U.S. patent application number 15/666261 was filed with the patent office on 2018-06-21 for jatropha curcas processing method and products.
The applicant listed for this patent is AGROILS TECHNOLOGIES SRL. Invention is credited to Roberto Crea.
Application Number | 20180169165 15/666261 |
Document ID | / |
Family ID | 48172699 |
Filed Date | 2018-06-21 |
United States Patent
Application |
20180169165 |
Kind Code |
A1 |
Crea; Roberto |
June 21, 2018 |
JATROPHA CURCAS PROCESSING METHOD AND PRODUCTS
Abstract
A process for preparing a food or feed composition from J.
curcas is disclosed. The method involves adding an acidified
aqueous solution to J. curcas components, to a final pH of between
1 and 5, incubating the acidified mixture for a period for a period
of at least 1 hour, and centrifuging the incubated mixture to
separate the mixture into three physically distinct fractions: (i)
a light, upper fraction containing oil, (ii) an aqueous fraction
containing soluble acid-extracted components and breakdown
products, and (iii) a substantially detoxified solid cake which
forms or is used in forming the food or feed composition. The
acidified aqueous solution added may be acidified olive vegetation
water having a ratio of hydroxytyrosol to oleuropein of between 5:1
to 100:1. Also disclosed are a food or feed composition, and oil
and aqueous fractions formed by the method.
Inventors: |
Crea; Roberto; (Hayward,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AGROILS TECHNOLOGIES SRL |
Florence |
|
IT |
|
|
Family ID: |
48172699 |
Appl. No.: |
15/666261 |
Filed: |
August 1, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13282106 |
Oct 26, 2011 |
9750779 |
|
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15666261 |
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61406719 |
Oct 26, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C11B 1/10 20130101; C11B
1/06 20130101; Y02P 60/877 20151101; A61K 2236/30 20130101; A23K
50/75 20160501; Y02P 60/87 20151101; C11B 1/04 20130101; A23L 25/00
20160801; A61K 36/47 20130101; A23K 50/10 20160501; A23K 10/37
20160501; A23L 11/34 20160801; A23K 50/80 20160501; A23K 50/30
20160501 |
International
Class: |
A61K 36/47 20060101
A61K036/47; C11B 1/10 20060101 C11B001/10; C11B 1/06 20060101
C11B001/06; C11B 1/04 20060101 C11B001/04; A23L 11/30 20160101
A23L011/30; A23K 50/10 20160101 A23K050/10; A23L 25/00 20160101
A23L025/00; A23K 10/37 20160101 A23K010/37; A23K 50/80 20160101
A23K050/80; A23K 50/75 20160101 A23K050/75; A23K 50/30 20160101
A23K050/30 |
Claims
1-69. (canceled)
70. An oil fraction from J. curcas formed by the steps of: (a)
crushing or grinding one or more J. curcas components to form a
slurry, wherein the one or more J. curcas components is a J. curcas
plant part selected from the group consisting of leaves, hulls,
fruit, or seeds or pre-formed cake thereof; (b) acidifying the
slurry of step (a) to a pH of 1-5 by adding an acidified aqueous
solution to form an acidified slurry; (c) incubating the acidified
slurry for a period of at least 1 hour; (d) separating the
incubated acidified slurry by centrifuging or decanting into three
distinct fractions: (i) a fraction containing oil, (ii) an aqueous
fraction comprising unoxidized phorbol esters and curcin, and (iii)
a detoxified solid cake; and (e) isolating the oil fraction in step
(d).
71. The oil fraction of claim 70, wherein the acidified aqueous
solution contains citric acid.
72. The oil fraction of claim 70, wherein the acidified aqueous
solution contains 1% citric acid.
73. The oil fraction of claim 70, wherein the acidified aqueous
antioxidant solution is olive vegetation water.
74. The oil fraction of claim 73, wherein the olive vegetation
water has a ratio of hydroxytyrosol to oleuropein of between 5:1 to
100:1.
75. The oil fraction of claim 73, wherein the olive vegetation
water further comprises 5-10% (v/v) of an organic solvent.
76. The oil fraction of claim 70, wherein the fraction containing
oil is removed from the detoxified solid cake and aqueous fraction,
and the detoxified cake and aqueous fraction is further separated
to provide three physically distinct fractions.
77. The oil fraction of claim 70, wherein the fraction containing
oil, the detoxified solid cake and the aqueous fraction are
simultaneously separated from each other.
78. The oil fraction of claim 70, wherein the acidified slurry is
acidified to a pH of 2-4.
79. A detoxified solid cake fraction from J. curcas formed by the
steps of: (a) crushing or grinding one or more J. curcas components
to form a slurry, wherein the one or more J. curcas components is a
J. curcas plant part selected from the group consisting of leaves,
hulls, fruit, or seeds or pre-formed cake thereof; (b) acidifying
the slurry of step (a) to a pH of 1-5 by adding an acidified
aqueous solution to form an acidified slurry; (c) incubating the
acidified slurry for a period of at least 1 hour; (d) separating
the incubated acidified slurry by centrifuging or decanting into
three distinct fractions: (i) a fraction containing oil, (ii) an
aqueous fraction comprising unoxidized phorbol esters and curcin,
and (iii) a detoxified solid cake; and (e) isolating the detoxified
solid cake fraction in step (d).
80. The detoxified solid cake fraction of claim 79, wherein the
acidified aqueous solution contains citric acid.
81. The detoxified solid cake fraction of claim 79, wherein the
acidified aqueous solution contains 1% citric acid.
82. The detoxified solid cake fraction of claim 79, wherein the
acidified aqueous antioxidant solution is olive vegetation
water.
83. The detoxified solid cake fraction of claim 82, wherein the
olive vegetation water has a ratio of hydroxytyrosol to oleuropein
of between 5:1 to 100:1.
84. The detoxified solid cake fraction of claim 82, wherein the
olive vegetation water further comprises 5-10% (v/v) of an organic
solvent.
