiments, the amount of the pea pepton is between 250-400 mg / 1.

[0098] In some embodiments, the growth medium further comprises pea pepton; and one or more of vitamin and or cytokine selected from the group consisting of glycine, myo inositol, nicotinic acid, pyridoxine, biotin, thiamine, folic acid, 2,4-D, and 2iP.

[0099] In some embodiments, the amount of the nicotinic acid, thiamine, biotin and folic acid is as follows: 2-8 mg / 1 nicotinic acid, 0.2-lmg / l thiamine HC1, 0.02-0.1 biotin and 0.2-lmg / l folic acid. In some embodiments, pea pepton is added to the growth medium. In some embodimewnts, the amount of 2, 4-D is between 0.3-0.6 mg / 1, the amount of 2iP is between 0.07-0.2 mg / 1, the amount of glycine is between0.5-6 mg / 1, the amount of myo inositol is between 50-200 mg / 1, the amount of pyridoxine HC1 is between 0.1-lmg / l.

[0100] In some embodiments, the amount of the pea pepton is 50-700 mg / 1. In some embodiments, the amount of the pea pepton is 100-500 mg / 1. In some embodiments, the amount of the pea pepton is 200-300 mg / 1.

[0101] According to further emb odiments, pea peptone i s included in the cell growth medium. According to further embodiments growth hormones may be included in the cell growth medium. According to further embodiments, the growth medium includes hormones.

[0102] In an embodiment of the invention, the concentration of the sucrose added to the growth medium is between 2 to 5%. In another embodiment, the concentration of the sucrose added to the growth medium is about 4%.

[0103] According to some other exemplary embodiments, the olive cells and the medium are continuously mixed during the first step. According to further embodiments, the olive cells and the medium are mixed occasionally during the first step. According to some embodiments, the temperature during the first step is between about 20°C and 30°C. According to some embodiments, the temperature during the first step is between about 22°C and 28°C. According to some embodiments, the temperature during the first step is about 25°C. According to some embodiments, the olive cells are grown in the first step for more than 3 days. According to some embodiments, the olive cells are grown in the first step for more than 5 days. According to some embodiments, the olive cells are grown in the first step for more than 3 days and less than 3 weeks.

[0104] According to some exemplary embodiments, the bioreactor used in the process of the invention includes an inlet through which the olive cells from the previous step, the medium and any additional materials are placed into the bioreactor. According to further embodiments, the bioreactor used in the process of the invention includes an outlet for removing any materials desired. According to some embodiments, the outlet includes a gas outlet, designed to relieve the bioreactor of excess gases. According to some embodiments, the gas outlet is operated manually. According to other embodiments, the gas outlet is operated automatically, wherein gases are let out of the bioreactor once the atmosphere in the bioreactor reaches a predefined pressure.

[0105] According to some embodiments, the gas outlet is a separate entity from the outlet for removing liquids / solids. In some embodiments, the bioreactor has an air flow of more than 800 liter / h, 1000 liter / h, 1200 liter / h, 1500 liter / h or more.

[0106] The inventors found that the OCR medium was the most effective medium and led to the unique Olive Cells Product as described herein. The combination of the OCR medium with an O-type bioreactor that was designed to provide optimal aeration of 1500 liter / h or more resulted in high amount of verbascoside and a high cell growth.

[0107] Once the first step of the olive cell growth is concluded, according to some exemplary embodiments, the olive cells are inoculated into a small scale bioreactor, which is termed here also the first bioreactor, for the second step of the large scale process. According to some embodiments, the small scale bioreactor is a 4L reactor. According to further embodiments, the small scale bioreactor is a 3-5L reactor. According to further embodiments, the small scale bioreactor is a 2-10L reactor. According to further embodiments, the small scale bioreactor is a 4-9L reactor.

[0108] The small scale bioreactor may be prepared from any appropriate material, such as, plastic and / or any type of polymer. According to some embodiments, the small scale bioreactor is disposable. If the small scale bioreactor is not disposable, according to some embodiments, it is cleaned and sterilized between uses by any appropriate means.

[0109] As described above, the production of secondary metabolites, including polyphenols, is known to be significantly reduced with increasing bioreactors volumes, in comparison to the amount of the same metabolites in small scale productions, using, e.g., glass flasks, such as Erlenmeyers. However, the large scale process detailed herein provides olive cells in which the amount of the secondary metabolites is not reduced when grown in bioreactors. Further, the production of certain secondary metabolites may even be amplified.

[0110] Thus, according to embodiments of the invention, the relative amounts of the secondary metabolites in olive cells grown in the small scale bioreactor are not significantly reduced in comparison to their relative amounts in the first step of the process. According to some embodiments, the components described above for use in the growth medium in the first step may be used also in the second step of the process. According to some embodiments, the growth medium used in the small scale bioreactor is the same as used in the first step of the large scale process. According to someembodiments, the relative amounts of the different components found in the growth medium in the second step, is the same as in the first step. According to other embodiments, the relative amounts of the different components found in the growth medium in the second step, differ from the relative amounts used in the first step. According to some embodiments, additional materials are added to the growth medium in the second step of the process.

[0111] According to some embodiments, the small scale bioreactor includes an inlet through which the fruit cells from the first step, air, the medium and any additional materials are placed into the bioreactor. According to further embodiments, the small scale bioreactor includes an outlet for removing any materials desired. According to some embodiments, the outlet includes a gas outlet, designed to relieve the bioreactor of excess gases. According to some embodiments, the gas outlet is operated manually. According to other embodiments, the gas outlet is operated automatically, wherein gases are let out of the bioreactor once the atmosphere in the bioreactor reaches a pre-defined pressure. According to some embodiments, the gas outlet is a separate entity from the outlet for removing liquids / solids. In some embodiments, the bioreactor has an air flow of more than 800 liter / h, 1000 liter / h, 1200 liter / h i, 1500 liter / h or more.

[0112] According to some embodiments, the olive cells and the medium are continuously mixed during the second step.. According to some embodiments, the temperature during the second step is between about 20 to 30°C. According to some embodiments, the temperature during the second step is between about 22 to 28°C. According to some embodiments, the temperature during the second step is about 25°C. According to some embodiments, the olive cells are grown in the second step for more than a week and less than two weeks. According to some embodiments, the olive cells are grown in the second step for less than a week. In some embodiments of the invention, the olive cells are grown between 4-30 days before being inoculated into the next bioreactor.

[0113] According to some embodiments, for the third step of the large scale process, the harvested olive cells are placed into a medium scale bioreactor. According to some embodiments, the medium scale bioreactor is a 30-50L reactor. According to further embodiments, the medium scale bioreactor is a 40-60L reactor. According to further embodiments, the medium scale bioreactor is a 30-70L reactor. According to further embodiments, the medium scale bioreactor is a 20-100L reactor.

