Note: Descriptions are shown in the official language in which they were submitted.
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B&P File No. 9369-130/MG
Title: OIL BODY BASED PERSONAL CARE PRODUCTS
FIELD OF THE INVENTION
T'he present invention provides novel emulsions which
comprise oil bodies. The invention also provides a method for preparing
the emulsions. and the use of the emulsions in various domestic and
industrial corr~positions.
BACKGROUND OF THE INVENTION
Emulsions are mixtures which are prepared from two
mutually insoluble components. It is possible to generate mixtures of
homogenous macroscopic appearance from these components through
proper selection and manipulation of mixing conditions. The most
common type of emulsions are those in which an aqueous component
and a lipophi.lic component are employed and which in the art are
frequently referred to as oil-in-water and water-in-oil emulsions. In oil-
in-water emulsions the lipophilic phase is dispersed in the aqueous
phase, while in water-in-oil emulsions the aqueous phase is dispersed in
the lipophilic phase. Commonly known domestic examples of
emulsion-based formulations include mayonnaise, margarine, ice cream,
cosmetics and paint. Emulsion systems are also extensively applied in
industries such as the pharmaceutical and the agrochemical industries,
where it is often desirable to formulate active ingredients in emulsions.
Generally emulsions are prepared in the presence of a
multiplicity of other substances in order to achieve a desirable balance of
emulsification; viscosity, stability and appearance. For example, the
formulation of emulsions usually requires at least one, and frequently a
combination of several, emulsifying agents. These agents facilitate the
dispersal of one immiscable phase into the other and assist in stabilizing
the emulsion. Emulsifiers comprise a wide variety of synthetic and
natural components. For example, monoglycerides and chemical
derivatives thereof, are widely used as emulsifiers in food applications
such as margarines and baked products. An example of a natural
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emulsifier is lecithin, a phospholipid present in egg yolk, which is
commonly used in the preparation of mayonnaise. It is also possible to
entrap active ingredients in emulsions. This is especially desirable in
compositions comprising active agents which are difficult to dissolve in
aqueous solui:ions, such as certain vitamins and nucleotides. Active
ingredients are also frequently formulated as emulsions in order to
enhance their stability. One example of an emulsion system comprising
a pharmaceutical agent is documented in US patent 5,602,183 which
discloses a wound healing composition containing an anti-inflammatory
agent. The foregoing exemplifies only a few of the myriad of
components ~-hich are included in formulations of emulsions known in
the art. A comprehensive overview of emulsifying agents and their
applications may be found in Becher, P. Encyclopedia of Emulsion
Technology, D~ekker Ed., 1983.
l:n the seeds of oilseed crops, which include economically
important crops, such as soybean, rapeseed, sunflower and palm, the
water insoluble oil fraction is stored in discrete subcellular structures
variously known in the art as oil bodies, oleosomes, lipid bodies or
spherosomes (Huang 1992, Ann. Rev. Plant Mol. Biol. 43: 177-200).
Besides a mixture of oils (triacylglycerides), which chemically are defined
as glycerol esters of fatty acids, oil bodies comprise phospholipids and a
number of associated proteins, collectively termed oil body proteins.
From a structural point of view, oil bodies are considered to be a
triacylglyceride matrix encapsulated by a monolayer of phospholipids in
which oil body proteins are embedded (Huang, 1992, Ann. Rev. Plant
Mol. Biol. 43: 177-200). The seed oil present in the oil body fraction of
plant species is a mixture of various triacylglycerides, of which the exact
composition depends on the plant species from which the oil is derived.
It has become possible through a combination of classical breeding and
genetic engineering techniques, to manipulate the oil profile of seeds and
expand on the naturally available repertoire of plant oil compositions.
For an overview of the ongoing efforts in his area, see Designer Oil
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Crops/Breeding, Processing and Biotechnology, D. J. Murphy Ed., 1994,
VCH Verlags~;esellschaft, Weinheim, Germany.
Plant seed oils are used in a variety of industrial
applications, notably in the food, detergent and cosmetics industries. In
order to obtain the plant oils used in these applications, seeds are crushed
or pressed and subsequently refined using processes such as organic
extraction, de~;umming, neutralization, bleaching and filtering. Aqueous
extraction of plant oil seeds has also been documented (for example,
Embong and (elen, 1977, Can. Inst. Food Sci. Technol. J. 10: 239 - 243).
Since the objective of the processes taught by the prior art is to obtain
pure oil, oil bodies in the course of these production processes lose their
structural inte;~rity. Thus, the prior art emulsions formulated from plant
oils do not generally comprise intact oil bodies.
Although there are many applications where mineral oil
based products dominate the market, in other applications, oils derived
from plant sources and fossil sources are in direct competition. Lauric
oils, for example, which are widely used in the manufacture of
detergents, are obtained from mineral oil as well as from coconut oil and
more recently from genetically engineered rapeseed (Knauf, V. C., 1994,
Fat. Sci. Techn. 96: 408). However, there is currently an increasing
demand for biodegradable sources of raw materials. The plant oil body
based emulsions of the present invention offer an advantage over
similar mineral oil based formulations, in that the oil fraction is derived
from a renewable and environmentally friendly source.
United States patents 5,683,740 and 5,613,583 disclose
emulsions comprising lipid vesicles that have been prepared from
crushed oleagenous plant seeds. In the course of the crushing process, oil
bodies substantially lose their structural integrity. Accordingly, these
patents disclose that in the crushing process, 70% to 90% of the seed oil is
released in the form of free oil. Thus the emulsions which are the
subject matter of these patents are prepared from crushed seeds from
which a substantial amount of free oil has been released while the
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structural integrity of the oil bodies is substantially lost. In addition, the
emulsions disclosed in both of these patents are prepared from relatively
crude seed extracts and comprise numerous endogenous seed
components including glycosylated and non-glycosylated non-oil body
seed proteins. It is a disadvantage of the emulsions to which these
patents relate that they comprise contaminating seed components
imparting a variety of undesirable properties, which may include
allergenicity and undesirable odour, flavour, colour and organoleptic
characteristics, to the emulsions. Due to the presence of seed
contaminants, the emulsions disclosed in these patents have limited
applications.
SUMMARY OF THE INVENTION
T'he present invention relates to novel emulsion
formulations which contain oil bodies. The emulsion formulations of
the subject invention are obtainable in non-toxic and food grade forms.
In addition, the emulsion formulations are advantageously prepared
from an oil body preparation which is creamy in texture and thus may be
readily applied in a variety of domestic and industrial applications. The
present inventors have found that the oil body fraction of living cells is
useful in the formulation of a variety of novel emulsion-based food,
personal care, pharmaceutical and industrial products. Broadly stated,
the present invention provides an emulsion formulation comprising
washed oil bodies derived from a cell.
The invention also provides methods for preparing the
emulsion formulations and the use of the emulsion formulations in
various domestic and industrial compositions.
Accordingly, the present invention provides a method for
preparing emulsion formulations comprising: 1) obtaining oil bodies
from a cell; 2) washing the oil bodies; and 3) formulating the washed oil
bodies into an emulsion.
In a preferred embodiment of the invention, the washed oil
body preparation is obtained from plant seeds, including seeds obtainable
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from flax, ~;afflower, rapeseed, soybean, maize and sunflower.
Accordingly, the invention provides a method for preparing the
emulsion formulations from plant seeds comprising:
(a) grinding plant seeds to obtain ground seeds comprising
substantially intact oil bodies;
(b) removing solids from the ground seeds;
(c) separating the oil body phase from the aqueous phase;
(d) washing the oil body phase to yield a washed oil body
preparation;
(e) formulating the washed oil body preparation into an
emulsion.
In a preferred embodiment of th.e invention, a liquid phase
is added to the seeds prior to or while grinding the seeds.
In a further preferred embodiment of the invention,
formulating th.e emulsion comprises adding a liquid phase to the washed
oil body preparation.
The emulsions of the present invention can be used in a
wide range of applications including in the preparation of food and feed
products, pharmaceutical products, personal care products, and industrial
products. T:he emulsion formulation of the present invention is
especially suited for the preparation of food-grade products as it is non-
toxic, creamy in texture and biodegradable.
Additional objects, advantages and features of the present
invention will become apparent after consideration of the accompanying
drawings and the following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a Coomassie blue stained gel of a washed oil body
preparation from white mustard, rapeseed (Brassica napus), soybean,
peanut, squasr~, flax, sunflower, safflower and maize.
Figure 2A-C are Coomassie blue stained gels showing the
protein profiles of various seed fractions obtained from Brassica napus
(Canola) (A), sunflower (B), and maize (C). The gels show the following
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fractions (1) total seed protein (TSP), (2) decanted liquid phase (DL), (3)
unwashed oil bodies (LP1), (4) three washes with water (LP4), (5) four
washes with v~~ater and one wash with 100 mM Na2C03 (Washed).
DETAILED DESCRIPTION OF THE INVENTION
As hereinbefore mentioned, the present invention relates to
emulsion forrrmlations comprising oil bodies derived from a cell. In one
embodiment, the present invention provides an emulsion formulation
comprising washed oil bodies.
In another embodiment, the present invention provides a
method for preparing an emulsion formulation comprising: 1) obtaining
oil bodies from a cell; 2) washing the oil bodies; and 3) formulating the
washed oil bodies into an emulsion.
The cell can be any cell that contains oil bodies (or oil-body
like structures) including plant cells, animal cells, fungal cells and
bacterial cells. In a preferred embodiment of the invention the oil bodies
are obtained from a plant cell. The oil bodies may be obtained from a
plant cell by rupturing the plant cell membrane and cell wall using any
method which releases the cells constituents without substantially
compromising the structural integrity of the oil bodies. More preferably,
the oil bodies are obtained from plant seeds. Accordingly, the present
invention further provides a method for preparing an emulsion
formulation comprising:
(1) obtaining oil bodies from plant seeds by a method that
comprises:
(a) grinding plant seeds to obtain ground seeds
comprising substantially intact oil bodies;
(b) removing solids from the ground seeds; and
(c) separating the oil body phase from the aqueous
phase;
(2) washing the oil body phase to yield a washed oil body
preparation; and
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(3) formulating the washed oil body preparation into an
emulsion.
In a preferred embodiment of the invention, a liquid phase
is added to the seeds prior to or while grinding the seeds.
In a further preferred embodiment of the invention,
formulating the emulsion comprises adding a liquid phase to the washed
oil body preparation.
T'he term "grinding" as used herein means milling,
crushing, chopping or granulating the seeds and these terms may be used
interchangeably throughout this application. In the process, the seed
cells are broken open while the oil bodies remain substantially intact.
The term "substantially intact" as used herein means that the oil bodies
have not released greater than 50% (v/v) of their total seed oil content in
the form of free oil. Preferably, grinding of th.e seeds results in release of
less than about 50% (v/v) of the total seed oil content in the form of free
oil, more preferably less than about 20% (v/v) and most preferably less
than about 10°0 (v/v).
The term "solids" as used herein means any material that is
not soluble in the aqueous phase or in the oil body phase, such as seed
hulls.