85. The detoxified solid cake fraction of claim 79, wherein the
fraction containing oil is removed from the detoxified solid cake
and aqueous fraction, and the detoxified cake and aqueous fraction
is further separated to provide three physically distinct
fractions.
86. The detoxified solid cake fraction of claim 79, wherein the
fraction containing oil, the detoxified solid cake and the aqueous
fraction are simultaneously separated from each other.
87. The detoxified solid cake fraction of claim 79, wherein the
acidified slurry is acidified to a pH of 2-4.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for processing
Jatropha curcas plants and products formed by the processing
method.
BACKGROUND OF THE INVENTION
[0002] Jatropha curcas L. is a multipurpose shrub of significant
economic importance because of its several potential industrial and
medicinal uses. Jatropha curcas L. or physic nut (or purging nut)
is a drought resistant large shrub or small tree, belonging to the
genus Euphorbiaceae, producing oil containing seeds. The species
has its natural distribution area in the Northeastern part of South
America. (Heller, 1996) and central Africa and several countries in
Asia. The seeds of physic nut are a good source of oil, which can
be used as a diesel substitute. They are used also in medicines,
and soap and cosmetics manufacture in various tropical
countries.
[0003] The fruit of J. curcas is green/yellow when fresh and
contains seed. The seed and seed products of J. curcas are
potentially a source of high nutritional value, e.g., as animal
feed. The levels of essential amino acids, except lysine, in the
seed cake are higher than that of the FAO/WHO reference protein for
a five year old child in all the meal samples on a dry matter
basis. The major fatty acids found in the oil samples were oleic
(41.5-48.8%), linoleic (34.6-44.4%), palmitic (10.5-13.0%) and
stearic (2.3-2.8%) acids. The residual protein-rich seed cake,
remaining after extraction of the oil, could form a protein-rich
ingredient in feeds for poultry, pigs, cattle and even fish if it
could be detoxified.
[0004] Like the oil, the seed cake is toxic and therefore only
suitable as animal feed after processing. The toxicity of J. curcas
is based on several components (phorbol esters, curcains, trypsin
inhibitors and others) which make complete detoxification a
complicated process. Detoxification has been successful at
laboratory scale (Gross et al., 16 1997; Martinez Herrera et al.,
2006), but since the process is complicated, it is not suitable for
small scale and local use. Large scale feed production, however,
has to compete on a global market with high quality demands.
Therefore, detoxification must be complete, constant and
guaranteed, and is thus expected to be expensive. Hence, a
successful penetration of J. curcas seed cake as feed to the market
at a profitable price seems doubtful.
[0005] Toxic components. The main toxic components are phorbol
esters, although in Mexico accessions without, or with low content
of phorbol esters have been found (Rivera Lorca & Ku Vera,
1997; Martinez Herrera et al., 2006; Basha & Sujatha, 2007).
The seed cake of this so called `non` or `low` toxic variety might
be suitable for use as animal feed, but it still contains minor
quantities of toxic components and resistance on the feed market
towards this product is to be expected.
[0006] On the other hand, the seed cake is nutrient rich and
therefore very suitable as fertilizer (Table 3). Together with the
fruit coats, the major part of the nutrients can be recycled. When
no fertilizers are used, which is assumed to be the case in the use
of J. curcas as a low input crop, this recycling is necessary to
maintain soil fertility, especially on non fertile marginal lands.
Patolia (2007a) reported total above ground dry matter increase of
24% after 2 years.
[0007] Because of unavoidable inefficiencies, recycling nutrients
will only be effective at a certain production level that allows a
high dynamic nutrient cycle to take place. Initiating a plantation
on low or non fertile soils therefore implies the need to use other
fertilizers, at least at the start, to boost crop growth and seed
production in the initial stages. The harvested part of J. curcas
is the fruit, mostly containing three seeds. The seeds make up
about 70% of the total weight of the fruit (30% fruit coat); the
mature fruits have a moisture content of circa 15%, the seeds circa
7%. The oil is stored in the interior of the seed: the kernel,
which makes up circa 65% of the total mass of the seed. The
moisture contents are circa 10% for the hull and circa 5% for the
kernel.
[0008] Oil Fraction and Quality.
[0009] The seed of J. curcas contains a viscous oil, highly
suitable for cooking and lighting by itself and for the production
of biodiesel. The total fraction of oil, fats and carbohydrates is
circa 30 to 35% for the seed and, since 99% of the oil is stored in
the kernel, circa 50 to 55% for the kernel (Table 1).
[0010] The oil contains very little other components and has a very
good quality for burning. Cetane number of J. curcas oil (23-41) is
close to cottonseed (35-40) and better than rapeseed (30-36),
groundnut (30-41) and sunflower (29-37) (Vaitilingom &
Liennard, 1997). The toxicity of J. curcas is mainly based on
phorbol esters and curcains, which give no pollution when burnt.
The oil is also very suitable for transesterification into
biodiesel (Mohibbe Azam et al., 2005).
[0011] The absence of sulphur dioxide (SO.sub.2) in exhaust from
diesel engines run on J. curcas oil shows that the oil may have a
less adverse impact on the environment (Kandpal & Madan, 1995).
As J. curcas oil has a higher viscosity than diesel oil (53 versus
8 cSt at 30 C), blending J. curcas oil up to 50% with diesel oil is
advised for use in a Compression Ignition (C.I.) engine without
major operational difficulties (Pramanik, 2003). Other publications
mention much lower values for viscosity (17.1 cSt at 30 C), which
would reduce the necessary blending fraction of diesel oil
(Akintayo, 2004), however, conventional engines can be operated by
blending biomethanol or bioethanol (with gasoline) or bio-diesel
(with diesel) from 3-20%. Some report that J. curcas oil should
only be used as ignition accelerator (Forson et al., 2004).
[0012] Seed Cake.