[0114] The medium scale bioreactor may be prepared from any appropriate material, such as plastic and / or any type of polymer. According to some embodiments, the medium bioreactor is disposable. If the medium scale bioreactor is not disposable, according to some embodiments, it is cleaned and sterilized between uses by any appropriate means.

[0115] Similarly to the small scale bioreactor, according to embodiments of the invention, the relative amounts of the secondary metabolites in olive cells grown in the medium scale bioreactor are not significantly reduced in comparison to their relative amounts in any of the previous steps of the process. According to some embodiments, the components described above for use in the growth medium in any of the previous steps may be used also in the third step of the process. According to some embodiments, the growth medium used in the medium scale bioreactor is the same as used in any of the previous steps of the medium scale process. According to some embodiments, the relative amounts of the different components found in the growth medium in the third step, is the same as in any of the previous steps of the process. According to other embodiments, the relative amounts of the different components found in the growth medium in the third step, differs from the relative amounts used in any of the previous steps of the process. According to some embodiments, additional materials are added to the growth medium in the third step of the process.

[0116] According to some embodiments, the medium scale bioreactor includes an inlet through which the olive cells from the second step, the medium, air and any additional materials are placed into the bioreactor. According to further embodiments, the medium scale bioreactor includes an outlet for removing any materials desired. According to some embodiments, the outlet includes a gas outlet, designed to relieve the bioreactor of excess gases. According to some embodiments, the gas outlet is operated manually. According to other embodiments, the gas outlet is operated automatically, wherein gases are let out of the bioreactor once the atmosphere in the bioreactor reaches a pre-defined pressure. According to some embodiments, the gas outlet is a separate entity from the outlet for liquids / solids.

[0117] According to some exemplary embodiments, the olive cells and the medium are continuously mixed during the third step. According to further embodiments, the olive cells and the medium are mixed occasionally during the third step. According to some embodiments, the temperature during the third step is between about 20 and 30°C. According to some embodiments, the temperature during the thirdstep is between about 22 and 28°C. According to some embodiments, the temperature during the third step is about 25°C. According to some embodiments, the olive cells are grown in the third step for about two to three weeks. According to some embodiments, the olive cells are grown in the third step for less than one week. According to some embodiments, the olive cells are grown in the third step for about one to two weeks. According to some embodiments, the olive cells are grown in the third step for about three to five weeks. According to some embodiments, the olive cells are grown in the third step for about 5 to 30 days.

[0118] Once the third step of the olive cell growth is concluded, the olive cells may be inoculated from the medium scale bioreactor typically by any appropriate means. For the fourth exemplary step of the large scale process, the harvested olive cells are placed into a larger scale bioreactor. According to some embodiments, the larger scale bioreactor is a lOOOL reactor. According to further embodiments, the larger scale bioreactor is a 200-500L reactor. According to further embodiments, the large scale bioreactor is a 500- WOOL reactor. According to further embodiments, the large scale bioreactor is a 1000-1500L reactor. According to further embodiments, the large scale bioreactor is a 500-1100L reactor. According to further embodiments, the large scale bioreactor is a 500-2000L reactor. According to further embodiments, the large scale bioreactor is a 2000-10000L reactor. According to further embodiments, the large scale bioreactor is a 10000-20000L reactor.

[0119] According to some embodiments, the first bioreactors may be a 0.5-10-liter bioreactor. According to some embodiments, the first bioreactors may be a 3- 9-liter bioreactor. According to some embodiments the larger bioreactor may be 40- 65-liter bioreactor. A ccording to some embodiments, the larger bioreactor may be a 40 -200-liter bioreactor. According to some embodiments, the larger bioreasctor may be a 200-400-liter bioreactor. According to some embodiments, the larger bioreasctor may be 200-1100-liter bioreactor. According to some embodiments, the larger bioreasctor may be 1000-2000-liter bioreactor. According to some embodiments, the larger bioreactor may be 2000-5000-liter bioreactor. According to some embodiments, the larger bioreasctor may be 2000-10000-liter bioreactor. According to some embodiments, the larger bioreasctor may be 10000-20000-liter bioreactor or more.

[0120] The larger scale bioreactor may be prepared from plastic and / or any type of polymer. According to some embodiments, the larger scale bioreactor is disposable.

[0121] Similarly to the small scale bioreactors, according to embodiments of the invention, the relative amounts of the secondary metabolites in olive cells grown in the larger scale bioreactor are not significantly reduced in comparison to their relative amounts in the previous steps of the process. According to some embodiments, the components described above for use in the growth medium in any of the previous steps may be used also in the fourth step of the process. According to some embodiments, the growth medium used in the larger scale bioreactor is the same as used in any of the previous steps. According to some embodiments, the relative amounts of the different components found in the growth medium in the fourth step, is the same as in any of the previous steps. According to other embodiments, the relative amounts of the different components found in the growth medium in the fourth step, differs from the relative amounts used in any of the previous steps. According to some embodiments, additional materials are added to the growth medium in the fourth step of the process.

[0122] According to some embodiments, the larger scale bioreactor includes an inlet through which the olive cells from the third or second step, the medium and any additional materials are placed into the bioreactor. According to further embodiments, the larger scale bioreactor includes an outlet for removing any materials desired. According to some embodiments, the outlets includes a gas outlet, designed to relieve the bioreactor of excess gases. According to some embodiments, the gas outlet is operated manually. According to other embodiments, the gas outlet is operated automatically, wherein gases are let out of the bioreactor once the atmosphere in the bioreactor reaches a pre-defined pressure. According to some embodiments, the gas outlet is a separate entity from the outlet for liquids / solids. In some embodiments, the bioreactor has an air flow of more than 800 liter / h, 1000 liter / h, 1200 liter / h, 1500 liter / h or more.

[0123] According to some embodiments, here and in any other appropriate bioreactor, the bioreactor may include two or more inlets and / or outlets. Each inlet and / or outlet may be designated for the passage of a certain type of material or otherwise, various materials may pass through the same inlet / outlet. The various materials may pass through the inlet / outlet together or separately from one another. Any bioreactor related to herein may further include two or more inlets / outlets designated for the passage of at least one type of material.

[0124] According to some embodiments, the olive cells and the medium are continuously mixed during the fourth step. According to further embodiments, the olivecells and the medium are mixed occasionally during the fourth step. According to some embodiments, the temperature during the fourth step is between about 20 to 30°C. According to some embodiments, the temperature during the fourth step is between about 22 to 28°C. According to some embodiments, the temperature during the fourth step is about 25°C. According to some embodiments, the olive cells are grown in the third or fourth step until they reached a cell biomass of 10% to 70% w / w of the entire mass of the medium.

