U.S. patent application number 16/072886 was filed with the patent office on 2019-08-01 for olive derived cell culture and methods for preparing and using the same.
This patent application is currently assigned to BIO HARVEST LTD.. The applicant listed for this patent is BIO HARVEST LTD.. Invention is credited to Malkit AZACHI, Yoheved HAGAY.
Application Number | 20190231837 16/072886 |
Document ID | / |
Family ID | 59397547 |
Filed Date | 2019-08-01 |
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United States Patent
Application |
20190231837 |
Kind Code |
A1 |
HAGAY; Yoheved ; et
al. |
August 1, 2019 |
OLIVE DERIVED CELL CULTURE AND METHODS FOR PREPARING AND USING THE
SAME
Abstract
The present application describes a large 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 and a method of treating metabolic
syndrome disorders, such as, high cholesterol level, comprising
administering an effective amount of the composition. The cell line
callus culture of olive cells manufactured according to the process
of the invention includes high level of hydroxytyrosoll, tyrosol,
oleoropein and verbascoside.
Inventors: |
HAGAY; Yoheved; (Rehovot,
IL) ; AZACHI; Malkit; (Rehovot, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIO HARVEST LTD. |
Rehovot |
|
IL |
|
|
Assignee: |
BIO HARVEST LTD.
Rehovot
IL
|
Family ID: |
59397547 |
Appl. No.: |
16/072886 |
Filed: |
January 26, 2017 |
PCT Filed: |
January 26, 2017 |
PCT NO: |
PCT/IL2017/050098 |
371 Date: |
July 26, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62287453 |
Jan 27, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0025 20130101;
C11B 1/00 20130101; A01H 4/00 20130101; A61P 3/00 20180101; A61K
9/14 20130101; C12M 21/00 20130101; A01H 5/10 20130101; A61K 36/63
20130101; A01H 5/08 20130101 |
International
Class: |
A61K 36/63 20060101
A61K036/63; A01H 5/08 20060101 A01H005/08; C11B 1/00 20060101
C11B001/00; A01H 5/10 20060101 A01H005/10; A61P 3/00 20060101
A61P003/00; A61K 9/14 20060101 A61K009/14; C12N 5/00 20060101
C12N005/00 |
Claims
1. A large scale process for the in vitro production of an olive
cell culture of olive cells grown comprising: growing olive cells
in a flask; inoculating the olive cells from the flask into a first
bioreactor; inoculating the olive cells from the first bioreactor
into a second bioreactor; optionally inoculating the olive cells
from the second bioreactor into a last bioreactor; and harvesting
the olive cells from the last bioreactor; wherein the second
bioreactor is a last bioreactor or an intermediate bioreactor and
wherein the olive cells harvested from the last bioreactor are
dried.
2. The large scale process of claim 1, wherein the size of each
bioreactor used in the process is larger than the one in which the
olive cells were previously grown.
3. The large scale process of claim 1, wherein if the second
bioreactor is an intermediate bioreactor, an additional step of
inoculating the olive cells to another intermediate bioreactor or
to the last bioreactor is performed.
4. The large scale process according to claim 1, further including
additional steps of inoculating the olive cells from the second
bioreactor into any number of sequential intermediate
bioreactors.
5. The large scale process of claim 1, wherein any one of the
bioreactors is a 4-10 liter bioreactor.
6. The large scale process of claim 1, wherein any one of the
bioreactors is a 10-50 liter bioreactor.
7-8. (canceled)
9. The large scale process of claim 1, any one of the bioreactors
is a 200-1000 liter bioreactor.
10. The large scale process of claim 1, wherein the olive cells are
grown in a modified MS growth medium.
11. The large scale process of claim 10, wherein the growth medium
is enriched with sucrose, casein hydrolysate, myo inositol,
1-naphthaleneacetic acid (NAA), kinetin, 2,4,D, [[NAA,]] BA, 2iP,
or any combination thereof.
12. The large scale process of claim 1, wherein at least one
bioreactor is a disposable bioreactor.
13. The large scale process of claim 12, wherein the disposable
bioreactor is made from one or more layers of polyethylene.
14. (canceled)
15. The large scale process of claim 10, wherein the growth medium
does not include plant hormones.
16. (canceled)
17. The large scale process of claim 10, wherein the growth medium
is enriched with 1-6% sucrose.
18. A composition in a form of a powder comprising olive fruit/leaf
cells grown in vitro, whereby the olive cells are derived from one
or more of section: olive pulp, olive seed, olive petiole or olive
leaf.
19. The composition according to claim 18, wherein the olive fruit
cells include polyphenols selected from hydroxytyrosol, tyrosol,
oleuropein, verbascoside and pinoresinol.
20. The composition of claim 18, wherein the olive cells grown in
vitro in a large scale process.
21. (canceled)
22. A method of treating metabolic syndrome such as affecting
cholesterol level, comprising administering an effective amount of
a composition comprising a powder comprising olive fruit cells
grown in vitro, whereby the olive cells are derived from one or
more of olive skin, olive lamellae and olive seeds.
23. (canceled)
24. The composition of claim 18, wherein the olive fruit/leaf cells
grown in vitro Olive calli culture comprising comprise verbascoside
in the amount of at least 4.1 mg/gr dry weight.
25. (canceled)
26. The large scale process of claim 10, wherein the modified MS
growth medium for growing olive cells in vitro in bioreactor
comprises: 1.1-8.8 gr/L MS medium, 0.5 mg/L kinetin, 0.5 mg/L
2,4,D, and 40 gr/L sucrose; 1.1-8.8 gr/L MS medium, 1.3 mg/L 2,4,D,
0.06 mg/L BA, 30 gr/L and sucrose; 1.1-8.8 gr/L MS medium, 2.6 mg/L
2,4,D, 0.12 mg/L BA and 30 gr/L sucrose; 1.1-8.8 gr/L MS medium,
0.1 mg/L 2,4,D and 20 gr/L sucrose; 1.1-8.8 gr/L MS medium, 0.5
mg/L 2,4,D, 0.1 mg/L 2iP and 45 gr/L sucrose; or 1.1-8.8 gr/L MS
medium, 2.0 mg/L NAA, 0.5 mg/L 2iP and 30 gr/L sucrose.
27-28. (canceled)
Description
FIELD OF THE INVENTION
[0001] 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 same.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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.
[0004] 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.
[0005] 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 productions of the secondary
metabolites. Since active ingredients, such as polyphenols, are
secondary metabolites their production in large-scale processes is
complex.
[0006] 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.
[0007] Nonetheless, dietary consumption of polyphenols was shown to
be inversely related to morbidity and mortality from coronary heart
disease (CHD). Moreover, an inverse association between polyphenols
intake and subsequent occurrence of ischemic heart disease, or
cerebrovascular disease was shown. Over the last decade, studies
indicated that olive polyphenolic compounds have potent antioxidant
properties and their therapeutic properties further include
anti-inflammatory and anti-microbial activities, treatment and
prevention of cardiovascular disease, promotion of healthy
lifestyle in the context of prevention of cancer and age-related
processes such as Alzheimer's disease (Farr et al,. J. Alzheim.
Dis. 2012, 28, 81-92, Luccarini et al., Neurobiology of Aging 2014
1-16). Another potential application is the effect of olive
polyphenols on gut microbial balance, affecting lipid metabolism.
Other potential applications include the effect on obesity-related
gene expression (Martin et al. Mol. Nutr. Food Res. 2013, 00,
1-12).
[0008] Thus, there is a need in the art for a large scale process
for preparing plant cells from natural ingredients, which includes
the production of both 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] Tyrosol, hydroxytyrosol, oleuropein and verbascoside are all
known for their potent antioxidant capacity. These compounds are
the major polyphenols in olive, and are responsible for the high
antioxidant activity of olives and olive oil.
[0011] 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.