The term "washing the oil bodies" as used herein means
any process that removes cellular contaminants from the oil body phase,
in particular any contaminant which imparts undesirable properties to
the emulsion formulation, such as allergenic properties, undesirable
colour, odour', flavour or organoleptic characteristics or any other
undesirable property. Examples of methods of washing include
gravitation based separation methods such as centrifugation and size
exclusion based separation techniques such as membrane ultrafiltration
and crossflow microfiltration. Washing methods and conditions are
30 selected in accordance with the desired purity of the oil body preparation.
T:he term "washed oil body preparation" as used herein
means a prep<~ration of oil bodies from which a significant amount of
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cellular material has been removed including contaminants which
impart undesirable properties to the emulsion formulation, such as
allergenic properties, undesirable colour, odour, taste or organoleptic
characteristics or any other undesirable property. Preferably, the washed
oil body preparation contains less than about 75% (w/w) of all
endogenously present non-oil body seed proteins, more preferably the
washed oil body preparation contains less than about 50% (w/w) of
endogenously present non-oil body seed proteins and most preferably
less than about 10%(w/w) of endogenously present non-oil body seed
proteins.
By "formulating the oil bodies into an emulsion", it is
meant that the washed oil body preparation is mixed or homogenized, if
necessary, until an emulsion is formed. In a preferred embodiment, an
additional ingredient is added, such as a liquid phase, and the washed oil
body preparation and the liquid phase are mixed until a homogenous
mixture is attained.
The washed oil body preparations are particularly suitable
for the formulation of emulsions due to advantageous properties
outlined below.
PROPERTIES OF THE OIL BODIES
The emulsion formulations of the present invention
comprise intact washed oil bodies of approximately uniform size, shape
and density. When viewed under the electron microscope, oil bodies are
found to be more or less spherically shaped structures (see: Example
Murphy, D. j. and Cummins L, 1989, Phytochemistry, 28: 2063-2069; Jacks,
T. J. et al., 1990, JAOCS, 67: 353-361). Typical sizes of oil bodies vary
between 0.4~.m for and 1.5~,m (Murphy, D. J. and Cummins L, 1989,
Phytochemistry, 28: 2063-2069). When analyzed using a Malvern Size
Analyzer, it was found that oil bodies in a washed oil body preparation
isolated from rapeseed were symmetrically and unimodally distributed
around l ~ m. Using a Malvern Size Analyzer a washed oil body
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preparation could be clearly distinguished from commercially obtainable
oil-in-water emulsions including soymilk, mayonnaise (Kraft Real
Mayonnaise) and two coconut milk preparations (Tosca, Aroy-D). The
exact size and density of the oil bodies depends at least in part on the
precise proteiin/phospholipid/triacylglyceride composition which is
present. Preparing washed oil bodies according to the present invention
does not result: in a substantive alteration in the shape of the oil bodies in
comparison with those present in whole seed when viewed under the
electron microscope.
Upon breaking open a cell containing oil bodies, the oil body
fraction may he rapidly and simply separated from aqueous solutions
since in aqueous solutions the oil body fraction will float upon
application of centrifugal force. In solutions, where the density of the oil
body fraction its greater than that of the solvent, such as 95% ethanol, the
oil bodies will sediment under the same conditions. The oil body
fraction may also be separated from the aqueous fraction through size-
exclusion based separation techniques, such as membrane filtration,
which may be advantageous in that more uniformly sized oil bodies may
be acquired.
The oil bodies present in the washed oil body preparations
of the present invention are resistant to exposure to strong acids and
bases, including prolonged exposure to acidic conditions at least as low as
pH 2 and alkaline conditions at least as high as pH 10. When exposed to
pH 12, a slight loss of oil was observed, indicating a loss of integrity of
the
oil body structure. In addition, extraction with various organic solutions,
including methanol, ethanol, hexane, isopropyl alcohol and ethyl acetate,
does not or only slightly compromise the integrity of the oil bodies
present in the washed oil body preparation. T'he oil bodies present in the
washed oil body preparation were also found to withstand mixing with
the anionic detergent, sodium dodecyl sulfate (SDS), the cationic,
detergent he~:adecyl trimethyl bromide and Tween-80, a non-ionic
detergent. Boiling of the washed oil body preparation in the presence of
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SDS was found to result at least partly in disintegration of the oil body
structure. The oil bodies present in the washed oil body preparation are
stable when maintained for 2 hours up to at least 100°C. A slow freeze
and thaw of washed oil body preparations resulted in a change in their
physical appearance characterized by the formation of clumps as opposed
to a homogeneous emulsion. Oil body clumping following a freeze-thaw
could also be prevented to a large degree by either a) flash freezing in
liquid nitrogen instead of slow freezing at -20°C or b) adding glycerol
in
excess of 5% (v/v) to the oil body preparation prior to freezing. The
resistance to relatively harsh chemical and physical conditions, is a
unique characteristic of the oil bodies present in the washed oil body
preparation of the subject invention.
The present invention provides emulsion formulations
comprising oil bodies from which a significant amount of seed
contaminants have been removed. These contaminants include
proteins, volatiles and other compounds which may impart undesirable
colour, odour, flavour, organoleptic characteristics or other undesirable
characteristics. A number of seed proteins have been reported to cause
allergenic reactions. For example, Ogawa et al. (1993, Biosci. Biotechnol.
Biochem., 57:1030-1033) report allergenicity of the soybean glycoprotein
P34 (alternatively referred to as Gly m Bd 30K). Allergenic reactions
against rapeseed, wheat and barley seed proteins have also been reported
(Armentia et al., 1993., Clin. Exp. Allergy 23: 410-415; Monsalve et al.,
1993, Clin. Exp. Allergy 27: 833 - 841). Hence removal of contaminating
seed proteins is advantageous. Washing conditions may be selected such
that a substantially pure oil body preparation is obtained. In that case,
only the oil body proteins are substantially present in the preparation.
For many applications, it is also considered desirable that a
purer better defined oil body preparation is obtained, as this allows more
control over the formulation process of the final emulsion. In order for
the washed oil body preparation to be included in a diverse set of
emulsions it is desirable that volatiles are kept to a minimum and the
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colour is preferably light or white. Washing of the oil body preparation
results in a lighter coloured preparation. In addition, a substantial
amount of volatiles is removed. Also removed by washing are
compounds which promote the growth of microorganisms as it was
observed that a washed oil body preparation had a longer shelf life than
an unwashed preparation. Other compounds which are removed by
washing include anti-nutritional glucosinilates and/or breakdown
products thereof and fibrous material. When heat treated to 60°C or
80°C, it was observed that larger quantities of water remained absorbed
by
the washed oil body preparation when compared with an unwashed
preparation. Upon cooling down to room temperature and
centrifugation,, it was observed that the washed oil body preparation
remained stable, while phase separation occurred in the unwashed
preparation. Given the enhanced stability of washed oil bodies, they are
preferred where the formulation process involves the application of
heat. When heated to 40°C, the washed oil body preparation was able to
absorb a larger quantity of exogenously added water without resulting in
phase separation. Thus in the formulation of aqueous emulsions,
washed oil bodies are preferred. The capacity to absorb exogenously
added oils was. also compared between a preparation of washed oil bodies
and an unwashed preparation. Larger amounts of exogenous oil could be
added to the ~Nashed oil body preparation before an unstable emulsion
was formed. This is advantageous in formulations where exogenous oils
or waxes are added in the formulation process such as where lubricants
or personal care products are prepared. When viscosity was compared
between a washed oil body preparation and an unwashed preparation it
was found that the washed preparation was more viscous. A more
viscous preparation of oil bodies is desirable as this eliminates the need
for the addition of thickening agents in the formulation process.
Tlhus the washed oil body preparation provided here is
superior to an unwashed preparation in many respects. The washed oil
body preparation of the present invention is a better defined preparation
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with a longer shelf life and more preferable colour, odour and viscosity
characteristics.. The washed oil body preparation also has superior water
and oil absorption characteristics. Finally due to the removal of a
significant amount of seed proteins, allergenic reactions are less likely to
occur. ThesE~ characteristics allow the use of the washed oil body
preparation in the formulation of a variety of domestic and industrial
emulsions.
The above observations were made using washed and
unwashed oil body preparations obtained from rapeseed and prepared as
detailed in Example 2 of the present application. It is believed that
resistance to relatively harsh chemical and physical conditions will be a
characteristic of the oil bodies present in the washed oil preparation of
the subject invention regardless of the source of the oil bodies. However
one or more of the hereinbefore documented properties for rapeseed oil
bodies may vary depending on the cells from which the washed oil
bodies preparation is obtained. Nevertheless it is to be clearly understood
that the subject invention is drawn to an oil body preparation which may
be obtained from any cell comprising oil bodies.
In one embodiment of the present invention, the oil bodies
are obtained from plant seeds. The presence of intact oil bodies in the
emulsion and the described characteristics of these oil bodies clearly
distinguish the subject emulsion formulation from other materials
which may be prepared from plant seeds.
SOURCES AND PREPARATION OF THE OIL BODIES
T:he washed oil body preparation of the subject may be
obtained from any cell containing oil bodies or oil body-like organelles.
This includes animal cells, plant cells, fungal cells, yeast cells (Leber, R.
et
al., 1994, Yeast 10: 1421-1428), bacterial cells (Pieper-Fiirst et al., 1994,
J.
Bacterol. 176: 4328 - 4337) and algae cells (Rossler, P.G., 1988, J. Physiol.
(London) 24: 394-400). In preferred embodiments of the invention the oil
bodies are obtained from a plant cell which includes cells from pollens,
spores, seed and vegetative plant organs in which oil bodies or oil body-
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like organelles. are present (Huang, 1992, Ann. Rev. Plant Physiol. 43: 177-
200). More preferably, the washed oil body preparation of the subject
invention is obtained from a plant seed and most preferably from the
group of plant species comprising: rapeseed (Brassica spp.), soybean
(Glycine max), sunflower (Helianthus annuus), oil palm (Elaeis
guineeis), cotfionseed (Gossypium spp.), groundnut (Arachis hypogaea),
coconut (focus nucifera), castor (Ricinus communis), safflower
(Carthamus ~'inctorius), mustard (Brassica spp. and Sinapis alba),
coriander (C.'oriandrum sativum), squash (Cucurbita maxima),
linseed/flax (Linum usitatissimum), Brazil nut (Bertholletia excelsa).
jojoba (Simmondsia chinensis) and maize (Zea mat's). Plants are grown
and allowed to set seed using agricultural cultivation practises well
known to a parson skilled in the art. After harvesting the seed and if
desired removal of material such as stones or seed hulls (dehulling), by
for example sieving or rinsing, and optionally drying of the seed, the
seeds are subsequently processed by mechanical pressing, grinding or
crushing. In a preferred embodiment, a liquid phase is added prior to or
while grinding; the seeds. This is known as wet milling. Preferably the
liquid is water although organic solvents such as ethanol may also be
used. Wet milling in oil extraction processes has been reported for seeds
from a variety of plant species including: mustard (Aguilar et al 1990,
Journal of Texture studies 22:59-84), soybean (US Patent 3,971,856; Carter
et al., 1974, J. Am. Oil Chem. Soc. 51:137-141), peanut (US Patent 4,025,658;
US Patent 4,362,759), cottonseed (Lawhon et al., 1977, J. Am. Oil, Chem.