[0125] According to some embodiments, the large scale process is terminated after the olive cells are grown in the larger scale bioreactor. According to such embodiments, the olive cells are grown in the larger scale bioreactor until they reach a cell biomass of 10% to 70% w / w. Once the cell biomass of 10% to 70% w / w is reached, the olive cells are harvested from the large scale bioreactor by any appropriate means and are further processed. According to some embodiments, the olive cells are further processed by any appropriate type of drying, lyophilization, Freeze-Drying, fluidized bed air drying and Spray Drying. According to some embodiments, the processing of the olive cells does not include the extraction of active ingredients therefrom.

[0126] According to some embodiments, the processing of the olive cells include the extraction of active ingredients therefrom.

[0127] According to some embodiments, the large scale process may include one step of inoculating the cells from a flask into a bioreactor, which can be in any size, and harvesting the cells. According to other embodiments, the olive cells may be inoculated in a series of bioreactors wherein each of the bioreactors is typically larger than the previous bioreactor used. Any number of additional steps is performed according to the large scale process. The additional steps include possible intermediate steps in which the cells are harvested or inoculated and placed in a larger bioreactor and grown there until being harvested or inoculated and transferred to a larger bioreactor. According to further embodiments, the process includes additional steps for growing the olive cells harvested from the large scale bioreactor.

[0128] In an embodiment of the invention, there is provided a pharmaceutical or nutraceutical composition or a food additive comprising the olive cells manufactured in the large scale process of the invention. The pharmaceutical or nutraceutical composition or a food additive may be administered to the subject by oral administration.

[0129] In some embodiments, there is provided an Olive Cell Product comprising dry cell line culture of olive cells manufactured in vitro in a large scale in accordance to the process described herein, comprising total polyphenols in an amount of 50-220 g / kg dry weight, wherein the total polyphenols comprises verbascoside in an amount of 40 -200 g / kg dry weight, 1-O-Synapoylglucose and Beta- Hydroxyverbascoside and the product does not comprise detectable levels of tyrosol, hydroxytyrosol, and oleuropein, wherein the Olive Cell Product demonstrates anti oxidative activity.

[0130] As is shown in the Examples, the Olive Cells Product of the invention showed dose dependent effectiveness is reducing steatosis and fibrosis in in vitro cellular models to non-alcoholic fatty liver disease (NAFLD). The fibrosis assay was conducted in an in vitro model of NAFLD using transforming growth factor beta (TGF P) that has been identified as the main inducer of transdifferentiate hepatic stellate cells into myofibroblast like cells exhibiting proliferative, contractile and fibrogenic properties, becoming the major source of fibrillary collagens in the fibrotic liver. The level of collagen type 1 in LX-2 cells was assessed to characterize liver fibrosis in this study. The steatosis assay was based on lipid accumulation in HepG2 cells in response to oleic acid (OA) treatment that can be effectively quantified using a Nile Red colorimetric assay. This technique involves staining of intracellular lipid droplets with Nile Red, followed by stain extraction and measurement of optical density that is proportional to the intracellular lipid content. Assay results were able to reflect the dose-dependent uptake of OA and appeared entirely consistent with lipid droplets stained with Nile Red, as visualized using microscopy.

[0131] In some embodiments, there is provided a method of treating or preventing a fatty liver disease, such as, non-alcoholic fatty liver disease (NAFLD). Hepatic lipid accumulation can progress from simple steatosis to non-alcoholic steatohepatitis (NASH), which includes hepatocellular injury, inflammation and fibrosis. In more severe cases, complications such as liver cirrhosis and hepatocellular carcinoma may arise by administering the Olive Cells Product of the invention.

[0132] In some embodiments, there is provided a method of treating or preventing steatosis by administering the Olive Cells Product of the invention.

[0133] In some embodiments, there is provided a method of treating or preventing liver fibrosis by administering the Olive Cells Product of the invention.

[0134] The methods comprises administering to a subject in need a pharmaceutical or nutraceutical composition or a food additive comprising the olive cell culture, wherein the culture is manufactured according to the large scale process detailed herein.

[0135] As used herein, the phrase "pharmaceutical composition" refers to a preparation of olive cell culture, as further described hereinabove, with or without other chemical components, such as physiologically suitable carriers and excipients.

[0136] As used herein the term “treating” refers to the prevention of some or all of the symptoms associated with an inflammatory disease, a condition or disorder. The term “treating” also refers to alleviating the symptoms or underlying cause of a fatty liver disease, prolongation of life expectancy of patients having a disease, as well as complete recovery from a disease.

[0137] Various aspects of the invention are described in greater detail in the following Examples, which represent embodiments of this invention, and are by no means to be interpreted as limiting the scope of this invention.EXAMPLES

[0138] The present invention will be further understood by reference to the following non-limiting examples. The examples herein refer to the process of producing olive cells that are grown in vitro in a large scale, i.e. in bioreactors of 1, 5, 10, 50, 100, 200, 500, 700, 1000 liter or more. The aim of the invention was to obtain in a large scale process a unique Olive Cells Product of polyphenols that contains a large amount of verbacoside and is devoid of detectable amounts of other polyphenols, such as, ol europin, hy droxtyrosol and tyrosol. The experiments herein show that specific composition of the growth medium as well as using a unique bioreactor having a structure that enables air flow of more than 800 liter / h yield in a large scale process in vitro grown olive cells with verbacoside in an amount of between 60g / kg to 220 g / kg DW and undetectable amounts of other polyphenols, such as, oleuropin, hydroxtyrosol and tyrosol.Example 1Manufacturing- Industrial level Scaling up

[0139] The production process encompasses propagation of olive cells that was initiated from olive fruits and or leaves (Olea europaea L.') ofNabali, Manzanilla, Souri and Bamea cultivars, including all leaves, petioles, fruits and kernels, in a progressively process having four stages. Starting from propagation of olive cells in petri dish to an Erlenmeyer shake flasks for further propagation in a small- and large- scale disposable bioreactors. The critical key factor was to create a unique product containing olive cells grown in vitro in bioreactors with high level of verbascoside and undetectable amount of other olive polyphenols, such as, oleuropin, hydroxtyrosol and tyrosol and to maintain a high level of the verbascoside in the olive cells during the propagation in different bioreactors scale which ranges from 1 liter to 2000 liters or more. At the end of the last large-scale stage of propagation, at the required biomass, the cells are harvested and dried to produce a fine powder yielding a biomass of olive dried cells product having a large amount of verbascoside, which has strong antioxidant activity.