[0013] Like the oil, the seed cake is toxic and therefore only
suitable as animal feed after processing. The toxicity of J. curcas
is based on several components (phorbol esters, curcains, trypsin
inhibitors and others) which make complete detoxification
complicated. Detoxification has been successful at laboratory scale
(Gross et al., 16 1997; Martinez Herrera et al., 2006), but since
the process is complicated, it is not suitable for small scale and
local use. Large scale feed production, however, has to compete on
a global market with high quality demands. Therefore,
detoxification must be complete, constant and guaranteed, and is
thus expected to be expensive. Hence, a successful penetration of
J. curcas seed cake as feed to the market at a profitable price is
challenging. The main toxic, but potentially medicinal, components
are phorbol esters, although in Mexico accessions without, or with
low content of phorbol esters have been found (Rivera Lorca &
Ku Vera, 1997; Martinez Herrera et al., 2006; Basha & Sujatha,
2007). The seed cake of this so called `non` or `low` toxic variety
might be suitable for use as animal feed, but it still contains
minor quantities of toxic components and resistance on the feed
market towards this product is to be expected.
[0014] On the other hand, the seed cake is nutrient rich and
therefore very suitable as fertilizer. Together with the fruit
coats, the major part of the nutrients can be recycled. When no
fertilizers are used, which is assumed to be the casein the use of
J. curcas as a low input crop, this recycling is necessary to
maintain soil fertility, especially on non fertile marginal lands.
Patolia (2007a) reported total aboveground dry matter increase.
Because of unavoidable inefficiencies, recycling nutrients will
only be effective at a certain production level that allows a high
dynamic nutrient cycle to take place. Initiating a plantation on
low or non fertile soils therefore implies the need to use other
fertilizers, at least at the start, to boost crop growth and seed
production in the initial stages.
[0015] The by-products of J. curcas, such as fruit coats, seed
hulls and the remaining de-oiled seed cake after pressing, may be
used for organic fertilization, or for the production of more
energy. Seed hulls can be burnt and the seed cake and fruit pulp
can be used for the production of biogas by anaerobic fermentation
(Lopez et al., 1997; Staubmann et al., 1997; Vyas & Singh,
2007). By burning, most nutrients will be lost, but after
fermentation, most nutrients will remain in the effluent that can
still be used as a fertilizer to recycle nutrients. To maintain J.
curcas production at a sustainable level, it is important to be
aware that a huge amount of nutrients are removed if J. curcas
byproducts are exploited for additional valorization. However, the
range in the reported nutrient values only comes from a few sources
(Table 3), with clear variation. This indicates that environmental
and management conditions have a large effect on the eventual
nutrient content of the various plant parts. Soil organic matter
content decreases in a production system where nutrients are
removed and not replenished by fertilization.
[0016] Oil Extraction.
[0017] For J. curcas oil extraction at small scale, various oil
presses have been developed and modified from presses for other oil
seed crops. They have in common that they vary in design and are
non-standardized, as they were originally developed for other
(edible) seeds and need to be optimized for J. curcas seeds.
Bielenberg Ram (Hand) Presses handle 7-10 kg seed h-1 and spindle
presses handle 15 kg seed h-1 (Mbeza et al., 2002). Commercially
available pressing systems claim processing 500 kg seed h-1 (FIG.
15).
[0018] The recoverable oil fraction is clearly affected by pressing
technology. For hand powered small scale pressing (such as the
Bielenberg (Hand) Ram Press), an oil yield of only 19% of the seed
dry weight or 30% of the kernel was reported (Foidl & Eder,
1997; Augustus et al., 2002; Akintayo, 2004; Henning, 2004; Francis
et al., 2005), which is about 60% of the total extractable amount.
With mechanized pressing equipment about 75% of the oil can be
recovered. Commercially available pressing systems used for
large-scale de-oiling of e.g. soybean and rapeseed reach up to
90%.
[0019] Modern extraction techniques can substantially raise the
extractable oil fraction. Industrial extraction with organic
solvents (mainly hexane) yield near 100% of the oil content, while
extractions on water basis can yield from 65-97% of the oil,
depending on, (a.o.) the composition of the extract solvent, the
acidity (pH) and the temperature of the solvent (Shah et al., 2004;
Shah et al., 2005).
[0020] Toxicity of the Cake.
[0021] A wide variation in toxic, but potentially medicinal,
constituents, e.g. trypsin inhibitor in defatted kernels (18.4-27.5
mg g-1; Makkar et al., 1997) was observed, as well as a wide
variation in saponins (1.8-3.4%; Makkar et al., 1997) and phytate
(6.2-10.1%; Makkar et al., 1997). Phorbol esters are predominantly
present, but are sometimes at low levels or not detected in
provenances from Mexico. Phorbol ester content ranged from
0.87-3.32 mg g-1 of kernel weight in 17 provenances (Makkar et al.,
1997; 3.85 mg g-1: Martinez Herrera et al., 2006).
[0022] Much attention to various aspects and tests of toxic
components (phorbol esters and curcain) in J. curcas was reported
at the `Jatropha 97` Symposium in Managua, Nicaragua (Chapter 4 in
G bitz et al., 1997), including experiences for using proteins from
toxic and `low toxic` J. curcas seeds for livestock feed (Makkar
& Becker, 1997). Toxic constituents were found to be effective
against a wide variety of pests (Solsoloy & Solsoloy, 1997; Rug
& Ruppel, 2000). A 100% mortality rate was obtained against
mosquito (Culex quinque fasciatus Say), when petroleum extracts of
J. curcas leaves were used as a larvicide (Karmegam et al., 1997).
The toxicity of J. curcas is based on several components (phorbol
esters, curcains, trypsin inhibitors and others) that are present
in considerable amounts in all plant components (including the
oil), which make complete detoxification a complicated process.
[0023] Since the detoxification of J. curcas organic material is
such a complicated process, it has--so far--only been successful at
laboratory scale, and seems not to be suitable for small scale and
local application. Like other J. curcas plant components, the seed
cake is toxic and the prospect for successful penetration of the
feed market with a detoxified product is challenging. The seed cake
(either as remainder of the pressing process, or as a complete
meal) is nutrient rich and therefore very suitable as
fertilizer.
