U.S. patent application number 16/534999 was filed with the patent office on 2020-01-02 for flowable concentrated phospholipid krill oil composition.
The applicant listed for this patent is Pharmalink International Limited. Invention is credited to Charles Edward John Hodgson.
Application Number | 20200000857 16/534999 |
Document ID | / |
Family ID | 56194538 |
Filed Date | 2020-01-02 |
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United States Patent
Application |
20200000857 |
Kind Code |
A1 |
Hodgson; Charles Edward
John |
January 2, 2020 |
FLOWABLE CONCENTRATED PHOSPHOLIPID KRILL OIL COMPOSITION
Abstract
The present invention is related to methods of making crustacean
oil compositions. In particular, the crustacean oil compositions
are krill oil compositions. In some embodiments, the krill oil
compositions are concentrated in phospholipids. These concentrated
phospholipid krill oil compositions have a sufficient flowability
to permit successful encapsulation at phospholipid concentrations
that is currently unattainable in the art. Such phospholipid krill
oil compositions are capable of encapsulation even though they may
have a phospholipid concentration ranging between approximately
60%-99% and a viscosity ranging between 100,000-3,000,000 cP. Such
concentrated phospholipid krill oil compositions may be created
using a small molecule organic solvent/water extraction mixture
and/or sub-critical or super-critical fluid extraction at low
temperatures followed by a drying process to remove water and
organic solvent (e.g., for example, ethanol).
Inventors: |
Hodgson; Charles Edward John;
(Nelson, NZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pharmalink International Limited |
Hong Kong |
|
CN |
|
|
Family ID: |
56194538 |
Appl. No.: |
16/534999 |
Filed: |
August 7, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15845932 |
Dec 18, 2017 |
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16534999 |
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15163127 |
May 24, 2016 |
10328105 |
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15845932 |
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62281974 |
Jan 22, 2016 |
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62166872 |
May 27, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/612 20130101;
A61K 9/4825 20130101; A61K 9/4858 20130101; A61K 31/685 20130101;
A61K 45/06 20130101; A61K 31/683 20130101; A61K 31/683 20130101;
A61K 2300/00 20130101; A61K 31/685 20130101; A61K 2300/00
20130101 |
International
Class: |
A61K 35/612 20060101
A61K035/612; A61K 31/683 20060101 A61K031/683; A61K 9/48 20060101
A61K009/48; A61K 31/685 20060101 A61K031/685; A61K 45/06 20060101
A61K045/06 |
Claims
1.-64. (canceled)
65. A semi-solid krill oil comprising a phospholipid content of
approximately 61.7% (w/w), a water content of approximately 1.9%
(w/w) and an ethanol content of approximately 0.8%, wherein said
semi-solid krill oil is at a temperature of at least 40.degree.
C.
66. The krill oil of claim 65, wherein said krill oil further
comprises a viscosity of approximately 140,000 cP.
67. The krill oil of claim 65, wherein said phospholipid content
comprising phosphatidylcholine of approximately 47% (w/w), alkyl
acyl phosphatidylcholine of approximately 4.5% (w/w),
phosphatidylinositol in a range of approximately 0.75% (w/w),
phosphatidylserine (PS) in a range of approximately 0.5%,
lysophosphatidylcholine in a range of approximately 2.9%, lyso
alkyl acyl phosphatidylcholine of approximately 0.65%,
phosphatidylethanolamine of approximately 2.8%, alkyl acyl
phosphatidylethanolamine of approximately 0.75%,
cardiolipin+N-acylphosphatidylethanolamine of approximately 1.5%,
lysophosphatidylethanolamine of approximately 0.35%, and lyso alkyl
acyl phosphatidylethanolamine of <0.1%.
68. The krill oil of claim 65, wherein the krill oil comprises a
fatty acid content of approximately 42.9% (w/w).
69. The krill oil of claim 65, wherein the krill oil comprises an
astaxanthin content of approximately 40.5 mg/100 g.
70. The krill oil of claim 65, wherein the krill oil comprises a
trimethylamine (TMA) content of approximately 8 mg/100 g.
71. The krill oil of claim 65, wherein the krill oil comprises a
trimethylamine oxide (TMAO) content of approximately 1174 mg/100
g.
72. The krill oil of claim 65, wherein said krill oil is a gently
dried krill oil.
73. The krill oil of claim 72, wherein said gently dried krill oil
is a lyophilized krill oil.
Description
FIELD OF THE INVENTION
[0001] The present invention is related to methods of making
crustacean oil compositions. In particular, the crustacean oil
compositions are krill oil compositions. In some embodiments, the
krill oil compositions are concentrated in phospholipids. These
concentrated phospholipid krill oil compositions have a sufficient
flowability to permit successful encapsulation at phospholipid
concentrations that is currently unattainable in the art. Such
phospholipid krill oil compositions are capable of encapsulation
even though they may have a phospholipid concentration ranging
between approximately 60%-99% and a viscosity ranging between
100,000-3,000,000 cP.
[0002] Such concentrated phospholipid krill oil compositions may be
created using a small molecule organic solvent/water extraction
mixture and/or a sub-critical or super-critical fluid extraction at
low temperatures followed by a drying process to remove water and
organic solvent (e.g., for example, ethanol).
BACKGROUND
[0003] Krill are marine crustaceans (Class Malacostraca, Order
Euphausiacea) comprising approximately 86 species, a majority of
which are free swimming, and are considered plankton. Krill
sometimes form dense swarms that can extend over several square
kilometers and represent a biomass of thousands or even millions of
tons.
[0004] There are currently several active krill fisheries but these
are dominated by two; one based in Antarctica for E. superba and
the other based predominantly in Japan (but also Canada) targeting
E. pacifica. Together these two fisheries (E. superba and E.
pacifica) represent at least 97% of the total krill landed. The low
levels of environmental pollutants in the Antarctic krill is a
benefit for the utilization of the krill for health products.
Currently available krill products for human consumption is mainly
based on krill oil in where the protein fraction is removed.
[0005] To utilize the whole krill for nutritional supplements or
for pharmaceuticals (clinical nutrition) there is a need for
compositions and formulations in where most of the nutrients and
the bioactive components from krill are kept intact and where both
lipid soluble and lipid insoluble micronutrients which are required
can be in mixed in a feasible way.
[0006] While the overall beneficial effects of krill compositions
(e.g., for example, krill oil) have been suggested, compositions
are unavailable that provide an effective therapeutic treatment
without repeated daily doses. Due to this, there is a great need
for effective nutritional supplements that contain sufficient
concentrations of therapeutic krill oil components such that a only
a single daily dose is required.
SUMMARY OF THE INVENTION
[0007] The present invention is related to methods of making
crustacean oil compositions. In particular, the crustacean oil
compositions are krill oil compositions. In some embodiments, the
krill oil compositions are concentrated in phospholipids. These
concentrated phospholipid krill oil compositions have a sufficient
flowability to permit successful encapsulation at phospholipid
concentrations that is currently unattainable in the art. Such
phospholipid krill oil compositions capable of encapsulation even
though they may have a phospholipid concentration ranging between
approximately 60%-99% and a viscosity ranging between
100,000-3,000,000 cP. Such concentrated phospholipid krill oil
compositions may be created using a small molecule organic
solvent/water extraction mixture and/or a sub-critical or
super-critical fluid extraction at low temperatures followed by a
drying process to remove water and organic solvent (e.g., for
example, ethanol).
[0008] In one embodiment, the present invention contemplates a
krill oil comprising a phospholipid content ranging between
approximately 60%-99% (w/w), a water content ranging between 1-4%
(w/w) and an organic solvent content of less than 1%. In one
embodiment, the organic solvent is ethanol. In one embodiment, the
krill oil comprises a viscosity ranging between approximately
100,000-3,000,000 cP. In one embodiment, the krill oil is a gently
dried krill oil. In one embodiment, the gently dried krill oil is a
lyophilized krill oil. In one embodiment, the krill oil is
encapsulated. In one embodiment the krill oil is a semi-solid at a
temperature of at least 40.degree. C. In one embodiment, the krill
oil further comprises a viscosity modifier. In one embodiment, the
krill oil further comprises a thixotropic carrier. In one
embodiment, the phospholipid content comprises phosphatidylcholine
in a range of approximately 35-55% (w/w), alkyl acyl
phosphatidylcholine in a range of approximately 3.0-6.0% (w/w),
phosphatidylinositol in a range of approximately 0.5-0.9% (w/w),
phosphatidylserine (PS) in a range of approximately 0.3-0.6%,
lysophosphatidylcholine in a range of approximately 1.5-4.0%, lyso
alkyl acyl phosphatidylcholine in a range of approximately
1.0-0.25%, phosphatidylethanolamine in a range of approximately
2.0-4.0%, alkyl acyl phosphatidylethanolamine in a range of
approximately 0.25-1.25%,
cardiolipin+N-acylphosphatidylethanolamine in a range of
approximately 0.5-2.5%, lysophosphatidylethanolamine in a range of
approximately 0.2-0.6%, and lyso alkyl acyl
phosphatidylethanolamine of <0.1%. In one embodiment, the krill
oil is encapsulated with a capsule that includes but is not limited
to a soft gel capsule and a hard gelatin capsule.
[0009] In one embodiment, the present invention contemplates a
capsule comprising a krill oil having a phospholipid content
ranging between approximately 60%-99% (w/w), a water content
ranging between 1-4% (w/w) and an organic solvent content of less
than 1%. In one embodiment, the organic solvent is ethanol. In one
embodiment, the krill oil comprises a viscosity ranging between
approximately 100,000-3,000,000 cP. In one embodiment, the krill
oil is a gently dried krill oil. In one embodiment, the gently
dried krill oil is a lyophilized krill oil. In one embodiment, the
krill oil is encapsulated. In one embodiment, the krill oil is a
semi-solid at a temperature of at least 40.degree. C. In one
embodiment, the capsule further comprises a viscosity modifier. In
one embodiment, the capsule further comprises a thixotropic
carrier. In one embodiment, the phospholipid content comprises
phosphatidylcholine in a range of approximately 35-55% (w/w), alkyl
acyl phosphatidylcholine in a range of approximately 3.0-6.0%
(w/w), phosphatidylinositol in a range of approximately 0.5-0.9%
(w/w), phosphatidylserine (PS) in a range of approximately
0.3-0.6%, lysophosphatidylcholine in a range of approximately
1.5-4.0%, lyso alkyl acyl phosphatidylcholine in a range of
approximately 1.0-0.25%, phosphatidylethanolamine in a range of
approximately 2.0-4.0%, alkyl acyl phosphatidylethanolamine in a
range of approximately 0.25-1.25%,
cardiolipin+N-acylphosphatidylethanolamine in a range of
approximately 0.5-2.5%, lysophosphatidylethanolamine in a range of
approximately 0.2-0.6%, and lyso alkyl acyl
phosphatidylethanolamine of <0.1%.
[0010] In one embodiment, the present invention contemplates a
method, comprising: a) providing; i) a krill oil; ii) a mixture
comprising a small molecule organic solvent and water; a
temperature controlled reaction vessel; h) mixing said krill oil
and said mixture in said reaction vessel; d) incubating said
mixture and said lain oil in said reaction vessel such that a phase
separation comprising a triglyceride-rich insoluble fraction and a
concentrated polar lipid krill oil is created; e) isolating said
concentrated polar lipid krill oil from said triglyceride-rich
insoluble fraction; and f) gently drying said concentrated polar
lipid krill oil to evaporate said small molecule organic solvent
and said water from said concentrated polar lipid krill oil to
create a concentrated polar lipid semi-solid krill oil comprising a
water content ranging between approximately 1-4% (w/w) and an
organic solvent content of less than 1%. In one embodiment, the
organic solvent is ethanol. In one embodiment, the concentrated
polar lipid krill oil comprises a viscosity ranging between
approximately 100,000-3,000,000 cP. In one embodiment, the
concentrated polar lipid krill oil is encapsulated. In one
embodiment, the concentrated polar lipid krill oil is a semi-solid
at a temperature of at least 40.degree. C. In one embodiment, said
concentrated polar lipid krill oil comprises between approximately
60-99% polar lipids. In one embodiment, the concentrated polar
lipid krill oil comprises approximately 63% polar lipids. In one
embodiment, the concentrated polar lipid krill oil comprises
approximately 72% polar lipids. In one embodiment, the small
molecule organic solvent is selected from the group consisting of
ethanol, subcritical carbon dioxide, supercritical carbon dioxide
and acetone. In one embodiment, the temperature ranges between
approximately 0.degree. C. to -25.degree. C. In one embodiment, the
reaction mixture comprises a ratio of the small molecule organic
solvent and the water ranging between 100:0 to 1:99. In one
embodiment, the ratio of the small molecule organic solvent and the
water ranges between 100:0 to 90:10. In one embodiment, the ratio
of the small molecule organic solvent and the water is 94:6. In one
embodiment, the polar lipids comprise a combination of
phosphatidylethanolamine, phosphatidylcholine and
lysophosphatidylcholine.
[0011] In one embodiment, the present invention contemplates a
method comprising: a) providing; i) a krill oil composition
comprising phospholipids ranging between 60%-99% (w/w), water and
an organic solvent; ii) a means for gentle drying; and iii) an
empty capsule; b) gently drying the krill oil composition under
conditions such that a gently dried krill oil product comprising a
water content between approximately 1-4% (w/w) and an organic
solvent content less than 1% is created; and c) filling the empty
capsule with the gently dried krill oil at a temperature of at
least 40.degree. C. In one embodiment, the means for gentle drying
is a lyophilizer In one embodiment, the means for gentle drying is
an oven. In one embodiment, the means for gentle drying is a
nitrogen stream. In one embodiment, the gently dried krill oil is a
semi-solid at a temperature of at least 40.degree. C. In one
embodiment, the filling is performed with a capsule filling
machine. In one embodiment, the krill oil further comprises a
viscosity modifier. In one embodiment, the krill oil further
comprises a thixotropic carrier. In one embodiment, the
phospholipid content comprises phosphatidylcholine in a range of
approximately 35-55% (w/w), alkyl acyl phosphatidylcholine in a
range of approximately 3.0-6.0% (w/w), phosphatidylinositol in a
range of approximately 0.5-0.9% (w/w), phosphatidylserine (PS) in a
range of approximately 0.3-0.6%, lysophosphatidylcholinc in a range
of approximately 1.5-4.0%, lyso alkyl acyl phosphatidylcholine in a
range of approximately 1.0-0.25%, phosphatidylethanolamine in a
range of approximately 2.0-4.0%, alkyl acyl
phosphatidylethanolamine in a range of approximately 0.25-1.25%,
cardiolipin+N-acylphosphatidylethanolamine in a range of
approximately 0.5-2.5%, lysophosphatidylethanolamine in a range of
approximately 0.2-0.6%, and lyso alkyl acyl
phosphatidylethanolamine of <0.1%. In one embodiment, the krill
oil is a collodial krill oil. In one embodiment, the krill oil is a
homogeneous krill oil.
[0012] In one embodiment, the present invention contemplates a
method, comprising: a) providing: i) a patient exhibiting at least
one symptom of a medical disorder; ii) a krill oil comprising a
phospholipid concentration ranging between approximately 60%-99%, a
water content ranging between approximately 1-4% and an organic
solvent content of less than 1%; b) administering said krill oil to
said patient under conditions such that said at least one symptom
is reduced. In one embodiment, the krill oil comprises a viscosity
ranging between approximately 100,000-3,000,000 eP. In one
embodiment, the krill oil is a semi-solid at a temperature of at
least 40.degree. C. In one embodiment the medical disorder
comprises an age-related medical disorder. In one embodiment, the
age-related medical disorder includes, but is not limited to, a
lack of homeostatic control, macular degeneration, diabetes, or
inflammation. In one embodiment, the medical disorder comprises
malnutrition. In one embodiment, the medical disorder comprises an
ocular disorder. In one embodiment, the medical disorder comprises
a cardiovascular disorder.
[0013] In one embodiment, the medical disorder comprises a skeletal
medical disorder. In one embodiment, the medical disorder comprises
a central nervous system disorder. In one embodiment, the central
nervous system disorder comprses a mental disorder. In one
embodiment, the mental disorder includes, but is not limited to
infancy, childhood or adolescence disorders, cognitive disorders,
substance-related disorders, psychotic disorders including but not
limited to schizophrenia, mood disorders including but not limited
to depression, anxiety disorders, somatoform disorders, factitious
disorder, dissociative disorders, sexual disorders, eating
disorders, sleep disorders, impulse-control disorders, adjustment
disorders or personality disorders. In one embodiment, the medical
disorder comprises a muscular disorder. In one embodiment the
medical disorder comprises cachexia. In one embodiment, the medical
disorder comprises digestive tract medical disorder. In one
embodiment, the medical disorder comprises a dyslipidemic medical
disorder. In one embodiment, the medical disorder comprises a hair
disorder. In one embodiment, the medical disorder comprises a nail
disorder. In one embodiment, the medical disorder comprises a skin
disorder. In one embodiment, the krill oil is encapsulated. In one
embodiment, the krill oil is encapsulated in a hard gelatin
capsule. In one embodiment, the krill oil is encapsulated in a soft
gel capsule. In one embodiment, the krill oil further comprises an
additional ingredient including, but not limited to, minerals,
lipid soluble vitamins, lipid insoluble vitamins, bioactive health
ingredients and/or omega-3 oils. In one embodiment, the
administered composition ranges between 0.005-0.50 grams per day
per kilogram of said patient's body weight. In one embodiment, the
encapsulated krill oil comprises approximately 600 mg
phospholipids.
Definitions
[0014] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly dictates otherwise, e.g., reference to "a method"
includes a plurality of such methods.
[0015] The words "comprise", "comprises", and "comprising" are to
be interpreted inclusively rather than exclusively.
[0016] The term "optionally" means here the same as "possibly". For
example, compositions disclosed herein as "optionally comprises
excipients", means that the composition may or may not comprise
excipients, in other words the composition possibly comprises
excipients.