Soc. 63:533-534) and coconut (Kumar et al., 1995, INFORM 6 (11):1217-
1240). It may .also be advantageous to imbibe the seeds for a time period
from about fifteen minutes to about two days in a liquid phase prior
grinding. Imbibing may soften the cell walls and facilitate the grinding
process. Imbibition for longer time periods may mimic the germination
process and result in certain advantageous alterations in the composition
of the seed constituents. Preferably the added liquid phase is water.
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T'he seeds are preferably ground using a colloid mill, such as
the MZ130 (:Fryma Inc.). Besides colloid mills, other milling and
grinding equipment capable of processing industrial scale quantities of
seed may also be employed in the here described invention including:
flaking rolls, disk mills, colloid mills, pin mills, orbital mills, IKA mills
and industrial scale homogenizers. The selection of the mill may depend
on the seed throughput requirements as well as on the source of the seed
which is employed. It is of critical importance that seed oil bodies remain
substantially intact during the grinding process. Grinding of the seeds
therefore results in the release of preferably less than about 50% (v/v) of
the total seed oil content in the form of free oil, more preferably less than
about 20% (v/v) and most preferably less than about 10% (w/w). Any
operating conditions commonly employed in oil seed processing, which
tend to disrupt oil bodies are unsuitable for use in the process of the
subject invention. Milling temperatures are preferably between 10°C and
90°C and more preferably between 26°C and 30°C, while the
pH is
preferably maintained between 2.0 and 10.
Solid contaminants, such as seed hulls, fibrous material,
undissolved carbohydrates and proteins and other insoluble
contaminants, are removed from the crushed seed fraction. Separation
of solid contaminants, may be accomplished using a decantation
centrifuge, such as a HASCO 200 2-phase decantation centrifuge or a
NX310B (Alpha Laval). Depending on the seed throughput
requirements, the capacity of the decantation centrifuge may be varied by
using other models of decantation centrifuges, such as 3-phase decanters.
Operating conditions vary depending on the particular centrifuge which
is employed and must be adjusted so that insoluble contaminating
materials sediment and remain sedimented upon decantation. A partial
separation of the oil body phase and liquid phase may be observed under
these conditions.
Following the removal of insoluble contaminants, the oil
body phase i;> separated from the aqueous phase. In a preferred
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embodiment of the invention a tubular bowl centrifuge is employed. In
other embodiments, hydrocyclones, disc stack centrifuges, or settling of
phases under natural gravitation or any other gravity based separation
method may lbe employed. It is also possible to separate the oil body
fraction from the aqueous phase employing size exclusion methods, such
as membrane ultrafiltration and crossflow microfiltration. In preferred
embodiments the tubular bowl centrifuge is a Sharpies model AS-16
(Alpha Laval) or a AS-46 Sharpies (Alpha Laval). A critical parameter is
the size of the ring dam used to operate the centrifuge. Ring dams are
removable rings with a central circular opening varying, in the case of
the AS-16, from 28 to 36 mm and regulate the separation of the aqueous
phase from the oil body phase thus governing the purity of the oil body
fraction which is obtained. In preferred embodiments, a ring dam size of
29 or 30 mm is employed when using the .AS-16. The exact ring dam size
employed depends on the type of oil seed which is used as well as on the
desired final consistency of the oil body preparation. The efficiency of
separation is further affected by the flow rate. Where the AS-16 is used
flow rates are typically between 750-1000 ml/min (ring dam size 29) or
between 400-n00 ml/min (ring dam size 30) and temperatures are
preferably maintained between 26°C and 30°C. Depending on the
model
centrifuge used, flow rates and ring dam sizes must be adjusted so that an
optimal separ;~tion of the oil body fraction from the aqueous phase is
achieved. These adjustments will be readily apparent to a skilled artisan.
Separation of solids and separation of the aqueous phase
from the oil body fraction may also be carried out concomitantly using a
gravity based separation method such as 3-phase tubular bowl centrifuge
or a decanter or a hydrocyclone or a size exclusion based separation
method.
T:he compositions obtained at this stage in the process,
generally are relatively crude and comprise numerous endogenous seed
proteins, which includes glycosylated and non-glycosylated proteins and
other contaminants such as starch or glucosinilates or breakdown
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products thereof. The present invention comprises the removal of a
significant arrtount of seed contaminants. 'To accomplish removal of
contaminating; seed material, the oil body preparation obtained upon
separation from the aqueous phase is washed at least once by
resuspending the oil body fraction and centrifuging the resuspended
fraction. This process yields what for the purpose of this application is
referred to as a washed oil body preparation. The number of washes will
generally depend on the desired purity of the oil body fraction.
Depending on the washing conditions which are employed, an
essentially pure oil body preparation may be obtained. In such a
preparation the only proteins present would be .oil body proteins. In
order to wash the oil body fraction, tubular bowl centrifuges or other
centrifuges such hydrocyclones or disc staclG centrifuges may be used.
Washing of oil. bodies may be performed using water, buffer systems, for
example, sodium chloride in concentrations between 0.01 M and at least 2
M, 0.1 M sodium carbonate at high pH (11-12), low salt buffer, such as 50
mM Tris-HC1 pH 7.5, organic solvents, detergents or any other liquid
phase. In pre:Ferred embodiments the washes are performed at high pH
(11-12). The liquid phase used for washing as well as the washing
conditions, such as the pH and temperature, rrtay be varied depending on
the type of seed which is used. Washing at a number of different pH's
between pH 2 and pH 11-12 may be beneficial as this will allow the step-
wise removal of contaminants, in particular proteins. Preferably
washing conditions are selected such that the washed oil body
preparation comprises less than about 75%(w/w) of all endogenously
present non-oil body seed proteins, more preferably less than about 50%
(w/w) of endogenously present non-oil body seed proteins and most
preferably less than about 10% (w/w) of endogenously present non-oil
body proteins. Washing conditions are selected such that the washing
step results in the removal of a significant amount of contaminants
without compromising the structural integrity of the oil bodies. In
embodiments where more than one washing step is carried out, washing
CA 02290278 1999-11-24
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conditions may vary for different washing steps. SDS gel electrophoresis
or other analytical techniques may conveniently be used to monitor the
removal of endogenous seed proteins and other contaminants upon
washing of the oil bodies. It is not necessary to remove all of the aqueous
phase between washing steps and the final washed oil body preparation
may be suspended in water, a buffer system, for example, 50 mM Tris-HC1
pH 7.5, or any other liquid phase and if so desired the pH may be adjusted
to any pH between pH 2 and pH 10.
The process to manufacture the washed oil body preparation
may be performed in batch operations or in a continuous flow process.
Particularly when tubular bowl centrifuges are used, a system of pumps
operating between steps (a) and (b), (b) and (c), and (c) and (d) a
continuous flow throughout the processing system is generated. In a
preferred embodiment, the pumps are 1 inch MZ Wilden air operated
double diaphragm pumps. In other embodiments, pumps, such as
hydraulic or peristaltic pumps may be employed. In order to maintain a
supply of homogenous consistency to the decantation centrifuge and to
the tubular bowl centrifuge, homogenizers, such as an IKA homogenizer
may be added between the separation steps. In-line homogenizers may
also be added. in between various centrifuges or size exclusion based
separation equipment employed to wash the oil body preparations. Ring
dam sizes, buffer compositions, temperature and pH may differ in each
washing step from the ring dam size employed in the first separation
step.
In embodiments of the invention where the oil bodies are
isolated from softer tissues, for example the mesocarp tissue of olives, the
techniques applied to break open the cell may vary somewhat from those
used to break harder seeds. For example, pressure-based techniques may
be preferred aver crushing techniques. The methodology to isolate oil
bodies on a small scale has been reported for isolation of oil bodies from
mesocarp tissues in olive (Oleo europaea) and avocado (Persea
americana) (boss et al., Plant Science, 1993, 93: 203-210) and from
CA 02290278 1999-11-24
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microspore-derived embryos of rapeseed (Brassica napus) (Holbrook et
al., Plant Physiol., 1991, 97: 1051-1058).
In embodiments of the invention where oil bodies are
obtained from non-plant cells, the washed oil body preparation is isolated
following similar procedures as outlined above. The methodology to
isolate oil bodies from yeast has been documented (Ting et al., 1997,
Journal Biol. C'hem. 272:3699-3706).
The chemical and physical properties of the oil fraction may
be varied in at least two ways. Firstly, different plant species contain oil
bodies with different oil compositions. For example, coconut is rich in
lauric oils (C12), while erucic acid oils (C22) are abundantly present in
some Brassica spp. Secondly, the relative amounts of oils may be
modified within a particular plant species by applying breeding and
genetic engineering techniques known to the skilled artisan. Both of
these techniques aim at altering the relative activities of enzymes
controlling the metabolic pathways involved in oil synthesis. Through
the application of these techniques, seeds with a sophisticated set of
different oils are obtainable. For example, breeding efforts have resulted
in the development of a rapeseed with a low erucic acid content (Canola)
(Bestor, T. H., 1994, Dev. Genet. 15: 458) and plant lines with oils with
alterations in the position and number of double bonds, variation in fatty
acid chain length and the introduction of desirable functional groups
have been generated through genetic engineering (Topfer et al., 1995,
Science, 268: 681-685). Using similar approaches a person skilled in the
art will be able to further expand on the presently available sources of oil
bodies. Variant oil compositions will result in variant physical and
chemical properties of the oil bodies. Thus by selecting oilseeds or
mixtures thereof from different species or plant lines as a source for oil
bodies, a broad repertoire of emulsions with different textures and
viscosities may be acquired.
CA 02290278 1999-11-24
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FORMULATL~1G THE EMULSION
The washed oil body preparation may be formulated into an
emulsion using techniques known in the art. Preferably, at least one
additional ingredient is added to the washed oil body preparation. The
additional ingredient may be added as a solution, suspension, a gel or
solid and qu<~ntities of the additional ingredient will depend on the
formulation. The additional ingredient may upon formulation become
associated with the oil bodies for example by the formation of non-
covalent bonds with the oil body, remain suspended in solution, or form
a suspension i:n which the oil bodies are dispersed. The ingredient may
also penetrate the phospholipid monolayer surrounding the oil body or
the triacylglyceride matrix. Ingredients which may penetrate the oil body
include oils, waxes and the colorant Nile Red. In a preferred
embodiment, the additional ingredient is a liquid phase. In a further
preferred embodiment the liquid phase is water. Water may be added
either directly or through moisture associated with another ingredient.
The final amount of water is not critical, as long as upon mixing of the
ingredients, a stable emulsion is formed. Generally, the compositions
will contain at least 1% of water and up to 99% water. Usually mixing
will be required to provide an adequate emulsion and it may be necessary
to apply heat or pressure.
In another preferred embodiment the additional ingredient
is an oil or a wax. Oils or waxes may partition to the triacyl glyceride
matrix of the oil bodies and in this manner lipid soluble ingredients,
such as lipid soluble vitamins may be delivered to the oil body matrix.
Where oils or waxes comprise the added ingredient, the oil bodies may
remain suspended in the lipophilic phase or double emulsions may be
formed.
The final compositions may be in solid or in liquid form or
of any other desired viscosity. The viscosity of the emulsion may be
modified using a viscosity modifier such as cetyl alcohol. The emulsion
may be thickened using gelling agents such as cellulose and derivatives,
CA 02290278 1999-11-24
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Carbopol and derivatives, carob, carregeenans and derivatives, xanthane
gum, sclerane gum, long chain alkanolamides, bentone and derivatives,
Kaolin USP, Veegum Ultra, Green Clay, Bentonite NFBC, typically
present in concentrations less than 2% by weight.