[0024] Phorbol esters of J. curcas decompose quickly as they are
very sensitive to elevated temperatures, light and atmospheric
oxygen (NIH, 2007); they decompose completely within 6 days (Rug
& Ruppel, 2000).
[0025] To maintain J. curcas production at a sustainable level, it
is important to take notion of the huge amount of nutrients that
are removed from the soil if J. curcas by-products are exploited
for additional uses, including the bio-refinery concept.
SUMMARY OF THE INVENTION
[0026] The invention includes, in one aspect, a process for
preparing a food or feed composition from J. curcas. The method
includes the steps of:
[0027] (a) forming a mixture containing J. curcas components, with
addition of acid to a final pH of the mixture of between 1 and
5,
[0028] (b) incubating the mixture for a period of at least 1 hour,
and
[0029] (c) centrifuging the incubated mixture to separate the
slurry into three physically distinct fractions: (i) a light, upper
fraction containing oil, (ii) an aqueous fraction containing
soluble acid-extracted components and breakdown products, and (iii)
a substantially detoxified solid cake which forms or is used in
forming the food or feed composition.
[0030] In one embodiment, step (a) in the method includes crushing
J. curcas to form a slurry, and acidifying the slurry to a pH of
between 1-5. The slurry may be acidified by addition of acidified
antioxidant solution. The acidified antioxidant solution may be
added before, during, or after crushing the J. curcas components.
The antioxidant solution may be olive vegetation water having ratio
of hydroxytyrosol to oleuropein of between 5:1 to 100:1. In certain
embodiments, the olive vegetation water comprises at least 0.1%
(w/v) polyphenols. In other embodiments, the olive vegetation water
comprises 5-10% (v/v) of an organic solvent. Preferred embodiments
of organic solvents include methanol and ethanol.
[0031] In another embodiment, step (a) in the method includes
crushing J. curcas to form a slurry, centrifuging the slurry to
separate the slurry into three physically distinct fractions: (i) a
light, upper fraction containing oil, (ii) an aqueous fraction
containing water-soluble components, and (iii) a first cake, and
forming a cake slurry by addition of an acidified aqueous solution
to the first cake, at a pH of between 1 and 5. The slurry may be
formed by addition to the first cake of acidified antioxidant
solution. The antioxidant solution may be olive vegetation water
having ratio of hydroxytyrosol to oleuropein of between 5:1 to
100:1. In this embodiment, the light upper oil fraction from step
(a) may be combined with the light upper oil fraction obtained in
step (d), and the aqueous fraction from step (a) may be combined
with the aqueous fraction obtained in step (d).
[0032] In still another embodiment, step (a) in the method includes
adding an acidic aqueous solution to a first cake prepared from
crushed J. Curcas, to form a cake slurry having a pH between 1-5.
The cake slurry may be formed by addition to the first cake of
acidified olive vegetation water having ratio of hydroxytyrosol to
oleuropein of between 5:1 to 100:1. In certain embodiments, the
olive vegetation water comprises at least 0.1% (w/v) polyphenols.
In other embodiments, the olive vegetation water comprises 5-10%
(v/v) of an organic solvent. Preferred embodiments of organic
solvents include methanol and ethanol.
[0033] Acid or an acidic aqueous solution or acidified olive
vegetation water may be added to the cake components in step (a) to
a final pH of between 2-4, and an exemplary acidifying agent is a
weak organic acid, such as citric acid.
[0034] The incubating step (c) may be carried out at room
temperature for a period of at least one day, for a period of at
least 10 days, or for a period of at least 30 days or longer.
[0035] The process may further include extracting soluble
components from the aqueous fraction obtained in step (c), and/or
concentrating the aqueous fraction by removal of water.
[0036] In the preceding embodiments, the J. curcas components are
selected from the fruit, the seed, or an already formed cake of J.
curcas. Also in the preceding embodiments, the olive vegetation
water may comprise at least 0.1% (w/v) polyphenols. In other
embodiments, the olive vegetation water comprises 5-10% (v/v) of an
organic solvent. Preferred embodiments of organic solvents include
methanol and ethanol.
[0037] In another aspect, the invention includes a food or feed
comprising J. curcas from which have been removed, toxic components
that are extracted and/or degraded by incubation of components in
an acidified aqueous slurry at pH 1-5 for at least one day.
[0038] The composition may be prepared by the methods disclosed
above.
[0039] Also disclosed is an oil fraction from J. curcas formed by
the steps of:
[0040] (a) pressing J. curcas components to form a cake and oil and
aqueous fractions,
[0041] (b) after removing the oil and aqueous fractions, adding an
acidified aqueous solution to the cake to form a slurry having a
final pH of between 1 and 5,
[0042] (c) incubating the slurry for a period for a period of at
least 24 hours,
[0043] (d) centrifuging the incubated slurry to separate the slurry
into three physically distinct fractions: (i) a light, upper
fraction containing additional oil, (ii) an aqueous fraction
containing soluble acid-extracted components and breakdown
products, and (iii) a substantially detoxified solid cake which can
be used as an animal feed, and
[0044] (e) isolating the light upper fraction obtained in step
(d).
[0045] In one embodiment, step (a) includes adding the acidified
antioxidant solution before, during, or after pressing the J.
curcas components. In another embodiment, step (b) may be carried
out by adding to the cake, in forming a slurry, acidified olive
vegetation water having ratio of hydroxytyrosol to oleuropein of
between 5:1 to 100:1. In certain embodiments, the olive vegetation
water comprises at least 0.1% (w/v) polyphenols. In other
embodiments, the olive vegetation water comprises 5-10% (v/v) of an
organic solvent. Preferred embodiments of organic solvents include
methanol and ethanol. The oil fraction may also includes the oil
fraction obtained in step (a).