[0140] As will be shown in here, many experiments were done in which cells were grown in different media. The composition of each of the assessed media is provided in Table 1 below. The inventors found that the OCR medium was the most effective medium and led to the unique Olive Cells Product as described herein. The combination of the OCR medium with an O-type bioreactor that was designed to provide optimal aeration of 1500 liter / h or more resulted in high amount of verbascoside and a high cell growth.Table 1: list of ingredients in the MS, OCR and Gamborg B5 mediaStage 1: Erlenmeyer, shake flasks

[0141] Olive cells are grown in suspension under continuous fluorescent light (1000 lx) at 26 + 3 °C, in one (1) liter Erlenmeyer flasks on an orbital shaker in growing MS medium containing vitamins and minerals supplemented with 200-300 mg / 1 pea protein peptone, 2-5 % sucrose, 0.3-0.6 mg / 1 2,4-D and 0.07-0.2 mg / 1 2iP (pH 5.8). The cells are sub-cultured every 5-10 days.Stage 2: Small scale bioreactor

[0142] Small scale bioreactor culturing is performed by inoculating a 5 to 10 old day cell suspensions grown in the Erlenmeyer of stage 1 into a 3— 6-liter disposable bioreactor at 26 + 3°C. The cells are grown in the suspension under continuous fluorescent light (1000 lx). The growing OCR medium contains vitamins and minerals supplemented with 200-300 mg / 1 pea protein peptone, 2-6 % sucrose, 0.3-0.6 mg / 1 2,4- D, 0.07-0.2 mg / 1 2iP(pH 5.8). The cells are sub-cultured every 6-14 days.Stage 3: Large scale bioreactor

[0143] The cell suspension grown in a small-scale bioreactor are inoculated into a 30-80-liter disposable bioreactor. The cells are grown in a suspension under continuous fluorescent light (1000 lx) at 26 + 5°C. The growing OCR medium contains vitamins and minerals supplemented with 200-300 mg / 1 pea peptone, 2-6 % sucrose, 0.3-0.6 mg / 1 2,4-D, 0.07-0.2 mg / 1 2iP (pH 5.8). The cells are sub-cultured every 14-25 days.Stage 4: Larger scale bioreactor

[0144] The cell suspension grown in a small- or large-scale bioreactor are inoculated into a 300-2000-liter disposable bioreactor. The cells are grown in a suspension under continuous fluorescent light (1000 lx) at 26 + 5°C. The growing OCR medium contains vitamins and minerals supplemented with 200-300 mg / 1 pea protein peptone, 2-6 % sucrose, 0.3-0.6 mg / 1 2,4-D, 0.07-0.2 mg / 1 and 2iP, (pH 5.8). The cells are sub-cultured every 14-25 days.Stage 5: Harvesting

[0145] The cells are harvested once they reach a cell biomass of 10 % to70% (w\v). The harvested cells are dried to produce a fine powder, with a typical composition, taste and odor.Stage 6: Drying process

[0146] These harvested cells are then subjected to a drying process. This process results in a fine powder of dried olive cells.Example 2The effect of medium composition on the growth and verbascoside level in olive cells grown in a large-scale disposable bioreactor

[0147] Olive cells were grown in large and larger disposable bioreactor as described on Example 1 stages 3, 4 in different medium compositions.

[0148] The effect of medium composition on olive cells growth, verbascoside production and total polyphenols levels was studied using large-scale and larger-scale disposable bioreactors. The cells were grown in MS, Gamborg B5 medium and OCR medium containing vitamins and minerals as mentioned above andsupplemented with pea peptone, sucrose and hormones. Table 2 shows that OCR medium, significantly boosted cell growth by 150%. The concentration of verbacoside was 282 mg / L in olive cells grown in MS medium, 96.8 mg / L in Gamborg B5 medium and 1048 mg / L in olive cells grown in OCR medium. Additionally, the experiment shows that OCR medium is essential to produce high levels of total polyphenols including verbascoside. Specifically, the total polyphenol content in olive cells grown on a large scale in OCR medium is 350% higher, and the verbascoside level is 366% higher compared to cells grown in MS medium. In addition, the total polyphenol and verbascoside content in olive cells grown on a large scale in OCR medium is 1000% higher compared to cells grown in Gamborg B5 medium. In addition, the biomass as demonstrated by fresh weight of olive cells grown in OCR medium was 56% higher than in MS and 750% higher than in Gamborg B5 medium.Table 2 - Levels of verbascoside, total polyphenols and fresh weight of olive cells grown in large scale bioreactors using different media*The data are the mean of at least two experiments.** The data are the mean of at least ten experiments.Example 3Evaluating Verbascoside, Total Polyphenols, and Fresh Weight in Olive Cells Across Different Growth Stages: From Erlenmeyer Shake Flask to Large-Scale Bioreactor

[0149] Olive Cells were grown in different scale stages from Erlenmeyer shake flask to a large-scale disposable bioreactor in the presence of OCR medium as described on Example 1, stages 2 to 6. The results in Table 3 show that olive cells grew in Erlenmeyer and large-scale bioreactors and synthesized high amount of verbascoside. When the cells were grown in either 50 liter, 300 to 1000 liters scale in disposable bioreactors, a higher growth rate achieved in comparison to the growth in an Erlenmeyer flask as revealed by fresh and dry weight of the cells, see the data in Table 3. When the cells were growing in Erlenmeyer and in either large size large scale (50-80 liter), larger scale (300 to 2000 liters scale) disposable bioreactors, a higher growth rate was achieved in large and larger scale bioreactors, 115, 125 gram / 1, respectively compared to 111 gram / L in Erlynmayer (Table 3). The dried weight was also higher in the large and larger scale bioreactor compared to Erlenmeyer due to higher level of polyphenols in the large / r scale. Moreover, the level of verbascoside in olive cells grown in the OCR medium, in large / r scale disposable bioreactors was higher than the level obtained in the Erlenmeyer 883 mg / 1, 1048 mg / 1 and 291 mg / 1, respectively (Table 3). In addition, the level of total polyphenols in olive cells grown in the OCR medium, in large / r scale disposable bioreactors was higher than the level obtained in the Erlenmeyer 930 mg / 1, 1138 mg / 1 and 321mg / l, respectively (Table 3). This is the first time to demonstrate successful growth of olive plant cells (originated from fruit or leaf) in a large-scale-disposable bioreactor, i.e about 1000 liters or more with high level of verbascoside, totalpolyphenols and fresh weight.

[0150] The large and larger-scale disposable bioreactors demonstrated superior production of verbascoside and total polyphenols, along with higher fresh and dry weights compared to Erlenmeyer flasks. This indicates the effectiveness of large- scale systems for optimizing olive cell growth and metabolite production.Table 3- Verbascoside, total polyphenols, and biomass levels in olive cells grown in shake flask and various scales of bioreactors* The data are the mean of at least of five experiments.Example 4Effect of plant peptone addition on olive cells growth in large scale disposable bioreactor

[0151] Olive cells were grown in a large-scale disposable bioreactor in the presence of OCR medium. An important factor for developing a process of successful growing of olive cells in large scale bioreactors is the ability to grow plant cells in the bioreactors and obtain high biomass.