5 The emulsion may further comprise surfactants to wet,
foam, penetrate, emulsify, solubilize, stabilize and or disperse a selected
material. For example anionic surfactants such as sodium coconut
monoglyceride sulphonate, cationic surfactants, such as lauryl trimethyl
ammonium chloride, cetyl pyridinium chloride and
10 trimethylammonium bromide, nonionic surfactants including
pluronics, and polyethylene oxide condensates of alkyl phenols, and
zwitterionic surfactants such as derivatives of aliphatic quaternary
ammonium, phosmomium and sulphonium compounds may all be
added as required.
15 C'.helating agents, capable of binding metal ions, such as
tartaric acid, EDTA, citric acid, alkali metal citrates, pyrophosphate salts
or
anionic polymeric polycarboxylates may be also included in the emulsion
formulation a~; desired.
Generally, the emulsion formulations will be treated such
20 that contamination by bacteria, fungi, mycoplasmas, viruses and the like
or undesired chemical reactions, such as oxidative reactions are
prevented thus allowing the preparation of a stable final product with a
shelf-life accepetable for the final formulation. In preferred
embodiments this is accomplished by the addition of preservatives, for
25 example sodium metabisulfite, Glydant Plus, Phenonip, methylparaben
propylparaben, German 115, Germaben II, phytic acid, or other chemical
additives, by irradiation, for example by ionizing radiation such as
cobalt-60 or cesium-137 irradiation or by ultraviolet irradiation or by heat
treatment for example by pasteurization in a constant temperature water
30 bath at approximately 65°C for 20 minutes. The pasteurization
temperature preferably ranges between 50°C and 90°C and the time
for
pasteurization preferably ranges between 7.5 seconds to 35 minutes.
CA 02290278 1999-11-24
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Oxidative reactions may be prevented by the addition of anti-oxidants
such as butyllated hydroxytoluene (BHT) or butylated hydroxyanisol
(BHA) or other anti-oxidants.
T'he physical stability of the formulation may be further
enhanced if desired by the addition of an emulsifier such as for example
Arlacel. Typically, emulsion stabilizers are added in small amounts (less
than 2% (w/w).
In addition, active agents may be added to the washed oil
body preparation. For example personal care compositions may be
formulated ;~s stable suspensions using the present emulsion
formulation a:nd vitamins and moisturizing agents may be included in
skin creams. One particularly advantageous way in which an active
ingredient may be included in emulsions of the subject invention, is
through construction of oleosin gene fusions as detailed in WO 96/21029.
Briefly stated, WO 96/21029 discloses a method of producing proteins and
peptides as fusion proteins of oleosins. These fusion proteins are created
by genetically linking the gene encoding oleosin to a gene encoding a
peptide or protein of interest. Expression of the fusion gene, in for
example an oilseed plant, results in synthesis of a fusion protein which is
then targeted to the oil body. Isolation of the oil body fraction results in
recovery of tlhe fusion protein. In principle any desired protein or
peptide may be produced using this technology. For example, it is
envisaged that polar fish antifreeze peptides (Davies, P.L. et al. 1990,
FASEB J. 4: 2460-2468) are produced as oleosin fusion proteins. The
washed oil body preparation may then be employed to prepare ice
creams, milkshakes or other frozen foodgrade materials with improved
freezing properties by inhibiting or preventing ice crystal formation. In
another example, a therapeutic protein may be produced as an oleosin
fusion. The oil bodies may then be used to formulate a desirable
suspension which may be for oral consumption, or for topical skin
application. This embodiment of the present invention is further
exemplified in. example 11 of the present invention where a fish food is
CA 02290278 1999-11-24
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prepared which comprises oil bodies comprising an oleosin - carp growth
hormone fusion.
An emulsion with film forming properties may also be
formulated. Such an emulsion when it is applied to a surface and dried
forms a coating. An example of an emulsion where a coated oil body
film is applied is in fish food, where oil bodies may be applied to the fish
food to enhance the dietary value. A film forming emulsion is
particularly useful in embodiments of the present invention where
controlled release of an active ingredient is desirable such as in delivery
of pharmaceuticals or volatiles such as fragrances. The release time of
the active agent from a film of emulsion, which occurs during drying,
depends, among other factors, on the thickness of the film. When a
thicker coating is applied a longer drying time will result in a slower
release of the active agent. In variant contemplated formulations, release
of the agent occurs only when the film is dry. Other factors, such as the
composition of the emulsion and the type and concentration of the
active ingredient also determine the characteristics of release. For
example, cosolvents, such as ethanol, may be included in the
formulation and influence the release time. Release of an active
ingredient is also desirable in food applications, where a flavorant
entrapped in an emulsion is released during consumption. The release
of the flavorant, depending on the exact formulation of the emulsion,
may elicit a sudden intense sensation or a more subtle blend of flavours
and essences.
The emulsion formulation may also be used in sprays and
aerosols. Preferably small sized oil bodies, for example lam or less in
diameter such as those found in B. napus, are used for this purpose.
Volatiles, such as alcohol and fragrances may be included in these sprays.
Emulsions of this type may also be sprayed onto the surface of dried food
preparations such as potato chips and dried soup. The emulsion might
CA 02290278 1999-11-24
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include a flavorant and add preservative value or assist in maintaining
the appropriai:e moisture levels of the food.
USES OF THE EMULSION FORMULATION
T'he subject invention is directed toward the production of
5 emulsions that are useful in industrial and domestic compositions. It is
noted that the emulsions may be applied in compositions which vary
widely in physical properties and use. Thus specific embodiments
include applications such as food and feed products, pharmaceutical
products, personal care products and industrial products.
10 Food and feed uses include non-dairy substitutes, such as
non-dairy cheese or yoghurt, margarines, mayonnaises, vinaigrettes,
icings, ice creams, salad dressings, synthetic mustards, candy, chewing
gum, pudding;, baking products, condiments, juice clouding agents, baby
formula, flavour carriers, texturing agents (shortening), pet food, fish
15 food and livestock feed. Personal care products applications include
soaps, cosmetics, skin creams, facial creams, night cream, day cream, a
face mask, skin cleanser, tooth paste, lipstick, perfumes, make-up,
foundation, blusher, mascara, eyeshadow, sunscreen lotions, hair
conditioner, and hair colouring. Pharmaceutical products which may be
20 formulated using the washed oil body preparation of the subject
invention incliude therapeutic agents, diagnostic agents and delivery
agents. As a therapeutic or diagnostic agent, the emulsion will
additionally contain an active ingredient. The active ingredient can be
anything that one wishes to deliver to a host. In one embodiment, the
25 active ingredient may be a protein or peptide that has therapeutic or
diagnostic value. Such peptides include antigens (for vaccine
formulations), antibodies, cytokines, blood clotting factors and growth
hormones. Industrial uses for the emulsions of the present invention
include paints, coatings, lubricants, films, gels, drilling fluids, paper
30 sizing, latex, building and road construction material, inks, dyes, waxes,
polishes and agrochemical formulations. In preferred embodiments, the
subject invention is directed to compositions which may be ingested by
CA 02290278 1999-11-24
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animals and humans. Since, these compositions may be ingested they
must be of foodgrade quality. The particular product and the particular
form in which the emulsion is applied, however is not of critical
importance anal may be as desired. It is to be clearly understood that the
emulsion formulated with the washed oil body preparation may be
applied in any domestic or industrial product.
T'he stability of the present emulsion formulation at low pH
may be exploited in formulations of acid emulsions. For example, the
emulsion formulation may be used in the preparation of a mayonnaise-
like foodproduct, which besides the washed oil body preparation
comprises a vegetable oil, mustard, vinegar and egg yolk, if desired.
Pourable emulsions, such as salad dressings may be prepared by
increasing the relative amount of vinegar and/or by the addition of
water.
15 An example of an application where heat may be applied
without apparent deleterious effects, is in the preparation of a savory
sauce such as a bechamel sauce or in sweet sauces such as chocolate
sauces. In these applications, the washed oil body preparation is
employed as a frying substitute. To prepare a bechamel sauce, to 1 part of
the heated washed oil body preparation, 1 part (w/w) of flour is added
and stirred until a thick suspension is formed. At moderate heat milk is
gradually added until a sauce with a desired viscosity is obtained.
T'he emulsion formulation rnay also be used as a butter
substitute. In this application, small amounts of water are added to the
25 washed oil body preparation, for example, less than 10% until a desired
viscosity is obtained. Natural butter flavours and thickeners may be
added as desired. The butter substitute may be used on sweet corn, bread,
in cake mixes or bread making. Salt, which contributes flavour and acts
as a preservative may be added typically to a level of about 2.5% (wt/vol).
30 Colour agents, for example, extracts of annatto seed or carotene may be
added to deepen the colour as desired. An advantage of this application
is that the oil body based butter does not comprise hydrogenated fatty
CA 02290278 1999-11-24
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acids, which are used in the formulations of margarines and the like to
achieve a desirable consistency, but are also with associated with
cardiovascular diseases.
Shortenings may be prepared to various degrees of stiffness,
from a foam to a pourable shortening. In this application, air is beaten
into the emulsion formulation and the emulsion formulation can be
considered to be dispersed into the continuous phase, air. Shortenings
may be applied to mixes where creaming and fluffing are desired. These
mixes include icings, synthetic creams, ice creams and cake batter.
An imitation fruit juice may be prepared from artificial or
natural flavours and nutrients. Such imitation juices do not have the
correct appearance and due to transparency appear to be weak or diluted.
By adding a small amount, for example 0.1 to 1% (v/v) of the washed oil
body preparation or an emulsion thereof clouding may occur to give the
juice a rich appearance. Thus the present oil body preparation may be
used as a clouding agent.
In another application involving juices, the washed oil body
preparation or an emulsion. thereof may be added to juices with settleable
solids, such as tomato juice. Adding a small amount of the washed oil
body preparation, for example 0.1 to 1% (v/v), may decrease the rate of
settling of the solids in the juice and assist in maintaining the rich
appearance.
The washed oil body preparation of the present invention
may also be used to prepare personal care products including cosmetic
and cosmeceutical products. In this embodiment the emulsion is
formulated as a dermatologically acceptable emulsion, which may for
example be err~ployed to apply facially and/or to the body skin, including
nails, teeth and lips. The formulation may have properties to combat
ageing of the skin, acne, pigmentation, hair loss, or promote hair
removal or facilitate wound healing and/or restructuring of the skin
tissue. The washed oil body preparation represents preferably 1-99% by
weight of the final composition. In order to formulate a personal care
CA 02290278 1999-11-24
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product it ma.y be advantageous to first formulate a base formulation
comprising oil bodies. The base formulation may subsequently be used
to formulate the final personal care product (See Examples 12 - 21).
The pesonal care compositions of the present invention
may comprise additional hydrocarbon compounds such as plant, animal,
mineral or synthetic oils or waxes or mixes thereof. They comprise
paraffin, petrolatum, perhydrosqualene, arara oil, almond oil,
calphyllum oil, avocado oil, sesame oil, castor oil, jojoba oil, olive oil, or
cereal germ oil. Esters may be included such as esters of lanolic acid, oleic
acid, lauric acid, stearic acid, myristic acid. It is also possible to include
alcohols for example, oleoyl alcohol, linoleyl alcohol or linolenyl
alcohol, isostearyl alcohol or octyl dodecanol, alcohol or polyalcohol.
