Note: Descriptions are shown in the official language in which they were submitted.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
1
METHOD OF MAKING MONOACYLGLYCERIDE OILS AND FOOD PRODUCTS
CONTAINING MONOACYLGLYCERIDE OILS
CROSS-REFERENCE TO RELATED APPLICATIONS
[001] The present application claims priority to U.S. Provisional Application
No. 62/794,412,
filed January 18, 2019, and U.S. Provisional Application No. 62/833,558 filed
April 12, 2019,
the entirety of each of which are incorporated herein by reference in their
entirety.
BACKGROUND
[002] Chronic deficiency in the secretion of digestive enzymes by the pancreas
is termed
Exocrine Pancreatic Insufficiency (EPI). Without these digestive enzymes,
patients suffering
from EPI cannot properly digest nutrients in food and may suffer from
malnutrition and
abdominal disorders. EPI is prevalent in individuals with chronic pancreatitis
and several other
chronic gastrointestinal disorders. EPI also manifests in patients suffering
from cystic fibrosis.
The effects of EPI can be mitigated by Pancreatic Enzyme Replacement Therapy
(PERT), in
which the individual administers enzyme capsules each time food is consumed.
Conventionally,
PERT treatment comprises pancreatic enzymes extracted from porcine pancreas.
[003] Lipids are energy-dense compounds that are the source of essential long
chain fatty acids.
Consumed lipids, typically comprising a high percentage of triacylglyceride
(TAG) are digested
with lipases secreted from the pancreas into free fatty acids (FFA) and
monoacylglyceride
(MAG). Blockage of lipase release from the pancreas results in very poor
digestion of
triacylglyceride-containing fats and oils. For patients suffering from EPI,
this can lead to
significant malnutrition because the calories, essential fatty acids and fat-
soluble nutrients are
trapped in the un-digested lipid particles and pass through the system.
[004] There is an unmet clinical need for alternative sources of nutrition
that can be consumed
without needing to supplement with PERT by individuals with EPI.
[005] Partially hydrolyzed fats and oils, in the form of MAGs are readily
absorbed by
individuals with EPI, without the requirement of PERT. MAG oil-based products
have been
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
2
evaluated in the clinic as capsule-based nutritional supplements; however,
capsules were utilized
to avoid the bad taste. For conventional sources of MAG oils, the starting oil
is treated
chemically or enzymatically to make MAGs, which are then extracted with
solvents and distilled
to fractionate the MAGs away from other components of the starting oils. These
MAG products
are sold as relatively pure products containing only negligible amounts of
contaminating free
fatty acid (FFA), (diacylglycerides) DAGs and TAGs, and with virtually no
other compounds.
Thus, conventional sources of MAG oil often lack the other natural compounds
found in the oil,
such as tocopherol.
[006] There is clinical need for nutritional products with very-high-energy
caloric density that
can be consumed by individuals with inefficient or compromised digestive
systems. In addition
to individuals with pancreas pathologies (e.g., cystic fibrosis, pancreatitis
and pancreatic cancer
patients) other patients with diagnosed or undiagnosed Exocrine Pancreatic
insufficiency (EPI)
would benefit from the products. In addition, individuals with bile
dysfunction (cholestasis) may
benefit from "pre-digested" fats that do not require bile acids for
emulsification. There are high
calorie "energy bars" and drinks on the market. But, these products are not
suitable for
individuals who are unable to digest (hydrolyze) the fats in the product.
Until now, no one has
formulated lipids into liquid (shakes) and solid (bar) forms that are suitable
for "PERT-free" use.
These formulations can be a source of "Complete Nutrition", suppling all the
caloric and
essential fatty acid requirements of individuals. Liquid nutrition can be in
the form of oral
nutritional supplements (ONS) products of products for enteral feeding.
[007] Accordingly, there is need for high caloric density foods that can be
consumed by
individuals with inefficient or compromised digestive systems. The present
application describes
a method to produce an edible enzyme-modified oil (EMO) that is substantially
free of TAGs.
[008] Monochloropropandiol (MCPD) and glycidyl esters are formed during the
refining of
edible oils. These compounds are toxic to humans and need to be minimized in
food products.
3-MCPD is currently classified as a possible human carcinogen (group 2B)
according to The
International Agency for Research on Cancer (IARC). Glycidol is categorized as
probably
carcinogenic for humans (group 2A) by IARC and the US National Toxicology
program. Current
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
3
standards recommend exposure of less than 2 ug/kg body weight per day, which
is less than 140
ug per day for a 70 kg man, and only 10 ug/day for a 5 kg (-10 lb) baby.
[009] MCPD compounds were first detected in acid (HC1) hydrolyzed protein, but
in 2008 the
presence of MCPD esters in refined vegetable oils was discovered. It turned
out the problem
was widespread. Many edible oils are processed to remove components that
negatively impact
appearance, taste, shelf stability, safety and consumer acceptance. Mono- and
di-acyl glycerols
(MAGs and DAGs) in the oils can react with chlorine ions in the deodorization
process to
produce 3-Monochloropropanediol (3-MCPD) esters and glycidyl esters (GEs).
[0010] Table 1 below shows the levels of MCPD compounds in a sampling of
edible oils in the
United States (from Food Additives & Contaminants: Part A, 2013 Vol. 30, No.
12, 2081-
2092).
Refined Oil mg/kg
Almond 2.14
Canola 0.39
Coconut 0.97
Corn 0.85
Cottonseed 0.93
Grape seed 2.88
Olive 1.04
Palm 6.69
Palm Kernel 0.64
Palm Olein 10.17
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
4
Palm Stearin 6.82
Peanut 0.98
Soybean 0.36
Sunflower 0.94
[0011] The estimated US infant exposures to 3-MCPD and glycidyl esters
consumption of infant
formula was recently reported (J. Spungen et al. Food Additives &
Contaminants: Part TA 2018,
VOL. 35, NO. 6,1085-1092). In this analysis, US FDA data on 3-MCPD and
Glycidyl Ester
concentrations (as 3-MCPD and glycidol equivalents, respectively) in a small
convenience
sample of infant formulas were used to estimate exposures from consumption of
formula by
infants 0-6 months of age. 3-MCPD and GE exposures based on mean
concentrations in all
formulas were estimated at 7-10 and 21.tg/kg bw/day, respectively. Estimated
mean exposures
from consumption of formulas produced by individual manufacturers ranged from
1 to 141.tg/kg
bw/day for 3-MCPD and from 1 to 3 1.tg/kg for GE.
[0012] Accordingly, there is need for methods to reduce and eliminate these
compounds in
edible oils. The method of the present application to produce an edible enzyme-
modified oil
(EMO) that is substantially free of TAGs and contaminants such as MCPD and
therefore
addresses this need.
[0013] Some vegetable oils are rich in oleic acid. These oils have
demonstrated health benefits.
Some of this can be attributed to the oleic acid and oils that are rich in
this fatty acid such as
olive oil, almond oil and canola (rapeseed) oil. Triolein is a TAG that is
very rich in oleic acid,
as illustrated below. Triolein is 100% oleic acid esterified to glycerol at
all three positions. The
three positions are defined as "sn-1, sn-2 and sn-3". Sn is "stereo number."
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
;
i Triolein 1
i%Cf= 'NI sn-1
,
Ls 9.
,
,
sn-2
1
a")
(
CH3
.NIAN3
sn-3
[0014] Given the health benefits of these oils, there is a need for
compositions and methods that
can provide the health benefits of these oils for individuals suffering from
EPI, and to enhance
and make the beneficial components of the oils more available.
SUMMARY
[0015] The present disclosure is directed to a product comprising a processed
oil derived from an
oil source. In one embodiment, the processed oil comprises a MAG content equal
to or greater
than 30% by weight of the total weight of the processed oil, a DAG content of
from about 10%
to about 30% by weight of the total weight of the processed oil, and a FFA
content of from about
5% to about 60% by weight of the total weight of the processed oil, wherein
the processed oil is
either free of TAGs or comprises a TAG content that is equal to or less than
5% by weight of the
total weight of the processed oil, and wherein the processed oil comprises non-
oil ingredients
derived from and naturally present in the oil source such that the non-oil
ingredients are not
added to the processed oil.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
6
[0016] In some embodiments, the oil source is from an origin selected from a
plant, an animal, a
fish, or mixtures thereof. In some embodiments, the oil source comprises MCPD
compounds. In
these embodiments, the processed oil is substantially free of MCPD compounds.
[0017] In some embodiments the non-oil ingredients of said product are
selected from
antioxidants, vitamins, and mixtures thereof
[0018] In some embodiments, said product comprises greater than 1% by weight
MAGs out of
the total weight of the product.
[0019] In some embodiments, said product comprises greater than 50% by weight
MAGs out of
the total weight of the product.
[0020] The present disclosure is also directed to a food product. In one
embodiment, the food
product comprises an oil and having a caloric density of from about 1
kcal/gram to about 5
kcal/gram, wherein from about 20% to about 50% of calories are derived from
said oil.
[0021] In some embodiments, the oil of said food product is a processed oil
derived from an oil
source, wherein the processed oil comprises a MAG content equal to or greater
than 30% by
weight of the total weight of the processed oilõ a DAG content of from about
10% to about 30%
by weight of the total weight of the processed oil, and a FFA content of from
about 5% to about
60% by weight of the total weight of the processed oil wherein the processed
oil is either free of
TAGs or comprises a TAG content that is equal to or less than about 5% by
weight of the total
weight of the processed oil, and wherein the processed oil comprises non-oil
ingredients derived
from and naturally present in the oil source such that the non-oil ingredients
are not added to the
processed oil.
[0022] In some embodiments, the oil source of said food product is from an
origin selected from
a plant, an animal, or a fish.
[0023] In some embodiments, the non-oil ingredients of said food product are
selected from
antioxidants, vitamins, and mixtures thereof
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
7
[0024] In some embodiments, said food product comprises greater than 1% by
weight MAGs out
of the total weight of the product.
[0025] In some embodiments, said food product comprises greater than 50% by
weight MAGs
out of the total weight of the product.
[0026] In some embodiments, said food product has a total weight from about 25
grams to about
3000 grams.
[0027] In some embodiments, said food product has a total calorie content from
about 1 kcals to
about 5 kcals per gram.
[0028] In some embodiments, said food product may further comprise a
carbohydrate source.
[0029] In some embodiments, said food product may further comprise a protein
source.
[0030] In some embodiments, said oil contributes from 5% to 95% of the total
calorie content of
the food product.
[0031] The present disclosure is also directed to a method for making a
monoacylglycerol-
enriched oil. In one embodiment, the method comprises mixing a starting oil
comprising
triacylglycerols (TAGs), a buffer solution and a first enzyme capable of
hydrolyzing said TAGs
to free fatty acids (FFAs) to yield a first reaction mixture; allowing said
reaction mixture to react
under conditions sufficient for said first enzyme to hydrolyze said TAGs for a
first period of time
to yield an aqueous phase and lipid (Free Fatty Acid) reaction product;
inactivating said first
enzyme in said reaction product; collecting said lipid reaction product;
mixing said lipid reaction
product and food-grade glycerol and a second enzyme capable of esterifying
FFAs to form a
second reaction mixture; allowing said second reaction mixture to react for a
second period of
time to yield a reaction product lipid oil phase and a glycerol phase;
inactivating said second
enzyme in said reaction product; adding salt to the reaction and separating
the lipid oil phase
from said glycerol phase; and collecting said lipid oil phase.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
8
[0032] In some embodiments, said starting oil is an oil derived from plant,
animal, marine, or
mixtures thereof. In some embodiments, the starting oil comprises MCPD
compounds and the
lipid oil phase is substantially free of MCPD compounds.
[0033] In some embodiments, said first enzyme is lipase AY.
[0034] In some embodiments, said first period of time is a period of time
sufficient to hydrolyze
at least 94% of the TAGs in said starting oil.
[0035] In some embodiments, said first period of time is between about 14
hours and 24 hours.
[0036] In some embodiments, said step of allowing said reaction mixture to
react under
conditions sufficient for said first enzyme to hydrolyze said TAGs is
performed at a temperature
between about 30 C and about 35 C.
[0037] In some embodiments, said steps of mixing a starting oil comprising
triacylglycerols
(TAGs), a buffer solution and a first enzyme capable of hydrolyzing said TAGs
to free fatty
acids (FFAs) and allowing said reaction mixture to react under conditions
sufficient for said first
enzyme to hydrolyze said TAGs to FFA are performed under a nitrogen
atmosphere.
[0038] In some embodiments, said second enzyme is lipase G.
[0039] In some embodiments, said second period of time is a period of time
sufficient to result in
enrichment of MAGs in the lipid oil phase of about 60% to 95%.
[0040] In some embodiments, said second period of time is between about 24
hours and about 72
hours.
[0041] In some embodiments, said step of allowing said second reaction mixture
to react for a
second period of time to yield a lipid oil phase and a glycerol phase is
performed at a
temperature between about 17 C and 23 C.
[0042] In some embodiments, the method further comprises drying said reaction
product by
applying a vacuum for a third period of time sufficient to remove at least a
portion of water from
the reaction product.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
9
[0043] In some embodiments, said step of drying said reaction product is
performed at a
temperature between 20 C-30 C.
[0044] In some embodiments, said drying step is applied throughout the second
period of time.
[0045] In some embodiments, said step of inactivating said second enzyme is
performed by
heating said reaction product.
[0046] In some embodiments, said heating is performed at a temperature of at
least 70 C for at
least 1 hour.
[0047] In some embodiments, said step of separating said lipid oil phase from
said glycerol
phase comprise adding sodium chloride to said reaction product.
[0048] In some embodiments, the final concentration of sodium chloride
comprises up to 0.3
weight percent sodium chloride.
[0049] In some embodiments, the method further comprises before mixing said
lipid reaction
product and food-grade glycerol and a second enzyme capable of esterifying
FFAs and glycerol,
re-establishing a nitrogen atmosphere over said lipid reaction product.
[0050] In some embodiments, said steps of removing at least a portion of said
aqueous phase and
replacing said at least a portion of said aqueous phase with about an
equivalent volume of water
and waiting a second period of time are repeated before performing said step
of collecting said
lipid reaction product.
[0051] In some embodiments, the method further comprises adding tocopherol to
said lipid oil
phase after collecting said lipid oil phase.
[0052] In some embodiments, a processed oil having a total fatty acid content
comprises oleic
acid monoglyceride (MOG) in an amount that contributes between about 5% and
about 75% by
weight of the total fatty acid content of the processed oil composition.
[0053] In some embodiments, a processed oil comprises oleic acid and linoleic
acid in a ratio of
between about 0.01 and about 5, the processed oil having greater than 50% by
weight
monoacylglyceride (MAGs) based on the total weight of the processed oil.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
[0054] In some embodiments, a processed oil comprises oleic acid and linolenic
acid in a ratio
between about 1 and about 100, the processed oil having greater than 50% by
weight
monoacylglyceride (MAGs) based on the total weight of the processed oil..
[0055] In some embodiments, a processed oil comprises oleic acid and linoleic
acid and has a
total fatty acid content, wherein the linoleic acid is present in an amount
from about 10% to
about 90% by weight out of the total fatty acid content of the processed oil.
[0056] In some embodiments, a processed oil has a fatty acid profile
substantially the same as
the fatty acid profile of the pre-processed oil from which the processed oil
was produced.
[0057] In some embodiments, a processed oil has a fatty acid profile
comprising oleic acid,
linoleic acid and linolenic acid, wherein the amount of oleic acid, linoleic
acid, and linolenic acid
is within about 10% of the amount of oleic acid, linoleic acid and linolenic
acid, respectively, in
the pre-processed oil from which the processed oil was produced.
[0058] In some embodiments, a processed oil has a fatty acid profile
comprising oleic acid,
linoleic acid and linolenic acid, wherein the amount of oleic acid, linoleic
acid, and linolenic acid
is within about 1% of the amount of oleic acid, linoleic acid and linolenic
acid, respectively, in
the pre-processed oil from which the processed oil was produced.