[0152] As can be seen in Table 4, the addition of peptone casein or pea peptone to the growth medium is crucial for getting higher biomass of olive cells. However, the type of the peptone also affects the growth. Fresh weight of olive cells that grew without any peptone in the medium in large scale bioreactor was low 54 gr / 1. Addition of casein peptone improved olive cells growth by % to 80gr / l. A significant higher level of olive cells growth was obtained with the addition of pea peptone, 126 gr / 1. The addition thereof results in a 133.3% increase compared to growing without any peptone and 57.5% compared to addition of casein peptone to the medium.

[0153] Plant peptone is the most effective at enhancing olive cell growth in large-scale disposable bioreactors, leading to a substantial increase in fresh weight compared to both casein peptone and no peptone conditions.Table 4: Effect of lant-based peptone on olive cell growth in large-scale disposable bioreactors compared to casein peptone and no peptone conditionsExample 5Effect of plant hormones addition on fresh weight of olive cells grown in large scale disposable bioreactor

[0154] Olive cells were grown in a large-scale disposable bioreactor in OCR supplemented with sucrose and pea peptone with or without hormones as described on Example 1, stage 3. As can be seen in Table 5, the fresh weight level in olive cells were similar when grown in disposable large-scale bioreactor with or without hormones: 0.3- 0.6 mg / 1 2,4-D and 0.07-0.2 mg / 1 2iP were used (Table 5).Table 5: Influence of Plant Hormone Addition on Fresh Weight of Olive Cells in Large-Scale Disposable Bioreactor*The data are the mean of three experimentsExample 6The effect of sucrose concentration on olive cells grown in large scale disposable bioreactor

[0155] Olive cells were grown in large disposable bioreactor as described onExample 1 stage 3, in OCR media with 2, 3, 4 and 6% sucrose and pea peptone. As revealed in Table 6 below, optimum cell growth and biomass is achieved when cells are grown with 2 to 4.5% sucrose (92 to 98 gram / L). Higher sucrose concentration such as 6% sucrose inhibits cell growth. Sucrose concentrations between 2% and 4% positively influence fresh weight.Table 6- Effect of Sucrose Concentration on Fresh Weight of Olive Cells in OCR MediumExample 7

[0156] The growth curves of olive cells grown in a large-scale and larger scale disposable bioreactor in OCR medium showed exponential growth yielding a 130- 160- gr / 1 fresh biomass at day 18.Example 8Effect of the bioreactor configuration, design and structure on olive cells grown in large- scale disposable bioreactor

[0157] An important factor influencing olive cell growth in large / larger disposable bioreactors is the design of the bioreactor that led to deliver optimal aeration to the cells. Supply of optimal level of air is critical for growth of olive cells in OCRmedium in bioreactor as they need oxygen and CO2 for their survival. Impaired design of the bioreactor with poor aeration results in low grow rate.The effect of OCR medium on the growth of olive cells and biomass produced was assessed in O-type large (>40 liters) and larger (> 300 liters) disposable bioreactor made from a sterilized, disposable, transparent plastic bioreactors with specific aeration design that allows good aeration of olive cells which is important for optimal growth and polyphenols production.

[0158] The unique composition of OCR medium has a significant improvement in the ability of olive cells to grow to high biomass and to produce high level of verbascoside.

[0159] The OCR media enhanced growth and productivity olive cells grown in OCR medium exhibited a significantly higher fresh weight (128 g / L) compared to MS medium (83g / L) and Gamborg B5 (16.9 gr / L).

[0160] In addition, OCR medium resulted in elevated levels of verbascoside(1048 mg / L) and total polyphenols (1138 mg / L) compared to MS medium, which had lower levels (286 mg / L and 323 mg / L, respectively) and to 96.8 gram / L verbascoside and 98.8 gram / L polyphenols in Gamborg B5 medium (see in Table 2).

[0161] Olive cells that are grown in OCR medium in O-type bioreactor that was designed to provide optimal aeration of 1500 liter / h reached to 128 g / 1 fresh weight which is 6-fold higher than the fresh weight of olive cells that were grow in bioreactor with low aeration 20 g / 1 (Table 7).

[0162] The results reveal that combination of OCR medium and O-type disposable bioreactor allows the olive cells to grow in a large and larger scale and reach a higher biomass than the biomass that was produced in MS or Gamborg B5 medium or in disposable bioreactor with low aeration. These results show that both the O-type specific design bioreactor and the OCR medium composition are required for maintaining high level of olive cells growth which is an important factor for the growing of olive cells in large scale bioreactors i.e. more than 30 liters and in larger scale bioreactors (>300 liters) for the production of Olive Cells Product that contains high level of verbascoside and undetectable levels of oleuropin , hydroxtyrosol and tyrosol.Table 7. The effect of bioreactor type and medium composition on olive cell growth

[0163] These results demonstarte that both the bioreactor type and the medium composition significantly influence the fresh weight in olive cells produced in a large and larger bioreactor of 30 L or more.The significant increase in fresh cell weight in larger bioreactors underscores the importance of both factors in achieving high-yield production.Example 9Lower level of fats in olive Cells grown in large scale disposable bioreactor compared to agriculture olive fruit and leaf

[0164] Olive leaves and fruits have a high antioxidant capacity because they are rich in polyphenols. These polyphenols possess significant antioxidant activity. The composition of olive cells grown in large scale disposable bioreactor according to the process of the invention is unique. The chemical composition of olive cells grown according to the process described herein is unique also because of the level of the fats, as can be seen in example 9.

[0165] The nutritional composition of sugar, protein, fibers in olive cells is comparable to olives grown using standard agricultural practices except from the level of total fats.

[0166] The amount of fats in olive cells that are grown in a large-scale and larger scale disposable bioreactor as described hereinin OCR medium is 20 to 40 fold lower compared to the level of fats from different types of olives fruit and leaves grown by agriculture means (table 8A and 8B).Table 8: Comparison of fat levels between olive cells grown in large and larger- scale disposable bioreactor in OCR medium calculated in g / lOOg fresh weight (Table 8A) and agricultural olives fruits and leaves as described in scientific literature (Table 8B)8A.Anabela Sousa, Susana Casal, Albino Bento, Ricardo Malheiro, M. Beatriz P.P.Oliveira, Jose Alberto Pereira, Chemical Characterization of “Alcaparras” Stoned Table Olives from Northeast Portugal, Molecules. 2011 Nov; 16(11): 9025- 9040. Published online 2011 Oct 26. doi: 10.3390 / moleculesl6119025Example 10Level of total polyphenols and verbascoside in olive cells grown in OCR medium in large scale disposable bioreactor compared to the level thereof in olives fruit and leaf as describe in the literature

[0167] The composition of polyphenols in olive cells that are grown in vitro in OCR medium in large scale bioreactors is different from the composition in olive fruit and leaf grown in the field. The level of total polyphenols in olive cells grown in OCR is similar (Tables 10, 11) to the level that are found in olives fruit and leaf. The level of verbascoside in olive cells is 22-38 fold higher (Tables 9, 10) than the level that are found in olives fruit and leaf.