[0046] Further disclosed is an aqueous fraction from J. curcas
formed by the steps of:
[0047] (a) pressing J. curcas components to form a cake and oil and
aqueous fractions,
[0048] (b) after removing the oil and aqueous fractions, adding an
acidified aqueous solution to the cake to form a slurry having a
final pH of between 1 and 5,
[0049] (c) incubating the slurry for a period for a period of at
least 24 hours,
[0050] (d) centrifuging the incubated slurry to separate the slurry
into three physically distinct fractions: (i) a light, upper
fraction containing additional oil, (ii) an aqueous fraction
containing soluble acid-extracted components and breakdown
products, and (iii) a substantially detoxified solid cake which can
be used as an animal feed, and
[0051] (e) isolating the aqueous fraction obtained in step (d).
[0052] The aqueous fraction may also include the aqueous fraction
of step (a). The aqueous fraction may be further treated to extract
medicinal components therefrom. In one embodiment, step (a)
includes adding the acidified antioxidant solution before, during,
or after pressing the J. curcas components.
[0053] These and other objects and features of the invention will
become more fully apparent when the following detailed description
is read below.
[0054] In another aspect, provided herein is a method of extracting
medicinal compounds from J. curcas, comprising the steps of:
[0055] (a) pressing J. curcas components to form a cake and oil and
aqueous fractions,
[0056] (b) removing the oil and aqueous fractions and then adding
an aqueous acid solution to the cake to form a slurry having a
final pH of between 1 and 5,
[0057] (c) incubating the slurry for a period of at least 24 hours,
and
[0058] (d) centrifuging the incubated slurry to separate the slurry
into three physically distinct fractions: (i) a light, upper
fraction containing additional oil, (ii) an aqueous fraction
containing medicinal compounds and breakdown products, and (iii) a
substantially detoxified solid cake.
[0059] In one embodiment, step (a) additionally comprises pressing
the J. curcas components in the presence of an aqueous acid
solution. In another embodiment, the aqueous acid solution is an
antioxidant solution. In yet another embodiment, step (a) includes
adding an acidified antioxidant solution before, during, or after
the pressing of the J. curcas components. In certain embodiments,
the antioxidant solution is olive vegetation water. The olive
vegetation water may comprise at least 0.1% (w/v) polyphenols. The
olive vegetation water may have a ratio of hydroxytyrosol to
oleuropein of between 5:1 to 100:1. The olive vegetation water may
comprise 5-10% (v/v) of an organic solvent. In a preferred
embodiment, the organic solvent is selected from methanol and
ethanol.
[0060] In another embodiment, the J. curcas components are selected
from the fruit, the seed, or an already formed cake of J. curcas.
In certain embodiments, the medicinal compounds are selected from
curcin and phorbol esters.
DESCRIPTION OF THE INVENTION
[0061] In the present invention, acidulated water, also referred to
as an acidic aqueous solution (e.g., citric acid 1%, chloridic acid
0.2 N or H.sub.2SO.sub.4 0.2 N) may be used as a medium for
extraction of hydrophobic compounds present in the cake. Among
these hydrophobic compounds are most of the toxic compounds which
make the cake poisonous. The aqueous extraction is carried at room
temperature for few hours to several days. The suspension or slurry
is then separated by a three phase centrifuge similar to than
commonly used by the olive oil industry.
[0062] Three phase centrifugation will produce a "light" phase
represented by the vegetable oil still trapped in the cake and thus
recoverable by this process, the "heavy" phase, represented by the
aqueous fraction containing the majority of the hydrophilic
compounds, which includes Trypsin inhibitors, sorbol esters and
lecitins (saponins), and the solid fraction (cake).
[0063] There are three different embodiments contemplated. In the
first, J. curcas components are crushed in the presence of an
acidified aqueous solution, to form a slurry, which is then
incubated, e.g., 1 hour to 30 days, to extract and/or detoxify
soluble compounds from the J. curcas cake components. After
incubation, the slurry is centrifuged to form the three fractions,
all of which form various aspects of the invention: an upper oil
phase, an intermediate aqueous fraction containing extractable
products, e.g., medicinal products, and a lower, detoxified cake,
which may be further processed into a food or feed composition. In
certain exemplary methods, the acidified aqueous solution that is
added to the crushed J. curcas is an acidified olive vegetation
water, that may be hydroxytyrosol-rich, having a pH preferably
between 1-5 and containing a ratio of hydroxytyrosol to oleuropein
of between 5:1 to 100:1. A suitable hydroxytyrosol-rich composition
is disclosed in co-owned U.S. U.S. Pat. No. 6,416,808, which is
incorporated herein in its entirety. Exemplary methods of obtaining
olive vegetation water are described in co-owned U.S. Pat. Nos.
6,165,475 and 6,197,308, each of which are expressly incorporated
herein by reference in their entirety. In certain embodiments and
examples disclosed herein, the olive vegetation water is
HIDROX.RTM. solution, an antioxidant solution prepared from
olives.
[0064] In a second general embodiment, a J. curcas component slurry
is first centrifuged to produce an upper oil fraction, an
intermediate aqueous fraction and a lower cake. This initial step
is preferably conducted under relatively neutral-pH conditions,
e.g., pH 5-8. The initial cake is then further treated by addition
of an acidified aqueous solution, e.g., the above acidified
hydroxytyrosol-rich olive vegetation water, to form an acidified
slurry, which is incubated as above, then centrifuged to form an
upper oil fraction, an intermediate aqueous fraction, and lower,
detoxified cake. The upper oil fraction may be combined with the
initial oil fraction, and the aqueous fraction may be combined with
the initial aqueous fraction. The aqueous fraction may be further
concentrated and/or used as a source of extractable medical or
other chemical components.
[0065] In a third general embodiment, an already formed J. curcas
cake is used as the starting material, and to this cake is added an
acidified aqueous solution, e.g., the above acidified
hydroxytyrosol-rich olive vegetation water, to form a cake slurry
which is incubated as above, then centrifuged to form an upper oil
fraction, an intermediate aqueous fraction, and a lower, detoxified
cake. In all of the preceding embodiments, the J. curcas components
may be the fruit, the seed, or an already formed cake of J.
curcas.
[0066] The presence of toxic/medicinal compounds in the aqueous
fraction has been confirmed by HPLC analysis. The toxicity of the
residual cake has been tested by animal toxicity studies conducted
by BioQuant, Inc. San Diego.