[0059] In some embodiments a method for promoting glucose homeostasis in a
subject in need
thereof includes the step of administering to the subject a composition
comprising a processed oil
comprising oleic acid monoglyceride, wherein at least 50% by weight of said
oleic acid
monoglyceride is 1-oley1 monoglyceride.
[0060] In some embodiments a method for treating type II diabetes in a subject
in need thereof
includes the step of administering to the subject a composition comprising a
processed oil
comprising oleic acid monoglyceride, wherein at least 50% by weight of said
oleic acid
monoglyceride is 1-oley1 monoglyceride.
[0061] In some embodiments, a method for promoting glucose homeostasis in a
subject in need
thereof includes the step of administering to the subject a composition
comprising a processed oil
of the present disclosure.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
11
[0062] In some embodiments, a method for treating diabetes in a subject in
need thereof includes
the step of administering to the subject a composition comprising a processed
oil of the present
disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS AND FIGURES
[0063] FIGURE 1 depicts the TLC separation of components of starting vegetable
oil,
intermediate FFAs, and final MAG oil.
[0064] FIGURE 2 depicts the TLC separation of components of starting vegetable
oil,
intermediate FFAs, and final MAG oil.
[0065] FIGURE 3 depicts the distribution of FFA, MAG, DAG, and TAG in "Ensure
Original
Nutritional Shake" and Enzyme Modified Oil product of the present disclosure
("GBFS").
[0066] FIGURE 4 depicts a block flow diagram of the process of manufacturing
enzyme
modified oil.
[0067] FIGURE 5 depicts the distribution of amino acids and peptides in ready-
to-drink
nutritional drinks and GBFS hydrolyzed pea protein.
[0068] FIGURE 6A depicts the NMR spectrum of authentic TAG (tristerin).
[0069] FIGURE 6B depicts the NMR spectrum for enzyme modified oil produced
from almond
oil.
[0070] FIGURE 7 depicts the increase in serum triglycerides following
ingestion of the EMO-
based ready-to-drink shake.
[0071] FIGURE 8 depicts the increase in serum triglycerides following
ingestion of MAG-based
RTDS without PERT.
[0072] FIGURE 9 depicts the serum glucose levels for patients (averaged)
following ingestion of
MAG-based RTDS without PERT or a standard nutritional supplement with PERT.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
12
[0073] FIGURE 10 depicts the serum triglyceride levels for patients (averaged)
following
ingestion of-based RTDS without PERT or a standard nutritional supplement with
PERT.
[0074] FIGURE 11 depicts the percentage of certain fatty acids in the canola
oil and canola
EMO of Example 13 and two commercial monoglyceride samples.
[0075] FIGURE 12A depicts the amount of various compounds in the canola oil
and canola
EMO of Example 14 based on LC/MS/MS analysis.
[0076] FIGURE 12B depicts the amount of various compounds in the canola oil
and canola
EMO of Example 14 based on LC/MS/MS analysis.
DETAILED DESCRIPTION OF THE DISCLOSED SUBJECT MATTER
[0077] It is to be understood that both the foregoing general description and
the following
detailed description are exemplary and explanatory only, and are not
restrictive of the invention,
as claimed. In this application, the use of the singular includes the plural,
the word "a" or "an"
means "at least one", and the use of "or" means "and/or", unless specifically
stated otherwise.
Furthermore, the use of the term "including", as well as other forms, such as
"includes" and
"included", is not limiting. Also, terms such as "element" or "component"
encompass both
elements or components comprising one unit and elements or components that
comprise more
than one unit unless specifically stated otherwise.
[0078] The section headings used herein are for organizational purposes only
and are not to be
construed as limiting the subject matter described. All documents, or portions
of documents,
cited in this application, including, but not limited to, patents, patent
applications, articles, books,
and treatises, are hereby expressly incorporated herein by reference in their
entirety for any
purpose. In the event that one or more of the incorporated literature and
similar materials defines
a term in a manner that contradicts the definition of that term in this
application, this application
controls.
[0079] "Enriched" in the context of this invention means with an amount higher
than in the
starting material. For example, a MAG-enriched oil is an oil having a MAG
content that is
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
13
greater than the starting MAG content prior to the enrichment process or that
the starting oil has
a greater percentage of MAG than the oil possessed prior to the enrichment
process. The
enrichment process can be by conversion of TAGs to MAGs thereby increasing the
MAG
content and percentage and decreasing the TAG content or percentage.
[0080] A triacylglycerol ("TAG"), also known as a triglyceride, is a glyceride
consisting of three
fatty acid chains covalently bonded to a glycerol molecule through ester
linkages. TAGs may
also be classified as having a long or medium chain length. Long chain TAGs
contain fatty acids
with 14 or more carbons, while medium chain TAGs contain fatty acids with 6 to
12 carbons.
Long chain TAGs can include omega-3 and omega-6 fatty acids. Medium chain TAGs
have
saturated fatty acids and thus do not contain omega-3 or omega-6 fatty acids.
Long chain TAGs
(LCT) and medium chain triglycerides (MCT) can serve as energy sources.
[0081] A diacylglycerol ("DAG"), also known as a diglyceride, is a glyceride
consisting of two
fatty acid chains covalently bonded to a glycerol molecule through ester
linkages.
[0082] A monoacylglycerol ("MAG"), also known as a monoglyceride, is a
glyceride consisting
of one fatty acid chain covalently bonded to a glycerol molecule through an
ester linkage
[0083] As used herein, the term "processed oil" refers to a non-naturally
occurring oil
composition substantially free TAGs or having a reduced amount of TAGs with
respect to the
pre-modified or pre-processed oil.
[0084] As used herein, the terms "enzyme-modified oil" or "EMO" refers to a
processed oil
wherein TAGs were enzymatically converted to MAGs, such as, for example, using
the
enzymatic conversion of the present disclosure.
[0085] As used herein, the term "food product" refers to a manufactured or non-
naturally
occurring food product. It should be understood that the food product referred
to herein, while
manufactured and non-naturally occurring as a whole, can comprise various
combinations of
natural ingredients where said combinations either do not occur in nature or
where said
combinations do exist in nature, they do not exist in the relative amounts
used in the food
product.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
14
[0086] The terms "patient," "individual," and "subject" are used
interchangeably herein, and
refer to a mammalian subject to be treated, with human patients being
preferred. In some cases,
the methods of the invention find use in experimental animals, in veterinary
application, and in
the development of animal models for disease, including, but not limited to,
rodents including
mice, rats, and hamsters, and primates.
[0087] "Treatment" is an intervention performed with the intention of
preventing the
development or altering the pathology or symptoms of a disorder. Accordingly,
"treatment"
can refer to both therapeutic treatment and prophylactic or preventative
measures. Those in
need of treatment include those already with the disorder as well as those in
which the
disorder is to be prevented. In tumor (e.g., cancer) treatment, a therapeutic
agent may
directly decrease the pathology of tumor cells, or render the tumor cells more
susceptible to
treatment by other therapeutic agents, e.g., radiation and/or chemotherapy.
[0088] As used herein, a "non-oil ingredient" is an ingredient that is
naturally present in an oil
source that is not a MAG, DAG, TAG, FFA or lipid.
[0089] In some embodiments, the starting oils may comprise, by way of example
but not
limitation, oils derived from plants such as olive oil, almond oil, canola
oil, coconut oil,
cottonseed oil, palm kernel oil, palm olein oil, palm stearin oil, peanut oil
flaxseed oil, sunflower
seed oil, corn oil, grapeseed oil, palm oil, soybean oil, or oil derived from
animals such as fish
oil, sardine oil, or anchovy oil, or algal oil, or mixtures of any of the
foregoing plant oils and/or
animal oils. In one aspect, the starting oil comprises a blend of olive oil,
sunflower seed oil, and
flaxseed oil, wherein the from about 50% to about 80% by weight of the total
weight of the
starting oil is olive oil, from about 10% to about 30% by weight of the total
weight of the starting
oil is suflower seed oil, and from about 5% to about 20% by weight of the
total weight of the
starting oil is flaxseed oil. In another aspect, from about 50% to about 80%
by weight of the
total weight of the starting oil is olive oil, from about 10% to about 30% by
weight of the total
weight of the starting oil is flax seed oil, and from about 5% to about 20% by
weight of the total
weight of the starting oil is sunflower seed oil.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
[0090] In some embodiments, the starting oils comprise MCPD compounds. The
MCPD
compounds may be in an amount from about 0.30 mg/kg to about 12.0 mg/kg, or
from about 1.0
mg/kg to about 11.00 mg/kg, or from about 2.00 mg/kg to about 10.50 mg/kg.
Examples of
starting oils comprising MCPD compounds includes almond, canola, coconut,
corn, cottonseed,
grape seed, olive, palm, palm kernel, palm olein, palm stearin, peanut,
soybean, and sunflower.
[0091] In some embodiments, the process of making a product enriched in MAGs
comprises a
first step of hydrolyzing TAGs. By way of example but not limitation, the
hydrolysis of TAGs
may be carried out by a lipase such as lipase AY (Amano Enzymes, USA Elgin IL,
USA), or any
non-regiospecific lipase that cuts at the sn-1, sn-2 and sn-3 positions.
[0092] In some embodiments, the first step of hydrolyzing TAGs may be carried
out at a
temperature of about 30 C to 35 C. By way of example but not limitation, the
first step of
hydrolyzing TAGs may be carried out at a temperature of 30 C to 35 C, 31 C to
35 C, 32 C to
35 C, 33 C to 35 C, 34 C to 35 C, 30 C to 34 C, 31 C to 34 C, 32 C to 34 C, 33
C to 34 C,
30 C to 33 C, 31 C to 33 C, 32 C to 33 C, 30 C to 32 C, 31 C to 32 C, 30 C to
31 C, or 30 C,
31 C, 32 C, 33 C, 34 C, or 35 C.
[0093] In some embodiments, the first step of hydrolyzing TAGs may be carried
out for about 14
hours to 24 hours. By way of example but not limitation, the first step of
hydrolyzing TAGs may
be carried out for 14 hours to 20 hours, 14 hours to 16 hours, 18 hours to 24
hours, 22 hours to
24 hours, 18 hours to 20 hours, or about 14 hours, 15 hours, 16 hours, 17
hours, 18 hours, 19
hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours.
[0094] In some embodiments, the first step of hydrolyzing TAGs results in
hydrolysis of
substantially all TAG. By way of example but not limitation, the first step of
hydrolyzing TAGs
results in hydrolysis of 94% to 100%, 95% to 100%, 96% to 100%, 97% to 100%,
98% to 100%,
99% to 100%, 94% to 99%, 95% to 99%, 96% to 99%, 97% to 99%, 98% to 99%, 94%
to 98%,
95% to 98%, 96% to 98%, 97% to 98%, 94% to 97%, 95% to 97%, 96% to 97%, 94% to
96%,
95% to 96%, or 94% to 95% TAG, or at least 94%, 95%, 96%, 97%, 98%, 99%, or
100% of
TAG.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
16
[0095] In some embodiments the process of making a product enriched in MAGs
comprises a
second step of esterification with glycerol to enrich MAG oil content. By way
of example but not
limitation, this second step of esterification may be carried out by a lipase
such as lipase G,
(Amano Enzymes, USA Elgin IL, USA), or any regiospecific lipase that catalyzes
esterification
at the sn-1 position but does not effectively catalyze formation of the second
or third ester on
glycerol (to make DAGs and TAGs).
[0096] In some embodiments the second step of esterification with glycerol to
enrich MAG oil
content results in enrichment of MAGs in the product by about 70% to 95%. By
way of example
but not limitation, the MAG oil content may be enriched by 70% to 95%, 75% to
95%, 80% to
95%, 85% to 95%, 90% to 95%, 70% to 90%, 75% to 90%, 80% to 90%, 85% to 90%,
70% to
85%, 75% to 85%, 80% to 85%, 70% to 80%, 75% to 80%, 70% to 75%, or 70%, 75%,
80%,
85%, 90%, or 95%.
[0097] In some embodiments, the second step of esterification with glycerol
may be carried out
at a temperature of about 17 C to 23 C. By way of example but not limitation,
the esterification
with glycerol may be carried out at a temperature of 17 C to 23 C, 18 C to 23
C, 19 C to 23 C,
20 C to 23 C, 21 C to 23 C, 22 C to 23 C, 17 C to 22 C, 18 C to 22 C, 19 C to
22 C, 20 C to
22 C, 21 C to 22 C, 17 C to 21 C, 18 C to 21 C, 19 C to 21 C, 20 C to 21 C, 17
C to 20 C,
18 C to 20 C, 19 C to 20 C, 17 C to 19 C, 18 C to 19 C, 17 C to 18 C, or 17 C,
18 C, 19 C,
20 C, 21 C, 22 C, or 23 C.
[0098] In some embodiments, the second step of esterification with glycerol
may be carried out
for about 24 hours to 72 hours. By way of example but not limitation, the
second step of
esterification with glycerol may be carried out for 24 hours to 72 hours, 36
hours to 72 hours, 48
hours to 72 hours, 60 hours to 72 hours, 24 hours to 60 hours, 36 hours to 60
hours, 48 hours to
60 hours, 24 hours to 48 hours, 36 hours to 48 hours, 24 hours to 36 hours, or
24 hours, 30 hours,
36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, or 72 hours.
[0099] In some embodiments, the process of making a product enriched in MAGs
comprises a
third step of lipase inactivation and phase separation.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
17
[00100] The resulting product from the above embodiments results in a
processed oil
having a MAG content of equal to or greater than 40% by weight based on the
total weight of the
processed oil. In certain aspects, the MAG content is from about 40% to about
99% by weight
based on the total weight of the processed oil. In certain aspects, the MAG
content is from about
50% to about 99% by weight based on the total weight of the processed oil. In
certain aspects,
the MAG content is from about 60% to about 99% by weight based on the total
weight of the
processed oil. In certain aspects, the MAG content is from about 70% to about
99% by weight
based on the total weight of the processed oil. In certain aspects, the MAG
content is from about
80% to about 99% by weight based on the total weight of the processed oil. In
certain aspects,
the MAG content is from about 50% to about 80% by weight based on the total
weight of the
processed oil. In any of the above aspects, the TAG content is equal to or
less than 5% by
weight based on the total weight of the processed oil. In any of the above
aspects, the TAG
content is equal to or less than 4%, equal to or less than 3%, equal to or
less than 2%, equal to or
less than 1% by weight based on the total weight of the processed oil.
[00101] The resulting product from the above embodiments results in a
processed oil that
is substantially free of MCPD compounds generally, but also when the starting
oils have more
than 0.30 mg/kg MCPD compounds and in some instances from about 1.0 mg/kg to
about 12.00
mg/kg. More specifically, the processed oil resulting from the above methods
comprises less
than 100 mg/kg MCPD and even to levels that are not detectable by standard
assays. As used
herein, the term "substantially free" with respect to the amount of MCPD
levels in a processed
oil of the present invention means levels that are below the limits of
detection for the assay
described in Example 11 below.
[00102] The resulting product from the above embodiments results in a
processed oil that
is enriched in MAGs, specifically 1-MAGs (MAGs esterified at the sn-1
position). In the case of
processed oils made from starting oils having oleic acid content, this results
in the production of
1-oley1 monoacylglyceride (1-OG). It has been found that manufacturing process
embodiments
of the present disclosure can efficiently convert even sn-2 oleic acid TAGs to
1-0G. For
example, starting with triolein, the manufacturing process embodiments of the
present disclosure
can yield three 1-0G molecules from a single triolein molecule, while
digestion of triolein by
CA 03127093 2021-07-16
WO 2020/150661
PCT/US2020/014182
18
pancreatic lipases can only yield one-third of the total oleic acid in
monoacylglycerol form,
specifically as 2-monoacylglycerol (2-OG) but not 1-OG, because the lipase
liberates the oleic
acid moieties at the sn-1 and sn-3 positions as free fatty acids. Thus, a
processed oil produced by
the manufacturing methods of the present disclosure can yield up to three
times the 1-0G content
compared to normal digestion processes. The below reaction schematic shows the
conversion of
triolein to 2-OG and two free oleic acid molecules (process A ¨ normal
digestion) and of triolein
to 3 molecules of 1-0G by the process of the present disclosure (process B).