[0168] The level of verbascoside in nine batches of olive cells grown in large scale disposable bioreactor as described on Example 1, stages 3 and 4, is in the range of between 6675-15940 mg / kg fresh weight compared to 172-2820 mg / kg in agricultural olive (Table 9, 10).

[0169] The levels of polyphenols and of verbascoside in olive cells were analyzed using HPLC coupled with UV / VIS detection at 280 nm.Table 9: Olive Cells batches from olive cells grown in vitro in OCR medium(mg / kg fresh weight)Table 10: Verbascoside and Total Polyphenolic compounds content in agriculture olive fruit and leaf (from literature)*Tekaya M et al., OCL (2022), 29, 35**Gomez-Rico A., Food Reseach International 41(4);443-440***N.S. A Malik Scienta Horticulturae 110 (2006), 274-278.***M.J. Amiot et al., J. Agriculture food Chemistry (1988), 34, 823-828****Talhaoui N. et al., J. Agriculture food Chemistry (2015), 63 (48)* * * * * http : / / phenol -expl or er , eu / contents / show / 2 / 457 / 750Example 11Polypenols composition of olive cell grown in vitro in OCR medium in O-type bioreactor

[0170] Olive cells dry powder originated from olive fruit and olive leaf that were grown in OCR medium in unique in O-type bioreactors demonstrated a new composition of polypnols and nutritional composition.

[0171] This composition contains three distinct polyphenols: verbascoside, beta-hydroxyverbascoside, and 1-O-synapoylglucose. Notably, other polyphenolscommonly found in olives fruits grown in the field, such as tyrosol, hydroxytyrosol, and oleuropein, are not detectable in the Olive Cell Product manuafactured in accordance with the process described herein. The verbascoside level in olive cells grown in vitro in OCR medium is significantly higher-between 5.7-38.8 times more than in olives that are grown in the field (See Tables I la and 11b). Additionally, the fat content in these cells is markedly lower compared to olive fruit (see Table 8). This unique polyphenol profile and reduced fat content are attributable to the combination of the OCR medium and the specific disposable bioreactor used in the production process.Table 11 A: Comparison of Polyphenol Levels in Olive Cell Product in manuafactured according to the process of the invention vs. Polyphenol Levels in Olive Fruit and Leaf.Table 11B: Levels of polyphenols in dried Olive Cell Product manuafactured according to the process of the inventionExample 12: Evaluation of Antioxidant Activity in Olive Cells (Trolox-equivalent antioxidant capacity assay)

[0172] The Trolox-equivalent antioxidant capacity (TEAC) assay measures the ability of antioxidants to scavenge a stable radical cation, that is, 2,2'-azino-bis-(3- ethylbenzothiazoline-6-sulfonic acid) (ABTS’+), The TEAC assay is a colorimetric method that can be adapted to microplate format.

[0173] The TEAC assay is based on the reduction in color (absorbance at735 nm) of radical cations of ABTS’+by antioxidants in test samples which converts the radical into its reduced colorless ABTS2-formThe degree of decolorization induced by a test compound is related to its antioxidant activity and it is calculated by using Trolox as a standard.

[0174] The dry olive cells as produced herein demonstrated high antioxidant activity range from 34.8to 118.2 TE / gram. This high anti-oxidant activity is proportional to the level of polyphenols in olive cells. Extra virgin olive is prepared from olive is known for their antioxidant activity. The results of antioxidant activity of the total phenolic compounds content of 30 samples of extra virgin olive oils were evaluated and presented in table 12. The antioxidant activity of olive cells grown in vitro is 5.5 fold higher than the activity of extra virgin olive oil whereas the polyphenols level is very similar.

[0175] Olive cells products grown in OCR medium demonstrated a superior antioxidant profile (100% higher) and higher verbascoside levels compared to the extracts from field-grown olive. This suggests that olive cells might offer enhanced antioxidant benefits and greater potential for use in various applications.Table 12- Antioxidant Activity of Olive Cells Compared to Extra Virgin Olive Oil (Antioxidant activity by 2,2-diphenyl-l-picrylhydrazyl (DPPH) assay)* The results are the mean of two determinations ± the standard deviation.**Ting Li et al., Antioxidant capacity of free and bound phenolic from olive leaves, Antioxidants 2023 12(12) 2033.*** Fanali et al., 2018. Extraction, Analysis, and Antioxidant Activity Evaluation of Phenolic Compounds in Different Italian Extra- Virgin Olive OilsExample 13Effect of medium composition on Verbascoside levels in Olive Cells grown in Erlenmeyer shake flask.

[0176] Olive Cells were grown in Erlenmeyer shake flasks as described inExample 1, stage 1, using different medium compositions: OCR, MS and Gamborg B5. The results, presented in Table 1, demonstrate that cells grown in the presence of OCR medium produce approximately 4 times higher levels of verbascoside and total polyphenols compared to those grown in MS and 2 times higher than those grown inGamborg B5 medium. In addition, the biomass as demonstrated by fresh weight of olive cells grown in OCR medium was higher than in MS and Gamborg B5 medium.

[0177] The results indicate that the OCR medium significantly enhances the production of verbascoside and total polyphenols in olive cells compared to the MS medium. The fresh weight of the cells grown in OCR medium is also higher.Table 13 -Effect of Medium Composition on Fresh Weight, Verbascoside, and Total Polyphenols in Olive Cells in olive cells grown in Erlenmeyer flaskExample 14Effect of Olive cells Product (OCP) in Steatosis model in HepG2 cell line

[0178] The liver is a vital organ that is highly susceptible to fat accumulation, resulting in a condition known as fatty liver disease or hepatic steatosis. Although chronic alcohol consumption is a major cause of fatty liver disease, non-alcoholic fatty liver disease (NAFLD) is also common and is strongly associated with central obesity, insulin resistance, hyperlipidemia and the metabolic syndrome. Insulin resistance causes increased lipolysis and thus leading to high levels of plasma free fatty acids (FFAs), as well as increased FFA uptake by hepatocytes, which results in the formation of intracellular lipid droplets. Hepatic lipid accumulation can progress from simple steatosis to non-alcoholic steatohepatitis (NASH), which includes hepatocellular injury, inflammation and fibrosis. In more severe cases, complications such as liver cirrhosis and hepatocellular carcinoma may arise.