[0012] Thus, there is a need in the art for a large scale process
for preparing olive plant cells, which includes both the primary
and the secondary metabolites of olive plant cells. There is
further a need for olive plant cells compositions that may be
prepared in a large scale process in which the amount of the active
ingredient/s is similar or higher than in olive plants, including
the leaves. Further, compositions prepared in a large scale process
would prove to be and be consistent and recurrent (e.g., clonal
preparations) and therefore, would be advantageous over olive
secondary metabolites obtained by other methods.
SUMMARY OF THE INVENTION
[0013] 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;
[0014] inoculating the olive cells from the flask into a first
bioreactor; [0015] inoculating the olive cells from the first
bioreactor into a second bioreactor; [0016] optionally inoculating
the olive cells from the second bioreactor into a last bioreactor;
and [0017] harvesting the olive cells from the last bioreactor;
[0018] wherein the second bioreactor is a last bioreactor or an
intermediate bioreactor and [0019] wherein the olive cells
harvested from the last bioreactor are dried.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] According to some embodiments, any one of the bioreactors is
a 4-10 liter bioreactor. According to some embodiments, any one of
the bioreactors is a 10-50 liter bioreactor. According to some
embodiments, any one of the bioreactors is a 50-200 liter
bioreactor. According to some embodiments, any one of the
bioreactors is a 200-500 liter bioreactor. According to some
embodiments, any one of the bioreactors is a 200-1000 liter
bioreactor.
[0024] According to some embodiments, the olive cells are grown in
bioreactors comprising a growth MS medium as defined in Table 1B
supplemented with different hormones and various sucrose
concentrations e.g. M-3, O-3, O-4, O-5, as defined in Table 1A, or
any combination thereof.
[0025] According to some embodiments, the olive cells are grown in
bioreactors comprising a growth MS medium comprising one or more of
kinetin, sucrose, 2,4,D, NAA, 2iP, BA or any combination
thereof.
[0026] According to some embodiments, the olive cells are grown in
bioreactors comprising a growth MS medium comprising agar and one
or more of kinetin, sucrose, 2,4,D, NAA, 2iP, BA or any combination
thereof.
[0027] The composition of each or those media, which in essence is
a modification of the MS medium, is as detailed in the table
below:
TABLE-US-00001 TABLE 1A Media composition of various MS plates used
for olive calli M-3 O-1 O-2 O-3 O-4 O-5 MS (g/L)* 4.4 (**Full 4.4
(**Full 4.4 (**Full 2.2 (***Half 4.4 (***Full 2.2 (***Half
strength) strength) strength) strength) strength) strength) Kinetin
(mg/L) 0.5 2,4,D (mg/L) 0.5 1.3 2.6 0.1 0.5 NAA (mg/L) 2 BA(mg/L)
0.06 0.12 2iP (mg/L) 0.1 0.5 Sucrose (g/L) 40 30 30 20 45 30
Charcoal (g/L) 0.25 Agar (g/L) 6 6 6 6 6 6 *Olive cultures are
grown on modified MS--Murashige and Skoog medium (Toshio Murashige
and Folke K). Skoog medium supplemented with different hormones and
various sucrose concentrations. **Full strength MS medium refers to
4.4 gr/L amount of MS powder added to the final medium ***Half
strength MS medium refers to 2.2 gr/L amount of MS powder added to
the final medium.
[0028] The composition of the MS medium is as follows:
TABLE-US-00002 TABLE 1B MS--Murashige and Skoog medium Composition
Concentrations mg/L Elements Min Max CoCl.sub.2.cndot.6H.sub.2O
0.0063 0.05 CuSO.sub.4.cndot.5H.sub.2O 0.0063 0.05 FeNaEDTA 9.18
73.4 H.sub.3BO.sub.3 1.55 12.4 KI 0.21 1.66
MnSO.sub.4.cndot.H.sub.2O 4.23 33.8
Na.sub.2MoO.sub.4.cndot.2H.sub.2O 0.06 0.50
ZnS0.sub.4.cndot.7H.sub.20 2.15 17.2 CaCl.sub.2 83.0 664.0
KH.sub.2PO.sub.4 42.5 340.0 KNO.sub.3 475 3800 MgSO.sub.4 45.1
361.1 NH.sub.4NO.sub.3 412.5 3300 Glycine 0.5 4.0 Myo-Inositol 25
200 Nicotinic acid 0.13 1.0 Pyridoxine HCl 0.13 1.0 Thiamine HCl
0.03 0.2
[0029] Thus, according to some embodiments, the olive calli cells
are grown in modified MS medium. According to some embodiments, the
modified MS medium comprises: [0030] a) (M-3) 1.1-8.8 gr/L MS
medium, 0.5 mg/L kinetin, 0.5 mg/L 2,4,D, 40 gr/L sucrose and 6
gr/L agar; [0031] b) (O-1) 1.1-8.8 gr/L MS medium, 1.3 mg/L 2,4,D,
0.06 mg/L BA, 30 gr/L sucrose and 6 gr/L agar; [0032] c)
(O-2)1.1-8.8 gr/L MS medium, 2.6 mg/L 2,4,D, 0.12 mg/L BA, 30 gr/L
sucrose, 0.25 gr/L charcoal, and 6 gr/L agar; [0033] d) (O-3)
1.1-8.8 gr/L MS medium, 0.1 mg/L 2,4,D, 20 gr/L sucrose, and 6 gr/L
agar; [0034] e) (O-4) 1.1-8.8 gr/L MS medium, 0.5 mg/L 2,4,D, 0.1
mg/L 2iP, 45 gr/L sucrose, and 6 gr/L agar; or [0035] f) (O-5)
1.1-8.8 gr/L MS medium, 2.0 mg/L NAA, 0.5 mg/L 2iP, 30 gr/L
sucrose, and 6 gr/L agar.
[0036] Thus, according to some embodiments, the olive cells growing
in vitro suspension are grown in modified MS medium. According to
some embodiments, the modified MS medium that can be used for the
suspension are same as those described for the growth of the olive
calli but do not comprise agar: [0037] a) (M-3) 1.1-8.8 gr/L MS
medium, 0.5 mg/L kinetin, 0.5 mg/L 2,4,D, and 40 gr/L sucrose;
[0038] b) (O-1) 1.1-8.8 gr/L MS medium, 1.3 mg/L 2,4,D, 0.06 mg/L
BA, 30 gr/L and sucrose; [0039] c) (O-2) 1.1-8.8 gr/L MS medium,
2.6 mg/L 2,4,D, 0.12 mg/L BA and 30 gr/L sucrose; [0040] d) (O-3)
1.1-8.8 gr/L MS medium, 0.1 mg/L 2,4,D and 20 gr/L sucrose; [0041]
e) (O-4) 1.1-8.8 gr/L MS medium, 0.5 mg/L 2,4,D, 0.1 mg/L 2iP and
45 gr/L sucrose; or [0042] f) (O-5) 1.1-8.8 gr/L MS medium, 2.0
mg/L NAA, 0.5 mg/L 2iP and 30 gr/L sucrose.
[0043] Thus, according to some embodiments, the olive calli cells
are grown in modified MS medium. According to some embodiments, the
modified MS medium comprises: [0044] a) 1.1-8.8 gr/L MS medium,
kinetin, 2,4,D, sucrose and agar; [0045] b) 1.1-8.8 gr/L MS medium,
2,4,D, BA, sucrose and agar; [0046] c) 1.1-8.8 gr/L MS medium,
2,4,D, BA, sucrose, charcoal, and agar; [0047] d) 1.1-8.8 gr/L MS
medium, 2,4,D, sucrose, and agar; [0048] e) 1.1-8.8 gr/L MS medium,
2,4,D, 2iP, sucrose, and agar; or [0049] f) 1.1-8.8 gr/L MS medium,
NAA, 2iP, sucrose, and agar.