[0017] The term "patient", as used herein, is a human or animal and
need not be hospitalized. For example, out-patients, persons in
nursing homes are "patients." A patient may comprise any age of a
human or non-human animal and therefore includes both adult and
juveniles (i.e., children). It is not intended that the term
"patient" connote a need for medical treatment, therefore, a
patient may voluntarily or involuntarily be part of experimentation
whether clinical or in support of basic science studies.
[0018] The term "animal" as used herein, means species including
but not limited to mammals, fish, crustaceans, amphibians, reptiles
etc. In particular, a "companion animal" refers to any non-human
animal kept by a human as a pet or any animal of a variety of
species that have been widely domesticated as pets, such as dogs
(Canis familiaris), and cats (Felis domesticus), whether or not the
animal is kept solely or partly for companionship. Companion
animals also include working animals including but not limited to
horses, cows, pigs, goats, sheep, dogs (i.e., for example,
livestock herding) and/or cats (i.e., for example, rodent
control).
[0019] The term "effective amount" as used herein, refers to a
particular amount of a pharmaceutical composition comprising a
therapeutic agent that achieves a clinically beneficial result
(i.e., for example, a reduction of symptoms). Toxicity and
therapeutic efficacy of such compositions can be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., for determining the LD50 (the dose lethal to 50% of
the population) and the ED50 (the dose therapeutically effective in
50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index, and it can be
expressed as the ratio LD50/ED50. Compounds that exhibit large
therapeutic indices are preferred. The data obtained from these
cell culture assays and additional animal studies can be used in
formulating a range of dosage for human use. The dosage of such
compounds lies preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity.
The dosage varies within this range depending upon the dosage form
employed, sensitivity of the patient, and the route of
administration.
[0020] The term "symptom", as used herein, refers to any subjective
or objective evidence of disease or physical disturbance observed
by the patient. For example, subjective evidence is usually based
upon patient self-reporting and may include, but is not limited to,
pain, headache, visual disturbances, nausea and/or vomiting.
Alternatively, objective evidence is usually a result of medical
testing including, but not limited to, body temperature, complete
blood count, lipid panels, thyroid panels, blood pressure, heart
rate, electrocardiogram, tissue and/or body imaging scans.
[0021] The term "disease", as used herein, refers to any impairment
of the normal state of the living animal or plant body or one of
its parts that interrupts or modifies the performance of the vital
functions. Typically manifested by distinguishing signs and
symptoms, it is usually a response to: i) environmental factors (as
malnutrition, industrial hazards, or climate); ii). specific
infective agents (as worms, bacteria, or viruses); iii) inherent
defects of the organism (as genetic anomalies); and/or iv)
combinations of these factors
[0022] The terms "reduce," "inhibit," "diminish," "suppress,"
"decrease," "prevent" and grammatical equivalents (including
"lower," "smaller," etc.) when in reference to the expression of
any symptom in an untreated subject relative to a treated subject,
mean that the quantity and/or magnitude of the symptoms in the
treated subject is lower than in the untreated subject by any
amount that is recognized as clinically relevant by any medically
trained personnel. In one embodiment, the quantity and/or magnitude
of the symptoms in the treated subject is at least 10% lower than,
at least 25% lower than, at least 50% lower than, at least 75%
lower than, and/or at least 90% lower than the quantity and/or
magnitude of the symptoms in the untreated subject.
[0023] The term "drug" or "compound" as used herein, refers to any
pharmacologically active substance capable of being administered
which achieves a desired effect. Drugs or compounds can be
synthetic or naturally occurring, non-peptide, proteins or
peptides, oligonucleotides or nucleotides, polysaccharides or
sugars.
[0024] The term "administered" or "administering", as used herein,
refers to any method of providing a composition to a patient such
that the composition has its intended effect on the patient. An
exemplary method of administering is by a direct mechanism such as,
local tissue administration (i.e., for example, extravascular
placement), oral ingestion, transdermal patch, topical, inhalation,
suppository etc.
[0025] The term "krill oil" as used herein, refers here to any
mixture of extracted lipids derived from any portion of a krill
organism. The term is not limited to any particular method of
making krill oil, but any method known in the art is contemplated.
Conventionally made krill oil is generally considered to be a
liquid having a viscosity that is proportional to phospholipid
concentration, where viscosity increases a phospholipid
concentration increases.
[0026] The term "semi-solid" as used herein, refers to the physical
nature of a substance or material that has characteristics of both
solids and liquids. While similar to a solid in some respects,
semisolids can support their own weight and hold their shapes, and
retains an ability to flow under pressure. Semisolids are also
known as amorphous solids because at the microscopic scale they
have a disordered structure unlike the more common crystalline
solids. Those in the art would understand that the words
quasisolid, semisolid, and semiliquid may be used
interchangeably.
[0027] The term "gently dried" as used herein, refers to an
evaporation process that selectively removes low molecular weight
molecules (e.g., water and/or organic solvents such as ethanol).
For example, heating in a oven, evaporation under a nitrogen stream
or lyophilization are examples of gentle drying techniques.
[0028] The term "gently dried krill oil" as used herein, refers to
any mixture of extracted lipids derived from a krill organism that
has been processed into a product having substantially reduced
water content and/or organic solvent content (e.g., for example,
ethanol). For example, a gently dried krill oil may be expected to
have a water content of between 1-4% (w/w) and/or an organic
solvent content of less than 1%. A gentle drying process produces
an oil that is a semi-solid and has flowable properties at
temperatures above forty (40) degrees Centigrade (40.degree.
C.).
[0029] The term "small molecule organic solvent" as used herein
refers to a non-cyclic aliphatic molecule comprising five (5)
carbon atoms or less. Preferably, such solvents are short chain
alcohols (e.g., ethanol) or oxidative products thereof (e.g.,
acetone).
[0030] The term "temperature controlled reaction vessel" as used
herein refers to any container of any size or shape configured to
maintain a desired stable temperature. Preferably, the temperature
is maintained between +45.degree. C. to -50.degree. C.
[0031] The term "viscosity" as used herein, refers to a measured
parameter (e.g., expressed as poise) describing the fluidic nature
of a material. For example, a material having high viscosity may
have a glutinous nature or consistency and is described as being,
for example, sticky, thick and/or adhesive. On the other hand, a
material having low viscosity may have a free flowing fluid nature
that is more comparable water or other aqueous substance.
[0032] The term "poise" as used herein, refers to a
centimeter-gram-second unit of viscosity, equal to the viscosity of
a fluid in which a stress of one dyne per square centimeter is
required to maintain a difference of velocity of one centimeter per
second between two parallel planes in the fluid that lie in the
direction of flow and are separated by a distance of one
centimeter. Generally, the units of expression are reported as
centipoise.
[0033] The term "medical disorders", as used herein, refers to any
biological condition diagnosed by medically trained personnel to
require treatment. For example, medical disorders may include, but
are not limited to, hair disorders, nail disorders, skin disorders,
skeletomuscular disorders, multiple sclerosis, or sexual
disorders.
[0034] The term "delusion", as used herein, refers to any mental
condition that results in the perception of an altered reality.
Specifically, delusion is contemplated to be, but not limited to,
"delusions of grandeur", psychoses or hallucinations.
[0035] The term "schizophrenia", as used herein, refers to any
idiopathic psychosis characterized by chronically disordered
thinking and emotional withdrawal often associated with paranoid
delusions and auditory hallucinations.
[0036] The term "mood disorder", as used herein, refers to any
mental condition that results in behavior patterns representing
alterations in mood. Specifically, mood disorders are contemplated
to be, but not limited to, unipolar depression or bipolar
depression.
[0037] The term "personality disorder", as used herein, refers to
any condition, that may or may not respond to medical intervention,
that include perversion and chronic dysfunction appearing in
multiple forms during a patient's life. In one embodiment,
characteristic symptoms include, but are not limited to, avoidance,
paranoia, withdrawal and dependency. More generally, another
embodiment reflects a pattern of behavior such as, but not limited
to, chemical dependency, deviant eating patterns, hypochondriasis
or antisocial behavior.
[0038] The term "deviant eating patterns", as used herein, refer to
any condition wherein a compulsive behavior pattern results in a
significant increase or decrease in food consumption. Specifically,
the present invention contemplates, but is not limited to,
conditions such as bulimia and anorexia nervosa.
[0039] The term "depression", as used herein, refers to any nervous
system disorder and/or mental condition characterized by, but not
limited to, the following symptoms: withdrawal, insomnia,
hypersomnia, loss of appetite, altered daily rhythms of mood,
activity, temperature and neuroendocrine function. For example,
dsythymia, seasonal affective disorder and the like.
[0040] The term "neuroses", as used herein, refers to any mild
psychiatric disorder wherein the ability to comprehend is retained
but suffering and disability are very severe. Other characteristics
of neuroses include, but are not limited to, mood changes (i.e.,
for example, anxiety, panic, dysphoria) or limited abnormalities of
thought (i.e., for example, obsessions, irrational fears) or of
behavior (rituals or compulsions, pseudoneurological or hysterical
conversion signs).
[0041] The term "psychoses", as used herein, refers to any severe
psychiatric disorder wherein there is a marked impairment of
behavior, a serious inability to think coherently, or to comprehend
reality. Psychoses may include organic conditions associated with a
definable toxic, metabolic, or neuropathologic change characterized
by confusion, disorientation, memory disturbances and behavioral or
intrapulmonary disorganization.
[0042] The term "anxiety state", as used herein, refers to any
human emotion, closely allied with appropriate fear, often serving
psychobiologically adaptive purposes that is a cardinal symptom of
many psychiatric disorders. Specifically, anxiety is commonly
associated with, but not limited to, neurotic depression, panic
disorder, phobias, obsessive-compulsive disorders and other related
personality disorders.
[0043] The term "improved performance", as used herein, refers to
any biological condition, where controlled medical testing measures
results that medically trained personnel would considered above the
expected norm. For example, improved performance may be measured
for physical or mental tests.
[0044] The term "effective amount" refers to any amount of a
supplement that improves the palatability of the food or feed.
[0045] The term "ingredient" or "supplement" refers to any
composition can be formulated to a suitable form, such as a tablet,
a granule, a pellet or powder. The composition may be formulated
also to a pet treat or a hard gelatin capsule (sprinkle capsule)
can be filled with the composition.
[0046] As used herein, the term "omega-3 fatty acid" refers to
fatty acids which have the final double bond between the third and
the fourth carbon atom counting from the methyl end of the carbon
chain. Omega-3 fatty acids mainly concerned in this disclosure are
the long chain polyunsaturated fatty acids eicosapentaenoic acid
(EPA) and docospentaenoic acid (DHA) as well as the minor omega-3
fatty acids including eicosatetraenoic acid (ETA) and
docosapentaenoic acid (DPA).
[0047] The term "excipients", as used herein, refer to any
substance needed to formulate the composition to the desired form.
For example, suitable excipients include but are not limited to,
diluents or fillers, binders or granulating agents or adhesives,
disintegrants, lubricants, antiadherants, glidants, wetting agents,
dissolution retardants or enhancers, adsorbents, buffers, chelating
agents, preservatives, colours, flavours and sweeteners. Typical
excipients are for example starch, pregelatinized starch,
maltodextrin, monohydrous dextrose, alginic acid, sorbitol and
mannitol. In general, the excipient should be selected from
non-toxic excipients (IIG, Inactive Ingredient Guide, or GRAS,
Generally Regarded as safe, Handbook of Pharmaceutical Excipients).
Typical excipients in particular for tableting are for example
magnesium stearate, stearic acid, talc, silic, cellulose,
microcrystalline cellulose, methyl cellulose, polyvinylpyrrolidone
and--commercial products, such as Aerosil.RTM., Kollidon.RTM. and
Explotab.RTM.. Excipients can be added into the direct powder
compression formula.
[0048] The term "clinical nutrition", as used herein, refers to the
study, treatment and/or prevention of nutritionally-related medical
disorders, including but not limited to malnutrition. The term
"fluoride" as used herein interchangeably and refer to any compound
containing an organofluoride and/or an inorganic fluoride.
[0049] The term "low fluoride" as used herein may refer to the
product of any method and/or process that reduced the fluoride from
the original material by approximately one third (i.e., for
example, from 1500 ppm to 500 ppm). For example, "a low fluoride
crustacean phospholipid-protein complex" comprises approximately
one third of the fluoride than "a hydrolyzed and disintegrated
crustacean material".
[0050] The term "low fluoride oil" as used herein refers to a
lipid-rich composition created by the extraction of a
phospholipid-peptide complex composition sub-fraction using a
selective extraction process, such as with a supercritical carbon
dioxide fluid. Such a process removes approximately ten-fold of the
fluoride from the raw hydrolyzed and disintegrated crustacean
material.
[0051] The term "phospholipid composition" as used herein refers to
a low fluoride composition comprising a high percentage of polar
lipids (e.g., approximately 75%) created by the extraction of a
de-oiled phospholipid-peptide complex using a co-solvent, such as
ethanol.
[0052] The term "peptide" as used herein, refers to any of various
amides that are derived from two or more amino acids by combination
of the amino group of one acid with the carboxyl group of another
and are usually obtained by partial hydrolysis of proteins. In
general, a peptide comprises amino acids having an order of
magnitude with the tens.
[0053] The term "pharmaceutically acceptable" or "pharmacologically
acceptable", as used herein, refer to molecular entities and
compositions that do not produce adverse, allergic, or other
untoward reactions when administered to an animal or a human.
[0054] The term, "pharmaceutically acceptable carrier", as used
herein, includes any and all solvents, or a dispersion medium
including, but not limited to, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof; and vegetable oils, coatings,
isotonic and absorption delaying agents, liposome, commercially
available cleansers, and the like. Supplementary bioactive
ingredients also can be incorporated into such carriers.
[0055] The term, "purified" or "isolated", as used herein, may
refer to a peptide composition that has been subjected to treatment
(i.e., for example, fractionation) to remove various other
components, and which composition substantially retains its
expressed biological activity. Where the term "substantially
purified" is used, this designation will refer to a composition in
which the protein or peptide forms the major component of the
composition, such as constituting about 50%, about 60%, about 70%,
about 80%, about 90%, about 95% or more of the composition (i.e.,
for example, weight/weight and/or weight/volume). The term
"purified to homogeneity" is used to include compositions that have
been purified to `apparent homogeneity" such that there is single
protein species (i.e., for example, based upon SDS-PAGE or HPLC
analysis). A purified composition is not intended to mean that some
trace impurities may remain.
[0056] As used herein, the term "substantially purified" refers to
amino acid sequences, that are removed from their natural
environment, isolated or separated, and are at least 60% free,
preferably 75% free, and more preferably 90% free from other
components with which they are naturally associated. An "isolated
peptide or protein" is therefore a substantially purified peptide
or protein.
BRIEF DESCRIPTION OF THE FIGURES
[0057] FIG. 1 presents exemplary data of phospholipid (PL)
content's effect on viscosity of concentrated krill oil
compositions. Viscosity (cP) was measured at 35.degree. C. in 3
batches of krill oil. Batch 8723-13-06-04, 8723-05-03 and
8723-13-07-04 contained 42.1%, 45.2% and 47.3% phospholipids (PL),
respectively.
[0058] FIG. 2 presents exemplary data of a predictable krill oil
viscosity (cP) level having an average PL content of 42.2%
(RSD=0.7%) (N=6).
[0059] FIG. 3 presents the packaging label showing compositional
analysis and patent protection of a commercially available krill
oil composition (Omenia, Acasti, Inc.)
[0060] FIG. 4 presents exemplary data showing equilibrium moisture
content of empty gelatin capsules shells stored at different
relative humidities for 2 weeks at 20.degree. C.
[0061] FIG. 5 presents exemplary data measuring shear stress and
shear rate of krill oil incubated at 40.degree. C.
[0062] FIG. 6 presents exemplary data measuring shear stress and
shear rate of krill oil incubated at 20.degree. C.
[0063] FIG. 7 presents a conventional industrial phospholipid
extraction process based upon acetone extraction.
[0064] FIG. 8 presents exemplary data showing the relationships
between triacylglycerol (TAG) solubility in alcohols with
temperature and water content. Shown is the solubility of
cottonseed oil in absolute ethanol and isopropanol and their
azeotropes, BP=boiling point. Lusas et al., "Final report: IPA as
an extraction solvent" INFORM 8(3):290-306 (1997). [0065] FIG.
9A-9B presents exemplary data showing a Principal Component
Analysis of acetone versus ethanol fractionation of krill oil.
[0066] FIG. 9A: PCA correlation loading plot based on process
optimization design and response variables given in Tables 9, 11
and 12. Figures represent correlation coefficients r1 and r2
between the input X- and Y-variables and the first 2 PCs t1
(abscissa) and t2 (ordinate). The two ellipses represents 50% and
100% explained variances. [0067] FIG. 9B: PCA score plot showing
similarities and differences in response based on the applied
combinations of process conditions. Markers represents
(ENo)-temperature (.degree. C.)--water content in the ethanol phase
(%). Abbreviations explained in Table 7.
[0068] FIG. 10A-10E presents exemplary data showing a neutral lipid
response surface and contour plots based on models described in
Table 9.
[0069] FIG. 11A-11F presents exemplary data showing a polar lipid,
sodium chloride and total yield response surface and contour plots
based on models described in Table 9.
[0070] FIG. 12 presents one embodiment of a krill oil production
workflow to produce various embodiments of the present
invention.
[0071] FIG. 13 presents exemplary data showing a representative
fatty acid analysis of krill oil extracted with a solvent
comprising subcritical carbon dioxide.
[0072] FIG. 14 presents exemplary data showing a representative
phospholipid analysis of krill oil extracted with a solvent
comprising subcritical carbon dioxide.
[0073] FIG. 15 presents exemplary data showing a comparison of a
subcritical liquid CO.sub.2 solvent to a supercritical CO.sub.2
solvent monitoring triglyceride (TG) extraction. Circles:
Supercritical TG extraction batches. Diamonds: Subcritical liquid
TG extractions. It can be seen that very little difference in
solubility is present between the two solvents.
[0074] FIG. 16 presents an embodiment of a two stage process where
the manufacture of krill meal is performed on board the krill
fishing ship and the extraction is performed in an extraction plant
on shore.