[0179] Oleic acid (OA)-induced lipid accumulation in HepG2 cells is commonly used as an in vitro model for studying NAFLD. Consistent with other studies, the present study demonstrates that lipid accumulation in HepG2 cells in response to OA treatment can be effectively quantified using a Nile Red colorimetricassay. This technique involves staining of intracellular lipid droplets with Nile Red, followed by stain extraction and measurement of optical density that is proportional to the intracellular lipid content. HepG2 intracellular lipid quantification using the staining technique provided reliable measurements of intracellular lipid-droplet levels. Assay results were able to reflect the dose-dependent uptake of OA and appeared entirely consistent with lipid droplets stained with Nile Red, as visualized using microscopy. Therefore, quantification of OA-induced HepG2 cell steatosis may act as a valuable model to study the pathogenesis of NAFLD and assess the effect of possible treatments for hepatic steatosis. In this study, an in vitro model of NAFLD was created using HepG2 cells exposed to oleic acid (OA), to induce lipid accumulation. The model was then utilized to assess the effect of OCP (Olive Cells Producton OA-induced lipid accumulation. In addition, the cytotoxic effect of OCP on HepG2 cells was assessed. Lipid accumulation following OA treatment was quantified using Nile Red staining, followed by extraction and optical density measurement to determine the intracellular lipid content.Experimental Procedure:Dilute cell suspension to.2.5x 105 cells / mL using plating medium and seed lOOpL per well of the cell suspension into coating-96 well cell plate by pipetting. Incubate plates for 6h at 37°C with 5% CO2.Replace the starving medium without FBS, and incubate cells overnight at 37°C with 5% CO2.Following starvation, discard the starving medium and then add lOOpL growth medium containing compound per well. Incubate the plates at 37° with 5% carbon dioxide (CO2) for 120 min.Add 50pL growth medium containing oleic acid per well and incubate cells overnight at 37°C with 5% CO2.Discard the media by Blue Washer and replace with lOOpL of 4% Paraformaldehyde Fix Solution per well. Incubate the plates at RT for 10 min. Discard the Fixing solution by Blue Washer, and washed three times with PBS. Add lOOpL PBS containing lOpg / mL Hoechst and lOpM nile red. Incubate the plates at RT for 30 min. Discard the PBS by Blue Washer, and washed three times with PBS. Add lOOpL PBS per well. Read plates by Operetta.Results: The effect of OCP treatment on in vitro model of OA-induced steatosis in HepG2 cells

[0180] To evaluate the effect of OCP on HepG2 cellular lipid accumulation, cultured HepG2 cells were exposed to increasing concentrations of OCP for 2 h before treatment with 400 pM OA. Subsequently, Nile Red staining and extraction for optical density determination were performed in order to quantify the OA accumulation intracellularly.

[0181] As demonstrated in Fig. 1, treatment with OCP resulted in a dosedependent reduction of oleic acid (OA)-induced lipid accumulation in HepG2 cells. The cells were pre-treated with varying concentrations of OCP, ranging from 0.0025 mg / mL to 7.5 mg / mL, for 2 hours prior to OA exposure. At concentration of 7.5 mg / ml, OCP achieved a 37% reduction in intracellular lipid accumulation, compared to the OA-treated control group These findings demonstrate the efficacy of OCP in mitigating lipid accumulation in this steatosis model.Example 15Effect of OCP in In vitro fibrosis model -TGF- i induced liver fibrosis in LX-2 cells

[0182] Liver fibrosis is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension and often requires liver transplantation. Activated hepatic stellate cells, portal fibroblasts, and myofibroblasts of bone marrow origin have been identified as major collagen - producing cells in the injured liver. These cells are activated by fibrogenic cytokines such as TGF-betal, angiotensin II, and leptin. Reversibility of advanced liver fibrosis in patients has been recently documented, which has stimulated researchers to develop antifibrotic drugs. Emerging antifibrotic therapies are aimed at inhibiting the accumulation of fibrogenic cells and / or preventing the deposition of extracellular matrix proteins. The present study used an in vitro model of NAFLD using transforming growth factor beta (TGF P) that has been identified as the main inducer of transdifferentiate Hepatic stellate cells into myofibroblast like cells exhibiting proliferative, contractile and fibrogenic properties, becoming the major source offibrillary collagens in the fibrotic liver. The level of collagen type 1 in LX-2 cells was assessed to characterize liver fibrosis in this study.Experimental Procedure:Dilute cell suspension to 2x 105cells / mL using plating medium and seed 200pL per well of the cell suspension into 96 well cell plate by pipetting. Let the plates seeded with cells stand at room temperature for 30 min. Incubate cells 6h at 37°C with 5% CO2. Replace the starving medium without FBS, and incubate cells overnight at 37°C with 5% CO2. Following cell starvation, discard the starving medium and add lOOpL starving medium containing compound per well. Incubate the plates at 37° with 5% carbon dioxide (CO2) for 20 min. Add lOOpL starving medium containing TGF-P per well. After pre-incubation, liver fibrosis was induced by 5 ng / mL TGF-ββ with indicated compound for 48h. Discard the media by Blue Washer and replace with 150pL of freshly prepared Fixing solution per well. Incubate the plates at RT for 20 min. Discard the Fixing solution by Blue Washer, and add 200 pL of Permeabilization solution to each well. Incubate the plates at RT for 10 min. Repeat the permeabilization step once. Discard the Permeabilization solution by Blue Washer and add 300 pL of Blocking buffer to each well. Cover the plates. Incubate the plates at RT for 2h. Remove Blocking buffer by Blue Washer and add 50 pL of freshly prepared Primary antibody solution to each well. Cover the plates. Incubate the plates at RT for about 2 h. Discard the Primary antibody solution by Blue Washer, wash each well 5 times using 200 pL of washing solution per well each time. Apply 50 pL of freshly prepared Secondary antibody solution to each well. Incubate the plates protected from light (use black lids) at RT for about 1 h. Discard the Secondary antibody solution by Blue Washer. Wash each well 5 times. Using 200 pL of washing solution per well each time. Add 200 pL of Washing solution per well and leave the solution until measurement. Protect the plates from light. Read plate by BIO-RAD GelDoc Go.Results:The effect of OCP was assessed in an In vitro fibrosis modelIn this assay the transforming growth factor beta (TGF P) was utilized as the main inducer of transdifferentiate Hepatic stellate cells (LX-2) (into myofibroblast like cells), which exhibit proliferative, contractile and fibrogenic properties making them the major source of fibrillary collagens in the fibrotic liver. The level of Collagen type I in the LX-2 cells following TGF p was used to characterize the liver fibrosis. In addition, the cytotoxic effect of OCP on LX-2 cells was assessed.