[0050] Thus, according to some embodiments, the olive cells growing
in suspension are grown in modified MS medium. According to some
embodiments, the modified MS medium comprises: [0051] a) 1.1-8.8
gr/L MS medium, kinetin, 2,4,D, and sucrose; [0052] b) 1.1-8.8 gr/L
MS medium, 2,4,D, BA, and sucrose; [0053] c) 1.1-8.8 gr/L MS
medium, 2,4,D, BA and sucrose; [0054] d) 1.1-8.8 gr/L MS medium,
2,4,D and sucrose; [0055] e) 1.1-8.8 gr/L MS medium, 2,4,D, 2iP and
sucrose; or [0056] f) 1.1-8.8 gr/L MS medium, NAA, 2iP and
sucrose.
[0057] According to further embodiments, the growth medium is
enriched with sucrose, casein hydrolysate, myo inositol,
1-naphthaleneacetic acid (NAA), kinetin, 2,4,D
(2,4-Dichlorophenoxyacetic acid), BA (benzyladenine), 2iP
(6-dimethylamino purine), or any combination thereof. According to
some embodiments, the growth medium does not include plant
hormones. According to other embodiments, the growth medium
includes plant hormones. According to some embodiments, the growth
medium is enriched with 1-6% sucrose.
[0058] According to some embodiments, at least one of the
bioreactors is disposable. According to some embodiments, the
disposable bioreactor is made from one or more layers of
polyethylene. According to some embodiments, the disposable
bioreactor includes an inner and an outer layer prepared from
polyethylene and a middle layer prepared from nylon.
[0059] Further embodiments of the invention are directed to a
composition in a form of a powder comprising olive fruit/leaf cells
grown in vitro, whereby the olive cells are derived from one or
more of section: olive pulp, olive seed, olive petiole or olive
leaf.
[0060] According to some embodiments, the olive fruit cells include
polyphenols, such as, hydroxytyrosol, tyrosol, oleuropein,
verbascoside and pinoresinol, but not limited only to these types.
According to some embodiments, the olive fruit cells prepared
include polyphenols selected from hydroxytyrosol, tyrosol,
oleuropein, verbascoside and pinoresinol.
[0061] According to some embodiments, the olive cells grown in
vitro in a large scale process. According to some embodiments, the
olive fruit cells are prepared according to the large scale process
of the invention, as detailed herein.
[0062] Further embodiments of the invention are directed to a
method of treating inflammation comprising administering an
effective amount of a composition comprising a powder comprising
olive fruit cells grown in vitro, whereby the olive cells are
derived from one or more of olive skin, olive lamellae and olive
seeds. According to some embodiments, the olive cells used in the
method are prepared according to the large scale process of the
invention, as detailed herein.
[0063] Additional embodiments of the invention are directed to
olive calli culture comprising verbascoside in the amount of at
least 8.2 .mu.g/mg dry weight.
[0064] In an embodiment of the invention, there is provided calli
culture grown in vitro for the in vitro production of an olive cell
culture of olive cells wherein the olive cell comprise Olive's
polyphenols and high amount, e.g. more than 8.2 .mu.g/mg DW, of
verbascoside.
[0065] Further, there is provided 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; [0066] inoculating the
olive cells from the flask into a first bioreactor; [0067]
inoculating the olive cells from the first bioreactor into a second
bioreactor; and harvesting the olive cells from the last
bioreactor; [0068] wherein the second bioreactor is a last
bioreactor or an intermediate bioreactor and [0069] wherein at
least one of the first and the second bioreactor is disposable and
wherein the olive cells harvested from the last bioreactor are
dried.
[0070] In some embodiments, the invention provides a composition in
a form of a powder comprising olive fruit/leaf cells grown in
vitro, whereby the olive cells are derived from one or more of
section: olive pulp, olive seed, olive petiole or olive leaf.
[0071] In further embodiments, the invention provides a method of
prevention and/or treatment of risk factors for cardiovascular
disease, development of atherosclerotic plaque, protection of LDL
particles from oxidative damage, maintenance of normal blood
HDL-cholesterol concentrations, decrease of high blood pressure and
to maintain normal haemostatic function. Olive polyphenols were
shown to have beneficial effects on learning and memory deficits
found in ageing and diseases, such as those related to the
overproduction of amyloid-beta peptide. In addition, olive
polyphenols were shown to influence gut microbial balance by
promoting growth of bacteria influencing lipid metabolism and
inhibition of pathogenic bacteria. The method comprising
administering an effective amount of a composition comprising
composition in a form of a powder comprising olive fruit/leaf cells
grown in vitro, whereby the olive cells are derived from one or
more of cross section, olive pulp, olive seed, olive petiole or
olive leaf. [0072] In some embodiments, the olive cells culture is
grown in vitro under dark conditions. [0073] In some embodiments,
the olive cells culture is grown in vitro under fully dark
conditions (24 hours a day).
BRIEF DESCRIPTION OF THE DRAWINGS
[0074] 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:
[0075] FIG. 1 presents an HPLC chromatogram at 280 nm of olive
polyphenols derived from olive cells grown in O4 medium (modified
MS medium); and
[0076] FIG. 2 presents olive cell growth in large bioreactors in O4
medium (modified MS medium).
DETAILED EMBODIMENTS OF THE INVENTION
[0077] 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.
[0078] 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, 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 includes tyrosol, hydroxytyrosol, oleuropein and
verbascoside in an amount of at least 0.5375, 0.14, 0.2 and 4.1
mg/gr dry weight (DW), respectively.
[0079] 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 fruit cells. Surprisingly, the produced fruit
cell cultures, manufactured in accordance with the large scale
process described herein, were shown to include high amounts of
polyphenols particularly, the secondary metabolites tyrosol,
hydroxytyrosol, oleuropein and verbascoside. The unique composition
of olive cells (OC), which, as an outcome of scale up process,
includes a whole matrix of polyphenols and other healthy
ingredients, naturally existing in OC of a plant, with higher
concentration, i.e., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90% or higher, of olive polyphenols, including tyrosol,
hydroxytyrosol, oleuropein and verbascoside, as described in table
5, which provides a summary of the values known from the
literature. 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.
[0080] 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 pulp, olive seed, olive petiole
or olive leaf.
[0081] Some embodiments are directed to a composition comprising
non-extracted, dry calli cells culture of olive fruit/leaf cells.
According to some embodiments, the calli cells culture is grown in
vitro. According to some embodiments, the cell culture comprises
both primary and secondary metabolites.
[0082] Some embodiments are directed to a method for the production
of polyphenols from a culture of olive cells. According to some
embodiments of the invention, although the amount of materials,
including polyphenols, may vary in different batches of fruit, the
use of a culturing protocol for preparing the fruit/leaf cell
cultures ensures the reproducibility of the preparation and its
contents. Thus, various batches of fruit cells, prepared from the
same culture have a typical HPLC fingerprint. According to some
embodiments, the concentrations of the various materials in each
batch may change; however, as mentioned above, if prepared from the
same culture, the HPLC fingerprint is consistent for all
batches.
[0083] 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,
as shown in Table 5 in the Examples Section. According to some
embodiments, the amount of certain polyphenols is amplified in the
prepared fruit/leaf cells, in comparison to their amount in the
agricultural olive fruit.
[0084] According to some embodiments, the amount of the secondary
metabolite hydroxytyrosol is more than about 0.14 mg/gr, after the
olive cell cultures are dried to a powder.
[0085] According to some embodiments, the amount of the secondary
metabolite tyrosol is more than about 0.5375 mg/gr, after the olive
cell cultures are dried to a powder.
[0086] According to some embodiments, the amount of the secondary
metabolite oleuropein is more than about 0.2 mg/gr, after the olive
cell cultures are dried to a powder.