1...W/N= Triolein Oleic
Acid
. . ----
[ \ 9
2-OG
.. )
v
A....õ..,,\,.,..õ.,..,,,....õ....Ø,,i
1 A 1 .Ø- ,..% Ne ..... ..õ )
ez " /-N...=="-N, . ' A
i Q. ( - === ,--
, ,
4. . .,,,....Qt i4A, :
Oleic Acid
rOs N
q
, ,...õ..õ--
,õ..,,,,........,,.......,ke
õ..-- ..., I --,..õ
C:44$1=\, \1'' ' B
1-OG 1-OG 1-OG
,..,..-\.,
i=ii.: 1 ii.F.."-\:: .s,"-
.õ,-,......".õ...- O. -."-../' "..." 0
c)
...,...,,,.....¨......a.H ,,õ=-=\:.,,,,A.:,õ."...A. 4. g ,,
v i -....---õõ---õ,":õ--.0").-0,i
oz e: )
1
k
4
[00103]
It has also been found that the manufacturing process embodiments of the
present
disclosure can preserve the fatty acid profile of the starting oil in the
processed oil.
Representative fatty acid profiles for various starting oils are shown in
Table 2 below which
provides percentages of total fatty acid methyl esters (FAMEs). Nd ¨ not
determined. SAF ¨
safflower, GRP- grape, SIL ¨ Silynum marianum, HMP ¨ hemp, SFL ¨ sunflower,
WHG ¨ wheat
germ, PMS ¨ pumpkin seed, SES ¨ sesame, RB ¨ rice bran, ALM ¨almond, RPS ¨
rapeseed
(canola), PNT ¨ peanut, OL ¨ olive, and COC ¨ coconut oils. Table 2 also
provides the ratios of
oleic acid/linoleic acid and oleic acid/linolenic acid for each oil.
Edible Oils
FAs [A] SAF GRP
SIL HMP SFL WHG PMS SES RB AIM RPS PNT .. OL .. COC
0
C6:0 nd nd nd nd nd nd nd
nd nd nd nd nd nd 0.52 t..)
o
t..)
C8:0 nd 0.01 nd nd nd nd nd
nd nd nd nd nd nd 7.6 =
,-,
C10:0 nd nd nd nd nd nd nd
nd nd nd 0.01 nd nd 5.5 u,
o
o
o
C12:0 nd 0.01 0.01 nd 0.02 0.07 nd nd nd 0.09 nd nd
nd 47.7
C14:0 0.1 0.05 0.09 0.07 0.09 nd 0.17 nd 0.39 0.07
nd 0.04 nd 19.9
C15:0 nd 0.01 0.02 nd nd 0.04 nd nd nd nd 0.02 nd
nd nd
m C16:0 6.7 6.6 7.9 6.4 6.2 17.4 13.1 9.7 20 6.8 4.6
7.5 16.5 nd
n
-I C17:0 0.04 0.06 0.06 0.05 0.02 0.03 0.13
nd nd 0.05 0.04 0.07 nd nd
-n
r7 1 C18:0 2.4 3.5 4.5 2.6 2.8 0.7 5.7
6.5 2.1 2.3 1.7 2.1 2.3 2.7
0 C20:0 nd 0.16 2.6 nd 0.21 nd 0.47 0.63 nd 0.09 nd
1.01 0.43 nd P
cn
1 C22:0 nd nd nd nd nd nd nd
0.14 nd nd nd nd 0.15 nd 0
,
m
m C16:1 (n-7) 0.08 0.08 0.05 0.11 0.12 0.21
0.12 0.11 0.19 0.53 0.21 0.07 1.8 nd ,
-I
70 C17:1 (1-7) nd nd 0.03 nd nd nd nd
nd nd nd nd nd nd nd " 0
"
,
,
c r C18:1cis (n-9)
0 ,
,
m Oleic 11.5 14.3 20.4 11.5 28 12.7 24.9 41.5 42.7
67.2 63.3 71.1 66.4 6.2 ,
c.o
1-. C18:1trans (n-9) nd nd nd nd nd nd nd
nd nd nd 0.14 nd nd nd
-
(.7) C20:1(n-9) nd 0.4 0.15 16.5 0.18 7.91 1.08 0.32 1.11
0.16 9.1 nd 0.3 nd
>
C18:2cis (n-6)
m
-0 Linoleic 79 74.7 63.3 59.4 62.2 59.7 54.2
40.9 33.1 22.8 19.6 18.2 16.4 1.6
C18:3 (n-3)
Iv
Linolenic 0.15 0.15 0.88 0.36 0.16 1.2 0.12 0.21 0.45
nd 1.2 nd 1.6 nd n
1-i
cp
t..)
0 leic/Linoleic
t..)
o
Ratio 0.15 0.19 0.32 0.19 0.45 0.21 0.46 1.01 1.29
2.95 3.23 3.91 4.05 3.88 O-
,-,
0 leic/Linolenic
cio
t..)
Ratio 76.7 95.3 23.2 31.9 175.0 10.6 207.5 197.6
94.9 nd 52.8 nd 41.5 nd
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
[00104] In particular, linoleic (omega-3 fatty acid) and linolenic acid
(omega-6 fatty acid)
are known to be essential for humans as they can be used in the creation of
longer and more
desaturated fatty acids otherwise known as long-chain polyunsaturated fatty
acids (LC-PUFA),
including eicosapentaenoic acid (EPA) and docosahexanenoic acid (DHA).
[00105] In some embodiments, a product comprises a processed oil of the
present
disclosure. In some embodiments, the processed oil is itself a stand-alone
product.
[00106] In some embodiments, the processed oil comprises a MAG content
equal to or
greater than 30% by weight of the total weight of the processed oil. By way of
example but not
limitation, the processed oil comprises a MAG content of about 30% to 95%, 40%
to 95%, 50%
to 95%, 60% to 95%, 70% to 95%, 80% to 95%, 90% to 95%, 30% to 90%, 40% to
90%, 50% to
90%, 60% to 90%, 70% to 90%, 80% to 90%, 30% to 80%, 40% to 80%, 50% to 80%,
60% to
80%, 70% to 80%, 30% to 70%, 40% to 70%, 50% to 70%, 60% to 70%, 30% to 60%,
40% to
60%, 50% to 60%, 30% to 50%, 40% to 50%, or about 30%, 35%, 40%, 45%, 50%,
55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, or 95% by weight of the total weight of the
processed oil.
[00107] In some embodiments, including the above embodiments regarding the
MAG
content, the processed oil comprises a DAG content equal to or greater than 5%
by weight of the
total weight of the processed oil. By way of example but not limitation, the
processed oil
comprises a MAG content of about 5% to 66%, 10% to 66%, 20% to 66%, 30% to
66%, 40% to
66%, 50 to 66%, 5% to 50%, 10% to 50%, 20% to 50%, 30% to 50%, 40% to 50%, 5%
to 40%,
10% to 40%, 20% to 40%, 30% to 40%, 5% to 30%, 10% to 30%, 20% to 30%, 5% to
20%, 10%
to 20%, 5% to 10%õ or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%,
60%, or 66% by weight of the total weight of the processed oil.
[00108] In some embodiments, including the above embodiments regarding the
MAG
and/or DAG content, the processed oil comprises a FFA content equal to or
greater than 5% by
weight of the total weight of the processed oil. By way of example but not
limitation, the
processed oil comprises a MAG content of about 5% to 66%, 10% to 66%, 20% to
66%, 30% to
66%, 40% to 66%, 50 to 66%, 5% to 50%, 10% to 50%, 20% to 50%, 30% to 50%, 40%
to 50%,
5% to 40%, 10% to 40%, 20% to 40%, 30% to 40%, 5% to 30%, 10% to 30%, 20% to
30%, 5%
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
21
to 20%, 1000 to 200 o, 500 to 150 o, 1000 to 150 0, 5 A to 10%õ or about 5%,
100 o, 150 o, 200 o, 25%,
30%, 350o, 40%, 450o, 50%, 550, 60%, or 66 A by weight of the total weight of
the processed
oil.
[00109] In some embodiments, including the above embodiments regarding the
MAG,
DAG and/or FFA content, the processed oil is either free of TAGs or comprises
a TAG content
that is equal to or less than 5 A by weight of the total weight of the
processed oil. By way of
example but not limitation, the processed oil comprises a TAG content that is
about 0% to 5%,
1 A to 5%, 2 A to 5%, 3 A to 5%, 4 A to 5%, 0% to 4%, 1% to 4%, 2 A to 4%, 3 A
to 4%, 0%
to 300, 100 to 300, 200 to 30, 000 to 200, 100 to 200, 000 to 100, or 000,
100, 200, 300, 400 or 50
by weight of the total weight of the processed oil. By way of further example
but not limitation,
the TAG content can be less than 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%.
[00110] In some embodiments, the processed oil comprises from about 30 A
to 80 A MAG
by weight of the total weight of the processed oil, from about 10% to about 30
A DAG by weight
of the total weight of the processed oil, from about 0% to about 5 A TAG by
weight of the total
weigh of the processed oil, and from about 5 A to about 60 A FFA by weight of
the total weight
of the processed oil.
[00111] In any of the embodiments herein, the processed oil comprises non-
oil ingredients
derived from and naturally present in the oil source such that the non-oil
ingredients are not
added to the processed oil. By way of example but not limitation, such non-oil
ingredients may
include antioxidants such as tocopherols, which include alpha-tocopherol, beta-
tocopherol, delta-
tocopherol, gamma-tocopherol, alpha-tocotrienol, beta-tocotrienol, delta-
tocotrienol, or gamma-
tocotrienol, and other vitamins such as Vitamin K and structurally similar 2-
methy1-1,4-
naphthoquinone (3-) derivatives. In some embodiments, the antioxidant is
selected from natural
(e.g., mixed tocopherols or ascorbic acid) and synthetic (e.g., 13utylated Hy
droxyAnisole or
Butylated HydroxyToluene) antioxidants. Further examples of non-oil
ingredients include
cerami de phosphates, mottogaiactosyl diacyglycerols, phosphatidyl methanol,
sitosteryl esters,
campesterol esters, sphingolipids, phosphatidyl glycerol, wax esters, and
sphingomyelin.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
22
[00112] In any of the embodiments herein, the processed oil comprises
oleic acid
monoglyceride (MOO) and has a total fatty acid content. The MOO can contribute
between
about 5% and about 75% by weight of the total fatty acid content of the
processed oil. In some
embodiments, by way of example, but not limitation, the MOO can contribute
between about
10% and about 75%, about 20% and about 75%, about 30% and about 75%, about 40%
and
about 75%, about 50% and about 75%, or about 60% and about 75% by weight of
the total fatty
acid content of the processed oil. In some embodiments, the processed oil
comprises oleic acid
in amount of about PA and about 75% by weight of the total fatty acid content
of the processed
oil. In some embodiments, by way of example, but not limitation, the oleic
acid can contribute
between about 10% and about 75%, about 20% and about 75%, about 30% and about
75%, about
40% and about 75%, about 50% and about 75%, or about 60% and about 75% by
weight of the
total fatty acid content of the processed oil.
[00113] In any of the embodiments herein, the processed oil comprises MOG
and the
MOO comprises at least 50% by weight 1-oley1 monoglyceride (1-00) out of the
total amount of
MOO. By way of example, but not limitation, the processed oil can comprise at
least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
95%, or at least 99% 1-
00 out of the total amount of MOG.
[00114] In any of the embodiments herein, the processed oil comprises
oleic acid in an
amount of about 10% to about 75% by weight out of the total fatty acid content
of the processed
oil. By way of example, but not limitation, the oleic acid can be present in
an amount of about
10% to about 75%, about 20% to about 75%, about 30% to about 75%, about 40% to
about 75%,
about 50% to about 75%, about 60% to about 75%, about 10% to about 20%, about
10% to about
30%, about 10% to about 40%, about 10% to about 50%, or about 10% to about 60%
by weight
out of the total fatty acid content of the processed oil. In any of the
embodiments herein, the oleic
acid is esterified at the sn-I position. By way of example, but not
limitation, the processed oil
can comprise at least 50%, at least 60%, at least 70%, at least 80%, at least
85%, at least 90%, at
least 95%, or at least 99% of oleic acid esterified at the sn-1 position out
of the total amount of
oleic acid.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
23
[00115] In any of the embodiments herein, the processed oil comprises
linoleic acid in an
amount of about 1.5% to about 90% by weight out of the total fatty acid
content of the processed
oil. By way of example, but not limitation, the linoleic acid can be present
in an amount of about
10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to
about 90%,
about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about
80% to about
90%, about 10% to about 25%, about 10% to about 20% by weight out of the total
fatty acid
content of the processed oil. In some embodiments, the processed oil comprises
oleic acid and
linoleic acid with a ratio of oleic acid to linoleic acid between about 0.01
and 5. By way of
example, but not limitation, the ratio of oleic acid to linolcic acid can be
between about 1 to
about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 1.5
to about 5, about 1.5
to about 4, about 1.5 to about 3, about 1.5 to about 2, about 2 to about 5,
about 2 to about 4,
about 2 to about 3, about 2.5 to about 5, about 2.5 to about 4.5, about 2.5 to
about 4, about 2.5 to
about 3.5, about 2.5 to about 3, about 3 to about 5, or about 3 to about 4.
[00116] In any of the embodiments herein, the processed oil comprises
linolenic acid in an
amount of about 0.01% to about 2% by weight out of the total fatty acid
content of the processed
oil. By way of example, but not limitation, the linolenic acid can be present
in an amount of
about 0.1 to about 2%, about 0.5 to about 2%, about 1% to about 2%, or about
1.5% to about 2%
by weight out of the total fatty acid content of the processed oil. In some
embodiments, the
processed oil comprises oleic acid and linolenic acid with a ratio of oleic
acid to linolenic acid of
about Ito about 100. By way of example, but not limitation, the ratio of oleic
acid to linolenic
acid can be between about 10 and about 100, about 20 and about 100, about 30
and about 100,
about 40 and about 100, about 50 and about 100, about 60 and about 100, about
70 to about 100,
about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to
about 60, about 10
to about 50, about 10 to about 40, about 10 to about 30, or about 10 to about
20. It should be
understood that reference to fatty acids in the claims should not be
understood as limited to the
free fatty acid only, and can refer to the fatty acid in glyceride form. In
processed oils of the
present disclosure, the fatty acid will predominantly be in the MAG form and
in the pre-
processed oil it will be predominantly in the TAG form. In certain instances,
the fatty acid to a
lesser extent can be in the free or DAG form. In the processed oil, the free
fatty acid will
typically be less than 10%.
RECTIFIED SHEET (RULE 91) ISA/EP
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
24
[00117] in any of the embodiments herein, the processed oil has a fatty
acid profile that is
substantially the same as the fatty acid profile of the pre-processed oil from
which the processed
oil was produced. By way of example, but not limitation, the fatty acid
profile of the processed
oil can be within about 10%, about 9%, about 8%, about 7%, about 6%, about 5%,
about 4%,
about 3%, about 2%, or about 1% for each component of the fatty acid profile.
In some
embodiments, the fatty acid profile comprises oleic acid, linoleic acid and
linoleic acid.
However, any combination of fatty acids may form the fatty acid profile. By
way of example,
but not limitation, the fatty acid profile can comprise any combination of
fatty acids listed in
Table 2 as well as EPA and
[00118] In any of the embodiments herein, the processed oil compii ses
three times the
amount of MAO molecules compared to the number of TAG molecules in the pre-
processed oil
from which the oil was produced. For example, on a molar basis, the amount
oleic acid in the
form of MAGs in the processed oil can be up to three times the amount of oleic
acid in TAGs in
the pre-processed oil. By way of example, but not limitation, the amount of
oleic acid in the
form of MAO in the processed oil can be 1,1, 1.25, 1.5, 1.75,2.0, 2.25, 2.5,
2.75 or 3 times the
amount of oleic acid in the form of TAGs in the pre-processed oil. For
example, each oleic acid
moiety in the form of TAGs in the pre-processed oil can be converted to an
oleic acid MAO,
such that up to three oleic acid moieties, as in triolein, would be converted
into three oleic acid
MAGs. in som.e embodiments, the amount of MAGs in the processed oil is three
times the
amount of TAGs in the pre-processed oil from which the processed oil was
produced. For
example, the number of MAGs produced corresponds to about three times the
number of TAGs
in the pre-processed oil. In some embodiments, the processed oil comprises
oleic acid in the
oleic acid monoglyceride form in an amount that is about the same as the
amount of oleic acid in
the pre-processed oil from which the processed oil was produced.