DETAILED DESCRIPTION OF THE INVENTION
[0075] The present invention is related to methods of making
crustacean oil compositions. In particular, the crustacean oil
compositions are krill oil compositions. In some embodiments, the
krill oil compositions are concentrated in phospholipids. These
concentrated phospholipid krill oil compositions have a sufficient
flowability to permit successful encapsulation at phospholipid
concentrations that is currently unattainable in the art. Such
phospholipid krill oil compositions are capable of encapsulation
event though they may have a phospholipid concentration ranging
between approximately 60%-99% and a viscosity ranging between
100,000-3,000,000 cP. Such concentrated phospholipid krill oil
compositions may be created using a small molecule organic
solvent/water extraction mixture and/or sub-critical or
super-critical fluid extraction at low temperatures followed by a
drying process to remove water and organic solvent (e.g., for
example, ethanol).
[0076] In one embodiment, the present invention contemplates a
crustacean oil composition (e.g., for example, a krill oil
composition) comprising a phospholipid content ranging between
approximately 60%-99% (w/w), a water content ranging between
approximately 1-4% (w/w) and an organic solvent content of less
than 1%. In one embodiment, the viscosity of the crustacean oil
composition ranges between approximately 100,000-3,000,000
centipoise, preferably between approximately 100,000-150,000 cP,
preferably between approximately 140,000-1,700,000 cP, preferably
between approximately 2,000,000-3,000,000 cP or preferably between
approximately 2,500,000-275,000,000.degree. P. In other
embodiments, the present invention contemplates concentrated krill
oil compositions comprising at least 60% (w/w) phospholipids
including, but not limited to, phosphatidylcholine in a range of
approximately 35-55% (w/w), alkyl acyl phosphatidylcholine in a
range of approximately 3.0-6.0% (w/w), phosphatidylinositol in a
range of approximately 0.5-0.9% (w/w), phosphatidylserine (PS) in a
range of approximately 0.3-0.6%, lysophosphatidylcholine in a range
of approximately 1.5-4.0%, lyso alkyl acyl phosphatidylcholine in a
range of approximately 1.0-0.25%, phosphatidylethanolamine in a
range of approximately 2.0-4.0%, alkyl acyl
phosphatidylethanolamine in a range of approximately 0.25-1.25%,
cardiolipin+N-acylphosphatidylethanolamine in a range of
approximately 0.5-2.5%, lysophosphatidylethanolamine in a range of
approximately 0.2-0.6%, and lyso alkyl acyl
phosphatidylethanolamine of <0.1%.
[0077] It has previously been reported that some conventional krill
oil extraction methods generally result in a phospholipid
concentration ranging between approximately 39-52%. One particular
method is directed towards producing crustacean oils that are low
in fluoride and/or trimethylamine (TMA)/trimethylamine oxide
(TMAO). Bruheim et al., WO 2013/102792.
[0078] Others have reported krill oil compositions that are
suggested to be concentrated therapeutic phospholipid
concentrations where the phospholipids are claimed in the range of
approximately 50%-99%. One report appears to suggest that
concentrated phospholipid compositions are not resultant from
natural extraction methods, but require addition of previously
purified phospholipids to the naturally extracted krill oil.
Sampalis et al., U.S. Pat. No. 8,586,567 (herein incorporated by
reference).
I. Conventional Phospholipid Extraction Methods
[0079] Separation of neutral and polar lipids in crude lipid
extracts have been obtained by use of acetone fractionation or
deoiling. Ziegelitz, "Lecithin processing possibilities" INFORM
6:1224-1230 (1995); and Joshi et al., "Modification of lecithin by
physical, chemical and enzymatic methods"Eur. J. Lipid. Sci.
Technol. 108:363-373. (2006). These processes are used on an
industrial scale to de-oil crude vegetable lecithin from degumming
of vegetable oils after hexane extraction. The principle is based
on the insolubility of phospholipids and glycolipids in acetone.
Cool acetone at 8-10.degree. C. is intensively mixed in excess with
the crude lipid extract and the separated lecithin glycolipid
mixture is decanted. The acetone is removed and the product
formulated. Crude vegetable lecithin is described to give a
phospholipid product after acetone fractionation that can be sieved
into granules and powder. A less focused drawback is the possible
formation of adducts between acetone and the aminophospholipids
phosphatidylethanolamine (PE) and -serine (PS). Kuksis et al.,
"Covalent binding of acetone to aminophospholipids in vitro and in
vivo" In: Baynes, J. W.; Monnier, V. M.; Ames, J. M., and Thorpe,
S. R., Eds., Maillard Reaction: Chemistry at the Interface of
Nutrition, Aging, and Disease. Annals of the New York Academy of
Sciences, pp. 417-439 (2005).
[0080] The use of alcohols has been extensively studied by the
vegetable oil industry to find a safer and environmental friendly
alternative to hexane. Lusas et al., "Final report: IPA as an
extraction solvent" INFORM 8(3):290-306 (1997). The biggest
disadvantages of alcohols as compared to hexane are believed to be
higher energy consumption for vapour recovery and a lower
solubility of vegetable oil of about half of is practical
achievable in counter current extraction based on hexane (33% oil).
However, this property can also be used to achieve a fractionation
of the triacylglycerides (TAG) in the oil. For example, it has been
reported that TAG solubility in alcohols is reduced by both
temperature and water content. See, FIG. 8.
[0081] Crude vegetable lecithins obtained by the conventional
methods described above generally contain a mixture of PC, PE and
PI. It has been reported that the PC and PI fractions have
different solubility when extracted using the lower aliphatic chain
alcohols (e.g., ethanol and/or methanol) or when using
ethanol/water mixtures at varying temperatures where the PC
fraction appears more soluble. Joshi et al., "Modification of
lecithin by physical, chemical and enzymatic methods" Eur. J.
Lipid. Sci. Technol. 108:363-373. (2006). These processes have also
obtained lecithin with a PC content of 35-50%. For example,
fractionation of sunflower lecithin by ethanol fractionation has
been reported where the main objective was to separate PC, PI and
PE by use of absolute ethanol with an ethanol:lecithin ratio of
between 2:1 and 3:1 within a temperature range of between
35-65.degree. C. A PC-enriched ethanol phase was obtained having up
to approximately 62.5% phospholipids. Cabezas et al., "Sunflower
Lecithin: Application of a Fractionation Process With Absolute
Ethanol" J. Am. Oil Chem. Soc. 86:189-196 (2009).
[0082] The use of fractional separation to concentrate PUFA and
phospholipids in an isopropanol (IPA) extracted marine lipids has
also been reported. Sola et al., "Process for enrichment of fat
with regard to polyunsaturated fatty acids and phospholipids, and
application of such enriched fat" EP 0 519 916 B1 (1993). Following
partial IPA evaporation, insoluble lipids were separated and
collected that contained concentrated PUFA levels. A combination of
low temperature (4.degree. C.) ethanol fractionation and
.beta.-cyclodextrine complexation was oberved to extract
triacylglycerols and cholesterol from egg yolk. Su et al. "Study on
a Novel Process for the Separation of Phospholipids,
Triacylglycerol and Cholesterol From Egg Yolk" Journal of Food.
Science and Technology-Mysore 52:4586-4592 (2015). In this study,
75.8% of the TAG was precipitated after 10 hours at 4.degree. C.
After .beta.-cyclodextrine cholesterol complexation, the residual
phospholipids in the ethanol phase was obtained after solvent
removal. Alternatively, ethanol extracted lipids from dried egg
yolk were cold temperature crystallized to remove TAG. A PL-level
of 77% phospholipids was obtained based on crystallization at
0.degree. C. and 4% water content in the ethanol phase. Nielsen et
al., "In Situ Solid Phase Extraction of Lipids From Spray-Dried Egg
Yolk by Ethanol With Subsequent Removal of Triacylglycerols by Cold
Temperature Crystallization" Food Science and Technology 37:613-618
(2004).
[0083] Supercritical (SC) carbon dioxide has been used in several
studies to concentrate phospholipids. The neutral lipid components
are soluble in the SC carbon dioxide leaving the polar
phospholipids behind. SC carbon dioxide mixed with ethanol as a
co-solvent has also been used for fractionation of phospholipids,
oil and cholesterol from egg-yolk. Sahena et al., "Application of
Supercritical CO.sub.2 in Lipid Extraction--a Review" Journal of
Food Engineering 95:240-253 (2009).
[0084] Membrane filtration technology has been extensively studied
as a more environmental friendly and cost-effective alternative to
conventional organic solvent oil extraction and refining methods
related to degumming, dewaxing, deacidification, pigment removal,
concentration of minor components and/or separation of emulsions,
exemplified by several reports of soy and rice bran lecithin
deoiling using ultrafiltration. Coutinho et al., "State of Art of
the Application of Membrane Technology to Vegetable Oils: a Review.
Food Research International" 42:536-550 (2009); Manjula et al.,
"Laboratory Studies on Membrane Deoiling of Lecithin" J. Am. Oil
Chem. Soc. 85:573-580 (2008); and Liu et al., "Preparation of
Deoiled Soy Lecithin by Ultrafiltration" Journal of the American
Oil Chemists Society 88:1807-1812 (2011). Alternatively, reverse
micelles have been prepared in hexane by the addition of lecithin
and water. Filtration was then run in a diafiltration mode and the
obtained final PL concentration was in the range 90-96% measured as
acetone insoluble matter. Both nonporous polymeric composite
hydrophobic membrane and ceramic membrane (5 nm) have successfully
been used. Generally, a ceramic membrane will be of advantage since
it is inert against solvents and avoids the possible swelling of
synthetic membranes with change in the physicochemical properties.
"Production of purified yolk lecithin" JPS62263192(4) (Priority
date 1987 Nov. 16); "Production of egg yolk lecithin"
JP2001072693(A) (Priority date 1999 Sep. 3); "Method of preparing
low impurity, clear and transparent food grade lecithin and
product" CN1948317 (A) (Priority date 2005 Oct. 13); and "A
producing method for food-level concentrated soybean phospholipid"
CN101006824 (A) (Priority date 2007 Jan. 22).
II. Relationships Between Phospholipid Concentration and Krill Oil
Viscosity
[0085] In general, a higher level of phospholipids makes a krill
oil on average, thicker and more viscous than fish oils, for
example. If the viscosity gets too high, it becomes problematic to
encapsulate the oil resulting in capsules that are leaking and
unusable. This results in high losses for the capsule producers.
Another problem is when the variation in viscosity is high. While
the high variability may be overcome, it is more tedious and time
consuming for the capsule producer.
[0086] Hence, the present invention provides an improved
concentrated phospholipid krill oil comprising a low water content
and a low organic solvent content that is a semi-solid composition
at a temperature of at least 40.degree. C. that provides efficient,
and commericially feasible encapsulation. The data presented herein
show that, using the provided methods, a concentrated phospholipid
krill oil composition is made having a high viscosity but can
efficiently undergo commercial encapsulation with automated capsule
filling machines.
[0087] A. Effect of Phospholipid Content on Krill Oil Viscosity
[0088] Preliminary data from krill oil compositions having
approximately 42% phospholipids demonstrated a viscosity of equal
to, or less than, 800 cP. Although it is not necessary to
understand the mechanism of an invention, it is believed that these
moderate phospholipid krill oils have a low viscosity that allow
for easy encapsulation with repeatable results and of high quality.
However, as the viscosity of the krill oil becomes higher and
higher as a result of increases in phospholipid content, the
concomitant increase in viscosity prevents efficient capsule
filling.
[0089] Surprisingly, the data presented herein shows that by
reducing both the water content and ethanol content in these
concentrated phospholipid krill oils, a semi-solid oil is created
having flowable characteristics at a temperature of at least
40.degree. C. These conditions allow efficient use of automated
capsule filling machines that is not possible with highly viscous
conventional high phospholipid content, high water content and high
organic solvent content krill oils, because these compositions are
solid even at a temperature of approximately 80.degree. C.
[0090] To demonstrate the relationship between krill oil
phospholipids and viscosity, krill oils with different phospholipid
content were prepared in accordance with Example I. The viscosity
was measured in accordance with Example II which contained
phospholipids in the amounts of 42.1 g, 45.1 g and 47.3 g per 100 g
respectively at the temperature of 35.degree. C. The data
demonstrate that viscosity increases in proportion with increased
concentration of phospholipids. For example, the viscosity was
highest for the krill oil that had the highest phospholipid content
around 47.3 g/100 g and lowest for batch 8723-13-07-04 which
contained 42.1 g/100 g phospholipids. See, FIG. 1.
[0091] B. Low Batch-to-Batch Krill Oil Viscosity Variation
[0092] Krill oils with similar phospholipid content were prepared
to determine the reliability of producing compositions having a
predictable viscosity. For example, viscosity was measured for the
batches of krill oil compositions labelled 8723-13-05-04,
8723-13-06-05, 8723-13-07-02, 8723-13-02-07, 8723-13-02-08 and
8723-13-04-03, which contained phospholipids in the amount of 42.1
g, 42.3 g, 41.8 g, 42 g, 42.6 g and 42.5 g per 100 g respectively
at the temperature of 35.degree. C. The results demonstrate a
repeatable low viscosity with an average of 655 cP with a relative
standard deviation (RSD) of 17%. See, FIG. 2.
[0093] C. Concentrated Phospholipid Krill Oils (e.g., >60%)
[0094] It has been reported that a krill oil having an approximate
52% phospholipid concentration can be successfully encapsulated
into soft gels. Even though this report suggested that 65%
phospholipid krill oil compositions might be obtainable, there is
no enabling data and the reference teaches away from the presently
contemplated invention by stating that encapsulation of krill oils
having phospholipid concentrations higher than 52% was difficult
and considered commercially unsuccessful due to an increased
viscosity of the 65% composition. Bruheim et al., Example 5 and
Tables 20A-C; WO 2008/117062.
[0095] While it has also been suggested that concentrated
phospholipid krill oil in excess of 60% can be encapsulated, there
is no guidance to one of ordinary skill in the art as how to
successfully perform encapsulation of a 60% concentrated
phospholipid krill oil compositions that overcome the known
problems in the art as discussed herein. Sampalis et al., U.S. Pat.
No. 8,586,567 (herein incorporated by reference). The '567 patent
reported some viscosity measurements, but only for the 47%
phospholipid krill oil composition which was reported to be 1323
centipoise. The reported composition analysis for 53%, 66%, 80%
phospholipid krill oil compositions viscosity were not listed as a
measured parameter. However, for the 90% phospholipid krill oil
composition, viscosity was listed as an intended measured parameter
but no data was presented.
[0096] In fact, even though the Sampalis et al. '567 patent is
currently assigned to Acasti, Inc. at the time of filing the
present application, Acasti Inc. has no commercial krill oil
composition with a phospholipid concentration of greater than 50%,
much less a commercial product sold as soft gel capsules. Acasti's
current commercial krill oil (Omenia.RTM.) is supported by Sampalis
et al., U.S. Pat. No. 8,030,348 (herein incorporated by reference).
See, FIG. 3. The '348 patent discloses a viscosity of approximately
1300 centipoise (cP) for a phospholipid krill extract composition
having approximately 40% total phospholipids and teaches only that
encapsulation may be performed by conventional means known at the
time of filing of the application.
[0097] The commercially available Acasti, Inc. soft gel krill oil
composition package label also refers to a patent teaching the
treatment of patients for cardiovascular disease with enzyme-free
krill oil extracts having an active phospholipid content of >5%.
Sampalis et al., U.S. Pat. No. 8,057,825 (herein incorporated by
reference). While a complete compositional analysis of the
enzyme-free krill oil extracts was not disclosed including
viscosity measurements, the reference further teaches that each
capsule for clinical administration was loaded with only 800 mg of
krill oil. The reference did not provide any technical details
regarding any filling procedures or methods of modulation krill oil
viscosity related to capsule preparation and/or filling.
Consequently, one of skill in the art would expect that the
viscosity of highly concentrated phospholipid krill oil
compositions having phospholipid concentrations in excess of 60%
would prevent encapsulation.
[0098] In one embodiment, the present invention contemplates a high
phospholipid krill oil composition (e.g., for example, between
approximately 60-99% (w/w) that can be subjected to a gentle drying
process (e.g., for example, lyophilization, oven heating, nitrogen
streaming) to remove excess water and organic solvent (e.g., for
example, ethanol), where a flowable, high viscosity krill oil
results when maintained at a temperature of at least 40.degree. C.
These krill oils can be extracted using either sub-critical fluids
or super-critical fluids in optional combinations with a polar
solvent (e.g., for example, ethanol). See, Table 1.