[0087] According to some embodiments, the amount of the secondary
metabolite verbascoside is more than about 4.1 mg/gr, after the
olive cell cultures are dried to a powder
[0088] According to some embodiments, the amount of the secondary
metabolite hydroxytyrosol between about 0.14-15.7 mg/gr, after the
olive cell cultures are dried to a powder. According to some
embodiments, the amount of the secondary metabolite hydroxytyrosol
between about 0.14-1.0 mg/gr, after the olive cell cultures are
dried to a powder. According to some embodiments, the amount of the
secondary metabolite hydroxytyrosol between about 1.0-2.0 mg/gr,
after the olive cell cultures are dried to a powder. According to
some embodiments, the amount of the secondary metabolite
hydroxytyrosol between about 2.0-3.0 mg/gr, after the olive cell
cultures are dried to a powder. According to some embodiments, the
amount of the secondary metabolite hydroxytyrosol between about
3.0-4.0 mg/gr, after the olive cell cultures are dried to a powder.
According to some embodiments, the amount of the secondary
metabolite hydroxytyrosol between about 4.0-5.0 mg/gr, after the
olive cell cultures are dried to a powder. According to some
embodiments, the amount of the secondary metabolite hydroxytyrosol
between about 5.0-6.0 mg/gr, after the olive cell cultures are
dried to a powder. According to some embodiments, the amount of the
secondary metabolite hydroxytyrosol between about 6.0-7.0 mg/gr,
after the olive cell cultures are dried to a powder. According to
some embodiments, the amount of the secondary metabolite
hydroxytyrosol between about 7.0-8.0 mg/gr, after the olive cell
cultures are dried to a powder. According to some embodiments, the
amount of the secondary metabolite hydroxytyrosol between about
8.0-9.0 mg/gr, after the olive cell cultures are dried to a powder.
According to some embodiments, the amount of the secondary
metabolite hydroxytyrosol between about 90-10.0 mg/gr, after the
olive cell cultures are dried to a powder. According to some
embodiments, the amount of the secondary metabolite hydroxytyrosol
between about 10.0-11.0 mg/gr, after the olive cell cultures are
dried to a powder. According to some embodiments, the amount of the
secondary metabolite hydroxytyrosol between about 11.0-12.0 mg/gr,
after the olive cell cultures are dried to a powder. According to
some embodiments, the amount of the secondary metabolite
hydroxytyrosol between about 12.0-13.0 mg/gr, after the olive cell
cultures are dried to a powder. According to some embodiments, the
amount of the secondary metabolite hydroxytyrosol between about
13.0-14.0 mg/gr, after the olive cell cultures are dried to a
powder. According to some embodiments, the amount of the secondary
metabolite hydroxytyrosol between about 14.0-15.7 mg/gr, after the
olive cell cultures are dried to a powder.
[0089] According to some embodiments, the amount of the secondary
metabolites tyrosol is between about 0.53-21.0 mg/gr, after the
olive cell cultures are dried to a powder. According to some
embodiments, the amount of the secondary metabolites tyrosol is
between about 0.53-1.0 mg/gr, after the olive cell cultures are
dried to a powder. According to some embodiments, the amount of the
secondary metabolites tyrosol is between about 1.0-3.0 mg/gr, after
the olive cell cultures are dried to a powder. According to some
embodiments, the amount of the secondary metabolites tyrosol is
between about 3.0-5.0 mg/gr, after the olive cell cultures are
dried to a powder. According to some embodiments, the amount of the
secondary metabolites tyrosol is between about 5.0-7.0 mg/gr, after
the olive cell cultures are dried to a powder. According to some
embodiments, the amount of the secondary metabolites tyrosol is
between about 7.0-9.0 mg/gr, after the olive cell cultures are
dried to a powder. According to some embodiments, the amount of the
secondary metabolites tyrosol is between about 9.0-11.0 mg/gr,
after the olive cell cultures are dried to a powder. According to
some embodiments, the amount of the secondary metabolites tyrosol
is between about 11.0-13.0 mg/gr, after the olive cell cultures are
dried to a powder. According to some embodiments, the amount of the
secondary metabolites tyrosol is between about 13.0-15.0 mg/gr,
after the olive cell cultures are dried to a powder. According to
some embodiments, the amount of the secondary metabolites tyrosol
is between about 15.0-17.0 mg/gr, after the olive cell cultures are
dried to a powder. According to some embodiments, the amount of the
secondary metabolites tyrosol is between about 17.0-19.0 mg/gr,
after the olive cell cultures are dried to a powder. According to
some embodiments, the amount of the secondary metabolites tyrosol
is between about 19.0-21.0 mg/gr, after the olive cell cultures are
dried to a powder.
[0090] According to some embodiments, the amount of the secondary
metabolites oleuropein is between about 0.2-10 mg/gr, after the
olive cell cultures are dried to a powder. According to some
embodiments, the amount of the secondary metabolites oleuropein is
between about 0.2-1.0 mg/gr, after the olive cell cultures are
dried to a powder. According to some embodiments, the amount of the
secondary metabolites oleuropein is between about 1.0-2.0 mg/gr,
after the olive cell cultures are dried to a powder. According to
some embodiments, the amount of the secondary metabolites
oleuropein is between about 2.0-3.0 mg/gr, after the olive cell
cultures are dried to a powder. According to some embodiments, the
amount of the secondary metabolites oleuropein is between about
3.0-4.0 mg/gr, after the olive cell cultures are dried to a powder.
According to some embodiments, the amount of the secondary
metabolites oleuropein is between about 4.0-5.0 mg/gr, after the
olive cell cultures are dried to a powder. According to some
embodiments, the amount of the secondary metabolites oleuropein is
between about 5.0-6.0 mg/gr, after the olive cell cultures are
dried to a powder. According to some embodiments, the amount of the
secondary metabolites oleuropein is between about 6.0-7.0 mg/gr,
after the olive cell cultures are dried to a powder. According to
some embodiments, the amount of the secondary metabolites
oleuropein is between about 7.0-8.0 mg/gr, after the olive cell
cultures are dried to a powder. According to some embodiments, the
amount of the secondary metabolites oleuropein is between about
8.0-9.0 mg/gr, after the olive cell cultures are dried to a powder.
According to some embodiments, the amount of the secondary
metabolites oleuropein is between about 9.0-10.0 mg/gr, after the
olive cell cultures are dried to a powder.
[0091] According to some embodiments, the amount of the secondary
metabolites verbascoside is between about 4.1-151.1 mg/gr, after
the olive cell cultures are dried to a powder. According to some
embodiments, the amount of the secondary metabolites verbascoside
is between about 4.1-10.0 mg/gr, after the olive cell cultures are
dried to a powder. According to some embodiments, the amount of the
secondary metabolites verbascoside is between about 10.0-20.0
mg/gr, after the olive cell cultures are dried to a powder.
According to some embodiments, the amount of the secondary
metabolites verbascoside is between about 20.0-30.0 mg/gr, after
the olive cell cultures are dried to a powder. According to some
embodiments, the amount of the secondary metabolites verbascoside
is between about 30.0-40.0 mg/gr, after the olive cell cultures are
dried to a powder. According to some embodiments, the amount of the
secondary metabolites verbascoside is between about 40.0-50.0
mg/gr, after the olive cell cultures are dried to a powder.
According to some embodiments, the amount of the secondary
metabolites verbascoside is between about 50.0-60.0 mg/gr, after
the olive cell cultures are dried to a powder. According to some
embodiments, the amount of the secondary metabolites verbascoside
is between about 60.0-70.0 mg/gr, after the olive cell cultures are
dried to a powder. According to some embodiments, the amount of the
secondary metabolites verbascoside is between about 70.0-80.0
mg/gr, after the olive cell cultures are dried to a powder.
According to some embodiments, the amount of the secondary
metabolites verbascoside is between about 80.0-90.0 mg/gr, after
the olive cell cultures are dried to a powder. According to some
embodiments, the amount of the secondary metabolites verbascoside
is between about 90.0-100.0 mg/g, after the olive cell cultures are
dried to a powder. According to some embodiments, the amount of the
secondary metabolites verbascoside is between about 100.0-110.0
mg/g, after the olive cell cultures are dried to a powder.