[00119] In some embodiments the product or food product can comprise at
least 1%
MAGs. By way of example, but not limitation, the food product can comprise at
least 1%, 2%,
3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%,
40%,
45%, 50% or more MAGs by weight of the product or food product and any range
or amount
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
therebetween. By way of further example, but not limitation, the product or
food product can
comprise between 5% to 15% weight MAGs by total weight of the product or food
product.
[00120] In some embodiments, the product or food product can comprise at
least 3% of
the processed oil of any of the embodiments herein by weight of the total
weight of the product
or food product. . By way of example, but not limitation, the food product can
comprise at least
3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,
20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more of the processed oil
of any of
the embodiments herein by weight of the product or food product and any range
or amount
therebetween.
[00121] In some embodiments, the food product may further comprise a
carbohydrate
source. By way of example but not limitation, such a carbohydrate source may
comprise simple
sugars such as glucose and fructose, derived from carbohydrate sources such as
fruit and agave
syrups. Other carbohydrate sources include other plant-based sugar syrups,
starches, and sugar
alcohols.
[00122] In some embodiments, the food product may further comprise a
protein source. In
certain aspects, the protein source can be hydrolyzed or partially hydrolyzed.
By way of
example but not limitation, such a protein source may comprise dairy protein
(casein and whey),
and other plant proteins including protein from soy, rice and rice bran,
lentils, chickpeas,
peanuts, almonds, spirulina (algal), quinoa, mycoprotein, chia seeds and hemp
seeds. The
hydrolyzed protein may be extensively hydrolyzed wherein the pea protein is
enriched in
peptides of 1 to 10 amino acids in length. In some embodiments, the protein is
enriched in
peptides of 1 to 10 amino acids in length by about 25% to 75% compared to
commercial partially
hydrolyzed protein and other whey-based hydrolysate products such as Peptamen
and Crucial.
By way of example but not limitation, the protein is enriched in peptides of 1
to 10 amino acids
in length by at least 25% to 75%, 35% to 75%, 45% to 75%, 55% to 75%, 65% to
75%, 25% to
65%, 35% to 65%, 45% to 65%, 55% to 65%, 25% to 55%, 35% to 55%, 45% to 55%,
25% to
45%, 35% to 45%, 25% to 35%, or 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, or
75%.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
26
[00123] In some embodiments, the food product can be a liquid, semi-solid
or solid. By
way of example but not limitation, a semi-solid can include a pudding, mousse,
popsicle or ice
cream-like product. By way of example but not limitation, a liquid could be a
shake or other
beverage. By way of example but not limitation, a solid could be a bar or
other solid food
product.
[00124] In some embodiments, the product or food product further comprises
a viscosity
altering agent. The viscosity altering agent can be, by way of example but not
limitation,
xanthan gum or gum acacia. In some embodiments, the product or food product
can further
comprises a structure or stability enhancing component such as, by way of
example but
limitation, gum Arabic, sunflower lecithin and xanthan gum. In some
embodiments, the product
or food product further comprises a fiber source, such as, by way of example
but limitation,
oligosaccharides. In some embodiments the product or food product can further
comprise a food
preservative such as, by way of example, but not limitation sodium benzoate or
potassium
sorbate.
[00125] In some embodiments, a product or food product further comprises a
flavor,
masker or blocker. By way of example, but not limitation, flavorings can
include chocolate,
vanilla, strawberry or other flavors.
[00126] In some embodiments, the food product has a total weight of at
least 25 grams.
By way of example, but not limitation, the food product can have a weight of
at least 25, 50, 100,
250, 500, 1000, 1500, 2000, 2500, 3000 grams or more. In some embodiments, the
food product
has a total weight from about 25 grams to about 3000 grams. By way of example
but not
limitation, the food product may have a weight from 25 grams to 3000 grams, 25
grams to 2500
grams, 25 grams to 2000 grams, 25 to 1000 grams, 25 grams to 500 grams, 50
grams to 3000
grams, 50 grams to 2500 grams, 50 grams to 2000 grams, 50 grams to 1500 grams,
50 grams to
1000 grams, 50 grams to 500 grams, 100 grams to 3000 grams, 100 grams to 2500
grams, 100
grams to 2000 grams, 100 grams to 1500 grams, 100 grams to 1000 grams, 100
grams to 500
grams, 250 grams to 3000 grams, 250 grams to 2500 grams, 250 grams to 2000
grams, 250
grams to 1500 grams, 250 grams to 1000 grams, 250 grams to 500 grams, 500
grams to 3000
grams, 500 grams to 2500 grams, 500 grams to 2000 grams, 500 grams to 1500
grams, 500
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
27
grams to 1000 grams, 1000 grams to 3000 grams, 1000 grams to 2500 grams, 1000
grams to
2000 grams, 1000 grams to 1500 grams, 1500 grams to 3000 grams, 1500 grams to
3000 grams,
2000 grams to 3000 grams, 2000 grams to 2500 grams, 25 grams to 250 grams, 50
grams to 250
grams, 100 grams to 250 grams, 25 grams to 100 grams, 50 grams to 100 grams,
25 grams to 50
grams, or a total weight that is less than or equal to 25 grams, 50 grams, 100
grams, 150 grams,
200 grams, 250 grams, 300 grams, 350 grams, 400 grams, 450 grams, 500 grams,
1000 grams,
1500 grams, 2000 grams, 2500 grams, or 3000 grams.
[00127] In some embodiments, the food product has a total calorie content
from about 200
kcals to 1000 kcals. By way of example but not limitation, the food product
may have a calorie
content from about 200 kcals to 1000 kcals, 400 kcals to 1000 kcals, 600 kcals
to 1000 kcals,
800 kcals to 1000 kcals, 200 kcals to 800 kcals, 400 kcals to 800 kcals, 600
kcals to 800 kcals,
200 kcals to 600 kcals, 400 kcals to 600 kcals, 200 kcals to 400 kcals, or an
amount less than or
equal to 200 kcals, 300 kcals, 400 kcals, 500 kcals, 600 kcals, 700 kcals, 800
kcals, 900 kcals, or
1000 kcals.
[00128] In some embodiments, about 20% to 75% of calories in the food
product are
derived from an oil or fat. In one aspect, the oil or fat is the processed oil
of any of the
embodiments herein. In yet another aspect, the processed oil has a MAG content
of equal to or
greater than 30% by weight of the total weight of the processed oil. In other
aspects, the
processed oil has a MAG content of from about 40% to about 99% by weight of
the total weight
of the processed oil. In yet another aspect, the processed oil has a TAG
content of less than 5%
by weight of the total weight of the processed oil. By way of example but not
limitation, 20% to
50%, 30% to 50%, 40% to 50%, 20% to 40%, 30% to 40%, 20% to 30%, 20% to 75%,
30% to
75%, 40% to 75%, 50% to 75%, 60% to 75%, 70% to 75%, 20% to 70%, 30% to 70%,
40% to
70%, 50% to 70%, 60% to 70%, 20% to 60%, 30% to 60%, 40% to 60%, 50% to 60%,
or about
20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,
35%,
36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,
51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,
67%,
68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75%, of calories in the food product are
derived from
any of the above described processed oils.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
28
[00129] In any of the above embodiments regarding the food product, about
20% to 50%
of calories in the food product are derived from the carbohydrate source. By
way of example but
not limitation, 20% to 50%, 30% to 50%, 40% to 50%, 20% to 40%, 30% to 40%,
20% to 30%,
or about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,
34%,
35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or
50% of
calories in the food product are derived from the carbohydrate source.
[00130] In any of the above embodiments regarding the food product, about
10% to 50%
of calories in the food product are derived from the protein source. By way of
example but not
limitation, 10% to 50%, 20% to 50%, 30% to 50%, 40% to 50%, 10% to 40%, 20% to
40%, 30%
to 40%, 10% to 30%, 20% to 30%, 10% to 20%, or about 10%, 11%, 12%, 13%, 14%,
15%,
16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,
31%,
32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,
47%,
48%, 49%, or 50% of calories in the food product are derived from the protein
source.
[00131] It should be understood that the above disclosed embodiments of
the food product
can be combined.
[00132] In some embodiments, a food product of the present disclosure
comprises a
processed oil of the present disclosure.
[00133] In some embodiments, the EMO-based products are consumed by
individuals
with poorly functioning digestive systems, by way of example but not
limitation, individuals
suffering from EPI or individuals who take PERT with food. In some
embodiments, the EMO-
based products are consumed by individuals who desire faster or more complete
conversion of
lipids to serum triglycerides. Thus, a method for feeding a human or animal
subject with a poorly
functioning digestive system is provided. The method comprises administering
to said patient a
food product described in any of the above embodiments or combinations of such
embodiments.
In one aspect, the human or animal subject is suffering from EPI. In other
aspects, the human or
animal subject is suffering from cystic fibrosis, pancreatitis, pancreatic
cancer, or cholestasis.
[00134] In some embodiments, a method is provided for promoting glucose
homeostasis
in a subject in need thereof, comprising administering to the subject a
composition comprising a
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
29
processed oil of the present disclosure. In some embodiments, the subject is
suffering from a
condition that affects glucose homeostasis. In some embodiments, the condition
is insulin
resistance or diabetes mellitus. In some embodiments, the condition is type II
diabetes.
[00135] In some embodiments, a method is provided for treating diabetes in
a subject in
need thereof, comprising administering to the subject a composition comprising
a processed oil
of the present disclosure. In some embodiments, the diabetes is type II
diabetes.
EXAMPLES
Example 1: Process of making a product enriched in MAG compared to starting
TAG-rich oil.
[00136] The method of making a product enriched in MAG compared to the
starting
TAG-rich oil involves 3 key steps: (1) A mild enzymatically-catalyzed reaction
to hydrolyze
triglycerides (TAGs) in a sequence that converts the natural oils to specific
combinations of
FFAs, MAGs, DAGs, and low residual TAGs; (2) an esterification with glycerol
to generate
predominantly high amounts of MAGs leaving low concentrations of FFAs; and (3)
isolation of
the modified lipid product; this is achieved by phase separation with or
without the aid of a
centrifuge.
[00137] Step 1. Enzymatic Conversion of Triacylglycerols
[00138] Preparation of the buffer solution. A sodium citrate solution (100
mM, pH 5.8)
was prepared in a stirred tank reactor. 11.1 L of deionized (DI) water was
placed in the mixing
vessel, the agitator was set to 200 RPM and 0.213 kg of citric acid
(anhydrous) were added.
After the powder was dissolved the pH was adjusted with sodium hydroxide
solution to pH = 5.8
(about 0.121 kg). 220 mL was removed for subsequent enzyme preparation.
[00139] Preparation of the enzyme solution. In a separate 250 mL bottle,
the enzyme
solution was prepared under gentle stirring: 200 mL of the citrate buffer
solution was placed in
the mixing vessel. 10 g of AMANO Lipase AY was added and the bottle was shaken
until the
enzyme is dissolved.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
[00140] Conversion of Triglyceride Oil Mixture. Three vegetable oils,
Olive Oil,
Flaxseed Oil and Sunflower seed Oil were added to the vessel to give a total
of 10 kg of plant oil
mixture. Vacuum was applied to drop the pressure to about 20 mmHg and degas
the material
(from any dissolved oxygen in particular). Agitation was set to 200 RPM and
the mixture was
heated to 33 C and agitated for about 15 Min to remove any dissolved gas. The
vacuum was
then replaced by a Nitrogen gas atmosphere. Once the mixture was sufficiently
dispersed, 220
mL of the enzyme preparation in buffer solution was added. Agitation was
continued and the
reaction monitored for 24 hours until conversion to FFA was complete based on
TLC analysis.
[00141] Reactor temperature was increased to 70 C and agitation resumed
for 1 hour to
inactivate the enzyme.
[00142] Agitation was stopped and the phases separated in about 60
Minutes.
[00143] The aqueous (lower) phase was removed along with a small amount of
the oil
phase to ensure residual protein at the interface was eliminated.
[00144] Step 2. Esterification with Glycerol. Typically when FFA are re-
esterified with
glycerol they produce a mixture of MAGs, DAGs and TAGs. We found that by
significantly
reducing the temperature (below 25 C) and removing the water (by evaporation)
as it is formed
in the reaction, that the proportion of MAGs in the product can be highly
enriched (at least 60%
but as high as 95%). This was un-expected.
[00145] In the reaction, the reaction product from step 1 (about 10L) was
cooled to about
30 C and agitated at 300 RPM. 10 kg of Food-grade glycerol were added to the
Lipid mixture,
and the temperature maintained at ¨ 30 C. The mixture was agitated to
generate a dispersion of
the oil and glycerol. To dry the reaction mixture a vacuum was applied: First
a vacuum (25
mmHg, Torr) was applied with receiver in place to collect water. Once
evaporation of the
residual water had stopped, 20g of Amano Lipase G dissolved in water (50 mL)
was added to the
reactor. The temperature was lowered to 23 C and the vacuum was changed to 5
mmHg using
an oil diaphragm pump and a cold trap to collect the water. The mixture was
stirred at 300 RPM
at 23 C under vacuum for 72 hrs, at which time the vacuum was broken and the
mixture was
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
31
blanketed with nitrogen gas. The mixture was analyzed after 72 hrs with TLC to
evaluate the
conversion to MAGs as shown in FIGURE 1.
[00146] Step 3. Lipase Inactivation and Phase Separation. After the
reaction was
complete, the lipase was inactivated by heating the mixture (under a Nitrogen
gas blanket) to
70 C for 1 hr. At this point the MAG oil and glycerol are well mixed and very
hard to separate
through traditional gravity or centrifugal methods. After considerable
experimentation, we found
that the lipid can be separated from the excess glycerol by adding 0.3%wt of
salt (NaCl) to the
reaction mixture under agitation. The product mixture was then allowed to cool
to about 60 C
and left without agitation for about 1 hr.
[00147] The lipid oil phase separated from the remaining heavier glycerol
phase. The
glycerol phase was removed: it contains some salt, residual water and the
dissolved inactivated
enzyme which is contained in the visible interface. The glycerol phase can be
reused after
membrane filtration and should be kept for recycle. Tocopherol (Vitamin E) was
added to give a
concentration of 200 ppm (0.02% wt) of the product oil. The hydrolyzed oil
(about 10 kg was
ready for use and could be stored under nitrogen blanket).
[00148] Product Storage. The final hydrolyzed products were transferred to
food-grade
containers with an overlay of nitrogen gas for storage and transportation.
Example 2: Characterization of oil produced in Example /.
[00149] Reaction products and the overall process can be evaluated using
Thin Layer
Chromatography and Gas Chromatography.
[00150] Thin-Layer Chromatography Testing. Components of the oil samples
were
separated using TLC plates (Analtech Uniplate Silica Gel GHL with inorganic
binder, 20 x 20
cm, 250 um). The solvent was Hexane: Diethyl Ether: Acetic Acid (70:30:1)
solution. Typical
sample sizes were 3 L. After the solvent front ran to near the top of the
plate (-1 cm), plates
were removed from the TLC tank and the solvent evaporated in a fume hood. The
components
were visualized with iodine vapors (at room temperature) in a TLC tank and
relative intensities
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
32
estimated by colorimetric imaging (Amersham 600 Imager). After 15 minutes in
the tank, plates
were removed and photographed. The intensity of the spots diminished after 30
¨ 60 minutes.
[00151] Physical properties determination was performed to establish
product consistency,
color, water content, fats & oils (miscible).
[00152] FIGURE 1 depicts the final results of steps 1 to 3 in the process
of making a
product enriched in MAG compared to starting TAG-rich oil as describe in
Example 1. FIGURE
2 illustrates that tocopherol initially present in the olive oil is preserved
following the steps
described in Example 1. In FIGURE 2, the tocopherol spot can be seen tracking
above the TAG
spot in all three lanes.