Further hydrocarbons which may be included are octanoates, decanoates,
ricinoleates, caprylic/capric triglycerides or C1~ to C22 fatty acid
15 triglycerides. Addition of these agents may result in the formation of
double emulsions.
Hydrogenated oils, which are solid at 25°C, such as
hydrogenated castor oil, palm oil or coconut oil, or hydrogenated tallow;
mono- di- tri- or sucroglycerides; lanolins; and fatty acids which are solid
at 25°C may <~lso be included in the personal care formulations of the
present invention. Among the waxes which may be included are animal
waxes such as beeswax; plant waxes such as carnauba wax, candelilla wax,
ouricurry wa;K, Japan wax or waxes from cork fibres or sugar cane;
mineral waxes, for example paraffin wax, lignite wax, microcrystalline
waxes or ozokerites and synthetic waxes.
Pigments may be included and may be white or coloured,
inorganic or organic and/or paerlescent. These pigments comprise
titanium dioxide, zinc oxide, ziriconium dioxide, black, yellow, red and
brown iron oxides, cerium dioxide, chromium oxide, ferric blue, carbon
black, barium, strontium, calcium and aluminum lakes and mica coated
with titanium oxide or with bismuth oxide.
CA 02290278 1999-11-24
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Active ingredients commonly employed in skin creams,
such as vitamins, for example as vitamin. A or C, E and alpha hydroxy
acids, such as. citric, glycolic, lactic, gamma-linolenic and tartaric, into
cosmetic and/or dermatological compositions may be included. For
example, US patent 5,602,183 teaches that vitamin C or ascorbic acid
promotes growth of connective tissue, particularly in the skin
strengthens the skin against external aggressions such as from smoke and
UV radiation. Moisturizing agents which may be included in personal
care products such as skin creams are for example glycerine, panthenol,
10 mineral oil a:nd urea. Antioxidants such as the naturally occurring
tocopherols and polyphenols, or butylated hydroxytoluene (BHT) and
hydroxyanisole (BHA), retinyl palmitate and vitamin E acetate may also
be also added. Emolients, such as Finsolv TN, Sesame oil,
isohexadecane, safflower oil, permethyl 101 A, Trivent OC-G, may be
added if desired. Soothing agents which may be added include for
example Allantoin. Skin protectants which may be used to formulate the
oil body based personal care products of the present invention include
dimethicone and SEE 839. A penetrating agent such as Trivalin SF and an
exfolient for example Palemol OL or glycolic acid may also be added.
20 Sunscreens such as octyl methoxycinnamate (Parsol MCX), 3-
benzophenone (Uvinul M40) and butylmethoxydibenzoylmethane
(Parsol 1789) may be employed to prepare a sun tanning lotion.
Pharmaceutically active ingredients which may be used to formulate
cosmetic compositions include for example antibiotics, fungicides and
anti-inflammatory agents.
T'he final personal care product may be in the form of a free,
poured or compacted powder (foundation, blusher or eyeshadow), a
relatively greasy product such as lipstick, mascara, or an oil or lotion for
the body or face.
T'he washed oil body preparation may also be used to serve
as an orally acceptable carrier in toothpaste which may further comprise
CA 02290278 1999-11-24
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silicas, surfactants, chelating agents, a fluoride, thickeners, sweeteners,
flavorants, for example as oil of peppermint, enzymes and biocides.
A,n example of an industrial product which may be
formulated is paint wherein the main resin, such as those based on
silicone type compounds, acrylic compounds, polyester, akyd, fluorine,
epoxy, polyurethane may be partly or entirely replaced by the washed oil
body preparation of the present invention. Further additives such as
pigments, dyes, glass flakes, and aluminum flakes, pigment dispersants,
thickeners, levelling agents, hardening catalysts, hardening agents such
as dioisocyanates, hardening catalysts, gelling inhibitors, ultraviolet
absorbing agents, free radical quenching agents etc. may be formulated in
paint compositions as required.
The washed oil body preparation may also be to formulate
lubricants. For example, the washed oil body preparation may be used to
partially or entirely replace the lubricating oils such as animal oils,
vegetable oils, petroleum lubricating oils, synthetic lubricating oils, or the
lubricating grease such as lithium grease, urea grease and calcium grease.
Other compositions employed in a lubricant formulation comprise
antioxidants, detergent dispersants, oilness agents, friction modifiers,
viscosity index improvers, pour point depressants, solid lubricant
material, rust inhibitors and antifoamers.
Waxes may also be prepared using the washed oil body
preparation of the present invention. These comprise rinse-wax types,
such as those providing a stable hydrophobic film-finish onto
automobiles and other protective coatings. Other compositions used in
the preparation of a wax comprise surfactants, mineral oils, such as
mixed paraffinic and aromatic/naphtenic oils, perfumes, biocides,
colouring agents which may be added in compatible amounts as desired.
Where industrial products, such as paints or lubricants are
formulated, purity of the oil body phase may be less critical and it may
not be necessary to subject the oil bodies to washing. An industrial
emulsion may be prepared by (i) obtaining oil bodies from a cell and (ii)
CA 02290278 1999-11-24
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formulating the oil bodies into an industrial emulsion. The oil bodies
may be obtained by (a) grinding plant seeds; (b) removing solids from the
ground seeds; and (c) separating the oil body phase from the aqueous
phase. The invention also includes an industrial emulsion comprising
oil bodies prepared according to the present invention.
T'he following non-limiting examples are illustrative of the
present invention:
EXAMPLES
Example 1
Obtaining a washed oil body preparation from oilseed rape,
soybean, sunflower, white mustard, peanut, squash, flax, safflower and
maize - laboratory scale. Dry mature seeds obtained from Brassica napus
cv Westar, soybean, sunflower, white mustard, peanut, squash, flax,
safflower and maize were homogenized in five volumes of cold grinding
15 buffer (50 mM Tris-HCI, pH 7.5, 0.4 M sucrose and 0.5 M NaCl) using a
polytron operating at high speed. The homogenate was centrifuged at 10
x g for 30 minutes in order to remove particulate matter and to separate
oil bodies from the aqueous phase containing the bulk of the soluble seed
protein. The oil body fraction was skimmed from the surface of the
supernatant ~~ith a metal spatula and added to one volume of grinding
buffer. In order to achieve efficient washing in subsequent steps it was
found to be necessary to thoroughly redisperse the oil bodies in the
grinding buffer. This was accomplished by gently homogenizing the oil
bodies in grinding buffer using a polytron at low speed. Using a syringe,
25 the redispersed oil bodies were carefully layered underneath five
volumes of cold 50 mM Tris-HC1 pH 7.5 and centrifuged as above.
Following centrifugation, the oil bodies were removed and the washing
procedure was repeated two times. The final washed oil body
preparation was resuspended in one volume of cold Tris-HC1 pH 7.5,
redispersed with the polytron.
CA 02290278 1999-11-24
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T'he oil body samples were dissolved in SDS sample buffer
and then analyzed by SDS gel electrophoresis. The results are shown in
Figure 1.
T'he material thus obtained material was then ready to be
employed in various formulations.
Example 2
Obtaining a washed oil body preparation from oilseed rape,
sunflower and maize on. a large scale. This example describes the
recovery of the oil body fraction from canola, sunflower and maize seed
on a large scale. The resulting preparation contains intact oil bodies and
is comparable in purity with a preparation obtained using laboratory scale
procedures.
C:rinding of seeds. A total of 10 - 15 kgs of dry canola seed
(Brassica napes cv Westar), sunflower (Helianthus annuus) or maize
(Zea mays) was poured through the hopper of a colloid mill (Colloid
Mill, MZ-130 (Fryma); capacity: 500 kg/hr), which was equipped with a
MZ-120 crosswise toothed rotor/stator grinding set and top loading
hopper. Approximately 50 - 75 1 water was supplied through an
externally connected hose prior to milling. Operation of the mill was at a
20 gap setting of 1R, chosen to achieve a particle size less than 100 micron
at
18°C and 30°C.. Following grinding of the seeds tap water was
added to
the seed slurry to a final volume of 90 litres.
Removal of solids. The resulting slurry, was pumped into a
decantation centrifuge (fiasco 200 2-phase decantation centrifuge
maximum operating speed 6,000 rpm) after bringing the centrifuge up to
an operating speed of 3,500 rpm. Transfer from the mill to the
decantation centrifuge at a flow rate of 360 L/hr was achieved using a 1
inch M2 Wilden air operated double diaphragm pump. In 15-20 minutes
approximately 15 kg of seed was decanted.
Oil body separation. Separation of the oil body fraction was
achieved using a Sharples Tubular Bowl Centrifuge model AS-16 (Alpha
Laval) equipped with a three phase separating bowl and removable ring
CA 02290278 1999-11-24
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dam series; capacity:150 L/hr; ringdam: 30 mm. Operating speed was at
15,000 rpm (13,200 x g). A Watson-Marlow (Model 704) peristaltic pump
was used to pump the decanted liquid phase (DL) into the tubular bowl
centrifuge after bringing the centrifuge up to operating speed. This
results in separation of the decanted liquid phase into a heavy phase (HP)
comprising water and soluble seed proteins and a light phase (LP)
comprising oi:i bodies. The oil body fraction which was obtained after
one pass through the centrifuge is referred to as an unwashed oil body
preparation. The oil body fraction was then passed through the
centrifuge three more times. Between each pass through the centrifuge,
concentrated oil bodies were mixed with approximately five volumes of
fresh water. The entire procedure was carried out at room temperature.
The preparations obtained following the second separation are all
referred to as the washed oil body preparation. Following three washes
much of the .contaminating soluble protein was removed and the oil
body protein profiles obtained upon SDS gel electrophoresis were similar
in appearance to those obtained using laboratory scale procedures.
The large scale oil body preparation may be pasteurized.
Pasteurization is achieved by initially thickening the washed oil bodies
with centrifugation to a water content of 30 to 60%, preferable between 35
and 50% weight and most preferable between 37 and 40% weight. The
thickened oil body solution can then be pasteurized in a constant
temperature water bath at approximately 65°C for 20 minutes. The
pasteurization temperature could range between 50 and 90°C and the
time for pasteurization could range between 15 seconds to 35 minutes. If
the oil bodies are used in a cosmetic formulation, then before
pasteurization, 0.1% Glydant Plus, 0.1% BHA and 0.1% BHT may be
added as a preservative and anti-oxidants respectively.
Example 3
Ii.emoval of seed proteins by washing the oil body phase.
This example describes the recovery of a washed oil body fraction from
canola, maize and sunflower seed. Using different washing conditions, it
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is shown that the washes result in the removal of significant amounts of
seed proteins from the oil body preparation. These proteins include
proteins which might be allergenic.
A total of 10 - 15 kgs of dry canola seed (Brassica napus cv
Westar), maize (Zea mays) or sunflower (Helianthus annuus) was
poured through the hopper of a colloid mill (Colloid Mill, MZ-130
(Fryma)), which was equipped with a MZ-120 crosswise toothed
rotor/stator grinding set and top loading hopper. Approximately 50 - 75 1
water was supplied through an externally connected hose prior to
milling. Operation of the mill was at a gap setting of 1R, chosen to
achieve a particle size less than 100 micron at 18°C and 30°C.