[0067] The aqueous extraction method has the advantage to:
[0068] (a) recover the residual oil trapped in the pressed
cake.
[0069] (b) extract and separate the toxic components present in the
cake which are either hydrolyzed and/or are highly hydrophilic, and
thus end up in the water fraction, and
[0070] c) render the solid fraction less or totally non-toxic as
confirmed by animal studies.
[0071] Thus, the cake become a very valuable food and feed
component which can be formulated in a variety of foods for human
and animals.
[0072] The aqueous fraction becomes a very valuable raw material
for further extraction and isolation of compounds of chemical and
pharmaceutical use, and can be further concentrated to reduce the
content in water. This can be easily accomplished by common steam
or vacuum evaporators generally used in the juice industry (orange
juice) as an example and then the water recycled for field
irrigation of other uses in water deficient areas of the world. By
performing the extraction of J. curcas with an acidified
antioxidant solution, the chemical compounds thereby extracted are
protected from decomposition during the extraction, storage and
concentration.
[0073] The concentrated juice can finally be sold as raw material
for the extraction and separation of valuable compounds for
medical, industrial and other uses based upon the active molecules
present in or isolated from the juice.
[0074] In one aspect, provided herein is a process for treating J.
curcas comprising:
[0075] (a) forming a mixture containing J. curcas components, with
addition of acid to a final pH of the mixture of between 1 and
5,
[0076] (b) incubating the mixture for a period of at least 1 hour,
and
[0077] (c) centrifuging the incubated mixture to separate the
mixture into three physically distinct fractions: (i) a light,
upper fraction containing oil, (ii) an aqueous fraction containing
soluble acid-extracted components and breakdown products, and (iii)
a substantially detoxified solid cake which forms or is used in
forming the food or feed composition.
[0078] In one embodiment, the process comprises the additional
step: repeating steps (a)-(c).
[0079] In another embodiment, the process comprises the additional
step: using the cake formed in step (c) as a food or feed
composition.
[0080] In another embodiment of the process, step (a) includes
crushing J. curcas components to form a slurry, and acidifying the
slurry to a pH of 1-5.
[0081] In another embodiment of the process, step (a) includes
acidifying the slurry by adding an acidified antioxidant solution.
In yet another embodiment, step (a) comprises adding an acidified
antioxidant solution before, during, or after crushing the J.
curcas components. In still another embodiment, the antioxidant
solution is olive vegetation water. In one embodiment, the olive
vegetation water comprises at least 0.1% (w/v) polyphenols. In
another embodiment, the olive vegetation water comprises 5-10%
(v/v) of an organic solvent.
[0082] In another embodiment of the process, step (a) includes
crushing J. curcas components to form a slurry, centrifuging the
slurry to separate the slurry into three physically distinct
fractions: (i) a light, upper fraction containing oil, (ii) an
aqueous fraction containing water-soluble components, and (iii) a
first cake, and forming a cake slurry by addition of an aqueous
acid solution to the first cake, to a pH of between 1 and 5. In
some embodiments of the process, the aqueous acid solution is an
antioxidant solution. In some embodiments, the antioxidant solution
is olive vegetation water.
[0083] In another embodiment of the process, the light upper oil
fraction from step (a) is combined with the light upper oil
fraction obtained in step (c).
[0084] In another embodiment of the process, the aqueous fraction
from step (a) is combined with the aqueous fraction obtained in
step (c).
[0085] In another embodiment of the process, the mixture formed in
step (a) has a final pH of 2-4.
[0086] In another embodiment of the process, the mixture formed in
step (a) is acidified by addition of a weak organic acid that
imparts a final pH of 2-4 to the slurry. In some embodiments, the
weak organic acid is citric acid.
[0087] In another embodiment of the process, the incubating step
(b) is carried out at room temperature for a period of at least one
day.
[0088] In another embodiment, the process further comprises
extracting soluble components from the aqueous fraction obtained in
step (c). In yet another embodiment, the process further comprises
concentrating the aqueous fraction by removal of water.
[0089] In another embodiment of the process, the olive vegetation
water comprises at least 0.1% (w/v) polyphenols. In yet another
embodiment, the olive vegetation water has a ratio of
hydroxytyrosol to oleuropein of between 5:1 to 100:1. In still
another embodiment, the olive vegetation water comprises 5-10%
(v/v) of an organic solvent.
[0090] In another embodiment of the process, the J. curcas
components are selected from the fruit, the seed, or an already
formed cake of J. curcas.
[0091] In another aspect, provided herein is a food or feed
composition prepared according to the preceding process, and
embodiments thereof.
[0092] In still another aspect, provided herein is an oil fraction
obtained according to the preceding process, and embodiments
thereof. In one embodiment, provided herein is the combined oil
fractions of steps (a) and (c).
[0093] In yet another aspect, provided herein is an aqueous
fraction obtained according to the preceding process, and
embodiments thereof. In one embodiment, provided herein is the
combined aqueous fractions of steps (a) and (c).
[0094] In one embodiment of the process, step (a) comprises:
(i) pressing J. curcas components to form a cake and oil and
aqueous fractions, and (ii) removing the oil and aqueous fractions,
and then adding an aqueous acid solution to the cake to form a
slurry having a final pH of between 1 and 5, and further comprising
the step of: isolating the aqueous fraction obtained in step (c).
In another embodiment, provided herein is the aqueous fraction
obtained according to the process. In one embodiment, provided
herein is the combined aqueous fractions of steps (a) and (c). In
another embodiment, the aqueous fraction or fractions are further
treated to extract medicinal compounds therefrom.
[0095] In another embodiment of the process, step (a)
comprises:
(i) pressing J. curcas components to form a cake and oil and
aqueous fractions, and (ii) removing the oil and aqueous fractions,
and then adding an aqueous acid solution to the cake to form a
slurry having a final pH of between 1 and 5, and further comprising
the step of: isolating the light upper oil fraction obtained in
step (c). In another embodiment, provided herein is the oil
fraction obtained according to the process. In one embodiment,
provided herein is the combined oil fractions of steps (a) and
(c).