TABLE-US-00001 TABLE 1 Compositional Analysis of A Lyophilized
Concentrated Phospholipid Krill Oil.sup.a Viscosity 140,000
Centipoise Phospholipids (g/100 g) Total 61.7 Phosphatidylcholine
(PC) 47.0 Alkyl Acyl Phosphatidylcholine (AAPC) 4.5
Phosphatidylinositol (PI) 0.75 Phosphatidylserine (PS) 0.5
Lysophosphatidylcholine (LPC) 2.9 Lyso Alkyl Acyl
Phosphatidylcholine (LAAPC) 0.65 Phosphatidylethanolamine (PE) 2.8
Alkyl Acyl Phosphatidylethanolamine (AAPE) 0.75 Cardiolipin +
N-acylphosphatidylethanolamine (CL/NAPE) 1.5
Lysophosphatidylethanolamine (LPE) 0.35 Lyso Alkyl Acyl
Phosphatidylethanolamine (LAAPE) <0.1 Astaxanthin (mg/100 g)
40.5 Fatty Acids (% w/w) Total n-3: 42.9 14:0 - 6.6 15:0 - 0.4 16:0
- 21.8 16:1 (n-9) - 0.6 16:1 (n-7) - 2.7 16:1 (n-5) - 0.6 i17:0 -
0.3 phytanic - 1.1 16:2 - 0.3 17:1 - 0.3 i18:0 - 0.3 16:4 (n-1) -
0.4 18:0 - 1.1 18:1 (n-9) - 8.1 18:1 (n-7) - 6.1 18:1 (n-5) - 0.4
18:2 (n-6) - 2.0 18:3 (n-3) - 1.7 18:4 (n-3) - 4.9 20:1 (n-9) - 0.5
20:1 (n-7) - 0.3 20:4 (n-6) - 0.4 20:4 (n-3) - 0.6 205 (n-3) EPA -
21.5 22:1 (n-9) - 0.6 21:5 (n-3) - 0.6 225 (n-3) - 0.5 22:6 (n-3)
DHA - 13.1 Others - 1.7 Flashpoint PMCC, .degree. C. 99 Specific
Gravity (@ 1.0038 15/15.degree. C.) Fecal Coliforms (grams) Not
Detected E. coli (grams) Not Detected Salmonella (grams) Not
Detected Aerobic plate count @ 35.degree. C. <10 (cfu/g) Total
yeast/mold (cfu/g) <10 Moisture @ 70.degree. C. (g/100 g) 1.9
Ethanol (mL/100 mL) 0.8 Peroxide (meq O.sub.2/kg fat) <0.1 TMA
(mg/100 g) 8 TMAO (mg/100 g) 1174 .sup.aavg: N = 2
Although it is not necessary to understand the mechanism of an
invention, it is believed that the krill oil composition detailed
in Table 1 having an approximate 60% phospholipid concentration and
a viscosity of 140,000 cP is capable of undergoing encapsulation
using commercially available capsule filling equipment while
maintained at a temperature of at least 40.degree. C. At this
temperature range, even though highly viscous, the lack of water
and organic solvent provides a semi-solid krill oil composition
that imparts flowability. Although it is not necessary to
understand the mechanism of the invention, it is believed that the
drying of the krill oil needs to be a gentle drying in order to
prevent oxidation believed responsible for the viscosity increase
in high phospholipid krill oils. Consequently, it is believed that
these gentle drying techniques, for example, freeze drying,
nitrogen streaming or oven heat, removes excess ethanol and water
as opposed to the standard oil drying methods such as falling film
and thin film evaporation.
[0099] In one embodiment, the present invention contemplates
encapsulating a gently-dried krill oil that was made by two step
sub-critical fluid extraction. Although it is not necessary to
understand the mechanism of an invention, it is believed that the
present method comprises removing triglycerides by precipitation
that concomitantly increases the krill oil phospholipid content to
above 60% and then gently drying the high phospholipid krill oil to
remove the excess water and organic solvent. In one embodiment, the
gently-dried krill oil was put into soft gel capsules after heating
to at least 40.degree. C.
III. Encapsulation Methods
[0100] A. Basic Concepts
[0101] The encapsulation of liquids and semi-solids (e.g., for
example, krill oil compositions) provides solutions for convenient
delivery through improved oral absorption of poorly water-soluble
drugs. Both hard and soft capsules can be considered and in each
case the capsule wall may comprise gelatin or some other suitable
polymer such as hypromellose. The choice of a hard or soft capsule
will depend primarily on the components of the formulation which
provides the best absorption characteristics as well as on the
physical characteristics, such as the viscosity of the formulation
and the temperature at which the product needs to be filled.
Numerous excipients are available for formulation of lipid-based
systems and their compatibilities with hard gelatin capsules have
been tested. The availability of new enhanced manufacturing
equipment has brought new opportunities for liquid-filled hard
capsules. Commercially available filling and sealing technologies
for hard capsules provides for scale-up capabilities.
[0102] When using compounds having higher molecular weights and
greater lipophilicity that increase viscosity conventional
formulation strategies are no longer adequate to achieve acceptable
bioavailability. Cole et al., "Challenges and opportunities in the
encapsulation of liquid and semi-solid formulations into capsules
for oral administration" Advanced Drug Delivery Reviews 60:747-756
(2008). Such lipophilic formulations make use of excipients which
are either liquid or semi-solid in nature and therefore the only
solid oral dosage form that has good patient acceptability is a
capsule.
[0103] Two types of capsules are commonly used and are classified
according to the nature and flexibility of the capsule shell. Soft
capsules are single unit solid dosage forms comprising a liquid or
semi-solid fill and are usually oblong or oval in shape. They are
formed, filled and sealed in one operation using a rotary die
process. The technology is currently available from a few
specialist companies.
[0104] Hard capsules are single unit dosage forms which are
manufactured separately and supplied empty for filling. E. T. Cole,
Hartgelatinekapseln, In: H. Sucker, P. Fuchs, P. Speiser (Eds.),
Pharmazeutische Technologie, Georg Thieme Verlag, Stuttgart, 1991,
pp. 319-320; and B. E. Jones, "Manufacture and properties of
two-piece hard capsules" In: F. Podczek, B. E. Jones (Eds.),
Pharmaceutical Capsules, Pharmaceutical Press, London, 2004, pp.
79-100. They are always cylindrical in shape, consist of a cap and
body and have domed ends. Soft capsule have been used as unit dose
containers for liquids for many years whereas hard capsules have
conventionally been used for delivery of solids in the form of
powders and pellets.
[0105] 1. Encapsulation of Water-Insoluble Compounds
[0106] One characteristic of concentrated phospholipid krill oil
compositions for which the liquid fill technology is applicable is
related to low water solubility and concomitant increases in
viscosity. The use of a capsule filled with a semi-solid
formulation may be advantageous in improving bioavailability. An
improvement in bioavailability may result the inclusion of
polysorbate 80 which ensures complete release in a finely dispersed
form and which was likely to facilitate solubilization by bile
acids.
[0107] 2. Physical Characteristics of Capsules
[0108] A useful polymer for the production of hard capsules
comprises a gelatin compound. Additional components of a capsule
shell includes, but is not limited to, water (which acts as a
plasticizer), coloring agents and/or opacifiers. If an alternate to
gelatin is required, hard capsules may be manufactured from
hydroxypropyl methylcellulose (HPMC). Recent advances made in the
HPMC capsule technology have resulted in the achievement of similar
in vitro dissolution rates to gelatin capsules. The composition of
a shell material for hard gelatin capsules for powder or liquid
filling is identical, as are the capsule sizes.
[0109] Soft shells are generally thicker than those of hard
capsules and are also most commonly manufactured from gelatin but,
in contrast to hard capsules, the plasticizer includes, but is not
limited to, glycerin, sorbitol and water. Soft shell capsules may
also include a coloring agent and/or an opacifier. Alternative
shell materials to gelatin that are either commercially available
or in development, include a combination of iota carrageenan and
hydroxypropyl starch, a specific potato starch and polyvinyl
alcohol and the advantages and disadvantages of alternative
materials to gelatin have been discussed. G. Reich, "Formulation
and physical properties of soft capsules" In: F. Podczek, B. E.
Jones (Eds.), Pharmaceutical Capsules, Pharmaceutical Press,
London, 2004, pp. 201-212. The presence of a plasticizer in the
soft gelatin shell can give a relatively high permeability to
oxygen and it has been reported that at relative humidities of
between 31 and 80%, the log of the oxygen permeability coefficient
decreases linearly with decreasing glycerin content. Horn et al,
"Soft gelatin capsules II: oxygen permeability study of capsule
shells" J. Pharm. Sci. 64:851-857 (1975). Therefore, it is likely
that the oxygen permeability of a sealed hard gelatin capsule will
be lower than that of a soft capsule. An assessment of the smell of
highly odorous products which were transferred from commercially
available soft capsules into hard capsules and scaled effectively
demonstrated this to be the case. Cade et al., "Liquid filled and
sealed hard gelatin capsules" Acta Pharm. Technol. 33:97-100
(1987).
[0110] In practice, soft gelatin capsules can perform well as
oxygen barriers by modification of the type and level of
plasticizer used. The primary function of the plasticizer in a soft
capsule shell is to maintain the flexibility of the shell wall. The
plasticizers are, however, hygroscopic and absorb moisture when
exposed and it has been shown that the sorption of water by soft
gelatin shells containing different plasticizers is considerably
higher than is the case with hard gelatin capsules. The commonly
used plasticizers for soft gelatin shells also have the ability to
solubilize water-soluble compounds.
[0111] Any formulation approach should consider a potential
interaction between the fill material and the capsule wall. To
illustrate this point the moisture content of a range of different
molecular weight PEGs at a relative humidity of 55% has been shown
to vary between 18.8% for PEG 200 and b1% for the solid PEGs.
Walters et al., "Moisture uptake of excipients for liquid filling
into hard gelatin capsules" Proceedings Pharmaceutical Technology
Conference (Utrecht) 18:97-101 (1999); and G. Rowley, "Filling of
liquids and semi-solids into hard two-piece capsules" In: F.
Podczek, B. E. Jones (Eds.), Pharmaceutical Capsules,
Pharmaceutical Press, London, 2004, pp. 169-194. Liquid PEGs can
thus only be used in low concentrations for filling hard gelatin
capsules.
[0112] 3. Fill Characteristics of Capsules
[0113] Fill formulations for hard gelatin capsules may be Newtonian
liquids, such as oils, thixotropic or shear thinning gels or
semi-solid matrix products that are filled at elevated temperatures
and in which the compound is either dissolved or suspended as a
fine dispersion. For example, a model system in which lactose was
dispersed in poloxamers of different viscosities revealed that the
limiting concentration of the dispersed phase decreased as particle
size decreased and as the molecular weight of the poloxamer
increased. Kattige et al., "Influence of rheological behaviour of
particulate/polymer dispersions on liquid-filling characteristics
for hard gelatin capsules" Int. J. Pharm. 316:74-85 (2006).
Satisfactory filling characteristics were achieved with poloxamer
F68 up to a concentration of 35% w/w when the mean particle size of
lactose was 22.6 .mu.m and 27.5% w/w when the mean particle size
was 15.3 .mu.m.
[0114] In principle, any formulation composition found to be
compatible with gelatin can be used provided that the viscosity of
the fill material conforms to the requirements of the filling
process. The uniformity of capsule fill weights was shown to
decrease as the viscosity of thermo-softened fill materials
increased. Saeed et al., "Rheological Characteristics of Poloxamers
and Poloxamer/Silicon Dioxide Gels in Relation to Liquid Filling of
Hard Gelatin Capsules" Proceedings Pharmaceutical Technology
Conference (Athens) 16:217-224 (1997); and Hawley et al., "Physical
and chemical characterisation of thermosoftened bases for
molten-filled hard gelatin capsule formulation" Drug Dev. Pharm.
18:1719-1739 (1992). The general guidelines for fill materials are
listed in Table 2.
TABLE-US-00002 TABLE 2 General Guidelines for filling
liquids/semi-solid fill materials into hard gelatin capsules
Parameter Recommendation Temperature of fill material Max.
~70.degree. C. Viscosity at the temperature of dosing 10-1000 cPs
Dosing characteristics Clean break from dosing nozzle Absence of
"stringing"
[0115] Compatible excipients have been categorized into three
groups and are summarized below. See, Tables 3, 4 and 5. The broad
categories are lipophilic liquid vehicles, semi-solid lipophilic
vehicles and viscosity modifiers for lipophilic liquid vehicles and
solubilizing agents, surfactants, emulsifying agents and absorption
enhancers.
TABLE-US-00003 TABLE 3 Lipophilic liquid vehicles compatible with
hard gelatin capsules Refined specialty oils Medium chain
triglycerides and related esters Arachis oil Caprylic/capric
triglycerides (Akomed E, Akomed R, Miglyol 810, Captex 355) Castor
oil Medium chain triglyceride (Labrafac CC) Cottonseed oil
Propylene glycol diester of caprylic/capric acid (Labrafac PG)
Maize (corn) oil Propylene glycol monolaurate (Lauroglycol FCC)
Olive oil Fractionated coconut oil (Miglyol 812) Sesame oil
Caprylic/capric/diglyceryl succinate (Miglyol 829) Soybean oil
Medium chain diesters of propylene glycols (Miglyol 840) Sunflower
oil Partial ester of diglycerides with natural fatty acids
(Softisan 645)
TABLE-US-00004 TABLE 4 Semi-solid lipophilic vehicles and viscosity
modifying substances compatible with hard gelatin capsules
Substance Tradename Arachis oil Groundnut 36 Castor oil Cutina HR
Cottonseed oil Sterotex Palm oil Softisan 154 Soybean oil Akosol
407 Aerosil Cetosteryl alcohol Cetyl alcohol Semi-synthetic
glycerides based on hydrogenated vegetable oils Gelucires 33/01,
39/01, 43/01 Glyceryl behenate Compritol 888 ATO Glyceryl
palmitostearate Precirol ATO 5 Hydrogenated coco-glycerides
Softisans 100, 142 Caprylic/capric/stearic triglycerid Softisan 378
Bis-diglyceryl/caprylate/caprate/stearate/adipate Softisan 649
Stearic acid Steryl alcohol
TABLE-US-00005 TABLE 5 Solubilizing agents, surfactants,
emulsifying agents and absorption enhancers compatible with hard
gelatin capsules Substance Tradename Propylene glycol monocaprylate
Capryol 90 Polyglycolized glycerides Gelucire 44/14, 50/13
Polyoxyl-40 hydrogenated castor oil Cremophor RH 40 Glycerol
monostearate/di-triglycerides + glycerin Imwitor 191 Glyceryl
monocaprylate Imwitor 308 a Glyceryl cocoate/citrate/lactate
Imwitor 380 Glyceryl mono-di-caprylate/caprate Imwitor 742
Isosteryl diglyceryl succinate Imwitor 780 K Glyceryl cocoate
Imwitor 928 Glyceryl caprylate Imwitor 988 Oleoyl macrogol-8
glycerides Labrafil M 1944 CS Linoleoyl macrogolglycerides Labrafil
M 2125 CS) PEG-8 caprylic/capric glycerides Labrasol) Lauric acid
Propylene glycol laurate Lauroglycol 90 Oleic acid PEG MWN4000
Polyglycerol dioleate Plurol Oleique CC 497
Polyoxyethylene-polyoxypropylene copolymer Poloxamer 124, 188
Partial glycerides of hydroxylated unsaturated Softigen 701 fatty
acids PEG-6 caprylic/capric glycerides Softigen 767 Polyoxyethylene
glyceryl trioleate Tagat TO Polyoxyethylene(20)sorbitan monooleate
Tween 80
[0116] 6. Capsule Filling Equipment
[0117] The equipment that is necessary to enable automatic filling
of hard gelatin capsules with either hot or cold liquid is
available in a range of filling rates, from laboratory to
production scale. The liquid to be filled is usually dispensed by
volume and the machines all meet the conventional requirements to
allow for the industrial manufacture of liquid-filled capsules.
Cole E., "Liquid-filled and -sealed hard gelatin capsule
technologies" In: M. J. Rathbone, J. Hadgraft, M. S. Roberts
(Eds.), Modified-Release Drug Delivery Technology, Marcel Dekker,
New York, 2003, pp. 177-188. A variety of commercially available
capsule filling machines are available. See, Table 6.
TABLE-US-00006 TABLE 6 Major capsule-filling machines for liquid
filling of hard gelatin capsules up to production scale Approximate
filling Machine type Filling action rate (capsules/h) Robert Bosch
GmbH GKF 1400 L Intermittent motion 60,000 GKF 701 L 36,000 Harro
Hoefliger GmbH KFM III-C Intermittent motion 5,000 IMA Zanasi
Division Zanasi 6/12 6000-12,000 Zanasi 25/40 25,000-40,000 Zanasi
Lab 8/16 All intermittent motion 8000-16,000 Zanasi Plus
32E/48E/70E/ 32,000-85,000 85E MG2 MG Compact 6000-96,000 MG Futura
All continuous motion 6000-96,000 Planeta 100 100,000 Qualicaps F-5
4000 F-40 All continuous motion 30,000 F-80 60,000 F-120 90,000
F-150 120,000 Schaefer Technologies Inc. LF-10 Semi-automatic
10,000-25,000 Bonapace IN-CAP Intermittent motion 3000
A capsule-filling machine for dosing hard capsules with high
viscosity pastes and which operates by extrusion of a cylinder of
material directly into a capsule body and an alternate system for
filling highly viscous materials has been developed that operates
by filling hot mixtures under high pressure by means of time
controlled pneumatic valves and which has been used in a production
environment for many years. Strickrodt J., "Fully automatic process
for filling high viscosity pastes into hard gelatin capsules"
Pharm. Ind. 52:1276-1279 (1990); and Bohne et al., "A new process
for filling hard gelatin capsules with semisolid materials.
Experiences in development and production" Pharm. Ind. 53:1127-4134
(1991).
[0118] B. Thixotropic Capsule Carriers for High Viscosity
Compositions
[0119] Due the above described problems regarding the ability to
fill capsules with high viscosity compositions, one proposed
solution in the art offered to solve this problem was to suspend
the high viscosity compositions in a thixotropic carrier to
facilitate capsule filling.
[0120] Conventional carrier compositions are either liquid at
ambient temperature or they become liquid with heating and they are
poured into the hard or soft capsules as a liquid. When these
carrier compositions are liquid at ambient temperature, the active
agents incorporated therein must be dissolved. Consequently, they
cannot be added at high loadings and it is difficult to maintain
them in a uniform distribution within the capsule. Carrier
compositions that are solid at ambient temperature require heating
before they can be poured into the capsules and the heat can damage
the capsule walls, reduce the activity of the active ingredient or
damage other heat sensitive ingredients.
[0121] Thixotropic carriers were suggested to alleviate the
problems encountered in filling capsules with highly viscous
compositions because active agents may be easily mixed together by
stirring and stable uniform dispersions with high loadings can be
achieved because the carrier becomes semi-solid when the stirring
is stopped. For example, one thixotropic carrier has been reported
that includes vegetable oil (84%-95%), a viscosity modifier (1%-9%)
and a surface active agent (1%45%). Viscosity modifiers may
include, but are not limited to, glyceryl palmitol stearate and
glyceryl behenate. Surface active agents may include, but are not
limited to, polyglyceryloleate.
[0122] A thixotropic gelatin carrier composition may be used as a
vehicle in the manufacture of soft or hard gelatin capsule. These
compositions may range from about 84% to 95% of a vegetable oil,
from about 1% to 9% of a viscosity modifier and from about 1% to
15% of a surface active agent. Usually, when a carrier composition
is stirred it becomes fluid and active solids may be dispersed in
the carrier during stirring. When the stirring is stopped, the
carrier becomes a semi-solid and maintains the active solids in a
stable uniform dispersion. Although it is not necessary to
understand the mechanism of an invention, it is believed that up to
about 50% by weight of active solids can be dispersed in a
thixotropic carrier composition and the active solids can include
vitamins, pharmaceuticals or nutriceuticals or combinations
thereof. Matthews J., "Thixotropic Gelatin Carrier Composition"
U.S. Pat. No. 6,365,181 (herein incorporated by reference).