Following
grinding of the seeds, tap water was added to the seed slurry to a final
volume of 60 -- 90 litres and a sample of the seed slurry was obtained for
SDS gel electrophoresis. The slurry was then pumped into a decantation
centrifuge (Masco 200 2-phase decantation centrifuge maximum
operating speed 6,000 rpm) after bringing the centrifuge up to an
operating speed of 3,500 rpm. Transfer from the mill to the decantation
centrifuge was achieved using a 1 inch M2 Wilden air operated double
diaphragm pump. In 15-20 minutes approximately 15 kg of seed was
decanted. A sample from the decanted liquid phase was obtained for SDS
gel electrophoresis. Separation of the oil body fraction was achieved
using a Sharples Tubular Bowl Centrifuge model AS-16 (Alpha Laval)
equipped with a three phase separating bowl and removable ring dam
series; capacity: 150 L/hr; ringdam: 29 mm. Operating speed was at 15, 000
rpm (13,200 x g). A Watson-Marlowe (Model 704) peristaltic pump was
used to pump the decanted liquid phase into the tubular bowl centrifuge
after bringing the centrifuge up to operating speed. The unwashed oil
body phase was obtained and mixed with approximately 5 volumes of
water. This procedure was repeated a total of three more times. The oil
body phase which was obtained following the first spin, is referred to as
an unwashed oil body preparation. All other preparations are washed oil
CA 02290278 1999-11-24
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body preparations. Samples for analysis by SDS gel electrophoresis were
obtained follo'ving the first and fourth separations.
Upon completion of the fourth wash a 0.9 ml sample of the
oil body preparation was homogenized in 0.1 ml 1 M Na2C03 and left at
room temperature for 30' with agitation. The washed oil body fraction
was then recovered following centrifugation, washed once with water
and prepared for SDS gel electrophoresis.
A.11 of the samples were dissolved in SDS sample buffer and
the samples ~~ere analyzed by SDS gel electrophoresis. The results are
shown in Figure 2.
Example 4
The effect of washing the oil body phase on water retention
characteristics. A washed oil body preparation and an unwashed oil
body phase ware prepared from rapeseed as in example 2. To determine
the difference in water retention capacity between the unwashed oil body
phase and the washed oil body preparation, 30 mls of oil body
preparations were thoroughly mixed using a vortex. The preparations
were then incubated for 2 hours in a water bath at 40, 60 or 80°C and
the
samples were .centrifuged at 1,500 x g for 20 minutes (undiluted samples).
Another set c>f samples was prepared by mixing 15 g of washed or
unwashed oil body preparation with 15 ml of water. The samples were
mixed on a vortex and then incubated at 40, 60 or 80° C for 2 hours and
the amount of water present in the samples was determined following
centrifugation at 1,500 x g for 20 minutes (diluted samples). Loss of mass
attributable to evaporation was measured at 80 °C and 60 °C.
A.,t 80°C, the undiluted preparations comprising oil bodies
lost significant amounts of water through evaporation. The preparation
of unwashed oil bodies lost 26% of their mass, while the washed
preparation lost 16%. Upon centrifugation the unwashed preparation
released approximately 2.5 ml of aqueous phase, while the washed oil
bodies remained in the same phase. Both diluted preparations absorbed
water. The volume of oil bodies increased in both cases to 18.5+1 ml.
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A.t 60°C, the undiluted preparations lost approximately 10%
of water through evaporation. Following centrifugation, the washed
preparation released about 0.5 ml of aqueous phase, while the washed oil
body preparation stayed in the same phase. Both diluted preparations
absorbed water. At 60°C, the volume of oil bodies increased in both
cases
to 18+ 1m1.
At 40°C, the undiluted samples both released approximately
2 ml of aqueous phase. When the diluted samples were compared, the
unwashed preparation absorbed about 3 ml of water, as was the case at 60
or 80° C. However the washed preparation absorbed 8 ml of water at
40°C.
These experiments demonstrate that in a washed oil body
preparation heated to 60°C or 80°C, water remains more tightly
associated
with the oil body preparation than in an unwashed preparation. When
15 cooled down the washed oil body preparation appeared to be more stable
than the unwashed emulsion. When heated to 40°C, the washed oil
body preparation was able to absorb a larger volume of exogenously
added water without resulting in phase separation offering greater
flexibility in preparing oil body based formulations.
Example 5
T'he effect of washing oil bodies on oil absorption
characteristics. A washed oil body preparation and an unwashed oil body
phase were prepared from rapeseed as in example 2. To determine the
difference in oil absorption capacity between the unwashed oil body
phase and the washed oil body preparation, 2 gr of the oil body
preparations 'vas dispersed into 12 ml of refined, bleached, deodorized
canola oil in a 50 ml tube. The contents were stirred for 30 seconds every
5 minutes for 30 min. The tubes were then centrifuged at 4,400 rpm for
25 min. The free oil was decanted and the percentage of absorbed oil was
determined by weight difference. Three preparations of washed oil
bodies were tested and three preparations of unwashed oil bodies were
tested.
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The oil absorption capacity of unwashed oil bodies was
found to vary significantly between the three batches and varied from
18.7% to 28%. Washed oil bodies had reproducible oil absorption of
32~1%. Thus the washed oil body preparation was found to be superior
since (1) a larger amount of oil was found to be absorbed and (2) the
absorption occurred in a more reproducible manner.
Example 6
Preparation of a mayonnaise-like emulsion comprising a
washed oil body preparation. A washed oil body preparation was
prepared from. rapeseed as in example 2 and a mayonnaise-like emulsion
was produced by mixing the following components using a domestic
electric blender.
Sunflower oil 78 gr
15 Egg yolk 8 gr
Vinegar 9 gr
Salt 0.5 gr
Washed oil bodies 5 gr
20 A product with a mayonnaise-like texture was obtained.
The mayonnaise-like product was stable for at least 1 day at 4°C.
Example 7
Preparation of a cholesterol-free mayonnaise-like emulsion.
A washed oil body preparation was prepared from rapeseed as in
25 Example 2 and a mayonnaise-like emulsion was produced by mixing the
following ingredients:
Sunflower oil 200 gr
Washed oil bodies 100 gr
30 Vinegar 30 ml
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A. product with a mayonnaise-like texture was obtained.
Since the mayonnaise is prepared without egg yolk, an ingredient
commonly employed in commercially obtainable mayonnaises, the
product prepared using washed oil bodies is free of cholesterol. The
mayonnaise was found to be as stable as a commercial mayonnaise when
stability was assessed using centrifugation.
Example 8
Preparation of a vinaigrette-like emulsion comprising a
washed oil body preparation. A washed oil body preparation was
prepared from. rapeseed as in example 2 and a vinaigrette-like emulsion
was produced by manual mixing of the following components.
Sunflower oil 17.5 gr
Mustard 0.4 gr
Vinegar 0.5 gr
Washed oil bodies 7.7 gr
A, product with a vinaigrette-like texture was obtained. The
vinaigrette-like product was stable for at least several days at 4° C.
Example 9
Preparation of a spreadable mustard-like product. A washed
oil body preparation was obtained from rapeseed as outlined in example
2. The following ingredients were mixed to obtain a mustard-like
product.
Mustard 70 gr
Washed oil bodies 30 gr
The resulting emulsion formulation is a mustard-like
product which may easily be spread and has creamier, less gritty taste
characteristics than mustard.
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Example 10
Preparation of a bechamel-like sauce. A washed oil body
preparation was obtained from rapeseed as outlined in example 2. The
washed oil body preparation was heated at moderate heat and an equal
part of flour was added and mixed with the heated washed oil body
preparation. While stirring manually, milk was gradually added to this
mixture.
Flour 50 gr
Washed oil bodies 50 gr
Milk 100 ml - 1 1.
A, bechamel-like sauce was obtained. The consistency of the
sauce may be as desired depending on the amount of milk which is
added. Additional flavorants also may be added as required. The absence
of hydrogenated fatty acids in this product gives it an advantage over a
sauce prepared from common domestic margarine.
Example 11
Preparation of a pharmaceutical emulsion for coating onto
fish food. A washed oil body preparation from. a transgenic B. napus
plant which expresses carp growth hormone (cGH) fused to oleosin,
wherein the fusion protein was targeted to the oil bodies, was obtained as
follows. A DNA fragment containing the cGH coding region lacking its
22 amino acid signal sequence was amplified from a plasmid containing
on an insert a~ common carp (Cyprinus carpio) growth hormone cDNA
(Koren et al., 7.989, Gene 67: 309-315) using the polymerase chain reaction
in combination with two cGH specific primers. The amplified cGH
fragment was fused in the correct reading frame and 3' to the Arabidopsis
thaliana oleosin using pOThromb (van Rooijen, 1993, PhD Thesis,
University of Calgary) as a parent plasmid and employing cloning
strategies known to a person skilled in the art. In pOThromb a thrombin
cleavage site was engineered 3' to the oleosin coding sequence. The
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oleosin-CGH fusion gene was introduced into the binary vector
pCGN1559 (McBride and Summerfelt, 1990, Plant Mol. Biol. 14: 269-276)
and the resulting construct was used to transform A. tumefaciens. The
agrobacterium. strain was employed to transform B. napus cv Westar
seedlings. Seeds from transgenic plants were obtained and oil bodies
were isolated from transgenic seed as outlined in example 1.
The oil bodies were subsequently taken up in a syringe and
sprayed onto fish food using approximately 2.5 ~g of oil body protein per
1 mg of fish food. The fish food coated with oil bodies was then left
overnight to dry. A total of 50 mg of fish food was then mixed with 10
ml of water and was incubated for 0, 30, 45 or 60 minutes. The food was
then collected and resuspended in 0.2 ml of 50 mM Tris-Cl (pH 7.5) and
prepared for <~nalysis by SDS gel electrophoresis upon boiling in 2.5%
SDS. The presence of the oil bodies on the fish food was assessed using
Western blotting and monoclonal antibodies against cGH.
Judging by the intensity of the signal of the single band
observed in each lane on the Western blot, the oil bodies comprising cGH
remained tightly associated with the fish food upon incubation of the oil
bodies in water. Fish food which was incubated for 30, 45 or 60 minutes
in water was shown to contain approximately the same amounts of cGH
as the control :Fish food which was not incubated in water.
This example demonstrates that a transgenic plant variety
can be prepared which imparts specific desirable properties to an
emulsion. The example further demonstrates that an emulsion can be
prepared from a washed oil body preparation which can be used as a
coating or film. Finally, this example demonstrates that the washed oil
body preparation may be employed to formulate a pharmaceutical
composition.
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Example 12
Preparation of base formulation emulsions comprising
washed safflower oil bodies for use in a personal care product. A washed
oil body preparation was prepared from safflower as in example 2. To the
washed oil body preparation was added: 0.1% Glydant Plus, 0.1%
Butylated Hydroxyanisole (BHA) 0.1% Butylated Hydroxytoluene (BHT)
and a base formulation for use in a cosmetic product was prepared as
follows. The oil bodies were transferred into a mixing pot and keltrol
was added and was hydrated at room temperature with a high-speed
propeller. Subsequently the glycerin was added. The mixture was then
heated to 45-50°C, at 50°C the BHA and BHT were added. Finally
the
Glydant Plus was added. The procedure for Base B and C was slightly
different as the temperature was subsequently increased to 60°C and the
Arlacel 165 is added and mixed until the texture is homogeneous. The
mixture was then cooled to 30°C quickly with moderate propeller mixing.