According to some embodiments, the amount of the secondary
metabolites verbascoside is between about 110.0-120.0 mg/g, after
the olive cell cultures are dried to a powder. According to some
embodiments, the amount of the secondary metabolites verbascoside
is between about 120.0-130.0 mg/g, after the olive cell cultures
are dried to a powder. According to some embodiments, the amount of
the secondary metabolites verbascoside is between about 130.0-140.0
mg/g, after the olive cell cultures are dried to a powder.
According to some embodiments, the amount of the secondary
metabolites verbascoside is between about 140.0-151.1 mg/g, after
the olive cell cultures are dried to a powder.
[0092] According to some embodiments, the amount of the secondary
metabolites hydroxytyrosol, tyrosol, oleuropein and verbascoside in
the olive cell cultures, is between about 0.14-15.7, 0.53-21.0,
0.2-10, 4.1-151.1 mg/gr, respectively after the olive cell cultures
are dried to a powder.
[0093] According to some embodiments of the invention, the amount
of total olive polyphenols including hydroxytyrosol, tyrosol,
oleuropein and verbascoside is more than about 1 mg/gr after the
olive cell cultures are dried to a powder. According to some
embodiments of the invention, the amount is more than about 10
mg/gr after the olive cell cultures are dried to a powder According
to some embodiments of the invention, the amount is more than about
15 mg/gr after the olive cell cultures are dried to a powder.
According to some embodiments of the invention, the amount is more
than about 25 mg/gr after the olive cell cultures are dried to a
powder. According to some embodiments of the invention, the amount
is more than about 50 mg/gr after the olive cell cultures are dried
to a powder. According to some embodiments of the invention, the
amount is more than about 100 mg/gr after the olive cell cultures
are dried to a powder. According to some embodiments of the
invention, the amount is more than about 150 mg/gr after the olive
cell cultures are dried to a powder. According to some embodiments
of the invention, the amount is more than about 200 mg/gr after the
olive cell cultures are dried to a powder. According to some
embodiments of the invention, the amount is more than about 250
mg/gr after the olive cell cultures are dried to a powder.
According to some embodiments of the invention, the amount is more
than about 300 mg/gr after the olive cell cultures are dried to a
powder. According to some embodiments of the invention, the amount
is more than about 350 mg/gr after the olive cell cultures are
dried to a powder. According to some embodiments of the invention,
the amount is more than about 400 mg/gr after the olive cell
cultures are dried to a powder.
[0094] 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. Fat refers to any of a group
of natural esters of glycerol and various fatty acids conjugated or
free or any combination thereof.
[0095] According to some embodiments, the olive cell cultures
contain less than about 5% w/v fat. According to some embodiments,
the olive cell cultures contain less than about 3% w/v fat.
According to some embodiments, the olive cell cultures contain less
than about 2% w/v fat. According to some embodiments, the olive
cell cultures contain less than about 1% w/v fat. According to some
embodiments, the olive cell cultures contain about 1% w/v fat, 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. According to some embodiments,
the materials are concentrated by a factor of about 5. According to
some embodiments, the materials are concentrated by a factor of
about 10. According to some embodiments, the materials are
concentrated by a factor of about 15. According to some
embodiments, the materials are concentrated by a factor of about
20. According to some embodiments, the materials are concentrated
by a factor of about 25. According to some embodiments, the
materials are concentrated by a factor of about 30.
[0096] 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;
[0097] inoculating the olive cells from the flask into a first
bioreactor; and harvesting the produced olive cells.
[0098] 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: [0099] growing olive
cells in a flask; [0100] 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 [0101] harvesting the olive cells from the last
bioreactor; [0102] wherein the olive cells harvested from the last
bioreactor are dried.
[0103] According to some embodiments, at least one of the
bioreactors is a disposable bioreactor.
[0104] 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.
[0105] 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.
[0106] In general there are two different approaches for
constructing single-use bioreactors, differing in the means used to
agitate the culture medium.
[0107] 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.
[0108] 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.
[0109] 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 each step 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.
[0110] According to some embodiments, there is provided a
composition comprising a complex of pholyphenols including tyrosol,
hydroxytyrosol, oleuropein and verbascoside, wherein the amount of
verbacoside in respect to the other polyphenols is higher than
1:20. In some embodiments, the ratio is higher than 1:10. In some
embodiments, the ratio is higher than 1:5. In some embodiments, the
ratio is higher than. In some embodiments, the ratio is higher than
1:3. In some embodiments, the ratio is higher than 1:2.
[0111] According to some embodiments, the composition is derived
from a natural source. 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.
[0112] 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.
[0113] 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 1.0 L
of an olive cell culture. According to further embodiments, first
step involves the preparation of up to 1.5 L of an olive cell
culture. According to further embodiments, first step involves the
preparation of up to 2.0 L of an olive cell culture.
[0114] 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.
[0115] 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.
[0116] According to further embodiments, the cell growth medium
includes O-5, O-4, O-3, M-3medium (as defined in Table 1A) or any
combination thereof. According to some embodiments, the cell growth
medium is supplemented with sucrose, casein hydrolysate,
myoinositol, 1-naphthaleneacetic acid (NAA), kinetin, 2,4,D,
written already BA, 2iP, or any combination thereof.
[0117] According to some embodiments, the cell growth medium is
supplemented with about 1-6% sucrose, about 0.2-0.3 g/L casein
hydrolysate, about 0.05-0.15 g/L myo inositol, about 0.05-0.15 mg/L
NAA and/or about 0.1-0.3 mg/L kinetin. According to some
embodiments, the pH of the medium is between about 4-6.
[0118] According to some embodiments, the growth medium comprises
salts such as magnesium, phosphate, nitrate or any combination
thereof. According to some embodiments of the invention, the growth
medium includes KNOB, MgSO.sub.4, NaH.sub.2PO.sub.4, or any
combination thereof. According to some embodiments, the medium
includes vitamins or any combination thereof. According to further
embodiments, the medium includes sugars such as sucrose or any
combination thereof.
[0119] In an embodiment of the invention, the concentration of the
sucrose added to the growth medium is between 1 to 6%. In another
embodiment, the concentration of the sucrose added to the growth
medium is about 3%.
[0120] According to further embodiments, casein, casein hydrolysate
or casein peptone may be 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. According to some embodiments the
olive cells are grown without the addition of hormones.
[0121] Examples of plant culture media that may be used according
to some embodiments in one stage or more of the process, include,
but are not limited to: Anderson (Anderson, In Vitro 14:334, 1978;
Anderson, Act. Hort., 112:13, 1980), Chee and Pool (Sci. Hort.
32:85, 1987), CLC/Ipomoea (CP) (Chee et al., J. Am. Soc. Hort. Sci.
117:663, 1992), Chu (N.sub.6) (Chu et al., Scientia Sinic. 18:659,
1975; Chu, Proc. Symp. Plant Tiss. Cult., Peking 43, 1978), DCR
(Gupta and Durzan, Plant Cell Rep. 4:177, 1985), DKW/Juglans
(Driver and Kuniyuki, HortScience 19:507, 1984; McGranahan et al.,
in: Bonga and Durzan, eds., Cell and Tissue Culture in Forestry,
Martinus Nijhoff, Dordrecht, 1987), De Greef and Jacobs (De Greef
and Jacobs, Plant Sci. Lett. 17:55, 1979), Eriksson (ER) (Eriksson,
Physiol. Plant. 18:976, 1965), Gresshoff and Doy (DBM2) (Gresshoff
and Doy, Z Pflanzenphysiol. 73:132, 1974), Heller's (Heller, Ann.
Sci. Nat. Bot. Biol. Veg. 11th Ser. 14:1, 1953), Hoagland's
(Hoagland and Arnon, Circular 347, Calif. Agr. Exp. Stat.,
Berkeley, 1950), Kao and Michayluk (Kao and Michayluk, Planta
126:105, 1975), Linsmaier and Skoog (Linsmaier and Skoog, Physiol.