[0096] In another aspect, provided herein is a method of extracting
compounds from J. curcas, comprising the preceding processes and
embodiments thereof. In one embodiment, the compounds are selected
from curcin and phorbol esters.
Experimental
[0097] I. Jathropa Curcas Processing from Seed
[0098] Procedure A: To 200 kg seeds, prior to crushing, add the
following solution A, made of 100 liters of 1% Citric Acid. Mix
thoroughly to have a loose slurry and pour the mix onto a grinding
machine. Grind mix into a wet pulp and pump slurry into kneading
tank. Stir for about 1 hour at 30.degree. C. Pump slurry into a
three phase decanter and separate the three components, Solid pulp,
oil and aqueous extract. Examine three components accordingly and
calculate yields in oil. Save the solid fraction in freezer, until
toxicity test is performed. Analyze aqueous fraction by HPLC.
[0099] Procedure B: To 200 kg seeds, prior to crushing, add the
following solution B, made of 100 liters of 0.5% polyphenols
extracted from the pulp of the olives in 1% citric acid. Mix
thoroughly to obtain a slurry and proceed as above.
[0100] Procedure C: 200 kg seeds are processed without any addition
of liquid. The solution A is added after the seeds are crushed into
a thick paste and pumped into a tank for 1 hr. kneading. Proceed
then as above in 1 and 2.
[0101] Procedure D: 200 kg seeds are processed without addition of
any liquid. The solution B is added after the seeds are crushed
into a thick paste and pumped into a tank for 1 hr. kneading.
Proceed then as above in 1 and 2.
[0102] Procedure E (Control experiment): One kilogram of seeds are
processed in a blender with addition of 500 ml water. The slurry is
left at room temperature for 1.5 hrs and then centrifuged to
separate liquid fraction from solid residue. Liquid is collected
separately and analyzed by HPLC. The samples are frozen until
further analysis is performed.
II. Processing from Solid Seed Cake
[0103] Procedure AI: To 200 kg dry cake add the following solution
A, made of 100 liters of 1% Citric Acid directly into kneading
tank. Stir for about 1.5 hour at 30.degree. C. Pump slurry into a
three-phase decanter and separate the three components: solid pulp,
crude oil and aqueous extract. Examine three components accordingly
and calculate yields in crude oil. Save the solid fraction in
freezer until toxicity test is performed. Analyze aqueous fraction
by HPLC.
[0104] Procedure BI: To 200 kg dry cake add the following solution
B. made of 100 liters of 0.5% polyphenols extracted from the pulp
of the olives in 1% citric acid. Mix thoroughly to obtain a slurry
in kneading tank for 1.5 hrs at 30.degree. C. and proceed as
above.
[0105] Procedure E2 (Control experiment): One kilogram of dry seed
cake is processed in a blender with addition of 500 ml water. The
slurry is left at room temperature for 1.5 hrs and then centrifuged
to separate liquid fraction from solid residue. Liquid is collected
separately and analyzed by HPLC. The samples are frozen until
further analysis is performed.
III. HPLC Jatropha Curca Processing and Detoxification.
[0106] I. HIDROX.RTM. 0.5% Liquid as antioxidant solution
containing olive polyphenols (e.g., hydroxytyrosol) was obtained
from Creagri, Inc. (Hayward, Calif.). The HPLC profile of
HIDROX.RTM. 0.5% liquid is characterized by the presence of a large
peak (RT=5 m) corresponding to hydroxytyrosol (HT) with a percent
area of approximately 40% of total UV absorbing materials (Total
Polyphenols, TP). A second small peak (RT=9.3 min.) corresponds to
tyrosol. The area is approximately 10% of the HT area, 4% of total
polyphenols (TP). The HPLC profile is then characterized by the
presence of late peaks (at least 4-5) that elute at high
concentration of methanol in Buffer A (RT from 19.5 m to 20.8 m).
These peaks correspond to oleuropein, verbascoside and their
aglycon derivatives, which contribute all together to 46-47% of the
TP. Total UV area=41.5 million units.
[0107] 2. Sample #1: Jatropha Curcas seeds (from Ghana) processed
in the presence of 1% citric acid solution: The peaks of these
chromatograms correspond to 100% compounds derived from the
Jatropha Curcas (JC) and soluble in water (hydrophilic fraction).
The front part of the spectrum is characterized by the presence of
a large peak (RT=2 m) representing ca. 16-17% of the total UV
areas, in a possible concentration of ca. 0.25% in weight of the
total compounds in the solution (as direct comparison with 0.5%
HIDROX.RTM. liquid). In addition, there are three additional peaks
of relevance: the first one elutes with RT=1.6 m (3.5%), the second
one with RT=2.4 m (3.8%) and the third one with RT=3.0 m (8.2%). A
second set of peaks (three detectable) elutes with RT between 19.2
m and 20.0 m with percent areas of 4.5%, 6.3% and 4.0%
respectively. Finally a third set of peaks (with two predominant
peaks at RT=21.5 m and 21.8 m) is visible with a total % area of
22% (11.5% and 11% respectively). Total UV area=15.5 million
units.
[0108] 3. Sample #2: Jatropha Curcas cake (from the same source in
Ghana) processed with HIDROX.RTM. 0.5% instead of 1% citric acid:
The spectrum should contain the total compounds of #1 and #2 in a
first approximation. The list of fast peaks eluting between RT=0
and RT=3.1 m include the large peak for JC (RT=2.0 m) which
represents 21.2% of the total UV absorbing material, the two peaks
at 5 m and 9.4 m (HT and Tyrosol (Ty) from HIDROX.RTM. 0.5%, the
first representing HT (15.6%) and the second at 9.5 m representing
Ty (1.7%). Also visible are the several peaks with low RT and high
RT. Total UV area=49 million units. Observations: The total
concentration of JC cake material in to HIDROX.RTM. 0.5% is
approximately 8 million units in a total of 49 million units, or
approximately 20%, assuming that the compounds in HIDROX.RTM. 0.5%
are neither consumed nor diluted. The increase percentage of the JC
peak at 2 m, (21.2%) vs. the HT peak area (15.6%), however seems to
indicate that more than 60-65% of the JC cake compounds contribute
to the total peak area of the extract. (Reduction of HT area from
37% to 15.6%, or 42% reduction). The Ty concentration is also
reduced from 3.64% to 1.76%, or 48% reduction). The 3 peaks from JC
cake are now present in 3.1%, 5.2% and 9.5%, which corresponds to
an increase of 73% and 86%.