Base A
Hydrated safflower oil bodies 96.95%
(0.1% Glydant Plus, 0.1% BHT,
0.1% BHA)
Glydant Plus 0.15%
BHT 0.1%
BHA 0.1%
Keltro:l 0.7%
Glycerine 2.0%
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Base B
Hydrated safflower oil 94.45%
bodies
(0.1% Glydant Plus, 0.1%
BHT,
0.1% B:E3A)
Glydant Plus 0.15%
BHT 0.1'%
BHA 0.1'%
Keltrol 0.7'%
Glycerine 2.0'%
Arlace:l 2.5'%
Base C
Hydrated safflower oil 94.75%
bodies
(0.1% C~lydant Plus, 0.1%
BHT,
0.1% B:~IA)
Glydant Plus 0.25%
BHT 0.1'%
BHA 0.1'%
Keltrol 0.7'%
Glycerine 2.0'%
Arlacel 2.5'%
5 The hydrated safflower oil body preparation alone and all
three bases ~n~ere shown to be stable with respect to oxidation and
microbial growth. Similarly, little or no change was observed in either
the colour or odor of these three base formulations.
The chemical analysis of the hydrated safflower oil body
10 preparation revealed that the sample contained 50.82% water and 49.18%
dry weight. Tlhe dry weight (DW) component consisted of 3.76 % protein,
93.56% oil and 2.68% other.
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Example 13
Preparation of base formulation emulsions for use in a
cosmetically elegant product. Base B was further modified for use in
cosmetically elegant topical formulations. The formulation was mixed to
an emulsion with the following procedure. Phase I is the water phase.
In this phase, the water in the main tank is charged. A propeller is used
to hydrate the keltrol, panthenol and allantoin with moderate agitation
at room temperature. The glycerin is then added with continued mixing.
The water phase is heated to a final temperature of 75°C to
77°C. Phase II,
the oil phase is mixed in a separate mixing pot with moderate agitation
and then subsequently heated up to 75 to 77°C. The oil phase
ingredients
include, Dimethicone 350, Cetyl Alcohol, Arlacel 165, Finsolv TN,
Sesame Oil, Vitamin E Acetate, and I'henonip. The final step of
emulsification includes the addition of the oil phase (Phase II) to the
water phase (Phase I). The two phases are mixed under high agitation
with a propeller or homogenizer for 15 minutes. After 15 minutes of
mixing the mixture is cooled slowly to 40°C. The agitation is decreased
as
the temperature decreases. At approximately 40°C Base Formulation B is
added slowly. The mixture is allowed to cool to room temperature.
Keltro~. 0.5"/
Panthenol 0.1"/
Allantoin 0.05%
Glycerin 2.0"/
Dimethicone 1.0"/
Arlacel 165 2.5"/
Cetyl Alcohol 2.0"/
Finsol'r TN 2.0%
Sesame Oil 1.0%
Vitamin E Acetate 0.05%
Phenonip 1.0%
Base Formulation B 10.0%
Water 77.8%
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Example 14
Preparation of base formulation emulsions for use in a
cosmetically elegant product. Base C was further modified for use in
cosmetically elegant topical formulations. The formulation was mixed to
an emulsion v~~ith the following procedure. Phase I is the water phase.
In this phase, the water in the main tank is charged. A propeller is used
to hydrate the keltrol, panthenol and allantoin with moderate agitation
at room temperature. The glycerin is then. added with continued mixing.
The water phase is heated to a final temperature of '75°C to
77°C. Phase II,
the oil phase is mixed in a separate mixing pot with moderate agitation
and then subsequently heated up to 75°C to 77°C. The oil phase
ingredients include, Dimethicone 350, Cetyl Alcohol, Arlacel 165, Finsolv
TN, Isohexadecane, Vitamin E Acetate, and Phenonip. The final step of
emulsification includes the addition of the oil phase (Phase II) to the
water phase (Phase I). The two phases are mixed under high agitation
with a propeller or homogenizer for 15 minutes. After 15 minutes of
mixing the mixture is cooled slowly to 40°(~. T'he agitation is
decreased as
the temperature decreases. At approximately 40°C Base Formulation C
are added slowly. The mixture is allowed to cool to room temperature.
Keltroll 0.5"/
Panthenol 0.1"/
Allantoin 0.05%
Glycerin 2.0%
Dimetlnicone 1.0%
Arlacel~ 165 2.5%
Cetyl Alcohol 2.0%
Finsol'~ TN 2.0%
Isohex;adecane 2.0%
Vitamin E Acetate 0.0,5%
Phenonip 1.0%
Base Formulation C 20.0%
Water 66.8%
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Example 15
Preparation of base formulation emulsions for use in a
cosmetically elegant product. Base B was further modified for use in
cosmetically elegant topical formulations. The formulation was mixed to
an emulsion with the following procedure. Phase one is the water phase.
In this phase, the water in the main tank is charged. A propeller is used
to hydrate the keltrol, panthenol and allantoin with moderate agitation
at room temperature. The glycerin is then added with continued mixing.
The water phase is heated to a final temperature of 75°C to
77°C. Phase II,
the oil phase is mixed in a separate mixing pot with moderate agitation
and then subsequently heated up to 75°C to 77°C. The oil phase
ingredients include, SEE 839, Cetyl Alcohol,, Arlacel 165, Finsolv TN,
Vitamin E Acetate, and I'henonip. The final step of emulsification
includes the addition of the oil phase (Phase II) to the water phase
(Phase I). The two phases are mixed under high agitation with a
propeller or homogenizer for 15 minutes. After 15 minutes of mixing
the mixture is cooled slowly to 40°C. The agitation is decreased as the
temperature decreases. At approximately 40°C Base Formulation B are
added slowly. The mixture is allowed to cool to room temperature.
Keltrol'~. 0.5"/
Panthenol 0.1"/
Allantoin 0.05%
Glycerin 2.0"/
SEE 83!a 1.0"/
Arlacel'~ 165 2.5"/
Cetyl Alcohol 2.0%
Finsol'~ TN 2.0%
Vitamin E Acetate 0.05%
Phenonip 1.0%
Base Formulation B 40.0%
Water 48.8%
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Example 16
Preparation of a sunscreen with a sun protection factor of 8.
Bases B was farther modified for use in cosmetically elegant sunscreen.
The formulation was mixed to an emulsion with the following
5 procedure. Phase I is the water phase. In this phase, the water in the
main tank is charged. A propeller is used to hydrate the keltrol,
panthenol and allantoin with moderate agitation at room temperature.
The glycerin is then added with continued mixing. The water phase is
heated to a final temperature of 75°C to 77°C. Phase II, the oil
phase is
10 mixed in a separate mixing pot with moderate agitation and then
subsequently heated up to 75°C to 77°C. The oil phase
ingredients
include, Dimethicone, Cetyl Alcohol, Arlacel 165, Finsolv TN, Sesame
Oil, Vitamin E Acetate, and Phenonip. The final step of emulsification
includes the addition of the oil phase (Phase II) to the water phase
15 (Phase I). The two phases are mixed under high agitation with a
propeller or h.omogenizer for 15 minutes. After 15 minutes of mixing
the mixture is cooled slowly to 40°C. The agitation is decreased as the
temperature decreases. At approximately 40°C Base Formulation B are
added slowly. The mixture is allowed to cool to room temperature.
Keltrol 0.5%
Panthenol 0.1%
Allantoin 0.05%
Glycerin 2.0%
Dimethicone 1.0%
Arlacel 165 2.5%
Cetyl Alcohol 2.0%
Finsol~~ TN 2.0%
Sesame Oil 1.0%
Vitamin E Acetate 0.05%
Phenonip 1.0%
Base :Formulation 10.0%
B
Water 77.8%
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Example 17
Preparation of a sunscreen with a sun protection factor of 8.
The formulation was mixed to an emulsion with the following
5 procedure. Phase I is the water phase. In this phase, the water in the
main tank is charged. A propeller is used to hydrate the Kaolin and the
Veegum Ultra with moderate agitation at room temperature. The
glycerin is then added with continued mixing. The water phase is heated
to a final temperature of 75°C to 77°C and methylparaben is
added. Phase
10 II, the oil phase is mixed in a separate mixing pot with moderate
agitation and then subsequently heated up to 75°C to 77°C. The
oil phase
ingredients include, Dimethicone 250, Cetyl Alcohol, Arlacel 165,
Propylparaben, Safflower Oil, Trivalin SF, Palemol OL and Parsol MCX.
The final step of emulsification includes the addition of the oil phase
15 (Phase II) to tile water phase (Phase I). The two phases are mixed under
high agitation with a propeller or homogenizer for 15 minutes. After 15
minutes of mixing the mixture is cooled slowly to 40°C. The agitation
is
decreased as the temperature decreases. At 40°C the German 115 is added
and when the temperature reaches about 37°C to 40°C the
safflower oil
20 bodies are added slowly. The mixture is allowed to cool to room
temperature and the color (red 33 solution) is added. The final pH was
6.0 and viscosity was 25,000 cps.
Purified Water 47.15%
25 Kaolin USP 2.50%
Veegum Ultra (Mg, A1 Sillicate) 5.00%
Glycerin 2.00%
Methylparaben 0.30%
Dimethicone 350 0.50%
30 C:etyl Alcohol 2.00%
Arlacel 165 (Glyceryl Stearate 2.50%
8c PEG-100 Stearate)
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Propylparaben 0.15%
Safflower Oil 2.00%
Trivalin SF (Ethyoxydiglycol) 2.00%
Palemol OL (Oleyl Lactate) 1.00%
Parsol MCX (Octyl Methoxycinnamate) 7.50%
Germall 115 (Imidazolidinyl Urea) 0.30%
Hydrated Safflower Oil Body (0.1% 25.00%
Glydant Plus, 0.1% BHT, 0.1/> BHA)
R.ed #33 1% 0.10%
Example 18
Preparation of a skin care cream containing a stable vitamin
A derivative, retinyl palmitate. Base C was further modified for use in
cosmetically Elegant skin care cream containing a stable vitamin A
derivative, retinyl palmitate. The formulation was mixed to an
emulsion with. the following procedure. Phase I is the water phase. In
this phase, the water in the main tank is charged. A propeller is used to
hydrate the Keltrol, Panthenol and Allantoin with moderate agitation at
room temperature. The glycerin is then added with continued mixing.
The water phase is heated to a final temperature of 75°C to
77°C. Phase II,
the oil phase is mixed in a separate mixing pot with moderate agitation
and then subsequently heated up to 75°C to 77°C. The oil phase
ingredients include Dimethicone 350, Cetyl Alcohol, Arlacel 165, Finsolv
TN, Permethyl 101A, Phenonip and Petinyl Palmitate. The final step of
emulsification includes the addition of the oil phase (Phase II) to the
water phase (Phase I). The two phases are mixed under high agitation
with a propeller or homogenizer for 15 minutes. After 15 minutes of
mixing the mi;tture is cooled slowly to 40°C. The agitation is
decreased as
the temperature decreases. At 40°C the Base Formulation C is added
slowly.