Plant. 18:100, 1965), Litvay's (LM) (Litvay et al., Plant Cell Rep.
4:325, 1985), Nitsch and Nitsch (Nitsch and Nitsch, Science 163:85,
1969), Quoirin and Lepoivre (Quoirin et al., C. R. Res. Sta. Cult.
Fruit Mar., Gembloux 93, 1977), Schenk and Hildebrandt (Schenk and
Hildebrandt, Can. J. Bot. 50:199, 1972), White's (White, The
Cultivation of Animal and Plant Cells, Ronald Press, NY, 1963),
etc.
[0122] 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.degree. C. and 30.degree. C. According to some embodiments, the
temperature during the first step is between about 22.degree. C.
and 28.degree. C. According to some embodiments, the temperature
during the first step is about 25.degree. 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.
[0123] 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 flask once the atmosphere in the
flask reaches a pre-defined pressure. According to some
embodiments, the predefined pressure up to 8 PSI. According to some
embodiments, the gas outlet is a separate entity from the outlet
for removing liquids/solids.
[0124] 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 4 L reactor. According to further embodiments, the small scale
bioreactor is a 3-5 L reactor. According to further embodiments,
the small scale bioreactor is a 3-10 L reactor. According to
further embodiments, the small scale bioreactor is a 4-8 L
reactor.
[0125] The small scale bioreactor may be prepared from any
appropriate material, such as glass, metal, 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.
[0126] 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.
[0127] 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 some embodiments, 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.
[0128] 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 predefined pressure is 8 PSI. According to some
embodiments, the gas outlet is a separate entity from the outlet
for removing liquids/solids.
[0129] According to some embodiments, the olive cells and the
medium are continuously mixed during the second step. According to
further embodiments, the olive cells and the medium are mixed
occasionally during the second step. According to some embodiments,
the temperature during the second step is between about 20 to
30.degree. C. According to some embodiments, the temperature during
the second step is between about 22 to 28.degree. C. According to
some embodiments, the temperature during the second step is about
25.degree. 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.
[0130] 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 30-50 L reactor. According to further
embodiments, the medium scale bioreactor is a 40-60 L reactor.
According to further embodiments, the medium scale bioreactor is a
30-70 L reactor. According to further embodiments, the medium scale
bioreactor is a 20-100 L reactor.
[0131] The medium scale bioreactor may be prepared from any
appropriate material, such as glass, metal, 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.
[0132] 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.
[0133] 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 predefined pressure is up to 8 PSI. according to
some embodiments, the gas outlet is a separate entity from the
outlet for liquids/solids.
[0134] 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.degree. C. According to some embodiments, the temperature during
the third step is between about 22 and 28.degree. C. According to
some embodiments, the temperature during the third step is about
25.degree. 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.
[0135] 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 1000 L reactor. According to further
embodiments, the larger scale bioreactor is a 200-500 L reactor.
According to further embodiments, the large scale bioreactor is a
500-1000 L reactor. According to further embodiments, the large
scale bioreactor is a 1000-1500 L reactor. According to further
embodiments, the large scale bioreactor is a 500-1100 L
reactor.
[0136] The larger scale bioreactor may be prepared from any
appropriate material, such as glass, metal, plastic and/or any type
of polymer. According to some embodiments, the larger scale
bioreactor is disposable. If the larger scale bioreactor is not
disposable, according to some embodiments, it is cleaned and
sterilized between uses by any appropriate means.
[0137] 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.
[0138] 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 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 predefined pressure is up to 8
PSI. According to some embodiments, the gas outlet is a separate
entity from the outlet for liquids/solids.
[0139] 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.
[0140] According to some embodiments, the olive cells and the
medium are continuously mixed during the fourth step. According to
further embodiments, the olive cells 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.degree. C. According to some embodiments, the temperature during
the fourth step is between about 22 to 28.degree. C. According to
some embodiments, the temperature during the fourth step is about
25.degree. 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.
[0141] 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 ant 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.
[0142] 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.
[0143] 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.
[0144] 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.
[0145] In an embodiment of the invention, there is provided a
method of treating an inflammatory as well as prevention and/or
treatment of risk factors for cardiovascular disease, development
of atherosclerotic plaque, protection of LDL particles from
oxidative damage, maintenance of normal blood HDL-cholesterol
concentrations to decrease blood pressure and to maintain normal
haemostatic function. Olive polyphenols were shown to have
beneficial effects on learning and memory deficits found in ageing
and diseases, such as those related to the overproduction of
amyloid-beta peptide. In addition, olive polyphenols were shown to
influence gut microbial balance by promoting growth of bacteria
influencing lipid metabolism and inhibition of pathogenic bacteria,
as well as increase bone density. The method 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 possibly manufactured
according to the large scale process detailed herein and is
possibly rich in secondary metabolites.
[0146] 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 an inflammatory
disease, prolongation of life expectancy of patients having a
disease, as well as complete recovery from a disease.
[0147] Examples of such diseases and conditions that may be treated
by the compositions of the invention are detailed herein.
[0148] Metabolic abnormalities are associated with obesity, insulin
resistance glucose intolerance, type II diabetes mellitus (DMII),
dyslipidemia fatty liver, steatohepatitis, and steatosis. These
abnormalities increase the risk of stroke cardio-vascular diseases.
The etiology of the metabolic syndrome is considered to be
multifactorial involving genetic and environmental effects.
[0149] Metabolic syndrome also known as "the deadly quartet" or
"Syndrome X" or the "Insulin Resistance Syndrome" is a cluster of
risk factors for various diseases, such as cardio-vascular
diseases, stroke and diabetes mellitus type II, i.e. insulin
resistance, hyperinsulinemia, abdominal obesity, (caused by an
accumulation of intra-abdominal fat), elevated serum lipids, and
high blood pressure. 25% of adults living in the United States are
diagnosed with metabolic syndrome. It is believed that the
pathophysiology of the metabolic syndrome is related to insulin
resistance. The risk factors include the following: elevated waist
circumference (.gtoreq.102 cm in man and 88 cm in women); elevated
triglycerides (>150 mg/dL); reduced high-density lipoprotein
(HDL) cholesterol (<40 mg/dL in men and 50 mg/dL in women);
elevated blood pressure (>130/85 mm Hg) and elevated fasting
glucose (>100 mg/dL. Other risk factors may contribute to the
metabolic syndrome as well. Additionally, the risk factors may vary
in different populations.
[0150] 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
Example 1
Generation of Olive Cell Lines (Calli) and Suspension Cultures in
Erlenmeyer
1. Material and Methods
Plant Material
[0151] Olive cell culture was initiated from olive fruits (Olea
europaea L.) of Nabali, Manzanilla, Souri and Barnea cultivars,
including all leaves, petioles, fruits and kernels.
Establishment of Calli from Olive Explants
[0152] Olive plant parts: young leaves and their petriols, immature
fruits (about 6 weeks from flowering) and kernels from immature
olives were rinsed under running water and sterilized by agitation
in 70% ethanol for three minutes and then 3% Na-hypochlorite
solution for 20 minutes, followed by three washes in sterile water.
Plant parts were dried under sterile conditions and further
dissected into .about.0.5 cm sections.
[0153] Olive plant explants were plated on MS Murashige and Skoog
medium basal medium (Table 1B) supplemented with sucrose and
various combinations of the auxines 2,4-dichlorophenoxyacetic acid
(2,4-D), 1-naphthaleneacetic acid (NAA) and the cytokinines
kinetin, 6-(.gamma.,.gamma.-Dimethylallylamino)purine (2iP) and
benzyladenine (BA) at various concentrations. One medium
composition (O-2) included active charcoal. The pH of the medium
was 5.8. The medium was solidified with agar (Gelrite, Duchefa or
Phytagel, Sigma). Plate compositions are specified in Table 1A.