[00153] Fatty Acid Profile Testing - Gas Chromatography. Lipid components
including C10:0 Capric Acid, C12:0 Lauric Acid, C14:0 Myristic Acid, C16:0
Palmitic Acid,
C18:0 Stearic Acid, C18:1 Oleic Acid, C18:2 Linoleic Acid, and C18:3 Alpha
Linolenic Acid
were analyzed after derivatization as the fatty acid methyl esters and
compared to standards. For
the derivatization, a sample (500 .1) was added to a 5-ml reaction tube
containing 2 ml boron
trifluoride solution (12% in methanol), 20 .1 dimethoxypropane and 100 11.1 of
a tridecanoic acid
internal standard solution (10 mg/ml). The reaction tube was vortexed and
incubated in a heating
block at 60 C for 30 minutes.
[00154] The reaction tube was removed from the heating block and allowed
to cool for 15
minutes. Then, 1 ml of distilled water was added to quench the reaction,
followed by 1 ml of
hexane. The reaction tube was vortexed for 60 seconds and the phases were
allowed to separate
for 3 minutes. The top (hydrophobic) phase was removed to a 1.5-ml tube
containing about 50
mg sodium sulfate (anhydrous). After vortexing for 60 seconds, the 1.5-ml tube
was centrifuged
and ¨500 .1 of the clarified, dried hydrophobic phase was transferred to a gas
chromatography
sample vial.
[00155] Samples were analyzed using an Agilent 7890A gas chromatograph
with Flame
ionization detector and Agilent Openlab CDS Chemstation software. GC Column:
Omegawax
100 (15m x 0.1 mm x 0.1 um) column. Results were converted to weight % by
internal standard
reference.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
33
[00156] FIGURE 3 depicts the distribution of FFA, MAG, DAG, and TAG in
Ensure and
a GBFS product of the present disclosure. Percent FFA, MAG, DAG and TAG in the
oils
determined by Thin Layer Chromatography.
Example 3: Another example of the process of making a product enriched in MAG
compared to
starting TAG-rich oil.
[00157] The following procedure is depicted by block flow diagram in
FIGURE 4.
[00158] Plant oil was added to citric acid and sodium hydroxide (caustic)
in DI water and
heated to 33 C +/- 2 C. A low vacuum was applied to de-gas to remove oxygen.
Lipase AY was
added. Hydrolysis of the mixture was performed for 14 ¨ 24 hours under a
Nitrogen blanket at a
temperature of 33 C +/- 2 C.
[00159] Lipase was inactivated at a temperature of 70 C +/- 2 C for 1
hour. The aqueous
phase with inactivated enzyme was drained. Glycerol was added. Then the
reaction was cooled
to 22 C +/- 2 C. Then Lipase G was added and water was evaporated under a
moderate
vacuum.
[00160] Re-esterification was carried out for 72 hours under high vacuum
(around 720
mmHg) at a temperature of 20 C +/- 2 C. Lipase G was inactivated at 70 C +/-
2 C for one
hour and salt was added to the reaction.
[00161] Enzyme inactivation and phase separation was carried out under a
Nitrogen
blanket for 1 hour at 70 C +/- 2 C. The aqueous phase with inactivated
enzyme, glycerol, and
salt was drained. The reaction was cooled to 60 C +/- 2 C. Antioxidant was
added. The final
product was stored at 4 C +/- 2 C under nitrogen.
Example 4: Ready-to-Drink formulation incorporating MAGs and hydrolyzed
protein
[00162] A product of the present disclosure was produced as a conventional
"milk shake"
formulation that includes a source of fats, proteins, carbohydrates, vitamins
and fiber in addition
to the traditional surfactants and stabilizing agents typically found in these
products. An
individual serving was 250 ml. The ingredients as would appear on the
ingredient label were as
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
34
follows: Water, Organic Agave Syrup, Hydrolyzed Pea Protein, Hydrolyzed Oil
Blend, Gum
Arabic, Sunflower Lecithin, Xanthan Gum, Oligosaccharides, Potassium Sorbate,
Sodium
Benzoate, Instant Coffee, Natural Organic Vanilla Flavor, Vitamin C, Vitamin E
Succinate,
Vitamin A PaImitate, Niacinamide, D-Calcium Pantothinate, Pyrodoxine HC1,
Thiamine HC1,
Riboflavin, Vitamin D3, Folic Acid, Cyanocobalamin, Vitamin K2.
[00163] Carbohydrates were supplied as simple sugars (glucose and
fructose) from fruit and
agave syrups.
[00164] Protein in the product was partially hydrolyzed pea protein (PURIS
Pea Protein
870H, World Food Processing LLC, Turtle Lake, WI 54889).
[00165] We also produced extensively hydrolyzed pea protein (EHP) with
peptides in a
size range more bio-available for transport across the intestinal wall. EHP
was produced by
further enzyme hydrolysis. For example, partially hydrolyzed pea protein (Puns
Pea Protein
870H described above) protein was dissolved in 100 mM Phosphate buffer to a
concentration of
25 mg/ml. Enzyme was added and the reaction incubated at 50 overnight. Three
different
commercial GRAS enzymes were evaluated: Alcalase, Thermoase and flavourzyme.
[00166] The average size and distribution of peptides in the protein
samples were evaluated
using size exclusion chromatography. Samples were dissolved in 100 mM
phosphate buffer, pH
6.8 to a concentration of 25 mg/ml and analyzed on a Shimadzu HPLC with UV
detector (214 nm)
using a Phenomenex Yarra 3 um SEC-2000 column (Eluted with 100 mM sodium
Phosphate
buffer (pH 6.8), Flow rate of 0.8 mL/min at room temperature). Samples were
compared to a
molecular weight standard (Phenomenex SEC standard part ALO-3042). Sizes were
estimated
using a calibration curve generated from known molecular weight standards. The
average size of
un-hydrolyzed pea protein was ¨200 amino acids. The average size of the Puns
Pea 870H partially
hydrolyzed material was 34 amino acids, with a substantial amount in the 2-40
amino acid range,
similar to other (whey-based) protein hydrolysate products.
[00167] FIGURE 5 depicts the distribution of amino acids and peptides in
ready-to-drink
nutritional drinks as reported in E. Phillips et at., 2005 Peptide-Based
Formulas: the
Nutraceuticals of Enteral Feeding? EPCN October:40-45 compared to the GBFS
extensively
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
hydrolyzed pea protein described above. FIGURE 5 depicts the percentage of
amino acids (Y-
Axis) of the size 1 amino acid, 2-4 amino acids, 9-10 amino acids, 10-40 amino
acids, or greater
than 40 amino acids in the products Peptamen, Perative, Crucial, Pivot, and
the GBFS of the
present disclosure (X-Axis). The average size of the GBFS EHP is 3-4 amino
acids, mostly 1 - 7
amino acids.
[00168] Fat is provided in the form of the re-structured Lipid MAG and
were produced
from a blend of olive oil (70%), sunflower oil (21%) and flax seed oil (9%) to
provide the energy
and other benefits of poly-unsaturated fatty acids (PUFA), omega-6 PUFA and
omega-3 PUFA.
The Omega-6/omega-3 ratio is ¨4/1.
[00169] Gum Arabic, sunflower lecithin, and xanthan gum are common GRAS
food
ingredients used to provides structure and stability for the drink.
Oligosaccharides provide non-
digestable fiber. Potassium Sorbate and Sodium Benzoate are common food
preservatives.
Instant coffee and Vanilla provide flavor.
[00170] A vitamin package including fat soluble and water-soluble vitamins
was included.
[00171] A prototype Product Nutritional label is shown in Table 3. In
Table 3, **
Percentage Daily Values (% DV) are based on a 2,000 calorie diet. t Daily
Value (DV) not
established.
Supplement Facts
Serving Size 250m1
Amount Per % DV **
Serving
Calories 379 kcal
Total Fat 19 g 24%
Cholesterol mg 0%
Total Carbohydrate 32 g 12%
Dietary Fiber 2 g 7%
Sugars 30
Protein 20 g 40%
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
36
Vitamin A 320 mcg 35%
Vitamin C 480 mg 35%
Vitamin D 7 mcg 35%
Vitamin E 5 mg 35%
Vitamin K 42 mcg 35%
Thiamine 400 mcg 35%
Riboflavin 450 mcg 35%
Niacin 6 mg 35%
Vitamin B6 600 mcg 35%
Folate 132 mcg 35%
Vitamin B12 1 mcg 35%
Biotin 10 mcg 35%
Pantothenic Acid 2 mg 35%
Calcium 480 mg 35%
Iron 6 mg 35%
Phosphorus 450 mg 35%
Chloride mg
Sodium 175 mg 7%
Potassium 1,600 mg 35%
[00172] Sensory Evaluation. FFAs produced from the vegetable oil blend
were found to
be unpalatable by a taste panel. Surprisingly, the MAG oil produced from this
blend was
palatable and similar in the taste and texture of the original triglyceride
oil. When the MAG oil
was formulated into the RTDS product described above, the flavor was
acceptable and
indistinguishable from similar commercial products with intact (un-digested)
lipids and proteins.
Example 5: Production of multiple batches of MO
[00173] The timing of the process described in Example 1 for manufacturing
EMO was
evaluated to generate oil that was greater than 70% MAG, greater than >85% MAG
+ FFA and
TAG content of 5% or less.
[00174] Table 4 depicts a scan of TLC plates illustrating the
concentrations of MAG and
FFA in the enzyme modified EMO produced from an olive oil/flax oil/sunflower
oil blend (ratio
of 7/2/1). In these experiments, step 2 was extended to 84 hours to establish
upper limits on
timing and temperature to avoid TAG formation.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
37
Experiment TAG DAG MAG FFA FFA + MAG
1 4 26 57 13 70
2 4 11 72 13 85
3 5 10 76 9 85
The reaction can be monitored in essentially real time with TLC analysis and
stopped at any point
during step 2 to yield to desired amounts of MAG, DAG, TAG and FFA. Reaction
time of 72
hours for step 2 was found to be a practical and productive stopping point and
illustrated in
Example 6.
Example 6: Evaluation of TAG-Free Enzyme Modified Almond Oil by NMR Analysis.
[00175] TAG-free EMO was produced using almond oil. Reaction conditions
were as
described in Example 1. Step 2 was 72 hours to minimize TAG production.
[00176] FIGURES 6A-6B depict the 13C-NMR analysis of the EMO produced from
almond oil. FIGURE 6A illustrates that 13C-NMR signal associated with
authentic TAG
(tristerin) and the characteristic peaks at 62.173 ppm and 68.921 ppm. FIGURE
6B illustrates
the 13C-NMR signal for the EMO. It shows that there is no discernable TAG
signals. Integration
of the actual signals indicates an acyl glycerol distribution between MAG:DAG
of 88%:12%.
Samples were analyzed on a JEOL model ECA600II NMR spectrometer operating at
600 MHz
proton and 150 MHz carbon. Samples were made up in 5 mm tubes and run locked
with CDC13
at ambient temperature.
Example 7: Clinical Testing of an MO-based Ready-To-Drink-Shake
[00177] A study was designed to show that patients with EPI can consume
food produced
with the TAG-free oils we have produced, without taking their enzyme
supplement medication,
and still absorb the lipids and produce TAGs in their serum.
[00178] The clinical study was a single center, randomized, double-blind,
cross-over trial
assessing an EMO-based ready-to-drink shake (RTDS) for blood lipid levels,
safety, tolerability
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
38
and palatability compared to a standard nutritional supplement used
concomitantly with
pancreatic enzyme replacement therapy capsules ("PERT").
[00179] Inclusion Criteria for the study included:
1. Provision of signed and dated informed consent form.
2. Stated willingness to comply with all study procedures and availability
for the
duration of the study.
3. Male or female, aged 12 years or older.
4. Diagnosis of CF
5. Currently receiving treatment with a commercially available pancreatic
enzyme
product for more than 3 months.
6. Clinically stable condition without evidence of acute respiratory
disease within 1
month of enrollment.
7. Stable body weight defined as no more than 5% decline within 3 months of
enrollment.
8. Females of child-bearing potential should agree to continue using a
medically
acceptable method of birth control throughout the study and for 30 days
immediately
after the last dose of study drug. Medically acceptable methods of birth
control include
bilateral tubal ligation or the use of either a contraceptive implant, a
contraceptive
injection (Depo-ProveraTm), an intrauterine device, or an oral contraceptive
taken within
the past 3 months where the subject agrees to continue using during the study
or to adopt
another birth control method, or a double-barrier method which consists of a
combination
of any two of the following: diaphragm, cervical cap, condom, or spermicide.
9. Ability to take oral medication and oral liquid nutritional supplements
and be
willing to adhere to the study interventions.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
39
10. Agreement to adhere to Lifestyle Considerations (see Section 5.3)
throughout the
duration of the study.
[00180] Exclusion Criteria for the study included:
1. Evidence of cardiovascular, respiratory (except underlying disease),
urogenital,
gastrointestinal/hepatic (except underlying disease), hematologic/immunologic,
head,
ears, eyes, nose, throat, dermatologic/connective tissue, musculoskeletal,
metabolic/nutritional (except underlying disease), endocrine (except
controlled diabetes
mellitus), neurologic/psychiatric, milk, nut or soy allergies, recent major
surgery, or other
relevant diseases as revealed by history, physical examination and/or
laboratory
assessments, which could limit participation in or completion of the study.
2. History of acute abdomen in the last year.
3. History of fibrosing colonopathy.
4. History of distal intestinal obstruction syndrome (DIOS) within 6 months
prior to
enrollment.
5. Solid organ transplant or surgery affecting the large bowel other than
appendectomy.
6. Small bowel surgery that significantly affected absorptive capacity
(e.g.
gastrectomy or pancreatectomy).
7. Intestinal inflammatory diseases including chronic diarrheal illness
unrelated to
pancreatic insufficiency.
8. Celiac disease or Crohn's disease.
9. Receiving enteral tube feeds for >50% of daily calorie intake.
10. Pregnancy or lactation.
11. Any type of malignancy involving the digestive tract in the last 5
years.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
12. Known allergy to pancreatin or inactive ingredients (excipients) of
pancreatin
capsules.
13. Suspected non-compliance or non-cooperation.
14. Intake of experimental drugs within 30 days prior to study start.
15. Mental disability or any other lack of fitness, in the Investigator's
opinion, to
preclude subject's participation in or ability to complete the study.
16. Diagnosis of human immunodeficiency virus in medical history.
17. Listed for lung transplantation or other solid organ transplant or
documented
forced expiratory volume (FEV) <25%.
18. Use of lipid lowering therapy including statins, fibrates, niacin, and
proprotein
convertase subtilisin kexin type 9 (PCSK9) inhibitors that cannot be held at
least 14 days
prior to Day 1 and through Day 15 of the study.Patients were male or female,
aged 12
years or older with a diagnosis of cystic fibrosis. Patients were also
currently receiving
treatment with a commercially available pancreatic enzyme product for more
than 3
months and had a clinically stable condition without evidence of acute
respiratory disease
within 1 month of enrollment.
[00181] Patients came to the clinic after an overnight fast and
standardized evening meal.
Those in Arm 1 (10 patients) were administered the RTDS along with a PERT
placebo and those
in Arm 2 (10 patients) were administered standard nutritional supplement with
PERT. Serial
blood samples over 6 hours (0, 1, 2, 3, 4, 5, 6 hours) were obtained from
patients in both study
arms, with no repeat serving of the RTDS or standard nutritional supplement.
Water could be
consumed during the study.
[00182] Patients returned to the clinic for Treatment 2 (cross-over
treatment) after
overnight fast and standardized evening meal. Patients in Arm 1 were
administered the standard
nutritional supplement with PERT, while patients in Arm 2 were administered
the RTDS along
with PERT placebo. Serial blood samples over 6 hours (0, 1, 2, 3, 4, 5, 6
hours) were obtained
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
41
from patients in both study arms, with no repeat serving of the RTDS or
standard nutritional
supplement.