IV. Gently Dried Krill Oils
[0123] In one embodiment, the present invention contemplates a
method comprising gentle drying a concentrated phospholipid krill
oil. In one embodiment, the gentle drying comprises
lypophilization. In one embodiment, the gentle drying comprises
oven heat. In one embodiment, the gently drying comprises a
nitrogen stream. Although it is not necessary to understand the
mechanism of an invention the gentle drying encompasses any process
that will efficiently remove water and organic solvent (e.g.,
ethanol) from a krill oil without inducing oxidation-induced
increases in viscosity.
[0124] In one embodiment, the present invention contemplates a
method for encapsulation of krill oil with a phospholipid content
of 60% or more subsequent to a gentle drying method. As described
in detail above, encapsulation of concentrated phospholipid krill
oils has previously proven difficult due to the high viscosity of
the oil. The present invention solves this problem by creating a
semi-solid concentrated phospholipid krill oil at a temperature of
at least 40.degree. C. having a physical characteristic of
flowability, even though the viscosity may be measured between
100,000-3,000,000 cP.
[0125] 1. Lyophilization
[0126] Freeze-drying, also known as lyophilisation, lyophilization,
or cryodesiccation, is a dehydration process typically used to
preserve a material or make the material more convenient for
handling (e.g., for example, capsule filling). Freeze-drying works
by freezing a material and then reducing the surrounding pressure
to allow the frozen water in the material to sublimate directly
from a solid phase to a gas phase.
[0127] a. The Freeze-Drying Process
[0128] It is generally accepted that there are at least four stages
in the complete drying process including, but not limited to,
pretreatment, freezing, primary drying, and secondary drying.
[0129] Pretreatment includes any method of treating the product
prior to freezing. This may include, but is not limited to,
concentrating the product, formulation revision (i.e., addition of
components to increase stability, preserve appearance, and/or
improve processing), decreasing a high-vapor-pressure solvent,
and/or increasing the surface area.
[0130] The freezing step is often done by placing a material in a
freeze-drying flask and rotating the flask in a bath, called a
shell freezer, which is cooled by processes including, but not
limited to, mechanical refrigeration, dry ice and methanol, and/or
liquid nitrogen. Alternatively, commercially available
freeze-drying machines are available (e.g., for example, a
lyophilizer). In this step, the material is cooled to below its
triple point, typically defined as the lowest temperature at which
the solid and liquid phases of the material can coexist. This
ensures that sublimation, rather than melting, will occur in the
following steps. Larger crystals are easier to freeze-dry. To
produce larger crystals, the product should be frozen slowly or can
be cycled up and down in temperature. This cycling process is
called annealing. Alternatively, the freezing may be done rapidly,
in order to lower the material to below its eutectic point quickly,
thus avoiding the formation of ice crystals. Usually, the freezing
temperatures are between -50.degree. C. and -80.degree. C.
Amorphous materials do not have a eutectic point, but they do have
a critical point, below which the product must be maintained to
prevent melt-back or collapse during primary and secondary
drying.
[0131] During the primary drying phase, the pressure may be lowered
to a range of a few millibars, and enough heat can be supplied to
the material for the ice to sublime. The amount of heat necessary
can be calculated using the sublimating molecules' latent heat of
sublimation. In this initial drying phase, about 95% of the water
in the material may be sublimated. This phase may be slow (e.g.,
for example, several days), because, if too much heat is added, the
material's structure could be altered. In this phase, pressure can
be controlled through the application of partial vacuum. Vacuum is
believed to speed up the sublimation, making it useful as a
deliberate drying process. Furthermore, a cold condenser chamber
and/or condenser plates provide a surface(s) for the water vapor to
re-solidify on. This condenser plays no role in keeping the
material frozen; rather, it prevents water vapor from reaching the
vacuum pump, which could degrade the pump's performance. Condenser
temperatures are typically below -50.degree. C. (-60.degree. F.).
Of note is that, in this range of pressure, the heat is brought
mainly by conduction or radiation; the convection effect is
negligible, due to the low air density.
[0132] A secondary drying phase aims to remove unfrozen water
molecules, since the ice was removed in the primary drying phase.
This part of the freeze-drying process may be governed by the
material's adsorption isotherms. In this phase, the temperature can
be raised higher than in the primary drying phase, and can even be
above 0.degree. C., to break any physico-chemical interactions that
have formed between the water molecules and the frozen material.
Usually the pressure can also be lowered to encourage desorption
(typically in the range of microbars, or fractions of a pascal).
However, there are products that benefit from increased pressure as
well. After the freeze-drying process is complete, the vacuum is
usually broken with an inert gas, such as nitrogen, before the
material is sealed. At the end of the operation, the final residual
water content in the product is extremely low, around 1% to 4%.
[0133] b. Properties of Lyophilized Products
[0134] If a freeze-dried substance is sealed to prevent the
reabsorption of moisture, the substance may be stored at room
temperature without refrigeration. Freeze-drying also causes less
damage to the substance than other dehydration methods using higher
temperatures. Freeze-drying does not usually cause shrinkage or
toughening of the material being dried.
[0135] In addition, flavors, smells and nutritional content
generally remain unchanged, making the process popular for
preserving edible substances (e.g., for example, krill oil
compositions). However, water is not the only chemical capable of
sublimation, and the loss of other volatile compounds such as
acetic acid (vinegar) and alcohols can yield undesirable
results.
[0136] Freeze-dried products can be rehydrated (reconstituted) much
more quickly and easily because the process leaves microscopic
pores. The pores are created by the ice crystals that sublimate,
leaving gaps or pores in their place.
[0137] 2. Lyophilization of Concentrated Phospholipid Krill Oil
Products
[0138] In some embodiments, the present invention contemplates a
method that effectively reduces the water content and organic
solvent content of a concentrated phospholipid krill oil product to
create a flowable semi-solid at a temperature of at least
40.degree. C. In one embodiment, the flowable concentrated
phospholipid semi-solid krill oil product is encapsulated in to a
capsule.
[0139] In general, the present invention may be practiced on any
krill oil composition regardless of the method of making.
Nonetheless, it is preferred that the concentrated phospholipid
krill oil is produced using fresh krill (e.g., generally on board
the trawler), where the whole krill is disintegrated and subjected
to hydrolytic enzymatic digestion, or disintegrated and subjected
to denaturation by heating. Both hydrolytic enzymatic digestion
and/or heat denaturation produces a phospholipid-peptide complex
(PPC) krill meal composition that is capable of krill oil
extraction.
[0140] Subsequent to hydrolytic enzyme digestion and/or heat
denaturation, the PPC may be subjected to a low speed
centrifugation (e.g., 2000 g) that separates the krill exoskeleton
from the peptide\lipid biomass (PPC). This process has the added
advantage of producing low fluoride krill oil compositions as the
vast majority of fluoride is located in the exoskeleton. The PPC
may then be used as a starting material for krill oil extraction
using either a polar solvent (e.g., ethanol), a sub-critical
gaseous fluid or a supercritical gaseous fluid (e.g., for example,
carbon dioxide). These extractions may be peformed individually or
in series and in any combination.
[0141] These process have been reported to result in the production
of concentrated phospholipid krill oil compositions having high
viscosity. See, Bruheim et al., United States Patent Application
Publication No. 2013/0225794 (herein incorporated by reference);
and Sampalis et at, U.S. Pat. No. 8,586,567 (herein incorporated by
reference). As discussed above, conventionally extracted krill oil
viscosity is proportional to phospholipid content. There are no
available reports of successful encapsulation of krill oil having a
phospholipid concentration of above 50% (e.g., Omenia.RTM., Acasti,
Inc.). Although it is not necessary to understand the mechanism of
an invention it is believed that the encapsulation of a 60%
phospholipid krill oil is not possible with commercially available
capsule filling equipment because the resulting viscosity results
in a solid composition at a temperature of at least 80.degree.
C.
[0142] Such concentrated phospholipid krill oil compositions may
then be lyophilized as described above into a low water content,
low organic solvent content semi-solid krill oil product that is
flowable at a temperature of at least 40.degree. C. This semi-solid
phospholipid concentrate krill oil composition may then
encapsulated using commercially available capsule filling
equipment.
V. Phospholipid Enrichment by Small Molecule Organic Solvent
Fractionation
[0143] A. Krill Oil Extraction from Krill Meal
[0144] Commercial extraction of krill oil from dried krill meal is
mainly performed by ethanol extraction. In the process,
triglycerides are co-extracted with the phospholipids and the
amount of polar lipids in the obtained krill oil decided by the
krill meal composition. In some embodiments, the present invention
contemplates a method for concentrating an ethanol-extracted krill
meal polar lipid fraction by a reduction in temperature during an
ethanol evaporation step. Although it is not necessary to
understand the mechanism of an invention, it is believed that the
precipitation of triglycerides from an extracted krill oil
composition results in a greater phospholipid percentage (w/w) as
the originally extracted concentrated phospholipid krill oil
composition.
[0145] Crude krill oil extracted from Antarctic krill (Euphausia
superba) contains typically around 40% phospholipids and 300 mg/kg
poly-unsaturated fatty acids (PUFAs; e.g., for example, omega-3
PUFAs). In one embodiment, the present invention contemplates a
krill oil composition comprising greater than 300 mg/kg omega-3
PUPA product and at least 60% phospholipids. Although it is not
necessary to understand the mechanism of an invention, it is
believed that by modifying krill oil extraction methods to increase
the phospholipid content, the omega-3 PUPA concentration
concomitantly increases.
[0146] Krill oil production based on ethanol extraction of krill
meal can be performed on-board fishing vessels. In one embodiment,
an ethanol evaporation step comprises separation and
crystallization of triacylglycerol (TAG). Although it is not
necessary to understand the mechanism of an invention, it is
believed that TAG separation and crystallization can be obtained by
controlling water content and temperature level. In one embodiment,
a >60% phospholipid ethanol soluble fraction is isolated
subsequent to removal of a non-soluble fraction.
[0147] The data provided herein illustrates a central composite
design within a temperature range of between 4.1.degree. C. and
-24.1.degree. C. showing effects on lipid and fatty acid
composition, sodium chloride (NaCl) and astaxanthin levels in
ethanol soluble lipids having a water content in the ethanol phase
of between 0-10%. These variables are futher assesed using response
surface models having an R.sup.2>0.74. Concentrations of a
combined polar lipid fraction (e.g., for example, PE+PC+lyso-PC)
were observed to be above 60%.
[0148] Optimal conditions to obtain increased polar lipid levels
were observed when the ethanol phase comprised 10% water at a
temperature of 249.degree. K (-24.1.degree. C.), where predicted
levels of polar lipids based on these conditions are approximately
73%. For example, an above 60% level of polar lipids can be
obtained by using: i) pure ethanol at a temperature level of
249.degree. K (-24.1.degree. C.); ii) a water content below 6.0% at
a temperature level of 277.3.degree. K (4.1.degree. C.); and iii)
combinations within the conditions specified in (i) and (ii).
Product yield is positively correlated to temperature (K) and
negatively to water content. Predicted levels for the above
specified conditions giving >60% polar lipid content is 66.0%
and 64.1%, respectively.
[0149] Astaxanthin content was observed to be strongly negatively
correlated to water content and the highest carry over to the polar
lipids is obtained using pure ethanol. Pure ethanol concentration
also coincides with lowest level of undesired compounds including,
but not limited to, sodium chloride (NaCl), cholesterol,
trimethylamine oxide (TMAO) and trimethylamine (TMA).
[0150] Omega-3 PUFA retention was maximal during extractions at low
temperature and a 1.5% water content. These fractional conditions
produced a solid fraction and a soft sediment fraction that
separated easily. In one embodiment, the soft sediment composition
comprises an ethanol insoluble phase including, but not limited to,
the separated and crystallized TAGs, <1% polar lipids, and
approximately 559 mg/kg astaxanthin (cf. 222 mg/kg astaxanthin in
the krill oil fraction). Although it is not necessary to understand
the mechanism of an invention, it is believed that the presently
disclosed ethanol fractionation method may be applied to
concentrate astaxanthin esters from a krill oil fraction.
[0151] It is further believed that water content in the ethanol
phase has a strong impact on the composition and yield of polar
lipids. For example, the extracted polar lipid content depends on
both ethanol water content and krill meal moisture content. In some
embodiments, the present invention contemplates an ethanol
fractionation method that controls ethanol water content and krill
meal moisture content to create a krill oil comprising at least 60%
polar lipids.
[0152] In one embodiment, the present invention contemplates an
organic solvent (e.g., for example, ethanol) fractionation method
that produces a concentrated phospholipid krill oil composition. In
one embodiment, the concentrated phospholipid krill oil composition
further comprises lipids, fatty acids, astaxanthin and sodium
chloride (NaCl). In one embodiment, the ethanol fractionation
method partitions out TMAO and TMA from the concentratred
phospholipid krill oil composition.
[0153] B. Phospholipid Enrichment: Effects of Ethanol Water Content
and Extraction Temperature
[0154] A two-factorial rotatable central composite design (CCD) was
used to study the effect of temperature and concentration of water
(w/w %) in the ethanol phase on yield and composition of the lipids
in the sediment and ethanol phase. Meyers et al., In: Response
Surface Methodology. Process and Product Optimization Using
Designed Experiments John Wiley & Sons, New York, N.Y. (USA)
(2002). A total of 11 experimental settings were used with three
replications of the centre point, See, Table 7.
TABLE-US-00007 TABLE 7 Coded and experimental values for the
experimental design variables. Water Temperature ENO. Coded value
(%) (.degree. C.) (K) 1 -1 -1 1.5 -20.0 253.2 2 1 -1 8.5 -20.0
253.2 3 -1 1 1.5 0.0 273.2 4 1 1 8.5 0.0 273.2 5 -1.41 0 0.1 -10.0
263.2 6 1.41 0 9.9 -10.0 263.2 7 0 -1.41 5.0 -24.1 249.0 8 0 1.41
5.0 4.1 277.3 9 0 0 5.0 -10.0 263.2 10 0 0 5.0 -10.0 263.2 11 0 0
5.0 -10.0 263.2
The distance (.alpha.) from the centre point to the axial points
(star points) was calculated based on the equation
.alpha.=(2k).sup.1/4, were k is the number (2) of independent
variables. The temperature was varied between 249.0 and 277.3 K
(+24.1.degree. C. and +4.1.degree. C., respectively), and the water
content was varied between 0.1% (w/w) and 9.9% (w/w), respectively.
For practical reasons, the experiments were run in blocks depending
on the temperature level.
[0155] 1. Acetone Fractionation of Krill Oil
[0156] The krill oil used as a starting material in this study
contained approximately equal levels of polar and neutral lipids.
Acetone fractionation was performed to establish the obtainable
level based on current industrial lecithin de-oiling practice. The
level of polar lipids could be increased to 72% with a reduction in
neutral lipids to 3.4%. The yield of acetone insoluble matter was
44.5%. In contrast, the acetone soluble fraction contained low
level of polar and high level of neutral lipids. See, Table 8.
TABLE-US-00008 TABLE 8 Composition (w/w %) and yield (% of starting
material) of lipids after acetone fractionation of Rimfrost krill
oil. Acetone Acetone Rimfrost insoluble soluble Batch# fraction
fraction Ash g/100 g 3.7 NA NA Astaxanthin esters.sup.a mg/kg 222
15 354 Free astaxanthin mg/kg <2 <2 2 TMA-N mg N/100 g 43 47
<1 TMAO-N mg N/100 g 73 44 2 NaCl g/100 g 1.4 NA NA Water g/100
g 0.23 2.1 NA TAG g/100 g 38 1.8 56 DAG g/100 g 0.7 <0.5 1.2 MAG
g/100 g <1 <1 <1 FFA g/100 g 2.7 <0.5 4.2 Cholesterol
g/100 g 2.2 0.9 2.8 Cholesterol esters g/100 g <0.5 <0.5
<0.5 PE g/100 g 1.8 2.2 <0.5 PI g/100 g <1 <1 <1 PS
g/100 g <1 <1 <1 PC g/100 g 41 67 11 Lyso-PC g/100 g 1.2
2.6 <0.5 Polar lipids g/100 g 44.4 72 11.3 Neutral lipids g/100
g 44 3.4 63.9 Sum lipids g/100 g 88.4 75.4 75.3 Yield w/w % -- 44.5
60.9 TAG--triacylglycerols; DAG--diacylglycerols;
MAG--monoacylglycerols; FFA--free fatty acids; Chol--cholesterol;
Chol-ester--cholesterol esters; PE--phosphatidylethanolamine;
PI--phosphatidylinositol; PS--phosphatidylserine;
PC--phosphatidylcholine; Lyso-PC--lyso phospahatidylcholine;
Polar--polar lipids = PE + PC + Lyso-PC; Neutral--neutral lipids
=TAG + DAG + FFA + Chol + Chol-ester; NA--not analyzed. .sup.aGiven
as astaxanthin equivalents.
Most of the astaxanthin esters and cholesterol followed the acetone
phase, with only 6.8% and 40.9%, respectively, of the initial level
found in the obtained polar lipid fraction. TMA and TMAO was less
soluble in the acetone and concentrated quantitatively in the polar
lipids. The observed levels indicate some loss of TMAO during the
processing, and might be linked to redox reactions and formation of
TMA. The latter compound is volatile and may, to some extent, be
removed during evaporation of the acetone after the fractionation
process.
[0157] 2. Ethanol. Fractionation of Krill Oil
[0158] Ethanol fractionation of krill oil for the concentration of
polar lipids was evaluated by use of a 2-factorial CCD within a
temperature range of between -24.1.degree. C. to 4.1.degree. C. and
an ethanol water content between 0.1% (w/w) and 9.9% (w/w). See,
Table 7. The starting material, Rimfrost krill oil, contained 0.23
g/100 g moisture and this was corrected for when adjusting the
water content in the ethanol phase. See, Table 8. Moisture content
in the krill oil was quantified and validated by spiking of samples
to a 1% and 2% level using the Karl Fischer technique. Krakeli et
al., "Matriksinterferenser i krillolje ved maling av vanninnhold
(KF)" Nofima notat. (2015). A retrieval degree of 101.7% and 98.2%,
respectively, confirmed a feasible use of the method to quantify
moisture content in this matrix.