Plates were kept at 25.degree. C. in the dark. When formed calli
reached a diameter of 1 cm, they were split in a 1:3 ratio onto a
similar medium. Calli were transferred to fresh plates every 4
weeks.
Calli Results
[0154] Culture growth: Fifty three calli were successfully
developed from olive explants (see Table 2). It is noted that the
calli development was similar for the Nabali, Manzanilla and Barnea
cultivar and lower for the Souri cv.
TABLE-US-00003 TABLE 2 The efficiency of olive calli production
from various olive fruit sections of Nabali, Manzanilla, Barnea and
Souri cultivars Total Calli Calli Calli Calli number from from from
from Cultivar of calli Leaf petiole fruit kernel Nabali 17 2 3 6 6
Souri 7 1 0 3 3 Manzanilla 15 2 0 11 2 Barnea 14 2 2 7 3 Total 53 7
5 30 14
[0155] The effect of media composition: Calli growth was detected
on MS plates including various media components M-3, O-1, O-2, O-3,
O-4 and O-5 (see Table 1A). However, as can be seen in Table 3, the
most callus development-promoting media compositions were the
following: Half-strength MS including 3% sucrose, 2 mg/L NAA and
0.5 mg/L 2iP (O-5, 31 calli) and to a lower extent on full-strength
MS including 4.5% sucrose, 0.5 mg/L 2,4,D and 0.1 mg/L 2iP (O-4,
eleven calli) and half-strength MS including 2% sucrose and 0.1
mg/L 2,4,D (O-3, eight calli).
TABLE-US-00004 TABLE 3 The efficiency of olive calli production
(number of well-established calli) on various MS plates Medium
composition M-3 O-1 O-2 O-3 O-4 O-5 Nabali 0 0 0 0 7 10 Souri 0 0 0
2 0 5 Manzanilla 0 0 0 3 3 9 Barnea 3 0 0 3 1 7 Total 3 0 0 8 11 31
number of calli
Establishment of Suspension Culture
[0156] Several different calli were transferred to liquid media for
the generation of suspension cultures. Olive suspension culture
growth was efficient in liquid, O-5, O-4, O-3 and M-3 medium
supplemented with vitamins, minerals, and sugar, as detailed in
Table 1A above, apart from the agar, which was not added to the
suspension medium; rather, Agar was added only to the medium in
which the olive calli were grown. The pH of the suspension medium
is between pH of between4-6.
[0157] The suspension cultures maintained stable growth in
suspension (50 to 1000 ml Erlenmeyer flasks). Cultures were
routinely subcultured every 7-20 days to fresh growth media.
Erlenmeyer, Shake Flasks
[0158] Olive cells were grown in suspension under continuous
fluorescent light (1000 1.times.) at 25+5.degree. C., in 1 liter
Erlenmeyer flasks on an orbital shaker.
Preparation of Olive Cell Powder
[0159] The cultured olive cells grown in liquid medium were
filtered through filter and dried by lyophilization or spray
dryer
Example 2
Expression Polyphenolic Compounds in Olive Cell Calli
Materials and Methods
[0160] Polyphenols extraction for HPLC analysis: Fresh olive Callus
cell cultures were extracted for analytical determination of
polyphenol content in the olive culture. About 1 gr of callus was
harvested and kept at -20.degree. C. for at least 16h before
analysis. The callus was extracted by 80% methanol/water solution
in a ratio of 0.4 ml methanol per 1 gr of cells. The suspension was
sonicated for 10 minutes at 30.degree. C. in a sonicator. The
solution was centrifuged and the supernatant was re-centrifuged,
filtered through a 0.45 .mu.m filter and used for the HPLC
analysis.
[0161] Olive tissues (fruits and leaves) were crushed under liquid
nitrogen and extracted as described above, the extract was analyzed
by HPLC and used as a reference for olive polyphenols content.
[0162] Olive calli extract was analyzed by LC-MS. The method is
described in: Food Chemistry 138 (2013) 1381-1391.
[0163] Olive calli samples were extracted in methanol (400 .mu.l/l
gr of calli's fresh weight), as described above. The content of
olive polyphenols was determined as follows: hydroxytyrosol,
oleuropein and pinoresinol were detected at 280 nm. Tyrosol was
detected at 275 nm and verbascoside was detected at 330 nm. Total
polyphenols were monitored at 280 nm using commercial Epicatechin
(Sigma) as a standard, and expressed as .mu.g epicatechin
equivalent.
HPLC Analysis
[0164] Polyphenolic compounds in olive-derived cultures were
characterized and quantitated by high performance liquid
chromatography (HPLC) analysis. Selected phenolic compounds were
identified by their UV absorbance spectra and retention times.
Their concentrations were determined by means of a calibration
curve using different external standards. Analysis was performed by
JASCO PU-2089 HPLC system using the operation software ChromNAV.
Total polyphenols were determined by summing AUC of peaks as
monitored at 280 nm, and quantified, based on epicatechin as a
standard. Total polyphenols is expressed as epicatechin
equivalents. Hydroxytyrosol, Oleuropein and pinoresinol were
analyzed based on characteristic absorption at 280 nm Similarly,
tyrosol was detected at 275 nm and verbascoside was detected at 330
nm. Compounds were quantified according to standard curves of
commercial standards, respectively.
Liquid Chromatography Mass Spectrometry (LC-MS) Analysis
[0165] LC-MS analysis of the sample was performed using an Accela
LC system coupled with an LTQ Orbitrap Discovery hybrid FT mass
spectrometer (Thermo Fisher Scientific Inc.) equipped with an
electrospray ionization source. The mass spectrometer was operated
in a negative ionization mode, wherein the ion source parameters
were as follows: spray voltage 3.5 kV, capillary temperature
300.degree. C., and ion-transfer optics parameters were optimized
using automatic tune option, sheath gas rate (arb) 35, and
auxiliary gas rate (arb) 15.
[0166] Mass spectra were acquired in the m/z 150-2000 Da range. The
LC-MS analysis was performed in data depending acquisition mode.
The LC-MS system was controlled and the data were analyzed using
Xcalibur software (Thermo Fisher Scientific Inc). Chromatographic
separation was achieved on Kinetex Hexyl-Phenyl column (2.6 .mu.m,
150.times.2.1 mm, Phenomenex) using an ACN/Water+0.1% AcOH (in
both) gradient.
Results
Polyphenolic Compound Expression
[0167] Expression of various polyphenolic compounds that are
produced in olive fruit was also detected in olive calli and in
suspension cultures. Amongst these compounds, hydroxytyrosol,
tyrosol, oleuropein, verbascoside and pinoresinol were detected, as
confirmed by LC-MS. (Table 4).
TABLE-US-00005 TABLE 4 Expression of olive polyphenols in
representative olive calli of four cultivars: Nabali (callus
originated from petiole), Souri (callus originated from leaf)
Manzanillo (callus originated from leaf) and Barnea (callus
originated from pulp) Phenolic compounds in olive calli and cell
culture (.mu.g/mg Dry Weight (DW). Nabali Souri Manzanilo Barnea
3,4-DHPEA 1.963 0.785 0.339 0.281 (hydrosytyrosol) p-HPEA (tyrosol)
2.557 4.191 1.075 3.034 Oleuropein 10.0 6 4 2 Verbascoside 37.785
15.925 24.037 8.212 (+)-pinoresinol 0.090 0.000 0.000 0.000
Active Polyphenolic Compound Quantitation in Olive Suspension
Culture
[0168] Hydroxytirosol content in various olive calli cultures
ranged from 0.281 to 1.963 .mu.g/mg of dry-weight (DW). Tyrosol
content ranged from 1.075 to 4.191 .mu.g/mg of DW. Oleuropein
content ranged from 2-10 .mu.g/mg (DW). Verbascoside content ranged
from 8.212 to 37.78 .mu.g/mg DW, and pinoresinol was detected only
in callus of Nabali cv. Origin, at 0.09 .mu.g/mg DW, considering a
drying factor of 20. The content of these compounds in different
parts of olive plant as compared to olive calli as well as in cell
suspension cultures and bioreactors is summarized in Table 5. The
concentrations of active compounds found in olive calli are within
the range of these compounds in green olives (Alagna et al., 2012,
phenol explorer database). Moreover, the production of high
verbascoside concentrations characterizes early maturation stages
of olive fruit, and was not shown in calli generated by others,
whereas the verbascoside expression in the calli presented here and
prepared according to this invention, is stable and is not limited
to any development stage.