[0183] As demonstrated in Fig. 2, OCP in the range of concentration range of 0.0025-7.5 mg / ml reduced the formation of collagen type I accumulation in LX -2 cells in a dose-dependent manner. Further, OCP at 7.5 mg / ml reduced collagen type I accumulation by 50%.

[0184] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

CLAIMS1. A process for the in vitro production of an olive cell culture of olive leaf, or olive fruit cells grown in vitro in a large scale 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 the size of each bioreactor used in the process is larger than the one in which the olive cells were previously grown; wherein the olive cells harvested from the last bioreactor are dried and wherein the olive cells are grown in bioreactors in a growth medium comprising between about 2-5% sucrose, 9-20 mg / 1, ZnSO4.7H2O, 7-20 mg / 1 H3BO30.1-1 mg / 1, CuSO45H2O, 400-900 mg / 1 MgSO4, 100-500 mg / 1 Ca(NO3)2mg / 1 and 150-500 mg / 1 KH2PO4, 350-650 mg / 1 KC1, 700-1800mg / l KN03or 200-1500mg / l NH4NO3, between about 0.2-lmg / l folic acid, between about 2-8 mg / 1 nicotinic acid, between about 0.2-lmg / l thiamine, between about 0.02-0.08 mg / 1 biotin and pea pepton, wherein the olive fruit / leaf cells grown in vitro comprises verbascoside in the amount of at least between 40 -200 g / kg dry weight and wherein the olive fruit / leaf cells grown in vitro do not comprise detectable levels of tyrosol, hydroxytyrosol, and oleuropein.

2. 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.

3. 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.

4. The large-scale process of claim 1, wherein any one of the bioreactors is a 3-9- liter bioreactor.

5. The large-scale process of claim 1, wherein any one of the bioreactors is a 30- 65-liter bioreactor.

6. The large-scale process of claim 1, wherein any one of the bioreactors is a 30 - 200-liter bioreactor.

7. The large-scale process of claim 1, wherein any one of the bioreactors is a 200- 400-liter bioreactor.

8. The large-scale process of claim 1, any one of the bioreactors is a 200-1100- liter bioreactor.

9. The large-scale process of claim 1, any one of the bioreactors is a 1000-2000- liter bioreactor.

10. The large-scale process of claim 1, any one of the bioreactors is a 2000-5000- liter bioreactor.

11. The large-scale process of claim 1, any one of the bioreactors is a 2000-10000- liter bioreactor.

12. The large-scale process of any one of the preceding claims, wherein at least one bioreactor is a disposable bioreactor having air flow of more than 800 liter / h, 1000 liter / h, 1200 liter / h, 1500 liter / h or more.

13. The large-scale process of claim 12, wherein the disposable bioreactor is made from one or more layers of polyethylene.

14. The large-scale process of claim 13, wherein the disposable bioreactor includes an inner and an outer layer prepared from polyethylene and a middle layer prepared from nylon.

15. The large-scale process of any one of the preceding claims, wherein the growth medium further comprises a pea pepton.

16. The large-scale process of claim 1, wherein the pea pepton is in amount of between 50 to 500g / l.

17. The large-scale process of claim 13, wherein the pea pepton is in amount of between 100 to 400g / l.

18. The large-scale process of claim 16, wherein the pea pepton is in amount of between 200 to 300g / l.

19. The large-scale process of any one of the preceding claims, wherein the growth medium is enriched with one or more of vitamin, cytokine, auxin, glycine, myo inositol, nicotinic acid, pyridoxine, biotin, thiamine, folic acid, 2,4-D, or 2iP.

20. The large -scale process of any one of the preceding claims wherein the amount of 2, 4-D is between 0.3-0.6 mg / 1.

21. The large -scale process of any one of the preceding claims, wherein the amount of 2iP is between 0.07-0.2 mg / 1.

22. The large -scale process of any one of the preceding claims, wherein the amount of glycine is between 0.5-6 mg / 1.

23. The large -scale process of any one of the preceding claims, wherein the amount of myo inositol is between 50-200 mg / 1.

24. The large -scale process of any one of the preceding claims, wherein the amount of nicotinic acid is between 2-8 mg / 1.

25. The large -scale process of any one of the preceding claims, wherein the amount of pyridoxine HC1 is between 0.1-lmg / l.

26. The large -scale process of any one of the preceding claims, wherein the amount of thiamine HC1 is between 0.2-0.8 mg / 1.

27. The large -scale process of any one of the preceding claims, wherein the amount of biotin is between 0.01-0.1 mg / 1.

28. The large -scale process of any one of the preceding claims, wherein the amount of folic acid is between 0.2-0.8 mg / 1.

29. A process for the in vitro production of an olive cell culture of olive leaf, or olive fruit cells grown in vitro in a large scale 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 the size of each bioreactor used in the process is larger than the one in which the olive cells were previously grown; wherein the olive cells harvested from the last bioreactor are dried and wherein the olive cells are grown in bioreactors in a growthone or more of vitamin, auxin, cytokine, glycine, sucrose, myo inositol, nicotinic acid, pyridoxine, biotin, thiamine and folic acid.

30. The process of claim 29, wherein the growth medium comprises:pea pepton; and one or more of vitamin, cytokine, auxin s glycine, sucrose, myo inositol, nicotinic acid, pyridoxine, biotin, thiamine and folic acid.

31. The process of claim 30, wherein the amount of pea peptone and the one or more of vitamin, auxin, cytokine, glycine, sucrose, myo inositol, nicotinic acid, pyridoxine, biotin, thiamine and folic acid is as follows:

32. The process of any one of claims 1-31, wherein the cells are grown in OCR medium at the stage of Erlenmeyers, wherein the medium is supplemented with pea pepton and sucrose.

33. The process of any one of claims 1-32, wherein the cells are grown in MS medium at the stage of Erlenmeyers, wherein the medium is supplemented with pea pepton and sucrose.

34. An Olive Cell Product comprising dry cell culture of olive cells manufactured in vitro in a large scale in accordance to any one of claims 1-33, comprising total polyphenols in an amount of 50-220 g / kg dry weight, wherein the total polyphenols comprises verbascoside in an amount of 40 -200 g / kg dry weight, 1-O-Synapoylglucose and Beta-Hydroxyverbascoside and the product does not comprise detectable levels of tyrosol, hydroxytyrosol, and oleuropein.

35. A method of treating or preventing a fatty liver disease comprising administering the Olive Cell Product of claim 34 to s subject in need thereof.

36. A method of treating or preventing a steaotosis comprising administering the Olive Cell Product of claim 34 to s subject in need thereof.

37. A method of treating or preventing a liver fibrosis comprising administering the Olive Cell Product of claim 34 to s subject in need thereof.

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