[0159] The data show an extraction of polar lipids above 60% (w/w)
based on several of the experimental conditions with the highest
level of 70.8% (e.g., ENo 2). See, Table 9.
TABLE-US-00009 TABLE 9 Composition (w/w %), yield (% of starting
material) and astaxanthin (mg/kg) in ethanol soluble components.
ENo FFA Chol Chol-ester MAG DAG TAG Neutral PE PI PS PC Lyso-PC
Polar NaCl Yield Asta 1 3.7 3.3 <0.5 <1 0.8 17 24.7 2.0 <1
<1 61 2.1 64.7 1.9 63.9 60 2 3.6 3.3 <0.5 <1 1.0 3.5 11.5
1.6 <1 <1 67 2.7 70.8 2.7 44.2 15 3 2.4 2.7 <0.5 <1 0.6
23 28.5 2.0 <1 <1 53 2.4 57.5 1.5 73.0 72 4 3.6 3.0 <0.5
<1 0.9 7.8 15.4 1.8 <1 <1 62 1.6 65.5 1.9 56.9 28 5 3.0
2.6 <0.5 <1 0.9 24 30.3 1.7 <1 <1 48 1.5 51.1 1.4 74.9
114 6 3.8 3.1 <0.5 <1 0.8 3.6 11.3 1.6 <1 <1 62 2.4
86.3 2.5 45.3 17 7 4.0 3.4 <0.5 <1 0.9 6.5 14.9 1.7 <1
<1 61 2.2 54.8 1.7 50.6 25 8 2.7 2.6 <0.5 <1 0.7 12 17.7
2.0 <1 <1 54 1.9 58.1 1.5 64.8 38 9 3.6 3.1 <0.5 <1 1.0
12 19.6 2.4 <1 <1 56 2.0 60.1 1.6 62.2 38 10 3.7 3.1 <0.5
<1 1.0 8.7 16.7 2.3 <1 <1 59 2.4 63.5 1.6 59.6 29 11 4.7
3.5 <0.5 <1 1.1 9.6 18.4 2.4 <1 <1 81 2.4 66.0 1.8 58.1
28 FFA--free fatty acids; Chol--cholesterol;
Chol-ester--cholesterol esters; MAG--monoacylglycerols;
DAG--diacylglycerols; TAG--triacylglycerols; Neutral - neutral
lipids = FFA + Chol + DAG + TAG; PE--phosphatidylethanolamine;
PI--phosphatidylinositol; PS--phosphatidylserine;
PC--phosphatidylcholine; Lyso-PC--lyso phospahatidylcholine; Polar
- polar lipids = PE + PC + Lyso-PC; Asta - Astaxanthin esters
expressed as astaxanthin equivalents
These polar lipid levels are close to levels obtained using acetone
fractionation (e.g., 72%, w/w). See, Table 8. The obtained yield
was also at approximately the same level (.about.44%) and confirms
that ethanol extraction may be used as an alternative fractionation
solvent to acetone.
[0160] Principal Component Analysis (PCA) of the combined
acetone/ethanol experimental settings and all measured responses
shows an explained variance by the first and second Principal
Component (PC) of 63% and 19%, respectively. See, FIG. 9A. The
third and fourth PC (not shown) explained 8% and 5%, respectively,
of the variance. The first PC explains the variance in polar and
neutral lipids and astaxanthin esters, and the second PC the
variance in PUFA. The loading plot shows a negative correlation
between yield and the level of polar lipids in the ethanol soluble
fraction. The level of polar lipids are associated with PC, TMAO,
TMA and NaCl, and the water content in the ethanol phase. The level
of neutral lipids in the ethanol soluble fraction are associated
with TAG, astaxanthin esters and n-6 PUFA. The latter might
indicate a higher level of n-6 PUFA in TAG compared to PL.
[0161] The temperature level is in part correlated to the level of
neutral lipids and astaxanthin. The data shown herein suggests that
lowering the exraction temperature appears to increase the
concentration of polar lipids in the ethanol extract (e.g.,
positively correlated with temperature). In contrast, a lowered
extraction temperature appears to decrease the concentration of
DAG, cholesterol and FFA, and in part to PUFA and n-3 PUFA in the
ethanol extract (e.g., negatively correlated with temperature). The
variance of lyso-PC and PE were explained by less than 50%. A score
plot shows groups of experimental conditions giving a comparable
extracted lipid fraction composition. FIG. 9B. To the left, ENo 3
and 5 experimental conditions provide the highest level (e.g., most
concentrated) of neutral lipids and astaxanthin esters. To the
right, ENo 2 and 6 experimental conditions provide the highest
level (e.g., most concentrated) of polar lipids and NaCl. At the
top, the ENo 11 experimental condition gave the highest level
(e.g., most concentrated) of DAG, cholesterol and FFA. The fatty
acid composition and TMAO/TMA levels were only analyzed under the
ENo 1-4 experimental conditions. However, the inclusion of these
parameters had only minor impact on the position of the respective
samples in the score plot, and improved the explained variance by
PC1 from 62% to 63% and PC2 from 15% to 19%. The ENo 11
experimental condition showed some deviations from the ENo 9 and
Eno 10 experimental conditions, however, Eno 11 was not identified
as an outlier based on the default Unscrambler software settings.
See, FIG. 9B.
[0162] Overall, a response surface modelling of the assessed
variables showed a complex and different behavior of the chemical
constituents depending on their polarity and solubility in the
ethanol phase. For most of the variables, satisfactory models with
R.sup.2>0.88 were achieved. See, Table 10 and FIG. 10.
TABLE-US-00010 TABLE 10 Response models after backward elimination
of nonsignificant (NS) regression coefficients representing
composition (w/w %), and yield (%) of ethanol soluble components.
FFA Chol Asta TAG Neutral PE Intercept -1.50E+02 6.43E+00 1.04E+02
-5.39E+01 -7.46E+00 -1.61E+02 T 1.25E+00.sup.a NS NS 2.98E-01
1.46E-01 1.23E+00 T.sup.e -2.54E-05 -5.38E-05 NS NS NS -2.54E-05 W
-2.18E+00 NS -2.12E+01 NS -8.39E+00 NS W.sup.2 -1.87E-02.sup.b
-1.11E-02.sup.c 1.31E+00 1.79E-01 1.49E-01 -2.73E-02 T .times. W
9.29E-03 5.01E-04 NS -1.46E-02 NS 9.38E-04 R.sup.2 0.918 0.775
0.925 0.980 0.968 0.935 Lyso-PC PC Polar NaCl Yield Intercept
1.95E+00 1.34E+02 1.35E+02 1.59E+00 -6.41E+01 T NS -3.10E-01
-2.99E-01 NS 5.22E-01 T.sup.e NS NS NS NS NS W 1.20E+00 NS NS
8.48E-01 -2.77E+00 W.sup.2 NS NS NS 1.74E-02 NS T .times. W
-4.43E-03.sup.d 4.73E-03 4.84E-03 -3.50E-03 NS R.sup.2 0.462 0.770
0.742 0.885 0.977 T - temperature in Kelvin; W - water content (%)
in ethanol phase; NS--not significant .sup.ap = 0.052; .sup.bp =
0.058; .sup.cp = 0.097; .sup.dp = 0.0502.
The TAG level in the ethanol soluble lipids was dependent on
temperature (T), a squared water content (W.sup.2) and a negative
interaction between the two variables. See, Table 10. The response
surface demonstrates a dominant effect of W within the tested
T-range. The solubility of TAG in ethanol is strongly dependent on
the water content and temperature. The ethanol azeotrope contains
4.5% by weight of water, close to the centre point in this study.
See, Table 7. The use of a 1:3 ratio between krill oil and ethanol
gives a TAG concentration of 9.5% in the experimental ethanol
phase. This is higher than the solubility of TAG in pure ethanol
and a water/ethanol azeotrope within the tested temperature range.
See, FIG. 7; and Lusas et. al., "Final report: IPA as an extraction
solvent" INFORM 8(3):290-306 (1997). Although it is not necessary
to understand the mechanism of an invention, it is believed that a
two-phase system may be formed during cool-down of the samples
after the initial conditioning at 35.degree. C. and, depending on
the temperature, some of the TAGs may crystallize and form a solid
phase that can be observed as a sediment after centrifugation of
the experimental samples.
[0163] A full model could be fitted to the FFA response, including
both negative and positive regression coefficients. However, this
suggested the use of a slightly higher criteria for the removal of
insignificant regressors, where the final model included variables
with a significance level up to p=0.058. See, Table 10. This
response surface demonstrated a curvature with a dominating effect
of temperature. The interaction effect gives a minor effect of
water at low compared to high temperature levels. The data suggest
that a maximum FFA level can be expected at conditions T=264 K.
(-9.degree. C.) and 7% water.
[0164] The cholesterol response model shows a negative squared
effect of both T and W in addition to a positive interaction. The
obtained model has a R.sup.2=0.77 and includes the W.sup.2
regressor with a significance level of 0.096. The response surface
shows a clear curvature for water. The net T-effect is
approximately linear, mainly caused by a very low T.sup.2
coefficient. The interaction effect gives less effect of water at
low compared to high temperature level. See, Table 10.
[0165] The astaxanthin response model shows no effect of T, and a
negative main and positive squared effect of W. See, Table 10.
Astxanthin mainly partitions in the apolar TAG phase and the shown
astaxanthin response is consistent with the TAG and neutral lipids
responses. In contrast, the acetone fractionation carried less
astaxanthin over to the polar lipids than the ethanol
fractionation. See, Table 8. However, even at the best experimental
condition (e.g., ENo 5) an astaxanthin reduction of 49% was
observed in the ethanol fractionation.
[0166] The neutral lipids response model show a positive effect of
T, a negative main and positive squared effect of W, and no
interaction. See, Table 10. The resulting response surface is very
similar to the TAG response and identifies the neutral lipids as a
dominating component.
[0167] The dominating polar lipid class (e.g., PC) response model
shows a simple behavior with a negative effect of T and a positive
interaction. See, Table 10. The data show a response surface having
a dominating effect of water, reflecting a reduced solubility of
TAG with increasing water content in an ethanol phase. See, FIG.
11. A less satisfactory model was obtained for lyso-PC with a
negative effect of W and positive interaction. PE shows a more
complex behavior to ethanol fractionation with a positive and
negative main and squared T effect, respectively, and a negative
squared W effect and positive interaction. The level of lyso-PC in
krill oil is very low which increases the analytical uncertainty
and also influences the obtainable quality of the fitted model. In
general, the total polar lipids response surface is dominated by
the PC response wherein the highest level is obtained by combining
the lowest T and highest W within the experimental range.
[0168] The sodium chloride response model shows a positive main and
squared effect of water content in the ethanol phase and a negative
interaction effect. See, Table 10. The response surface reflects a
dominating effect of water with minor effect of temperature. See,
FIG. 11.
[0169] The response model for the total yield of ethanol soluble
compounds is only dependent on T and W with no interaction. See,
Table 10. The model has a very high-explained variance of 97.7%.
The response surface reflects the linear effect of the main
variables with the highest effect of temperature within the tested
experimental range. See, FIG. 11. The highest yield is obtained
when combining the highest T and lowest W within the experimental
range. However, a higher yield also gives a higher TAG and lower PL
content in the final product.
[0170] The fatty acid composition was analyzed in the cube points
of the experimental design. See, Table 11.
TABLE-US-00011 TABLE 11 Fatty acid composition (g/100 g) in
Rimfrost, acetone insoluble and soluble lipids, and ethanol soluble
lipids ENo 1-4. Acetone fractionation Ethanol fractionation-soluble
Fatty acid Rimfrost Insoluble Soluble ENo1 ENo2 ENo3 ENo4 C14:0 5.8
1.6 8.5 2.7 1.7 4.2 2.3 C16:0 13.9 13.8 13.8 12.3 11.9 13.5 12.2
C16:1 n-7 2.4 0.7 3.8 1.6 0.9 1.9 1.1 C16:2 n-4 0.4 0.1 0.7 0.3 0.1
0.5 0.2 C16:3 n-4 0.2 0.1 0.3 0.1 0.1 0.1 0.1 C18:0 0.8 0.5 0.8 0.5
0.4 0.6 0.5 C18:1 n-9/7/5 10.2 6.3 12.8 7.6 5.6 8.3 6.1 C18:2 n-6
1.3 0.9 1.6 1.1 0.9 1.2 0.9 C18:3 n-3 2.6 2.1 3.1 2.4 2.1 2.5 2.1
C18:3 n-6 0.1 0.1 0.2 0.2 0.1 0.2 0.1 C18:4 n-3 5.6 2.6 8 4.9 3.4 5
3.5 C20:0 0.1 <0.1 0.1 <0.1 <0.1 0.1 <0.1 C20:1 n-9/7
0.7 0.4 0.8 0.5 0.4 0.5 0.4 C20:2 n-6 0.1 0.1 0.1 0.1 <0.1 0.1
0.1 C20:3 n-3 0.2 0.1 0.2 0.1 0.1 0.1 0.1 C20:3 n-6 <0.1 0.1
<0.1 0.1 0.1 0.1 0.1 C20:4 n-3 0.5 0.5 0.5 0.6 0.6 0.5 0.5 C20:4
n-6 0.2 0.2 0.1 0.2 0.2 0.2 0.2. C20:5 n-3 11.4 15.2 9.3 15.6 16.5
14.3 15.4 C21:5 n-3 0.5 0.6 0.5 0.7 0.7 0.6 0.6 C22:0 <0.1
<0.1 <0.1 <0.1 <0.1 <0.1 <0.1 C22:1 n-11/9/7 0.3
0.4 0.2 0.3 0.3 0.3 0.3 C22:4 n-6 <0.1 <0.1 <0.1 <0.1
<0.1 <0.1 <0.1 C22:5 n-3 0.3 0.4 0.3 0.4 0.4 0.3 0.3 C22:6
n-3 6.8 9.3 4.9 8.7 8.9 8.1 8.7 C24:1 n-9 <0.1 0.1 <0.1
<0.1 <0.1 <0.1 <0.1 Sum saturated 20.5 15.9 23.2 15.5
14 18.4 15 Sum monoenoic 19.6 7.9 17.6 10 7.2 11 7.9 Sum PUFA 30.1
32.4 29.8 35.5 34.2 33.6 32.9 Sum n-3 PUFA 27.8 30.8 26.8 33.4 32.7
31.4 31.2 Sum n-6 PUFA 1.7 1.4 2.0 1.7 1.3 1.8 1.4 Sum identified
fatty acids 64.2 56.2 70.6 61 55.4 63 55.8
The level of n-3 PUFAs in Rimfrost krill oil was increased after
ethanol fractionation (ENo 1) from 27.8 to 33.4% (e.g., W=1.5% and
T=-20.degree. C.). See, Table 7. This corresponds to an improvement
of 20.1%. Increasing the ethanol/water content to 8.5% (e.g., Eno
2) decreased PUFA levels level to 32.7%. Increasing the temperature
to 0.degree. C. further reduced PUFA levels to 31.4% and 31.2% at
water contents of 1.5% and 8.5%, respectively. In contrast, acetone
fractionation gave a level of polar lipids in the acetone insoluble
fraction of 72%; slightly higher than the upper level obtained
experimentally by ethanol fractionation (70.8% in ENo 2). However,
this higher acetone extraction yield was not reflected by the
obtained level of n-3 PUFA (30.8%). See, Table 11. Ethanol
fractionation provided a higher level of n-3 PUFAs at all tested
experimental conditions as compared to acetone fractionation. For
example, acetone fractionation gave a level of soluble fraction n-3
PUFAs of 26.8%. The acetone soluble lipid composition was dominated
by TAG and the results indicates a high level of PUFA in this lipid
fraction.
[0171] The data disclosed herein show that both the Rimfrost
starting material krill oil and acetone insoluble fraction
contained high levels of TMA and TMAO. Table 8. TMA has a strong
smell characterized as fishy and ammonia, and can be expected to
have a significant impact on the sensory characteristics of the
product. Both compounds were quantitatively carried over to the
acetone insoluble fraction, with a minor increase in the TMA level
of the obtained product. TMAO was somewhat reduced, probably
through redox reaction with unsaturated compounds during the
processing steps. The level of TMA in the ethanol soluble fraction
showed a comparable level to acetone insolubles. Cf, Table 8 and
Table 12.
TABLE-US-00012 TABLE 12 Levels (mg N/100 g) of trimethylamine (TMA)
and trimethylamine N-oxide (TMAO) in ethanol soluble lipids from
cube design treatments. TMA-N TMAO-N 1 42 105 2 78 142 3 45 91 4 58
130
It was also noted that the TMA/TMAO levels correlated to the water
content in the ethanol phase. FIG. 9. The level of TMAO was
increased compared to the krill oil level and is highly correlated
to the TMA level (R.sup.2=0.817).
[0172] After centrifugation of the ethanol fractionated samples a
solid, and easy to decant, sediment was obtained at the three
lowest temperature levels. At 0 and 4.1.degree. C. a softer
sediment was formed, where a separation funnel was used to control
the separation of the two phases. The solid structure and analyses
of the composition of the sediment confirmed the separation and
crystallization of TAGs in the ethanol insoluble phase. Table 13.
Some higher FFA and lower cholesterol concentration could also be
observed.
TABLE-US-00013 TABLE 13 Composition (w/w %), yield (% of starting
material) and astaxanthin (mg/kg) in the sediment after ethanol
fractionation. Chol- ENo FFA Chol ester TAG DAG MAG Neutral PE PI
PS PC Lyso-PC Polar NaCl Yield Asta 1 5.1 2.1 <0.5 75 3.5 <1
86.3 0.7 <1 <1 <1 <0.5 0.7 -- 38.9 454 2 4.1 2.2
<0.5 65 1.1 <1 72.5 1.8 <1 <1 15 <0.5 17 -- 59.0 356
3 4.9 0.7 <0.5 75 2.1 <1 83.0 0.9 <1 <1 <1 <0.5
0.9 -- 30.1 559 4 5.8 0.8 <0.5 77 1.0 <1 84.4 2.0 <1 <1
5.5 <0.5 7.5 -- 48.0 430
The polar lipids levels was <1% when using a 1.5% water content.