[0109] 4. Sample #3: Jatropha Curcas seeds processed with
HIDROX.RTM. 0.5%: The HPLC profile shows the presence of both peaks
from HIDROX.RTM. 0.5% and JC. Specifically, from HIDROX.RTM. 0.5%,
is well visible the HT peak RT=5.1 m (23.4%) and the Ty peak RT=9.4
m (2.1%). From the JC we clearly detect the peak at RT=2.0 m (7%)
and the 3 additional peaks at RT=1.7 m (2.3%), RT=2.4 m (3%) and
RT=3 m (9.7%). Total area: 31.5 million units.
[0110] 5. Conclusions: Extraction with an acidified aqueous
solution or an aqueous EtOH (ethanol) solution (5%) seem to provide
similar results. The extraction with the above solutions may
results in detoxification of both the oil and the biomass in that:
[0111] (a) some of the compounds detected by HPLC analysis
correspond to phorbol esters (commercially available). [0112] (b)
the curcin (toxic protein) solubilizes in aqueous solutions. In
order to avoid oxidation of the above molecules in aqueous
solution, it is necessary to introduce an antioxidant component,
like hydroxytyrosol or other commercially available antioxidants.
The antioxidants will perform better if the aqueous solution is
acidified (citric acid or other organic and non-organic acids). The
pH we have used is ranging between 3.0 and 5.0. The detoxifying
solution (water/antioxidant/acid and possibly some percentage of
EtOH (5%) can be added to the Jatropha Curcas seeds prior to the
milling and separation of the oil from the biomass (cake), or can
be used on the dry cake to extract hydrophilic molecules and
detoxify the biomass. Citric acid alone does not seem to protect
from oxidation as the aqueous extract develops a strong odor after
two-three months of storage. Experiments conducted at laboratory
scale and pilot plant (200 kg seeds/cake) confirm the above. HPLC
analysis of samples of the resulting aqueous fraction indicate that
ca. 70-80% of the compounds in the solution derive from the
extraction process. Subsequent use of the dry biomass as feed for
fish has confirmed the lack of toxicity of it.
IV. Quantization of HT (Hydroxytyrosol) in Freeze Dried Olive Juice
by HPLC-Gradient
[0113] Equipment and Reagents:
[0114] HPLC grade methanol, ddH.sub.2O, acetic acid and HIDROX.RTM.
were used.
[0115] Standard Preparation:
[0116] Accurately dilute stock solution of standard (100 mg/2 ml
HT; Cayman Chemical) 1:3 with mobile phase (Solvent A) into a 2 ml
micro tube. Mix well. The working concentration of the standard is
1.67 mg/ml.
[0117] Sample Preparation:
[0118] Accurately weigh 100 mg+/-0.5 mg of sample and transfer to a
15 ml conical centrifuge tube. Add 10 ml of mobile phase (Solvent
A) to the sample and mix well. Sonicate for 5 minutes then transfer
1 ml of dissolved sample to a 2 ml micro tube. Centrifuge the 1 ml
sample at 11,000.times.g for 10 minutes. Remove all but the small
pellet on the bottom to a new 2 ml micro tube.
[0119] Instrument Conditions: [0120] Mobile Phase: (Solvent A):
HPLC Grade ddH.sub.2O with 5% HPLC Grade Methanol and 3% HPLC Grade
Acetic Acid (pH 2.7-2.8). (Solvent B): 100% HPLC Grade Methanol
[0121] Flow Rate: 1.0 ml/min [0122] Gradient: Solvent A
(95.5%)/Solvent B (0.5%) isocratic for 20 min, then Solvent B
0.5-100% in 15 min. [0123] Wavelength: OD 280 mm [0124] Injection
Volume: 20 .mu.l [0125] Column: Beckman Coulter Ultrasphere RP-C18
[4.6.times.150 mm] [0126] Temperature: Column 20.degree.
C.+/-2.degree. C. [0127] Approximate Retention Times: [0128] HT
--5.9 minutes [0129] Tyrosol--11.5 minutes
[0130] Procedure:
[0131] Mix 920 ml of HPLC Grade ddH.sub.2O with 50 ml HPLC Grade
Methanol and 30 ml HPLC Grade Acetic Acid "Solvent A"). Filter
Solvent A with vacuum using a 0.45 micron Nalgene Filter. Condition
the analytical column for 30 minutes before beginning
calibration.
[0132] System Suitability:
[0133] Prepare a standard solution by thawing (from -20.degree. C.
freezer) a stock HIDROX.RTM. solution (1.67 mg/ml). Once thawed,
the standard is discarded. Inject the standard solution to
demonstrate presence of HT, retention time, peak area, peak height,
and plate number. Inject the standard solution 4 times to calibrate
and establish the precision of the chromatographic system. Compute
the relative standard deviation (% rsd) of the peak areas for HT.
The system is considered suitable for assay if the % rsd of the
four standard injections is <2%. As a further guide in assessing
column performance, the column should develop .about.9000
theoretical plates and the tailing factor should be less than 1.5.
At the completion of the analysis, inject the standard solution as
a calibration check. The calibration check should be +/-2% of the
expected concentration.
[0134] Calculation:
[0135] The concentration of HT is calculated as follows:
Asp/As.times.S.times.p.times.V.times.Ws=mg/g, wherein: [0136]
Asp=Area of sample peak [0137] As=Area of standard peak [0138]
S=working standard concentration in mg/ml [0139] P=purity of
standard [0140] V=Sample Volume [0141] Ws=Sample Weight
* * * * *