[00183] The standard nutritional supplement contained: Water, Glucose
Syrup, Sugar,
Vegetable Oil (Canola, High Oleic Sunflower, Corn), Milk Protein Concentrate,
and less than
2% of Soy Protein Isolate, Calcium Caseinate, Sodium Caseinate, Gum Acacia,
Fructooligosaccharides, Inulin (from Chicory), Soy Lecithin, Salt, Natural and
Artificial Flavor,
Carrageenan Potassium Citrate, Calcium Phosphate, Magnesium Phosphate,
Magnesium
Chloride, Sodium Ascorbate, Choline Bitartrate, DL-Alpha Tocopheryl Acetate,
Ascorbic Acid,
Potassium Chloride, Ferrous Sulfate, Zinc Sulfate, Niacinamide, Calcium
Pantothenate,
Manganese Sulfate, Copper Sulfate, Pyridoxine Hydrochloride, Thiamine
Hydrochloride, Beta-
Carotene, Vitamin A Palmitate, Riboflavin, Folic Acid, Chromium Chloride,
Biotin, Potassium
Iodide, Phytonadione, Sodium Selenite, Sodium Molybdate, Vitamin D3, Vitamin
B12.
[00184] The RTDS contained: Water, Organic Date Syrup, Enzymatically
Modified
Almond Oil, Enzymatically Hydrolyzed Pea Protein, Soluble Corn Fiber, Cocoa
Powder, Natural
Flavors, Salt, Vitamin C, Vitamin E (dl-alpha-tocopheryl acetate),
Niacinamide, Pantothenic
acid (calcium-D-pantothenate), Vitamin B6 (pyridoxine hydrochloride), Vitamin
A (retinyl
palmitate), Vitamin B2 (riboflavin), Vitamin B1 (thiamine hydrochloride), L-
methylfolate,
Vitamin K 1, Vitamin D3 (cholecalciferol), Biotin, Vitamin B12
(cyanocobalamin).
[00185] The nutritional supplement was 5.9% fat, 5.9% protein and 19%
carbs by weight
while the RTDS was 7.4% fat, 4.7% protein and 9.8% carbs by weight.
[00186] The dosage of lipid was 0.5g/kg body weight. The lipid in the
standard nutritional
supplement was a blend of TAGs from canola, high oleic sunflower and corn oil.
In the
interventional drink the MAGs were produced from almond oil. The dosage of
lipid for each
patient was based upon well-established "Lipid Tolerance Tests", which are
similar in design and
scope to well-known glucose tolerance tests. The recommended dosage for these
tests is 0.5 -1.0
g/kg administered over 20 -30 minutes. Samples were taken at the start of the
test and hourly for
6 hours. Serum triglyceride levels were measured using standard laboratory
methods.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
42
[00187] The PERT dosage used in the study followed the manufacturer's
recommended
dosage guidelines of 2,500 iu of lipase activity per gram of fat ingested.
This translated to 3-4
capsules during the crossover stage of the study.
[00188] FIGURE 7 depicts the increase in serum triglycerides following
ingestion of the
EMO-based ready-to-drink shake in two patients. In FIGURE 7, the dashed lines
represent
patients that received 0.5 g per Kg of body weight of enzyme-modified almond
oil and the solid
lines represent patients that received 0.5 g per Kg of body weight of canola,
high oleic sunflower
and corn oil incorporated into the RTDS consumed over a half-hour period.
FIGURE 7 illustrates
that the enzyme-modified oil in the RTDS was absorbed by the patients and
converted into
serum triglycerides.
[00189] FIGURE 8 depicts the increase in serum triglycerides in another
patient following
ingestion of the test drinks. In FIGURE 8, the dashed line represents serum
triglycerides
following ingestion of MAG-based RTDS without PERT and the solid line
represents standard
of care RTDS with PERT. The patients received 0.5 g per Kg of body weight of
enzyme-
modified almond oil or 0.5 g per Kg of body weight of corn oil, incorporated
in the RTDS
consumed over a half-hour period.
[00190] In this patient, the absorption of lipid and conversion to serum
triglycerides was
significantly faster following ingestion of the MAG-based RTDS without PERT
serum than from
the TAG-based (standard of care) product with PERT. This suggests that the
patient was
suffering from cholestasis (disruption of bile flow from the liver) in
addition to EPI and not just
suffering from lack of sufficient enzymes, and thus could not properly
emulsify the canola, high
oleic sunflower and corn oil in the standard of care drink. Emulsification of
the oil into very
small droplets is required to produce the surface area that the pancreatic
lipase need hydrolyze
the oil into the MAGs and FFA that are transported into the enterocytes. Bile
acids from the liver
are required for this activity. The TAGs in the standard of care drink could
not be emulsified
into the micro-emulsions required for optimal lipase activity in the small
intestine, and thus
serum triglycerides did not increase as quickly as with the MAG formulation,
which does not
require lipase activity.
CA 03127093 2021-07-16
WO 2020/150661
PCT/US2020/014182
43
[00191] The actual dosage of lipids per patient was the same in all cases:
0.5 g/kg body
mass.
[00192] Sugar intake for a hypothetical 65 kg patient in the study is
shown in Table 5
below for the standard nutritional supplement and for the RTDS. Most of the
sugar and
carbohydrates in the control drink are dextrose or starch, and are readily
converted to glucose. In
the study drink, the volume of drink the hypothetical 65 kg patient would
consume is less (the
study drink has a higher concentration of lipids) and the carbohydrates are
supplied in lower
amounts and as a 50:50 mixture of fructose and glucose. Thus, the actual
glucose consumed is
less.
Sugar Intake
Drink mL patient will Grams of carbs Sugar consumed Glucose
drink in the
consumed
study
Control drink 551 105 94 94
Test drink 439 43 29 14
(RTDS)
[00193] FIGURE 9 depicts the increase in serum glucose averaged from 8
patients from
each of Arm 1 and Arm 2 in the study over the six hour sampling period.
Patients who
consumed the RTDS showed a pronounced decrease in maximum post-prandial serum
glucose
and remained below the standard nutritional supplement cohort. These results
demonstrate that
the EMO-based nutritional supplement, which is high in 1-monoacylglycerols
such as 1-oley1
monoglycerol, was able to positively impact glucose homeostasis compared to
the standard
nutritional supplement with PERT which would only be expected to yield a small
amount of 2-
monoacylglycerols such as 2-oley1 monoglycerol. Based on the amount of glucose
administered,
a higher serum glucose level would have been expected for the RTDS cohort and
the effect
shown is unexpected.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
44
[00194] FIGURE 10 depicts the serum triglyceride level averaged from 8
patients from
each of Arm 1 and Arm 2 in the study over the six hour sampling period. Both
the test and
control groups showed similar triglyceride levels which indicate that the
patients absorbed the
same amount of lipid from either the standard nutritional supplement with PERT
or the RTDS
without PERT.
Example 8: High Calorie, PERT-free Ready-to-Drink Shake
[00195] A high calorie RTDS can be prepared as follows: Add DI water (-60%
of final
volume) into main mixing vessel, and heat water to ¨60 C. Mix in Hydrolyzed
Protein, then
add Agave syrup. Use hand mixer to combine. In a separate container, combine
warm (60 C)
EMO and lecithin. When lecithin has dissolved, add EMO/Lecithin to aqueous
phase & mix.
Add additional water to achieve final weight (volume). Emulsify with shear
blender. To prepare
a very high calorie RTDS, add higher levels of the components.
[00196] Following production of the beverage base, various flavors,
maskers and blockers
can be added to produce unique products such as chocolate, vanilla,
strawberry, etc.
[00197] This method can also be used to produce high calorie products in
semi-solid formats
such as puddings, mousses, "popsicles" and ice cream-like products using the
beverage base recipe
and adding viscosity altering agents such as xanthan gum and gum acacia.
[00198] Primary ingredients and nutritional values are shown in Table 6
for a high calorie
(1.5 kcal/mL) RTDS.
Components with significant calories Wt % kcal/g kcal/325 kcal/mL
mL drink
EMO 7.5 9 219 0.7
Agave sugars 14 4 182 0.6
Hydrolyzed Protein 5 4 65 0.2
pea fat 0.7 9 21 0.1
Sunflower lecithin 0.5 9 15 0.0
Total 28 502 1.5
[00199] Primary ingredients and nutritional values are shown in Table 7
for a very high
calorie (2.5 kcal/mL) RDTS.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
Components with significant calories Wt % kcal/g kcal/325 kcal/mL
mL drink
EMO 12 9 351 1.1
Agave sugars 20 4 260 0.8
Hydrolyzed Protein 10 4 130 0.4
pea fat 1.4 9 42 0.1
Sunflower lecithin 0.5 9 15 0.0
Total 44 797 2.5
Example 9: High Calorie, PERT-free Bar
[00200] A high calorie bar can be prepared as follows: Add DI water (-60%
of final
volume) into main mixing vessel, and heat water to ¨60 C. Mix in Hydrolyzed
Protein, then
add Agave syrup. In a separate container, combine warm (60 C) EMO and
lecithin with hand
mixer. When lecithin has dissolved, add EMO/Lecithin to aqueous phase & mix.
Add additional
water to achieve final weight (volume).
[00201] Primary ingredients and nutritional values are shown in Table 8
for a high calorie,
PERT-free bar.
Components with significant calories Wt % in kcal/g kcal/60 g
product bar
EMO 9 9 81
Agave sugars 15 4 60
Hydrolyzed Protein 19 4 76
pea fat 2.7 9 24
Sunflower lecithin 0.5 9 15
Total 46 256
[00202] Following production of the bar base, various flavors, maskers and
blockers can
be added prior to baking to produce unique products such as chocolate, vanilla
etc.
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
46
Example 10: Manufacturing of a Ready-to-Drink-Shake
[00203] Add DI water into main mixing vessel, and heat water to ¨60 +/- 2
C. Once the
water has reached 60 C, slowly add sugar syrup (such as agave or date syrup)
with low
agitation. Mix into solution. Weigh out the individual dry mass materials
(Vitamin/Mineral mix
and Hydrolyzed Protein) and combine in a separate container. Mix dry
ingredients thoroughly.
Slowly add mixed dry ingredients directly into main mixing vessel with sweep
agitation at low
agitation. Weigh out EMO in separate container. Heat the EMO to 60+/- 2 C C.
Slowly add
Sunflower lecithin to warm EMO and mix with moderate agitation (as needed)
until the
sunflower lecithin is completely mixed into solution. Slowly add EMO
/sunflower lecithin
mixture to main mixing vessel. Add distilled water to increase volume to 95%
total fluid mass,
return temperature at 70+/- 2 C. Slowly add flavors and color to main mixing
vessel. Slowly
add stabilizer (such as acacia gum) to main mixing vessel. Mix solution for 20
minutes (to allow
the viscosity to increase). Maintain temperature of solution at 70+/- 2 C .
QS solution with
distilled water to final volume. Pass material through pressure drop
homogenizer(s) to produce
stable emulsions. Pasteurize or sterilize material. Cool material to room
temperature. Fill product
into packaging.
Example 11: Removal of MCPD from Almond Oil
[00204] Enzyme modified almond oil was prepared as described in Example 6.
[00205] Samples of the enzyme-modified almond oil were sent to a third
party lab
(Eurofins Eurofins Scientific Inc. Nutrition Analysis Center 2200 Rittenhouse
Street, Suite 150
Des Moines, IA 50321) for analysis using the standard method described below.
[00206] AOCS Official Method Cd 29b-13 (Revised 2017) - 2- and 3-MCPD
Fatty Acid
Esters and Glycidol Fatty Acid Esters in Edible Oils and Fats by Alkaline
Transesterification
and GC/MS. This method is used for the determination of fatty acid esters of 2-
chloropropane-
1,3-diol (2-MCPD), 3-chloropropane-1,2-diol (3-MCPD) and glycidol in edible
oils and fats. See
also AOCS Official Methods Cd 29a-13 or Cd 29c-13. Bound glycidol is the sum
of all glycidyl
derivatives that are cleaved by alkaline-catalyzed alcoholysis. The content of
bound glycidol is
reported in milligrams per kilogram (mg/kg). Bound 2-MCPD is the sum of all 2-
MCPD-
derivatives that are cleaved by alkaline-catalyzed alcoholysis. The content of
bound 2-MCPD is
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
47
reported in milligrams per kilogram (mg/kg). Bound 3-MCPD is the sum of all 3-
MCPD-
derivatives that are cleaved by alkaline-catalyzed alcoholysis. The content of
bound 3-MCPD is
reported in milligrams per kilogram (mg/kg). This method describes a procedure
for the parallel
determination of glycidol together with 2-MCPD and 3-MCPD present in bound or
free form in
oils and fats. The method is based on alkaline-catalyzed ester cleavage,
transformation of the
released glycidol into monobromopropanediol (MBPD) and derived free diols
(MCPD and
MBPD) with phenylboronic acid (PBA). Though free MCPD and glycidol are only
present in
fats and oils in low to negligible quantities, significant content would
increase proportionately
the determination of bound analytes. This method is applicable to solid and
liquid fats and oils.
[00207] Results. The total 2-MCPD (free and bound) and 3-MCPD (free and
bound) in
the starting oil was 0.65 mg/kg and 1.17 mg/kg, respectively. The enzyme
modified almond oil
(Example 6) had a total 2-MCPD (free and bound) and 3-MCPD (free and bound)
each of <0.10
mg/kg, which is below the limit of quantification of the assay to measure MCPD
(0.10 mg/kg).
Thus, the sums of the total detected MCPD in the starting almond oil and in
the enzyme modified
oil was 1.82 mg/kg and <0.10 mg/kg, respectively.
[00208] Therefore, the present invention is well adapted to attain the
ends and advantages
mentioned as well as those that are inherent therein. While numerous changes
may be made by
those skilled in the art, such changes are encompassed within the spirit of
this invention as
illustrated, in part, by the appended claims.
Example 12: Fatty Acid Profiles in Starting Oils and MAG Oils
[00209] MCT, canola and almond oils were processed according to the
manufacturing
method of the present disclosure. Fatty acid profiles for each were measured
in both the starting
oil and the MAG oil as shown in Table 9 below.
[00210] Fatty acids were analyzed after derivatization as the fatty acid
methyl esters and
compared to standards. For the derivatization, a sample (500 pi) was added to
a 5-ml reaction
tube containing 2 ml boron trifluoride solution (12% in methanol), 20 Ill
dimethoxypropane and
100 pi of a tridecanoic acid internal standard solution (10 mg/ml). The
reaction tube was
vortexed and incubated in a heating block at 60 C for 30 minutes. The reaction
tube was
removed from the heating block and allowed to cool for 15 minutes. Then, 1 ml
of distilled water
was added to quench the reaction, followed by 1 ml of hexane. The reaction
tube was vortexed
for 60 seconds and the phases were allowed to separate for 3 minutes. The top
(hydrophobic)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
48
phase was removed to a 1.5-ml tube containing about 50 mg sodium sulfate
(anhydrous). After
vortexing for 60 seconds, the 1.5-ml tube was centrifuged and -500 [t1 of the
clarified, dried
hydrophobic phase was transferred to a gas chromatography sample vial. Samples
were analyzed
using an Agilent 7890A gas chromatograph with Flame ionization detector and
Agilent Openlab
CDS Chemstation software. GC Column: Omegawax 100 (15m x 0.1 mm x 0.1 urn)
column.
Results were converted to weight % by internal standard reference.