TABLE-US-00006 TABLE 5 Polyphenols content in olive plant parts
from prior art documents (source: phenol explorer) compared to the
olive cells of the invention - fresh weight (FW) and dry weight
(DW). Drying factor is 20. A. DATA regarding polyphenols
concentration in olives from scientific literature Content in green
olives (http://phenol-explorer.eu/) Content in jasmonate induced
(mg/g FW) Callus (Gentile et al., 2014) Mean Min. Max. (mg/g DW)
Hydrosytyrosol 0.5557 0.043 1.16 1.72 Tyrosol 0.0747 0 0.21 0.34
Verbascoside 0.172 0 0.665 0.56 Oleuropein 0.55 0 3.25 4.66
(+)-pinoresinol ND ND ND ND Luigi Gentile and Nicola A. Uccella,
(2014), Selected bioactives from callus cultures of olives (Olea
europaea L. Var. Coratina) by LC-MS, Food Research International
55: 128-136. B. Data regarding polyphenols concentration in the
invention olive cellsgrown in vitro Content in the callus culture,
cell suspension cultures and bioreactors of the invention mg/g FW
mg/g DW Min. Max. Min. Max. Hydrosytyrosol 0.007 0.78 0.14 15.7
Tyrosol 0.027 1.1 0.5375 21.0 Verbascoside 0.21 7.6 4.1 151.1
Oleuropein 0.010 0.5 0.2 10 (+)-pinoresinol 0.000 0.004 0.000
0.090
Example 3
[0169] Scale Up of Olive Culture in Bioreactors and Testing of the
Total Amount of the Polyphenols, including, Hydroxytyrosol,
Tyrosol, Oleuropein, Verbascoside and Pinoresinol Content in Olive
Cells Grown in Large Scale Bioreactors
Materials and Methods
Stage 1: Cells are Prepared and Grown as Described in Example
1.
Stage 2: Small Scale Bioreactor
[0170] Small scale bioreactor culturing is performed by inoculating
a 7 to 16 old day cell suspensions grown in the Erlenmeyer of Stage
1 into a 4-8 liter disposable bioreactor at 25+5.degree. C. The
cells were grown in the suspension under continuous fluorescent
light (1000 1.times.) in a growing medium containing modified MS
supplemented with sucrose 1-6% and the Kinetin, 2,4,D, NAA, BA, 2iP
and casein hydrolysate or a combination thereof
[0171] (pH 4.0-6.0)-. The cells were sub-cultured every 5-21
days.
Stage 3: Large Scale Bioreactor
[0172] The cell suspensions grown in a small scale bioreactor were
inoculated into a 30-50 liter disposable bioreactor. The cells were
grown in a suspension under continuous fluorescent light (1000
1.times.) at 25+5.degree. C. The growing medium containing
[0173] Modified MS supplemented with sucrose 1-6% and the Kinetin,
2,4,D, NAA, BA, 2iP and casein hydrolysate or a combination
thereof
[0174] (pH 4.0-6.000). The cells were sub-cultured every 5-30
days.
Stage 4: Larger Scale Bioreactor
[0175] The cell suspension grown in the small or large scale
bioreactor is inoculated into a 300-1000 liter disposable
bioreactor. The cells were grown in a suspension under continuous
fluorescent light (1000 1.times.) at 25+5.degree. C.
[0176] The growing medium contained modified MS supplemented with
sucrose 1-6% and the Kinetin, 2,4,D, NAA, BA, 2iP and casein
hydrolysate or a combination thereof
Stage 5: Harvesting
[0177] The cells were harvested once they reached a cell biomass of
10% to 70% (w\v). The harvested cells were dried (in a spray dryer
or lyophilized) to produce a fine green powder, with a typical
composition, taste and odor.
Example 4
4.1 The Effect of Medium Composition on the Growth of Olive Cells
Grown in Erlenmeyer Shake Flask.
[0178] Olive Cells were grown in an Erlenmeyer shake flask in
different medium compositions: O4 (modified MS supplemented with
sugar hormones),and Gamborg B5. The results are presented in Table
7, demonstrating that olive cells grown in O4 produce approximately
1.9 higher biomass than cells grown in Gamborg B5 medium.
TABLE-US-00007 TABLE 7 The effect medium composition on growth of
Olive cells Fresh weight Medium g/L O4 (modified MS) 134.9 Gamborg
B5 72.4
4.2 The Effect of Medium Composition on the Amount of Olive
Polyphenols Grown in Bioreactors.
[0179] Olive cells were grown in large bioreactors in different
medium compositions: O4 (modified MS) and Gamborg B5. The results,
obtained on day 5, are presented in Table 8. The results in Table 8
demonstrate that cells grown in the O4 medium produces three olive
polyphenols, i.e., tyrosol, hydroxytyrosol, and oleorupein, which
are not detected in the olive cells grown in Gamborg B5.
[0180] Table 8 further shows that olive cells grown in 0-4 medium
produce approximately 2.65 higher verbascoside levels than olive
cells grown in the Gamborg B5 medium.
[0181] Further, as shown in the fresh mass presented in Table 8,
olive cells grown in the O-4 medium resulted in a higher biomass in
large bioreactor after 5 days in comparison to the biomass obtained
for olive cells grown in the Gamborg B5 medium.
TABLE-US-00008 TABLE 8 The effect of medium composition on growth
of Olive cells in large bioreactor and their polyphenols levels as
measured by HPLC at 280 nm Fresh *weight Tyrosol Hydroxytyrosol
Oleuropein Verbascoside Medium g/L mg/L mg/L mg/L mg/L O4 (modified
76 12.54 4.71 2.36 949.24 MS) Gamborg B5 48 N.D N.D N.D 358.56
*MS--Murashige and Skoog medium (Toshio Murashige and Folke K.
Skoog in 1968)
4.3 The Effect of Light and Dark Conditions on the Growth of Olive
Cells Grown in Vitro In Bioreactors
[0182] The effect of growing in vitro the olive cells in dark
conditions was tested. Cell growth was assessed by the measurement
the fresh weight of the cells grown in vitro, demonstrating that
olive cells are growing in both conditions with slightly favorite
to dark condition (Table 9).
TABLE-US-00009 TABLE 9 The effect of growing conditions light and
Dark conditions on growth of olive cells in large bioreactors
Growing Fresh weight Conditions g/L O4 (modified MS) light 63 O4
(modified MS) dark 103
4.4 Olive Cell Culture Grown in Bioreactors.
[0183] Olive Cells were grown in O-4 medium, namely modified MS
medium with additional agents defined as O-4, in disposable large
bioreactors. The results revealethat cells undergo exponential
growth yielding a 200 gr/l fresh biomass at day 10 (FIG. 2). It is
noted that these cells continue to grow, and can reach higher
biomasses than presented.
[0184] This is the first time that the successful growth of olive
cells in a large scale disposable bioreactor, having a high level
of polyphenols production such as, Tyrosol, Hydrixityrosol,
oleoropein and verbascoside, has been demonstrated.
[0185] As described above, the production of secondary metabolites,
including polyphenols, such as tyrosol and verbascoside, is
expected to be significantly reduced when larger quantities of
olive cells are grown in bioreactors, 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.
[0186] 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.
* * * * *
References