(e.g., ENo 1 and ENo 3). Use of 8.5% water significantly increased
the polar lipid level. (e.g., ENo 2 and ENo 4). The astaxanthin
level was generally much higher than observed in the ethanol
soluble lipids with a maximum level of 559 mg/kg as compared to 222
mg/kg in the krill oil. Cf. Table 13: ENo3 and Table 7. The tested
processing conditions can thereby optionally be applied to
concentrate astaxanthin esters from the krill oil.
[0173] In one embodiment, the present invention contemplates a
method for concentrating a combination of polar lipids (e.g., for
example, PE+PC+lyso-PC) to above 60%. In one embodiment, the method
comprises ethanol fractionation of a krill extract (e.g., for
example, a krill oil). Although it is not necessary to understand
the mechanism of an invention, it is believed that the presently
disclosed method can easily be integrated in a downstream process
for removal of ethanol after extraction of lipids from krill meal.
In one embodiment, the method comprises an ethanol extraction
mixture ranging between 90%:10% ethanol/water and 100%:0%
ethanol/water. In one embodiment, the method comprises a 94%:6%
ethanol/water mixture. In one embodiment, the method is performed
at a temperature ranging between approximately 277.3.degree. K
(4.1.degree. C.) and 249.degree. K (-24.1.degree. C.). In one
embodiment, the method produces a concentrated phospholipid krill
oil ranging between approximately 60-73% polar lipids. FIG. 11.
[0174] Overall, the data suggest that ethanol fractionation results
in a product yield that is positively correlated to temperature and
negatively to water content. In some embodiments, ethanol
fractionation provides a krill oil having a polar lipid content of
>60% polar lipid content (e.g., 66.0% and 64.1%). FIG. 5. In
comparison, a level of 72% polar lipids could be obtained based on
acetone fractionation of the krill oil.
[0175] The data also show that astaxanthin content is strongly
negatively correlated to water content and the highest carry over
of astaxanthin from the starting krill oil material to the ethanol
fractionated concentrated polar lipid krill oil is obtained when
using pure ethanol (e.g., .about.114 mg/kg). A 100% ethanol
extraction also resulted in a concentrated polar krill oil having
the lowest level of unwanted compounds (e.g., for example, NaCl,
cholesterol, TMAO and TMA). Best retention of n-3 PUFA is obtained
at low temperature and water content.
[0176] From a practical perspective an easy to separate solid to
soft sediment was formed based on the used fractionation
conditions. The solid structure and analyses of the composition of
the sediment confirmed the separation and crystallization of TAGs
in the ethanol insoluble phase. The polar lipid levels were <1%
when using a 1.5% water content with astaxanthin levels of
approximately 559 mg/kg. In one embodiment, the present invention
contemplates a method comprising ethanol fractionation of krill oil
that creates a compposition comprises concentrated astaxanthin
esters.
[0177] Although it is not necessary to understand that mechanism of
an invention, it is believed that the water content in the ethanol
phase has a strong impact on the composition and yield of polar
lipids. In a processing plant, the polar lipid yield depends on the
water content of the used ethanol and the moisture content in the
krill meal. The need to control these parameters depends on the
target level of polar lipids after ethanol fractionation.
VII. Non-Supercritical Carbon Dioxide Krill Oil Extraction
[0178] In one embodiment, a krill lipid extract is obtained by
extraction process of dried meal derived from the marine crustacean
Euphausia superba (Antarctic Krill) with sub-critical liquid
CO.sub.2 and ethanol extraction (two steps). It is a two stage
process where the manufacture of krill meal is performed on board
the krill fishing ship and the extraction is performed in an
extraction plant on shore. The manufacturing process is presented
in FIG. 16. [0179] 1) The manufacturing process of the krill oil
starts on board a ship. Antarctic krill is immediately (maximally
20 minutes after catch) shredded through a knife cutter into pieces
of a particle size of 3-6 mm at a temperature of 1-2.degree. C.
[0180] 2) Fresh water and proteolytic enzymes are added and heated
to a temperature of 55-60.degree. C. The reaction is allowed to run
for 45 minutes [0181] 3) The material is then transferred to a
decanter separating the fluorine-containing fine particles and the
liquid proteinaceous fraction. [0182] 4) The material is then
heated to a temperature of 93.degree. C. in order to deactivate the
enzymatic activity. [0183] 5) The liquid proteinaceous fraction is
then transferred to a separation step by a specially designed
decanter, separating the solid phase containing insoluble proteins
and polar lipids concentrate (PPC) from the hydrolysate. [0184] 6)
The PPC is then dried in a thin film vacuum drier and packed in air
tight bags under nitrogen atmosphere. [0185] 7) The aqueous soluble
protein (hydrolysate) and neutral lipid phase are feed to a
separator separating the neutral lipid phase from the hydrolysate.
[0186] 8) The oil is stored in air tight containers under nitrogen
atmosphere. [0187] 9) The hydrolysate are continuously feed into a
flash evaporator for dewatering/concentration giving a concentrated
hydrolysate fraction (CHF) with dry weight of 55-70% and stored in
air tight containers under nitrogen atmosphere. [0188] 10) The
lipids and proteins are separated and extracted from the
hydrolysate via the addition of ethanol and sub-critical liquid
CO.sub.2. The hydrolysate and 100% ethanol are loaded into
extractors and kept there under elevated temperature and pressure
until extraction rate is reached and process completed
(approximately 12 hours). [0189] 11) Proteins and krill material
are removed from the lipid extract by precipitation and filtration.
[0190] 12) The ethanol and residual water are removed by subsequent
gentle drying evaporation steps (e.g., lyophilzation). The lipid
extract is then loaded into drums and stored at room temperature.
Use of controlled enzymatic hydrolysis step in production of krill
meal allows separation of fluorine containing exoskeleton and thus,
the reduction of the fluorine content of krill lipid extract.
[0191] In one embodiment, the present invention contemplates a
method for producing krill oil comprising extracting a krill meal
with a solvent comprising subcritical carbon dioxide, ethanol and
water to produce a concentrated phospholipid krill oil (e.g.,
OLYOIL) that was subsequently subjected to lyophilization. Table
14. In one embodiment, the method further comprises extracting the
krill meal with a solvent comprising supercritical carbon dioxide.
See, FIG. 12.
TABLE-US-00014 TABLE 14 Lyophilized Krill Oil Extracted With A
Subcritical Carbon Dioxide Solvent Astaxanthin 12-13 mg/100 gm
Esterified Astaxanthin 234 mg/1000 gm Ethanol 0.3 ml/100 gm Total
Fatty Acids.sup.a 68.7-72.1% Flash Point, PMCC 112-118.degree. C.
Specific Gravity @ 15/15.degree. C. 1.0011-1.0118 Total
Microbiology <10 cfu/g Moisture @ 70.degree. C. 3 g/100 g
Peroxide <0.1 Meq O.sub.2/1000 g fat Eicosapentaenoic Acid 20:5
(EPA) 15.5% (w/w) relative to total triglycerides Docosahexaenoic
Acid 22:6 (DHA) 9.4% (w/w) relative to total triglycerides Total
Omega 3 Fatty Acids 29.5% (w/w) relative to total triglycerides
Eicosapentaenoic Acid 20:5 (EPA) 21.8% (w/w) relative to total
fatty acids Docosahexaenoic Acid 22:6 (DHA) 13.2% (w/w) relative to
total fatty acids Total Omega 3 Fatty Acids 41.5% (w/w) relative to
total fatty acids Total Phospholipids.sup.b 60 g/100 g TMA 31-39
mg/100 g TMAO 618-661 mg/100 g Viscosity @ 35.degree. C. 1,700,000
Centiposie .sup.aIndividual fatty acid composition. FIG. 13
.sup.bIndividual phospholipid composition. FIG. 14.
Several runs comparing triglyceride (TG) removal using subcritical
liquid CO.sub.2 to supercritical CO.sub.2 were performed by
plotting the production of CO.sub.2 versus the production of free
fatty acids (FF). FIG. 15. As can be seen from the graph, the
solubility between these two extraction conditions was very
similar. Analysis of the TG oil samples indicated that omega 3
fatty acid loss is insignificant with subcritical CO2 extraction,
and intersample variation was observed to be between 5-10%. In one
embodiment, the present invention contemplates a method comprising,
providing a subcritical CO.sub.2, ethanol and water solvent and
extracting a high phospholipid krill oil with said solvent.
VI. Nutritional and Health Consequences of Aging
[0192] Nutritional needs are known to change as animals (e.g.,
mammals, such as humans) age. Reasons for these changes include,
but are not limited to, decreased absorption of essential nutrients
from diet, a more sedentary lifestyle, a decreased appetite and/or
a change in metabolism. These nutritional changes occur in both the
healthy elderly population and the elderly with exceptional
nutritional needs due to health problems or diseases. There is
growing scientific documentation that shows that certain essential
macro and micro nutrients can prevent the development of certain
diseases in the elderly population.
[0193] There has surprisingly been found that high phospholipid/low
viscosity krill oil compositions are effective in alleviation,
prevention or treatment certain diseases in the elderly population
or in groups of particular nutritional needs. It has also been
surprisingly found that high phospholipid/low viscosity krill oil
compositions enhance the absorption of some essential minerals and
lipid soluble health ingredients and that these krill compositions
are effective in prevention and treatment of health conditions
related to aging.
[0194] In one embodiment, the present invention contemplates a use
of a nutritional supplement composition by administering the
composition ranging between approximately 0.005-0.50 g krill meal
per day per kg of body weight of an animal for the treatment of a
degenerative joint disease. In one embodiment, the composition is
homogenous. In one embodiment, the composition further comprises at
least one omega-3 fatty acid. In one embodiment, the composition is
stable. In one embodiment, the homogeneity of the composition is
characterized by a lack of phase separation.
[0195] A. Homeostatic Control
[0196] Aging may be characterized by an inability of tissues to
maintain homeostasis. This leads to an impaired response to stress
and, as a consequence, an increased risk of morbidity and
mortality. The incidence of numerous debilitating chronic diseases,
such as cardiovascular disease, neurodegeneration, diabetes,
arthritis, and osteoporosis, increases almost exponentially with
age. Aging is thought to be driven, at least in part, by the
accumulation of stochastic damage in cells. This includes damage to
proteins, DNA, mitochondria, and telomeres, which is driven by
reactive oxygen species. Mitochondria are the major producers of
reactive oxygen species, which damage DNA, proteins, and lipids if
not rapidly quenched. Alterations in mitochondria have been noted
in aging, including decreased total volume, increased oxidative
damage, and reduced oxidative capacity. These biochemical and
bioenergetic changes are accompanied by perturbations in cellular
dynamics, such as a decrease in mitochondrial biogenesis and an
increase in mitochondrially mediated apoptosis. Peterson et al,
Journal of Aging Research, Epub Jul. 19, 2012.
[0197] A lack of homeostasis control can lead to an impaired
response to stress and, as a consequence, an increased risk of
morbidity and mortality. For example, reports suggest that the
incidence of numerous debilitating chronic diseases, such as
cardiovascular disease, neurodegeneration, diabetes, arthritis, and
osteoporosis, increases almost exponentially with age. Tilstra et
al., The Journal of Clinical Investigation 122(7)2601-2612 (2012).
Aging is associated with progressive loss of neuromuscular function
that often leads to progressive disability and loss of
independence. The term sarcopeniais now commonly used to describe
the loss of skeletal muscle mass and strength that occurs in
concert with biological aging. The prevalence of sarcopenia, which
may be as high as 30% for those over 60 years, will increase as the
percentage of the very old continues to grow in our populations.
The link between sarcopenia and disability among elderly men and
women highlights the need for continued research into the
development of the most effective interventions to prevent or at
least partially reverse sarcopenia. The aging process is also
believed to be a factor in the age-dependent occurrence of central
nervous system disabilities, such as dementia.
[0198] The ability of a cell to resist oxidant damage during
homeostatic imbalance is determined by a balance between the
generation of reactive oxygen species and the defensive capacity to
produce antioxidants. Glutathione
(.gamma.-glutamylcysteinylglycine) is the most abundant endogenous
intracellular antioxidant present in millimolar quantities within
cells. Glutathione plays a central role in antioxidant defenses,
and irreversible cell damage supervenes when the cell is unable to
maintain intracellular glutathione concentrations. Evidence from
several animal and human studies suggests that concentrations of
glutathione decline with aging. It has been shown that dietary
supplementation with the glutathione precursors cysteine and
glycine fully restores glutathione synthesis and concentrations and
lowers levels of oxidative stress and oxidant damages in elderly
persons. Rajagopal et al, Am J Clin Nutr 94:847-853 (2011). Other
naturally occurring bioactive compounds, such as pyrroloquinoline
quinone (PQQ), resveratrol, genistein, hydroxy-tyrosol, and
quercetin have also been reported to improve mitochondrial
respiratory control or stimulate mitochondrial biogenesis.
[0199] Cell permeable peptide antioxidants have been reported that
are very potent at reducing intracellular ROS and preventing cell
death. The peptides are tetrapeptides with alternating aromatic
residues and basic amino acids. Zhao et al, The Journal of
Biological Chemistry 279, 34682 (2004).
[0200] As many of the components in high phospholipid/low viscosity
krill oil compositions might work as antioxidants and/or affect the
antioxidative/inflammatory defense in the cell it would be
interesting to test the high phospholipid/low viscosity krill oil
compositions in biological systems that can measure the
antioxidative effect and the anti-inflammatory effect in vitro to
identify the most interesting composition. The compositions should
also be tested in bioavailability studies to identify if the
compounds of interest will be absorbed.
[0201] B. Age-Related Macular Degeneration
[0202] Age-related macular degeneration (AMD) is also a major cause
of disability in the elderly. More than 20 million people worldwide
are severely affected either by age-related macular degeneration or
cataracts. AMD is the leading cause of blindness in people over 55
years of age in the western world. Nearly 30% of Americans over the
age of 75 years have early signs of AMD and 7% have late stage
disease. There are currently no effective treatment strategies for
most patients with AMD, attention has focused on efforts to stop
the progression of the disease or to prevent the damage leading to
AMD.
[0203] C. Diabetes
[0204] Type 2 diabetes is the most common chronic metabolic disease
in the elderly, affecting .about.30 million individuals 65 years of
age or older in developed countries. The estimated economic burden
of diabetes in the United States is .about.$100 billion per year,
of which a substantial proportion can be attributed to persons with
type 2 diabetes in the elderly age group. Epidemiological studies
have shown that the transition from the normal state to overt type
2 diabetes in aging is typically characterized by a deterioration
in glucose tolerance that results from impaired insulin-stimulated
glucose metabolism in skeletal muscle. Petersen et al, Science
300(5622):1140-1142 (2003).
[0205] D. Inflammation
[0206] Recent scientific studies have advanced the notion of
chronic inflammation as a major risk factor underlying aging and
age-related diseases. Low-grade, unresolved, molecular inflammation
is described as an underlying mechanism of aging and age-related
diseases, which may serve as a bridge between normal aging and
age-related pathological processes.
[0207] Elevated oxidative stress has been linked to chronic
inflammation and several aging related illnesses. The ability of a
cell to resist oxidant damage is determined by a balance between
the generation of reactive oxygen species and the defensive
capacity to produce antioxidants. A central problem associated with
the assessment of free radical induced oxidative stress in disease
development has been the limitation in existing assay methods for
in vivo measurement of free radical generation. For example,
F2-isoprostanes, structural isomers of PGF2.alpha., are formed
during free-radical catalysed peroxidation of arachidonic acid. A
major F2-isoprostane, 8-iso-PGF2.alpha., is now a well-recognised
reliable indicator of oxidative stress in vivo. Basu S., Antioxid.
Redox Signal. 10:1405-1434 (2008).
[0208] The transcription factor NF-.kappa.B is a component of the
cellular response to damage, stress, and inflammation. Numerous
studies report increased NF-kB activity with aging, and NF-kB was
identified as the transcription factor most associated with
mammalian aging based on patterns of gene expression. Adam et al,
Genes and Development 3244 (2007). Chronic activation of
NF-.kappa.B is observed in numerous age-related diseases including,
but not limited to, muscle atrophy, multiplesclerosis,
atherosclerosis, heart disease, both type 1 and 2 diabetes,
osteoarthritis, dementia, osteoporosis, and cancer. Tilstra et al,
The Journal of Clinical Investigation 122(7):2601-2612 (2012).
NF-.kappa.B DNA binding is increased in skin, liver, kidney,
cerebellum, cardiac muscle, and gastric mucosa of old rodents
compared with that in young rodents. In to addition, NF-.kappa.B
was identified as the transcription factor most associated with
mammalian aging, based on patterns of gene expression. Furthermore,
chronic activation of NF-.kappa.B is observed in numerous
age-related diseases, including muscle atrophy, multiple sclerosis,
atherosclerosis, heart disease, both type 1 and 2 diabetes,
osteoarthritis, dementia, osteoporosis, and cancer.
[0209] Several scientific publications strongly suggest that
inhibitors of the IKK/NF-.kappa.B pathway may delay damage and
extend health span in patients with accelerated aging and chronic
degenerative diseases of old age.
[0210] Based on literature in the area of age related diseases, it
is apparent that oxidative damage and low grade inflammation are
central in the development of many of the age related diseases. The
transcription factor NF-.kappa.B is a central component for the
cellular response to these triggers. A scientific approach to the
development of nutritional supplements and drugs for the aging
population could be to focus on the development of effective
antioxidants to target these central biological mechanisms.