MCT Canola Almond
Compound MCT Canola Almond
MAG MAG MAG
C6:0 (Caproic acid) 5.43% 0.06%
C8:0 (Caprylic
acid) 58.41% 57.42%
C10:0 (Capric acid) 40.96% 41.96%
C11:0 (Undecanoic
acid) 0.07% 0.07%
C12:0 (Lauric acid) 0.32% 0.32%
C14:0 (Myristic
acid) 0.03%
0.03% 0.05% 0.06% 0.06% 0.06%
C16:0 (Palmitic
acid) 4.06%
4.06% 6.61% 6.65%
C16:1 Total
(Palmitoleic acid +
isomers) 0.30%
0.30% 0.57% 0.57%
C17:0 (Margaric
acid) 0.04%
0.04% 0.06% 0.06%
C17:1
(Heptadecenoic
acid) 0.05%
0.06% 0.11% 0.11%
C18:0 (Stearic
acid) 1.78%
1.78% 1.56% 1.57%
C18:1 (Vaccenic
acid) 3.28%
3.27% 1.38% 1.39%
C18:1, Total (Oleic
Acid + isomers) 0.04%
0.06% 62.78% 62.66% 64.79% 64.94%
C18:2, Total
(Linoleic Acid +
isomers) 0.04%
0.04% 19.07% 19.07% 25.28% 25.14%
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661
PCT/US2020/014182
49
C18:3, Total
(Linolenic Acid +
isomers) 8.26% 8.22% 0.29% 0.28%
C18:4 Total
(Octadecatetraenoic
Acid) 0.04% 0.04% 0.09% 0.08%
C20:0 (Arachidic
Acid) 0.57% 0.58% 0.08% 0.07%
C20:1 Total
(Gondoic Acid +
isomers) 2.10% 2.08% 0.14% 0.14%
C20:2
(Eicosadienoic
Acid) 0.11% 0.18% 0.12% 0.14%
C22:0 (Behenic
Acid) 0.31% 0.31% 0.03% 0.04%
C22:1 Total
(Erucic Acid +
isomers) 0.02%
C24:0 (Lignoceric
Acid) 0.15% 0.16%
C24:1 Total
(Nervonic Acid +
isomers) 0.16% 0.16%
[00211] As shown, both the starting oil and MAG oil contained substantially
the same
fatty acid profiles.
[00212] A separate experiment was run using almond and canola oil to obtain
the data in
Table 10 which show the same result for oleic, linoleic and linolenic acid.
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
FATTY ACID Almond Canola Almond Canola
MAG MAG
C18:1cis (n-9) Oleic 63.2 59.33 63.31 59.33
C18:2cis (n-6) Linoleic 25.28 19.07 25.14 19.07
C18:3 (n-3) Linolenic 0.29 8.26 0.2 8.22
Oleic/Linoleic Ratio 2.5 3.1 2.5 3.1
Oleic/Linolenic Ratio 218 7 317 7
[00213] These results demonstrate that the manufacturing process of the
present disclosure
is able to preserve the fatty acid profile of the starting oil in the MAG oil.
This result is different
than for conventional "distilled" oils whose fatty acid profile differs
significantly from the
starting oil based on the functional use of the distilled oil.
[00214] These results also demonstrate that the processed oils of the
present disclosure can
be used to provide "complete nutrition" by providing linoleic acid and
linolenic acid. Similar
results would be expected for oils high EPA and DHA, such as fish oils and
algal oils to provide
complete nutritional formulations.
Example 13: The EMO Process Conserves Essential Fatty Acids Found in Original
Oil
[00215] Canola EMO was produced as described in Example 1. Fatty acids were
measured as described in Example 2. Results of the fatty acid analysis are
shown in FIGURE
11. Canola oil and the EMO produced from the canola oil mtaining the integrity
of the fatty acid
profile and include the essential fatty acids linoleic and linolenic acids.
Similar results were
obtained with EMO from olive oil, sunflower oil and almond oil. Commercial
"Monostearin"
(Profood Products, Naperville, IL) contains little or no unsaturated fatty
acids, while "Capmul
GMO 50" (Abitec Corporation, Janesville, WI) contains predominantly the mono-
saturated fatty
acid oleic acid, little linoleic and virtually no linolenic or Gondoic (C20:1)
fatty acid.
Example 14: Beneficial Compounds are Preserved
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
51
[00216] Vegetable oils contain trace quantities of many lipids, steroids,
esters and
phenolic compounds that contribute to the taste and health benefits of the
oils. Many of these
compounds are preserved or enriched in the EMO process. This is illustrated in
FIGURES 12A-
12B, Canola EMO was prepared as described in Example 1. Lipid samples were
derivatized
with 3-picolylamide for LC/MS/MS analysis. In brief, 150 pL of standard (0.1-
100 pg/mL) and
20 ,uL of internal standard mixture were mixed and dried under nitrogen. To
the dried residue
was added 200 pL of oxalyl chloride (2 M in dichloromethane), and the mixture
was incubated at
65 C on a heating block for 5 min and then dried under nitrogen. To the
residue was added
150 duL of 3-picolylamine (1% in acetonitrile, v/v) to form the 3-picolylamide
(FA-PA). The
mixture was incubated at room temperature for 5 min, followed by drying under
nitrogen to give
the derivatized FAs. The dried FAs derivatives were dissolved in 1000 pL of
ethanol and further
diluted up to 10-fold with ethanol prior to LC¨MS analysis. LC-MS analysis was
in Ionization
mode: positive (total fatty acid & lipidomics) & negative (lipidomics)
ionization. Software used
for analysis: Thermo Scientific Freestyle & LipidSearch. Orbitrap fusion
method: data
dependent acquisition MS2 (total fatty acid) & MS.
[00217] Compounds that were preserved or enhanced include:
Glycosphingolipids (e.g.
Ceramide Phosphate), Glycoglycerolipids (e.g.
Monogalactodiacylglycerol),
Phosphatidylmethanol, Steroids (e.g. Sitosteryl ester), Neutral lipids (e.g.
Campesterol ester),
Sphingolipids, phosphatidyl glycerol ,wax esters and sphingomyelin. Coenzyme
was also found
to be enhanced.
[00218] These compounds are not retained in commercial (distilled)
monoglyceride
products.
[00219] In a first embodiment, a food product comprises an oil and has a
total caloric
content of from about 25 kcal to about 1,000 kcal, wherein from about 5% to
about 75% of the
total caloric content is derived from said oil, and wherein the oil comprises
less than 10% by
weight triacylglycerides (TAGs) based on the total weight of the oil, wherein
the oil is
substantially free of monochloropropandiol (MCPD).
[00220] In an aspect of the first embodiment, the oil comprises greater
than 50% by
weight monoacylglyceride (MAGs) based on the total weight of the oil.
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
52
[00221] In another aspect of the first embodiment, the oil comprises
greater than 60% by
weight MAGs based on the total weight of the oil.
[00222] In another aspect of the first embodiment, the oil comprises
greater than 70% by
weight MAGs based on the total weight of the oil.
[00223] In another aspect of the first embodiment, the oil comprises
greater than 80% by
weight MAGs based on the total weight of the oil.
[00224] In another aspect of the first embodiment, the oil comprises
greater than 90% by
weight MAGs based on the total weight of the oil.
[00225] In another aspect of the first embodiment or any of the foregoing
aspects of the
first embodiment, from about 10% to about 60% of the total caloric content is
derived from said
oil.
[00226] In another aspect of the first embodiment or any of the foregoing
aspects of the
first embodiment, from about 20% to about 50% of the total caloric content is
derived from said
oil.
[00227] In another aspect of the first embodiment or any of the foregoing
aspects of the
first embodiment, from about 25% to about 45% of the total caloric content is
derived from said
oil.
[00228] In another aspect of the first embodiment or any of the foregoing
aspects of the
first embodiment, from about 30% to about 40% of the total caloric content is
derived from said
oil.
[00229] In another aspect of the first embodiment or any of the foregoing
aspects of the
first embodiment, the food product further comprises a carbohydrate source
[00230] In another aspect of the first embodiment or any of the foregoing
aspects of the
first embodiment, the food product further comprises a carbohydrate source and
the carbohydrate
source comprises a fruit or agave syrup
[00231] In another aspect of the first embodiment or any of the foregoing
aspects of the
first embodiment, the food product further comprises a carbohydrate source and
the carbohydrate
source comprises simple sugars.
[00232] In another aspect of the first embodiment or any of the foregoing
aspects of the
first embodiment, the food product further comprises a carbohydrate source and
from about 20%
to about 50% of calories are derived from the carbohydrate source.
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661
PCT/US2020/014182
53
[00233] In
another aspect of the first embodiment or any of the foregoing aspects of the
first embodiment, the food product comprises a carbohydrate source and further
comprises a
protein source.
[00234] In
another aspect of the first embodiment or any of the foregoing aspects of the
first embodiment, the food product comprises a carbohydrate source and further
comprises a
protein source, and from about 10% to about 50% of calories are derived from
the protein source.
[00235] In
another aspect of the first embodiment or any of the foregoing aspects of the
first embodiment, the food product comprises a carbohydrate source and further
comprises a
protein source, and the protein source comprises an hydrolyzed or partially
hydrolyzed protein.
[00236] In
another aspect of the first embodiment or any of the foregoing aspects of the
first embodiment, the food product comprises a carbohydrate source and further
comprises a
protein source, and the protein source comprises an hydrolyzed or partially
hydrolyzed protein,
wherein the hydrolyzed protein is selected from hydrolyzed pea protein and a
whey-based
hydrolysate product.
[00237] In
another aspect of the first embodiment or any of the foregoing aspects of the
first embodiment, the food product further comprises a protein source.
[00238] In
another aspect of the first embodiment or any of the foregoing aspects of the
first embodiment, the food product further comprises a protein source, wherein
from about 10%
to about 50% of calories are derived from the protein source.
[00239] In
another aspect of the first embodiment or any of the foregoing aspects of the
first embodiment, the food product further comprises a protein source, wherein
the protein
source comprises an hydrolyzed or partially hydrolyzed protein.
[00240] In
another aspect of the first embodiment or any of the foregoing aspects of the
first embodiment, the food product further comprises a protein source, wherein
the protein
source comprises an hydrolyzed or partially hydrolyzed protein, and wherein
the hydrolyzed
protein is selected from hydrolyzed pea protein and a whey-based hydrolysate
product.
[00241] In
another aspect of the first embodiment or any of the foregoing aspects of the
first embodiment, the oil is a processed oil derived from an oil source.
[00242] In
another aspect of the first embodiment or any of the foregoing aspects of the
first embodiment, the oil is a processed oil derived from an oil source,
wherein the processed oil
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
54
comprises non-oil ingredients derived from and naturally present in the oil
source such that the
non-oil ingredients are not added to the processed oil.
[00243] In another aspect of the first embodiment or any of the foregoing
aspects of the
first embodiment, the oil is a processed oil derived from an oil source,
wherein the processed oil
comprises non-oil ingredients derived from and naturally present in the oil
source such that the
non-oil ingredients are not added to the processed oil, and wherein the non-
oil ingredients are
selected from antioxidants, vitamins, and mixtures thereof.
[00244] In another aspect of the first embodiment or any of the foregoing
aspects of the
first embodiment, the oil is a processed oil derived from an oil source,
wherein the processed oil
comprises non-oil ingredients derived from and naturally present in the oil
source such that the
non-oil ingredients are not added to the processed oil, wherein the non-oil
ingredients are
selected from antioxidants, vitamins, and mixtures thereof, and wherein the
antioxidant is
tocopherol.
[00245] In another aspect of the first embodiment or any of the foregoing
aspects of the
first embodiment, the oil is a processed oil derived from an oil source,
wherein the processed oil
comprises non-oil ingredients derived from and naturally present in the oil
source such that the
non-oil ingredients are not added to the processed oil, wherein the non-oil
ingredients are
selected from antioxidants, vitamins, and mixtures thereof, wherein the
antioxidant is tocopherol,
and wherein said tocopherol is selected from a-tocopherol, 3-tocopherol, 6-
tocopherol, y-
tocopherol, a-tocotrienol, P-tocotrienol, 6-tocotrienol, and y-tocotrienol.
[00246] In a second embodiment, a food product comprises a processed oil, a
carbohydrate source, and a protein source, and having a total weight from
about 25 grams to
about 500 grams with a caloric density of from about 1 kcal per gram to about
5 kcal per gram,
wherein the processed oil comprises from about 10% to about 50% of the total
caloric content,
and wherein the processed oil has a MAG content of equal to or greater than
40% by weight
based on the total weight of the processed oil and a TAG content of equal to
or less than 10% by
weight based on the total weight of the processed oil, and wherein the
processed oil has less than
0.10 mg/kg MCPD.
[00247] In a third embodiment, a product comprises a processed oil derived
from an oil
source, wherein the processed oil comprises a MAG content equal to or greater
than 40% by
weight of the total weight of the processed oil, wherein the processed oil is
either free of TAGs
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
or comprises a TAG content that is equal to or less than 10% by weight of the
total weight of the
processed oil, and wherein the processed oil comprises non-oil ingredients
derived from and
naturally present in the oil source such that the non-oil ingredients are not
added to the processed
oil, wherein the oil source comprise from about 1.00 mg/kg to about 12.00
mg/kg MCPD, and
wherein the processed oil comprises less than 0.100 mg/kg MCPD.
[00248] In an aspect of the third embodiment, said oil source is from an
origin selected
from a plant an animal, algae or fish.
[00249] In another aspect of the third embodiment, said oil source is of
plant origin.
[00250] In another aspect of the third embodiment said oil source is
selected from the
group consisting of olive oil, sunflower oil, corn oil, almond oil, rapeseed
oil, palm oil, soybean
oil, flaxseed oil, and mixtures thereof.
[00251] In another aspect of the third embodiment or any of the foregoing
aspects of the
third embodiment, the non-oil ingredients are selected from the group
consisting of antioxidants,
vitamins, and mixtures thereof.
[00252] In another aspect of the third embodiment or any of the foregoing
aspects of the
third embodiment, the non-oil ingredients are selected from the group
consisting of antioxidants,
vitamins, and mixtures thereof and the antioxidant is tocopherol.
[00253] In another aspect of the third embodiment or any of the foregoing
aspects of the
third embodiment, the non-oil ingredients are selected from the group
consisting of antioxidants,
vitamins, and mixtures thereof, the antioxidant is tocopherol, and the
tocopherol is selected from
the group consisting of a-tocopherol,13-tocopherol, 6-tocopherol, y-
tocopherol, a-tocotrienol, 13-
tocotrienol, 6-tocotrienol, and y-tocotrienol.
[00254] In another aspect of the third embodiment or any of the foregoing
aspects of the
third embodiment, the processed oil comprises a MAG content of from about 50%
to about 95%
by weight based on the total weight of the processed oil
[00255] In another aspect of the third embodiment or any of the foregoing
aspects of the
third embodiment, the processed oil comprises a MAG content of from about 50%
to about 95%
by weight based on the total weight of the processed oil, and the processed
oil comprises a TAG
content from about 50/0 to about 0.5% by weight based on the total weight of
the processed oil.
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
56
[00256] In another aspect of the third embodiment or any of the foregoing
aspects of the
third embodiment, the processed oil comprises a TAG content from about 5% to
about 0.5% by
weight based on the total weight of the processed oil.
[00257] In a fourth embodiment, a method for making a monoacylglycerol-
enriched oil,
comprises mixing a starting oil comprising triacylglycerols (TAGs) and from
about 1.00 mg/kg
to about 12.00 mg/kg of MCPD, wherein the TAGs are in an amount greater than
50% by weight
based on the total weight of the starting oil, a buffer solution and a first
enzyme capable of
hydrolyzing said TAGs to free fatty acids (FFAs) to yield a first reaction
mixture; allowing said
first reaction mixture to react under conditions sufficient for said first
enzyme to hydrolyze said
TAGs for a first period of time to yield an aqueous phase and a first lipid
reaction product
comprising FFAs; inactivating said first enzyme in said first lipid reaction
product; collecting
said first lipid reaction product by removing it from the aqueous phase;
mixing said first lipid
reaction product with a food-grade glycerol and a second enzyme capable of
esterifying FFAs to
form a second reaction mixture; allowing said second reaction mixture to react
for a second
period of time to yield a second lipid reaction product comprising a lipid oil
phase and a glycerol
phase; inactivating said second enzyme in said second lipid reaction product;
adding salt to the
reaction product and separating the lipid oil phase from said glycerol phase;
and collecting said
lipid oil phase, wherein the lipid oil phase is substantially free of MCPD.
[00258] In an aspect of the fourth embodiment, the starting oil is an oil
derived from plant,
animal or fish origin.
[00259] In another aspect of the fourth embodiment, the starting oil is a
plant oil or a
mixture of plant oils.
[00260] In another aspect of the fourth embodiment, the starting oil is a
plant oil selected
from the group consisting of olive oil, sunflower oil, corn oil, almond oil,
rapeseed oil, palm oil,
soybean oil, flaxseed oil, and mixtures thereof.