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K:eltrol 0.5%
Panthenol 0.1
A,llantoin 0.05%
5 Glycerin 2.0%
Dimethicone 1.0%
A,rlacel 165 2.5%
C'.etyl Alcohol 2.0%
Finsolv TN 2.0%
Permethyl 101A 2.0%
Phenonip 1.0%
Base Formulation C 50.0%
Water 35.85%
R.etinyl Palmitate 1.0%
Example 19
Preparation of a day cream. The formulation was mixed to
an emulsion with the following procedure. Phase I is the water phase.
In this phase, the water in the main tank is charged. A propeller is used
to hydrate the Kaolin and the Mg, A1 Silicate with moderate agitation at
room temperature. The glycerin is then added with continued mixing.
The water phase is heated to a final temperature of 75°C to
77°C. Phase II,
the oil phase is mixed in a separate mixing pot with moderate agitation
and then subsequently heated up to 75°C to 77°C. The oil phase
25 ingredients include, Dimethicone 350, Cetyl Alcohol, Arlacel 165,
Trivalin SF, and Palemol OL. The final step of emulsification includes
the addition of the oil phase (Phase II) to the water phase (Phase I). The
two phases .are mixed under high agitation with a propeller or
homogenizer for 15 minutes. After 15 minutes of mixing the mixture is
30 cooled slowly to 40°C. The agitation is decreased as the temperature
decreases. At 40°C the Germaben II is added and when the temperature
reaches about 37°C to 40°C the safflower oil bodies are added
slowly. The
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mixture is allowed to cool to room temperature. The final pH was
adjusted to 6.00 with a final viscosity of 25,060 cps.
Purified water 32.20%
Kaolin 2.50%
Veegum Ultra (Mg, A1 Silicate) 5.00%
Glycerin 2.00%
L~imethicone 350 0.50%
C'etyl Alcohol 2.00%
Arlacel 165 (Glyceryl Sterate 2.50%
& PEG-100 Stearate)
Trivalin SF (Ethoxydiglycol) 2.00%
Palemol OL (Oleyl Lactate) 1.00%
15 Germaben II (Diazolidinyl Urea) 0.30%
Hydrated Safflower Oil Body (0.1'% 50.00%
Glydant Plus, 0.1% BHT, 0.1°/'~ BHA)
Example 20
Preparation of a night cream. The formulation was mixed
to an emulsion with the following procedure. Phase I is the water phase.
In this phase, the water in the main tank is charged. A propeller is used
to hydrate the Kaolin and the Mg, Al Silicate with moderate agitation at
room temperature. The glycerin is then added with continued mixing.
25 The water phase is heated to a final temperature of 75°C to
77°C. Phase II,
the oil phase i_s mixed in a separate mixing pot with moderate agitation
and then subsequently heated up to 75°C to 77°C. The oil phase
ingredients include, Dimethicone 350, Cetyl Alcohol, Arlacel 165,
Trivalin SF, Palemol OL. The final step of emulsification includes the
30 addition of the oil phase (Phase II) to the water phase (Phase I). The two
phases are mixed under high agitation with a propeller or homogenizer
for 15 minutes. After 15 minutes of mixing the mixture is cooled slowly
to 60°C. The agitation is decreased as the temperature decreases. At
60°C
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Glycolic acid is added, at 50°C a 25% solultion of sodium
hydroxide is
added, at 40°C the German 115 is added and when the temperature
reaches about 37 to 40°C the safflower oil bodies are added slowly. The
final pH was adjusted to 3.64 with a final viscosity of 35,000 cps.
Purified water 24.20%
Kaolin 2.50%
V'eegum Ultra (Mg, A1 Silicate) 5.00%
Glycerin 2.00%
Dimethicone 350 0.50%
C'etyl Alcohol 2.00%
Arlacel 165 (Glyceryl Sterate 2.50%
8~: PEG-100 Stearate)
Trivalin SF (Ethoxydiglycol) 2.00%
Palemol OL (Oleyl Lactate) 1.00%
C~lycolic Acid 8.00%
Sodium Hydroxide (25% solution) qs pH 3.3-3.8
Germaben H (Diazolidinyl Urea) 0.30%
Hydrated Safflower Oil Body (0.1'% 50.00%
Glydant Plus, 0.1% BHT, 0.1°/'> BI~:A)
Example 21
Preparation of a facial mask. The formulation was mixed to
an emulsion v~ith the following procedure. Phase I is the water phase.
In this phase, the water in the main tank is charged. A propeller is used
to hydrate the Kaolin and the Mg, A1 Silicate with moderate agitation at
room temperature. The glycerin is then added with continued mixing.
The water ph<~se is heated to a final temperature of 75°C to
77°C and
methylparaber~, Green Clay and Bentonite NF BC are added. Phase II, the
oil phase is mixed in a separate mixing pot with moderate agitation and
then subsequently heated up to 75°C to 77°C. The oil phase
ingredients
include, Dimethicone 350, Trivent OC-G, Arlacel 165, Polyparabin and
CA 02290278 1999-11-24
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Safflower Oil. The final step of emulsification includes the addition of
the oil phase (Phase II) to the water phase (Phase I). The two phases are
mixed under high agitation with a propeller or homogenizer for 15
minutes. After 15 minutes of mixing the mixture is cooled slowly to
5 40°C. The agitation is decreased as the temperature decreases. At
40°C
the German 115 and Phytic acid is added and when the temperature
reaches about 37°C to 40°C the safflower oil bodies are added
slowly. The
final pH was 1.49 and viscosity is 45,000 cps.
Distilled Water 44.25%
Kaolin 2.50%
Glycerin 2.00%
Methylparaben 0.30%
Green Clay (Montmorillonate) 0.30%
Bentonite NF BC 10.00%
Dimethicone 350 0.50%
Trivent OC-G (Tricaprylin) 2.00%
Glyceryl Stearate & PEG-100 Stearate 2.00%
Propylparabin 0.15%
Safflower oil 1.00%
Germall 115 (Imidazolidinyl Urea) 0.30%
Hydrated Safflower oil body (0.1°~0 25.00%
Glydant Plus, 0.1% BHT, 0.1°/~ BHA)
Phytic Acid 5.00%
Example 22
C'.omparison of Washed Oil Bodies and Lipid Vesicles in the
Preparation of Cosmetic Formulations. Washed Oil bodies were
prepared as described in example 2, pasteurized and 0.1% BHT, 0.1% BHA
and 0.1% Glydant plus added. Lipid vesicles were prepared in accordance
with the specification of US Patent 5,683,140 except that they were
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prepared from safflower seed, pasteurized and 0.1% BHT, 0.1% BHA and
0.1% Glydant Plus was added.
The oil bodies and lipid vesicles were compared with respect
to emulsion stability, color changes, odor changes, viscosity, microbial
growth and cosmetic desirability parameters. To evaluate stability, the
samples were tested at 45°C, 4°C and room temperature (3 months
at 45°C
is equivalent fio approximately 2 year shelf life at room temperature). To
evaluate emulsion stability, 150 g of each sample was maintained at
45°C,
75 g of each sample was maintained at room temperature or at 4°C.
Emulsion stability was evaluated for emulsion separation, oil droplet
separation and coalescence. The 4°C sample was used as the reference
for
comparison. Color changes were evaluated by visual inspection. Color
was evaluated on the accelerated oven sample (45°C) and the room
temperature sample and compared to the 4°C: as a reference. Odor was
tested as with the color with the 4°C sample used as a reference point.
In
order to maintain consistency, the odor was judged by two individuals
who both agreed on the evaluation. Viscosity of each sample was
measured at :room temperature using a RVT Model viscometer with
Spindle E at l.Orpm. Microbial growth was measured on 10 g of each
sample. The sample was diluted and 1 ml of the sample is added to 49°C
Tryptic Soy A~;ar, swirled and allowed to cool. The plates were incubated
at 35°C for 48. hours and a colony count was taken. Finally, cosmetic
attributes were evaluated by 3 individuals, 2 individuals who were
familiar with oil bodies/lipid vesicles and 1 person who was not.
Cosmetic attributes include skin penetration, residue left on the skin
after the sample was rubbed in, dryness (lack of moisture) and oiliness.
Table 1 summarizes the results for the oil bodies. The pH
for the oil body sample was constant at 6.50 throughout the test at room
temperature and at 45°C. The oil body preparation, when applied to the
skin, distributed evenly on the skin, was fast penetrating and left almost
no residue on the skin surface. The oil body preparation was also stable
with respect to color, odor, viscosity and emulsion stability.
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Table 2 summarizes the results for the lipid vesicles. The
pH for the lipid vesicle sample is difficult to measure because of the total
separation but was approximately 6.8. The lipid vesicle preparation,
when applied to the skin, was very oily and left a film residue on the
skin. The lipid vesicle preparation was stable with respect to microbial
growth but was not stable with respect to color, odor and emulsion
stability.
The above results demonstrate that the oil washed oil body
preparation i~; clearly superior to lipid vesicles with respect to both
physical parameters (color, odor, stability) and cosmetic parameters
(penetration, residual residue, and oiliness). These parameters are
critical to the preparation of personal care products.
V~Jhile the present invention has been described with
reference to what are presently considered to be the preferred examples, it
is to be understood that the invention is not limited to the disclosed
examples. To the contrary, the invention is intended to cover various
modifications and equivalent arrangements included within the spirit
and scope of the appended claims.
A.11 publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as if each
individual publication, patent or patent application was specifically and
individually indicated to be incorporated by reference in its entirety.
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TABLE 1
Room Temperature
Time Color Odor StabilityViscosity Microbial
(days) (cps) Growth
0 Pale Very Mild No 3500+/-loo500
yellow separation
14 Pale No No 3500+/-100300
yellow change separation
25 Pale No No 3500+/-100<10
yellow change separation
45C
Time Color Odor StabilityViscosity Microbial
(days) (cps) Growth
0 Pale Very Mild No 3500+/-loo500
yellow separation
14 Pale Mild No 4000+/-100<20
yellow separation
25 Mildly Mild No 4000+/-100<10
yellow separation
4C
0 Pale Very Mild No 3500+/-100500
yellow separation
14 Pale Very Mild No 3500+/-100250
yellow separation
25 Pale Very Mild No 3500+/-100<10
yellow separation
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TABLE 2
Room Temperature
Time Color Odor StabilityViscosityMicrobial
(days) (cps) Growth
0 Dark Very Mild SeparationApprox. <20
yellow 4000
14 Dark Very Mild Total Sluggish <20
yellow Separation
25 Darker Very Mild Total Sluggish <10
yellow Separation
45C
Time Color Odor StabilityViscosityMicrobial
(days) (cps) Growth
0 Dark Neutral No 3500+/-loo<20
yellow separation
14 Brown Amine No 4000+/-100<10
Odor separ;rtion
25 Dark Fishy No 4000+/-zoo<10
brown separation
4C
0 Dark Neutral SeparationApprox. <20
yellow 4000
14 Dark Neutral No 3500+/-loo<10
yellow separ<~tion
25 Dark Neutral No 3500+/-100<10
yellow separation