VII. Treatment of Medical Disorders with Krill Oil
[0211] A. Central Nervous System Medical Disorders
[0212] In one embodiment, the medical disorder comprises a central
nervous system disorder. In one embodiment, the central nervous
system disorder comprses a mental disorder. In one embodiment, the
mental disorder includes, but is not limited to infancy, childhood
or adolescence disorders, cognitive disorders, substance-related
disorders, psychotic disorders including but not limited to
schizophrenia, mood disorders including but not limited to
depression, anxiety disorders, somatoform disorders, factitious
disorder, dissociative disorders, sexual disorders, eating
disorders, sleep disorders, impulse-control disorders, adjustment
disorders or personality disorders.
[0213] The central nervous system is particularly vulnerable to
oxidative insult on account of the high rate of O.sub.2
utilization, the relatively poor concentrations of classical
antioxidants and related enzymes, and the high content of
polyunsaturated lipids, the biomacromolecules most susceptible to
oxidation. In addition, there are regionally high concentrations of
redox-active transition metals capable of the catalytic generation
of ROS. Thus, it is not surprising that oxidative stress is a
common discussion point for neurodegenerative disease, where damage
to neurons can reflect both an increase in oxidative processes and
a decrease in antioxidant defenses.
[0214] Accumulating data indicate that oxidative stress (OS) plays
a major role in the pathogenesis of multiple sclerosis (MS).
Reactive oxygen species (ROS), leading to OS, generated in excess
primarily by macrophages, have been implicated as mediators of
demyelization and axonal damage in MS. ROS cause damage to main
cellular components such as lipids, proteins and nucleic acids
(e.g., RNA, DNA), resulting in cell death by necrosis or apoptosis.
In addition, weakened cellular antioxidant defense systems in the
central nervous system (CNS) in MS, and its vulnerability to ROS
effects may augmented damage. Thus, treatment with antioxidants
might theoretically prevent propagation of tissue damage and
improve both survival and neurological outcome. Miller et al, Pol
Merkur Lekarski. 27(162):499-502 (2009).
[0215] B. Ocular Medical Disorders
[0216] The use of herbal medicines and nutritional supplements in
ocular disorders including, but not limited to, age-related macular
degeneration (AMD), cataracts, diabetic retinopathy and glaucoma,
has recently been reviewed. Antioxidants and zinc have been used in
patients with certain forms of intermediate and advanced AMD.
However, there has been growing evidence regarding potential
significant adverse effects associated with the AREDS (Age-Related
Eye Disease Study) formula vitamins. However, whether the use of
antioxidants or herbal medications in the prevention or treatment
of cataracts, glaucoma or diabetic retinopathy would be beneficial
is inconclusive. It was recommended that further study of
nutritional supplements and herbal medicines in the treatment of
eye disease is needed to determine their safety and efficacy.
Wilkinson et al., "Use of herbal medicines and nutritional
supplements in ocular disorders: an evidence-based review" Drugs
71(18):2421-2434 (2011).
[0217] Some nutritional remedies have been tried for cataracts,
glaucoma, and retinal diseases (macular degeneration, diabetic
retinopathy, retinopathy of the newborn, and retinitis pigmentosa).
Specifically, some nutritional treatments were given for
asthenopia, blepharitis, chalazion, conjunctivitis (including giant
papillary conjunctivitis), gyrate atrophy of the choroid and
retina, keratoconus, myopia, sicca syndrome (dry eyes), and
uveitis. The data suggest that nutritional supplements may play
role the further of clinical therapy strategies to ocular
disorders. Gaby A R., "Nutritional therapies for ocular disorders:
Part Three" Altern Med Rev. 13(3):191-204 (2008).
[0218] C. Digestive Disorders
[0219] Functional digestive disorders can be characterized by
symptoms related to the digestive tract for which no pathological
causes can be found using routine diagnostic techniques. Recently,
several methods have been developed to the study digestive function
allow relation between in humans functional alterations, mainly
motor and sensory and to be related to functional digestive
symptoms. As a result of these advances, both motor and sensory
alterations have been identified in subgroups of patients with
functional digestive disorders. This knowledge should enable
current symptom-based classifications of these disorders to be
replaced with new classifications based on specific
physiopathologic mechanisms. This would allow more effective
therapies aimed at the specific mechanism causing the symptoms to
be developed. Serra J., "Clinical research techniques in functional
digestive disorders" Gastroenterol Hepatol. 29(4):255-62
(2006).
[0220] Functional dyspepsia and the irritable bowel syndrome (IBS)
are amongst the most widely recognised functional gastrointestinal
disorders. Symptom based diagnostic criteria have been developed
and refined for the syndromes (the Rome criteria) and these are now
widely applied in clinical research. Both functional dyspepsia and
IBS are remarkably prevalent in the general population, affecting
approximately 20% and 10% of persons, respectively. The prevalence
is stable from year to year because the onset of these disorders is
balanced by their disappearance in the population. Clinically
useful predictors of the course of these disorders have not been
identified. Approximately one third of persons with functional
dyspepsia concurrently have IBS. In most studies from Western
countries, it has been shown that only a minority with functional
dyspepsia and IBS present for medical care; the factors that
explain consultation behavior remain inadequately defined although
fear of serious disease and psychological distress may be
important. The majority of patients diagnosed as having functional
dyspepsia or IBS continue to have symptoms long term with a
significant impact on quality of life. The indirect costs of the
functional gastrointestinal disorders greatly outweigh the direct
costs but overall these conditions are responsible for a major
proportion of health care consumption. Rational management of the
functional gastrointestinal disorders will only follow a better
understanding of the natural history of these conditions. Talley
N.J., "Scope of the problem of functional digestive disorders" Eur
J Surg Suppl. 582:35-41 (1998).
[0221] D. Skeletal Disorders
[0222] Bone turnover, in which cells of the osteoclast lineage
resorb bone and cells of the osteoblast lineage deposit bone,
normally occurs in a highly regulated manner throughout life.
Perturbations to these processes underlie skeletal disorders, such
as osteoporosis, which are common, chronic and disabling, and
increase with age. On the basis of empirical observations or on
understanding of the endocrinology of the skeleton, excellent
bone-resorption inhibitors, but few anabolic agents, have been
developed as therapeutics for skeletal disorders. Goltzman D.,
"Discoveries, drugs and skeletal disorders" Nat Rev Drug Discov.
1(10):784-796 (2002). In some embodiment, the present invention
contemplates that crustacean meal compositions and other
ingredients are useful in treating these disorders.
[0223] Notch signaling mediates cell-to-cell interactions that may
be involved in embryonic development and tissue renewal. In the
canonical signaling pathway, the Notch receptor may be cleaved
following ligand binding, resulting in the release and nuclear
translocation of the Notch intracellular domain (NICD). NICD
induces gene expression by forming a ternary complex with the DNA
binding protein CBF1/Rbp-Jk, Suppressor of Hairless, Lag1, and
Mastermind-Like (Maml). Hairy Enhancer of Split (Hes) and Hes
related with YRPW motif (Hey) are also Notch targets. Notch
canonical signaling plays a central role in skeletal development
and bone remodeling by suppressing the differentiation of skeletal
cells. The skeletal phenotype of mice misexpressing Hes1
phenocopies partially the effects of Notch misexpression,
suggesting that Hey proteins mediate most of the skeletal effects
of Notch. Dysregulation of Notch signaling is associated with
diseases affecting human skeletal development, such as Alagillc
syndrome, brachydactyly and spondylocostal dysostosis. Somatic
mutations in Notch receptors and ligands are found in tumors of the
skeletal system. Overexpression of NOTCH1 is associated with
osteosarcoma, and overexpression of NOTCH3 or JAGGED1 in breast
cancer cells favors the formation of osteolytic bone metastasis.
Activating mutations in NOTCH2 cause Hajdu-Cheney syndrome, which
is characterized by skeletal defects and fractures, and JAG1
polymorphisms, are associated with variations in bone mineral
density. In conclusion, Notch is a regulator of skeletal
development and bone remodeling, and abnormal Notch signaling is
associated with developmental and postnatal skeletal disorders.
Zanotti et al., "Notch regulation of bone development and
remodeling and related skeletal disorders" Calcif Tissue Int.
90(2):69-75 (2012).
[0224] Genetic disorders involving the skeletal system may arise
through disturbances in the complex processes of skeletal
development, growth and homeostasis and remain a diagnostic
challenge because of their variety. The Nosology and Classification
of Genetic Skeletal Disorders provides an overview of recognized
diagnostic entities and groups them by clinical and radiographic
features and molecular pathogenesis. The aim is to provide the
Genetics, Pediatrics and Radiology community with a list of
recognized genetic skeletal disorders that can be of help in the
diagnosis of individual cases, in the delineation of novel
disorders, and in building bridges between clinicians and
scientists interested in skeletal biology. In the 2010 revision,
456 conditions were included and placed in 40 groups defined by
molecular, biochemical, and/or radiographic criteria. Of these
conditions, 316 were associated with mutations in one or more of
226 different genes, ranging from common, recurrent mutations to
"private" found in single families or individuals. Thus, the
Nosology is a hybrid between a list of clinically defined
disorders, waiting for molecular clarification, and an annotated
database documenting the phenotypic spectrum produced by mutations
in a given gene. The Nosology should be useful for the diagnosis of
patients with genetic skeletal diseases, particularly in view of
the information flood expected with the novel sequencing
technologies; in the delineation of clinical entities and novel
disorders, by providing an overview of established nosologic
entities; and for scientists looking for the clinical correlates of
genes, proteins and pathways involved in skeletal biology. Warman
et al., "Nosology and classification of genetic skeletal disorders:
2010 revision" Am J Med Genet A 155A(5):943-968 (2011).
[0225] E. Muscular Disorders
[0226] Skeletal muscle is the largest organ in the human body, and
plays an important role in body movement and metabolism. Skeletal
muscle mass is lost in genetic disorders such as muscular
dystrophy, muscle wasting and ageing. Chemicals and proteins that
restore muscle mass and function are potential drugs that can
improve human health and could be used in the clinic. Myostatin is
a muscle-specific member of the transforming growth factor
(TGF)-beta superfamily that plays an essential role in the negative
regulation of muscle growth. Inhibition of myostatin activity is a
promising therapeutic method for restoring muscle mass and
strength. Potential inhibitors of myostatin include follistatin
domain-containing proteins, myostatin propeptide, myostatin
antibodies and chemical compounds. These inhibitors could be
beneficial for the development of clinical drugs for the treatment
of muscular disorders. Bone morphogenetic protein (BMP) plays a
significant role in the development of neuromuscular architecture
and its proper functions. Modulation of BMP activity could be
beneficial for muscle function in muscular disorders. Tsuchida K.,
"The role of myostatin and bone morphogenctic proteins in muscular
disorders" Expert Opin Biol Ther. 6(2):147-154 (2006).
[0227] Currently, the diagnosis of muscular disorders is mainly
clinical, wherein myopathies can present with unusual or atypical
clinical features including, but not limited to, myotonia, periodic
paralysis, respiratory failure, swallowing difficulties, ptosis,
ophtalmoplegia, camptocormia, distal and/or asymmetrical limb
muscle weakness. Several recently discovered myopathies include,
but are not limited to, reducing body myopathy, X-linked myopathy
with postural muscle atrophy, Emery-Dreifuss muscular dystrophy,
and scapuloperoneal myopathy.
[0228] F. Cardiovascular Disorders
[0229] Dyslipidemias and insulin resistance constitute major risk
factors of cardiovascular diseases (CVD) and related-features.
Furthermore, oxidative stress impairment or altered antioxidant
status have been suggested as pivotal keys in the onset of certain
chronic diseases such as metabolic syndrome (MS), type 2 diabetes
and CVD. In this sense, oxidized low-density lipoprotein (ox-LDL),
a recognized oxidative stress marker, has been positively
associated with central obesity, metabolic syndrome manifestations
and subclinical atherosclerosis. Hermsdorff H., Nutrition &
Metabolism 8:59 (2011).
EXPERIMENTAL
Example I
Preparation of Conventional Phospholipid Krill Oil
[0230] Krill oil compositions contemplated herein were prepared
from freshly caught whole krill. A PPC material was obtained from
fresh krill using an enzymatic hydrolysis process, involving shell
removal, removal of water-soluble peptides and vacuum drying at a
low temperature followed by a 40% ethanol in water extraction. The
process is carried out immediately after catch to ensure that only
fresh krill is used, resulting in less degradation and higher
quality PPC. The phospholipid concentration of the extracted krill
oil ranged between approximately 60-99% and a viscosity ranging
between 100,000-3,000,000 cP.
Example II
Preparation of a Lyophilized Concentration Krill Oil
[0231] A mixture comprising krill oil, water and ethanol produced
in accordance with Example I is subjected to lyophilization using a
commercially available lyophilizer to produce a lyophilized krill
oil. Such a lyophilized krill oil comprises a semi-solid
composition including, but not limited to, phospholipids, fatty
acids, omega-3, EPA and DHA having flowability characteristics at a
temperature of at least 40.degree. C.
Example III
Krill Oil Viscosity Measurements
[0232] Krill oil viscosity was measured using a Brookfield Rotary
Dial viscometer at 35.degree. C. and the values were reported as
centipoise (cP).
Example IV
Krill Oil Phospholipid Measurements
[0233] The phospholipid levels were measured using the method
described in the reference with 31P-NMR spectroscopy. Amidon et
al., "A theoretical basis for a biopharmaceutics drug
classification: the correlation of in vitro drug product
dissolution and in vivo bioavailability" Pharm. Res. 12:413-420
(1995).
Example V
Preparation of a Lyophilized Concentrated Krill Oil
[0234] A krill oil produced in accordance with Example I is
subjected to lyophilization using a commercially available
lyophilizer to produce a lyophilized krill oil product in
accordance with Example II.
Example VI
Capsule Filling with a FlowableConcentrated Phospholipid Semi-Solid
Krill Oil
[0235] Five thousand capsules are filled with a
flowable/concentrated phospholipid semi-solid krill oil having a
phospholipid concentration ranging between 60-99%, a water content
between 1-4% and an organic solvent less than 1% using a
commercially available capsule filling machine (e.g., for example,
Robert Bosch GmbH GIF 1400 L). The capsule filling machine may
accommodate either hard gelatin capsules and soft gel capsules,
where the production run is completed with a leakage rate of
between approximately 0.1-3%
Example VII
Viscosity of Oxidized Krill Oil
Objectives
[0236] 1. To investigate if thermal treatment could increase the
viscosity of krill oil, which might be resulted from the increase
of lipid oxidation and subsequently increase also the
oxypolymerization.
Experimental Design
[0237] Krill oil (Rimfrost.RTM., Rimfrost AS (formerly Olympic
Seafoods, AS) were incubated at different temperatures (20 and
40.degree. C.) for approximately 1, 2, 3, 4 and 6 weeks, under
condition of constant stirring while being exposed to air
(semi-open air condition). Below is the experimental design from
previous experiment.
TABLE-US-00015 Storage time Temperature (.degree. C.) Weeks days 20
40 0 K2000 K4000 1 K2001* K4001* 3 K2003 K4003 1 7 K2007 K4007 2 14
K2014 K4014 3 21 K2021 K4021 4 28 K2028* K4028 6 48 K2048* K4042*
*Samples upon which viscosity was measured.
Method of Measurement
[0238] The viscosity of krill oil (1-2 g) was measured by rheometer
(Haake Mars Modular Advanced System, Thermo Fisher) at temperature
23.degree. C. by using Sensor plate P 35 Til S (serrated), with
shear rate (1/s) from 10-1000.
Overall Observation
[0239] The highest degree of viscosity index was obtained for krill
oil incubated at 40.degree. C. after 42 days of storage. The
increase of lipid oxidation seemed to increase the
oxypolymerisation in krill oil as illustrated by the increase of
viscosity index of krill oil. See, Table 15; FIGS. 5 and 6.
TABLE-US-00016 TABLE 15 Viscosity index of krill oil incubated at
different temperatures Samples Viscosity index (Pa s) Mean (Pa s)
K2001 1.847 1.846 .+-. 0.001 1.845 K2028 1.849 1.772 .+-. 0.180
1.594 K2042 1.821 1.807 .+-. 0.020 1.793 K4001 1.592 1.746 .+-.
0.140 1.775 1.872 K4042 2.085 2.083 .+-. 0.070 2.149 2.016
Example VIII
Comparison of Acetone and Ethanol Fractionation of Krill Oil
Materials
[0240] Commercial krill oil, Rimfrost.RTM., was obtained from
Olympic Seafood AS, Fosnavag, Norway. Pure ethanol (100%) was
purchased from Arcus Kjemi AS, Vestby, Norway. Acetone used in the
extraction process was of HPLC quality (LiChrosolv.RTM., 99.8%;
Merck, Damstadt, Germany). All solvents and reagents for the
analyses were of analytical grade.
Chemical Analyses
[0241] Lipid class analyses were performed by a HPLC system
(Perkin-Elmer, Waltham, USA) equipped with an ESA Corona.RTM. Plus
Charged Aerosol Detector (ESA Biosciences, Inc., Chelmsford, USA)
(Moreau, 2006). The samples were separated on a LiChrosphere.RTM.
100, 5 .mu.m diol column, 4.times.125 min (Merck KGaA, Germany). A
ternary gradient consisting of solvent A=isooctane,
B=acetone/diclormetane (1:2) and C=2-propanol/methanol/acetic
acid-ethanolamine-water (7.5 mM ethanolamine and 7.5 mM acetic
acid) (85:7.5:7.5) was used with the following profile: at 0 min,
100:0:0 (% A:% B:% C); at 1 min, 90:10:0; at 8 min 70:30:0; at 11
min 40:50:10; at 13 min 39:0:61; at 26.3 min 40:0:60; at 28.4 min
0:100:0; at 30.9 min 100:0:0. The lipid components were identified
by comparison to the retention time of commercial standards.
[0242] Sodium chloride (NaCl) content was determined based on water
soluble chloride. "Method 937.09"; In: Official Methods of
Analysis, 18th ed. Association of Official Analytical Chemists,
Gaithersburg, Md. (2005). Trimethylamine N-oxide (TMAO) and
trimethylamine (TMA) were determined based on a previously reported
micro-diffusion technique. Conway et al., "An absorption apparatus
for the micro-determination of certain volatile substances: The
micro-determination of ammonia" The Biochemical Journal
27(2):419-429 (1933). Lipid c