[00261] In another aspect of the fourth embodiment, said first enzyme is a
lipase.
[00262] In another aspect of the fourth embodiment, said first enzyme is
lipase AY.
[00263] In another aspect of the fourth embodiment, said buffer solution is
a sodium
citrate solution.
[00264] In another aspect of the fourth embodiment, said first period of
time is a period of
time sufficient to hydrolyze at least 94% of the TAGs in said starting oil.
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
57
[00265] In another aspect of the fourth embodiment, said first period of
time is between
about 14 and about 24 hours
[00266] In another aspect of the fourth embodiment, said step of allowing
said reaction
mixture to react under conditions sufficient for said first enzyme to
hydrolyze said TAGs is
performed at a temperature between about 30 C and about 35 C.
[00267] In another aspect of the fourth embodiment, said steps of mixing a
starting oil
comprising triacylglycerols (TAGs), a buffer solution and a first enzyme
capable of hydrolyzing
said TAGs to free fatty acids (FFAs) and allowing said reaction mixture to
react under conditions
sufficient for said first enzyme to hydrolyze said TAGs to FFA are performed
under a nitrogen
atmosphere.
[00268] In another aspect of the fourth embodiment, said second enzyme is a
lipase.
[00269] In another aspect of the fourth embodiment, said second enzyme is
lipase G.
[00270] In another aspect of the fourth embodiment, said second period of
time is a period
of time sufficient to result in enrichment of MAGs in the lipid oil phase of
about 60% to 95%.
[00271] In another aspect of the fourth embodiment, said second period of
time is between
about 24 hours and about 72 hours.
[00272] In another aspect of the fourth embodiment, said step of allowing
said second
reaction mixture to react for a second period of time to yield a lipid oil
phase and a glycerol
phase is performed at a temperature between about 17 C and 23 C.
[00273] In another aspect of the fourth embodiment or any of the foregoing
aspects of the
fourth embodiment, the method further comprises drying said second lipid
reaction product by
applying a vacuum for a third period of time sufficient to remove at least a
portion of water from
the second lipid reaction product.
[00274] In another aspect of the fourth embodiment or any of the foregoing
aspects of the
fourth embodiment, the method further comprises drying said second lipid
reaction product by
applying a vacuum for a third period of time sufficient to remove at least a
portion of water from
the second lipid reaction product, wherein said step of drying said second
lipid reaction product
is performed at a temperature between 20 C-30 C.
[00275] In another aspect of the fourth embodiment or any of the foregoing
aspects of the
fourth embodiment, the method further comprises drying said second lipid
reaction product by
applying a vacuum for a third period of time sufficient to remove at least a
portion of water from
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
58
the second lipid reaction product, wherein said drying step is applied
throughout the second
period of time.
[00276] In another aspect of the fourth embodiment or any of the foregoing
aspects of the
fourth embodiment, said step of inactivating said second enzyme is performed
by heating said
second lipid reaction product.
[00277] In another aspect of the fourth embodiment or any of the foregoing
aspects of the
fourth embodiment, said step of inactivating said second enzyme is performed
by heating said
second lipid reaction product, wherein said heating is performed at a
temperature of at least 70 C
for at least 1 hour.
[00278] In another aspect of the fourth embodiment or any of the foregoing
aspects of the
fourth embodiment, said step of separating said lipid oil phase from said
glycerol phase comprise
adding sodium chloride to said second lipid reaction product.
[00279] In another aspect of the fourth embodiment or any of the foregoing
aspects of the
fourth embodiment, said step of separating said lipid oil phase from said
glycerol phase comprise
adding sodium chloride to said second lipid reaction product, wherein the
final concentration of
sodium chloride comprises up to 0.3 weight percent sodium chloride.
[00280] In another aspect of the fourth embodiment or any of the foregoing
aspects of the
fourth embodiment, the method further comprises before mixing said first lipid
reaction product
and food-grade glycerol and a second enzyme capable of esterifying FFAs and
glycerol, re-
establishing a nitrogen atmosphere over said first lipid reaction product.
[00281] In another aspect of the fourth embodiment or any of the foregoing
aspects of the
fourth embodiment, the method further comprises adding tocopherol to said
lipid oil phase after
collecting said lipid oil phase.
[00282] In another aspect of the fourth embodiment or any of the foregoing
aspects of the
fourth embodiment, the lipid oil phase comprises MAGs in an amount from about
40% to about
99% by weight based on the total weight of the lipid oil phase and wherein the
lipid oil phase
either is free of TAGs or comprises TAGs in an about from about 0.1% to about
10% by weight
based on the total weight of the lipid oil phase.
[00283] In a fifth embodiment, a processed oil comprises oleic acid
monolgyceride
(MOG) and having a total fatty acid content, wherein said MOG contributes
between about 5%
and about 75% by weight of the total fatty acid content of the processed oil.
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661
PCT/US2020/014182
59
[00284] In an aspect of the fifth embodiment, the processed oil comprises
less than 10%
by weight triacylglycerides (TAGs) based on the total weight of the processed
oil.
[00285] In another aspect of the fifth embodiment, the processed oil
comprises greater
than 50% by weight monoacylglyceride (MAGs) based on the total weight of the
processed oil.
[00286] In another aspect of the fifth embodiment, the processed oil
comprises greater
than 60% by weight monoacylglyceride (MAGs) based on the total weight of the
processed oil.
[00287] In another aspect of the fifth embodiment, the processed oil
comprises greater
than 70% by weight monoacylglyceride (MAGs) based on the total weight of the
processed oil.
[00288] In another aspect of the fifth embodiment, the processed oil
comprises greater
than 80% by weight monoacylglyceride (MAGs) based on the total weight of the
processed oil.
[00289] In another aspect of the fifth embodiment, the processed oil
comprises greater
than 90% by weight monoacylglyceride (MAGs) based on the total weight of the
processed oil.
[00290] In another aspect of the fifth embodiment or any of the foregoing
aspects of the
fifth embodiment, the processed oil is processed oil derived from an oil
source.
[00291] In another aspect of the fifth embodiment or any of the foregoing
aspects of the
fifth embodiment, the processed oil is processed oil derived from an oil
source, and the
processed oil comprises non-oil ingredients derived from and naturally present
in the oil source
such that the non-oil ingredients are not added to the processed oil.
[00292] In another aspect of the fifth embodiment or any of the foregoing
aspects of the
fifth embodiment, the processed oil is processed oil derived from an oil
source, and the
processed oil comprises non-oil ingredients derived from and naturally present
in the oil source
such that the non-oil ingredients are not added to the processed oil, wherein
the non-oil
ingredients are selected from antioxidants, vitamins, and mixtures thereof.
[00293] In another aspect of the fifth embodiment or any of the foregoing
aspects of the
fifth embodiment, the processed oil is processed oil derived from an oil
source, and the
processed oil comprises non-oil ingredients derived from and naturally present
in the oil source
such that the non-oil ingredients are not added to the processed oil, wherein
the non-oil
ingredients are selected from antioxidants, vitamins, and mixtures thereof,
and wherein the
antioxidant is tocopherol.
[00294] In another aspect of the fifth embodiment or any of the foregoing
aspects of the
fifth embodiment, the processed oil is processed oil derived from an oil
source, and the
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661
PCT/US2020/014182
processed oil comprises non-oil ingredients derived from and naturally present
in the oil source
such that the non-oil ingredients are not added to the processed oil, wherein
the non-oil
ingredients are selected from antioxidants, vitamins, and mixtures thereof,
wherein the
antioxidant is tocopherol, and wherein said tocopherol is selected from a-
tocopherol, 13-
tocopherol, 6-tocopherol, y-tocopherol, a-tocotrienol, 13-tocotrienol, S-
tocotrienol, and y-
tocotrienol.
[00295] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, the processed oil is substantially free of
monochloropropandiol (MCPD).
[00296] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, the processed oil has less than 0.10 mg/kg
monochloropropandiol (MCPD).
[00297] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, said oil source is from an origin selected from a plant, an
animal, algae or fish.
[00298] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, said oil source is of plant origin.
[00299] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, said oil source is selected from the group consisting of
safflower oil, grape oil,
Silybum marianum oil, hemp oil, sunflower oil, wheat germ oil, pumpkin seed
oil, sesame oil,
rice bran oil, almond oil, rapeseed oil, peanut oil, olive oil, and coconut
oil.
[00300] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, said processed oil comprises a MAG content of from about 50%
to about 95%
by weight based on the total weight of the processed oil.
[00301] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, said processed oil comprises a TAG content from about 5% to
about 0.5% by
weight based on the total weight of the processed oil.
[00302] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, the MOG in said processed oil comprises at least 50% by
weight 1-oley1
monoglyceride out of the total amount of MOG.
[00303] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, the MOG in said processed oil comprises at least 60% by
weight 1-oley1
monoglyceride out of the total amount of MOG.
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661
PCT/US2020/014182
61
[00304] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, the MOG in said processed oil comprises at least 70% by
weight 1-oley1
monoglyceride out of the total amount of MOG.
[00305] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, the MOG in said processed oil comprises at least 80% by
weight 1-oley1
monoglyceride out of the total amount of MOG.
[00306] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, the processed oil further comprises linoleic acid, wherein
said linoleic acid is
present in said processed oil in an amount of about 1.5% to about 90% by
weight out of the total
fatty acid content of the processed oil
[00307] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, the processed oil further comprises linoleic acid, wherein
said linoleic acid is
present in said processed oil in an amount of about 10% to about 90% by weight
out of the total
fatty acid content of the processed oil
[00308] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, the processed oil further comprises linoleic acid, wherein
said linoleic acid is
present in said processed oil in an amount of about 20% to about 90% by weight
out of the total
fatty acid content of the processed oil,
[00309] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, the processed oil further comprises linoleic acid, wherein
said linoleic acid is
present in said processed oil in an amount of about 10% to about 25% by weight
out of the total
fatty acid content of the processed oil
[00310] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, the processed oil further comprises linolenic acid, wherein
said linolenic acid
is present in said processed oil in amount of about 0.01% to about 2% by
weight out of the total
fatty acid content of the processed oil
[00311] In
another aspect of the fifth embodiment or any of the foregoing aspects of the
fifth embodiment, wherein a ratio between the amount of oleic acid and
linoleic acid in said
processed oil is between about 0.01 and about 5.
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
62
[00312] In another aspect of the fifth embodiment or any of the foregoing
aspects of the
fifth embodiment, wherein a ratio between the amount of oleic acid and
linoleic acid in said
processed oil is between about 1 and about 4.
[00313] In another aspect of the fifth embodiment or any of the foregoing
aspects of the
fifth embodiment, wherein a ratio between the amount of oleic acid and
linoleic acid in said
processed oil is between about 3 and about 4.
[00314] In another aspect of the fifth embodiment or any of the foregoing
aspects of the
fifth embodiment, wherein a ratio between the amount of oleic acid and
linolenic acid in said
processed oil is between about 1 and about 100.
[00315] In another aspect of the fifth embodiment or any of the foregoing
aspects of the
fifth embodiment, wherein a ratio between the amount of oleic acid and
linolenic acid in said
processed oil is between about 10 and 100.
[00316] In another aspect of the fifth embodiment or any of the foregoing
aspects of the
fifth embodiment, wherein a ratio between the amount of oleic acid and
linolenic acid in said
processed oil is between about 10 and 30.
[00317] In another aspect of the fifth embodiment or any of the foregoing
aspects of the
fifth embodiment, wherein said processed oil has a fatty acid profile that is
substantially the same
as a fatty acid profile of the pre-processed oil from which the processed oil
was produced.
[00318] In an aspect of the preceding aspect, wherein said fatty acid
profile comprises
oleic acid, linoleic acid and linolenic acid.
[00319] In another aspect of the fifth embodiment or any of the foregoing
aspects of the
fifth embodiment, wherein the amount of oleic acid, linoleic acid and
linolenic acid in the
processed oil is within about 10% of the amount of oleic acid, linoleic acid
and linolenic acid,
respectively, in the pre-processed oil from which the processed oil was
produced.
[00320] In another aspect of the fifth embodiment or any of the foregoing
aspects of the
fifth embodiment, wherein the amount of oleic acid, linoleic acid and
linolenic acid in the
processed oil is within about 1% of the amount of oleic acid, linoleic acid
and linolenic acid,
respectively, in the pre-processed oil from which the processed oil was
produced.
[00321] In another aspect of the fifth embodiment or any of the foregoing
aspects of the
fifth embodiment, wherein the processed oil comprises three times the amount
of MAGs relative
to the amount of TAGs in the pre-processed oil from which the processed oil
was produced.
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
63
[00322] In another aspect of the fifth embodiment or any of the foregoing
aspects of the
fifth embodiment, wherein the processed oil comprises oleic acid in the form
of oleic acid
monoglyceride in an amount that is about the same as the amount of oleic acid
in the pre-
processed oil from which the processed oil was produced.
[00323] In a sixth embodiment, a processed oil has a fatty acid profile
comprising oleic
acid, linoleic acid and linolenic acid, wherein the amount of oleic acid,
linoleic acid and linolenic
acid in the processed oil is within about 10% of the amount of oleic acid,
linoleic acid and
linolenic acid, respectively, in the pre-processed oil from which the
processed oil was produced,
and wherein the processed oil comprises greater than 50% by weight
monoacylglyceride
(MAGs) based on the total weight of the processed oil.
[00324] In a seventh embodiment, a processed oil has a fatty acid profile
comprising oleic
acid, linoleic acid and linolenic acid, wherein the amount of oleic acid,
linoleic acid and linoleic
acid in the processed oil is within about 1% of the amount of oleic acid,
linoleic acid and
linolenic acid, respectively, in the pre-processed oil from which the
processed oil was produced,
and wherein the processed oil comprises greater than 50% by weight
monoacylglyceride
(MAGs) based on the total weight of the processed oil.
[00325] In an eighth embodiment, a processed oil comprises oleic acid and
linoleic acid,
wherein a ratio of oleic acid to linoleic acid in the processed oil is between
about 0.01 and about
5, and wherein the processed oil comprises greater than 50% by weight
monoacylglyceride
(MAGs) based on the total weight of the processed oil.
[00326] In an aspect of the eighth embodiment, the ratio of oleic acid to
linoleic acid in
the processed oil is between about 1 and about 4
[00327] In another aspect of the eighth embodiment, the ratio of oleic acid
to linoleic acid
in the processed oil is between about 3 and about 4.
[00328] In another aspect of the eighth embodiment or any of the foregoing
aspects of the
eighth embodiment, the processed oil further comprises linoleic acid, wherein
a ratio of oleic
acid to linolenic acid in the processed oil is between about 1 and about 100.
[00329] In another aspect of the eighth embodiment or any of the foregoing
aspects of the
eighth embodiment, the processed oil further comprises linoleic acid, wherein
a ratio of oleic
acid to linolenic acid in the processed oil is between about 10 and about 100.
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
64
[00330] In another aspect of the eighth embodiment or any of the foregoing
aspects of the
eighth embodiment, the processed oil further comprises linoleic acid, wherein
a ratio of oleic
acid to linolenic acid in the processed oil is between about 10 and about 30.
[00331] In a ninth embodiment, a processed oil comprises oleic acid and
linolenic acid,
wherein a ratio of oleic acid to linolenic acid in the processed oil is
between about 1 and about
100, and wherein the processed oil comprises greater than 50% by weight
monoacylglyceride
(MAGs) based on the total weight of the processed oil.
[00332] In a tenth embodiment, a processed oil comprises oleic acid and
linolenic acid,
wherein a ratio of oleic acid to linolenic acid in the processed oil is
between about 10 and about
100, and wherein the processed oil comprises greater than 50% by weight
monoacylglyceride
(MAGs) based on the total weight of the processed oil.
[00333] In an eleventh embodiment, a processed oil comprises oleic acid and
linolenic
acid, wherein a ratio of oleic acid to linolenic acid in the processed oil is
between about 10 and
about 30, and wherein the processed oil comprises greater than 50% by weight
monoacylglyceride (MAGs) based on the total weight of the processed oil.
[00334] In an aspect of the ninth, tenth or eleventh embodiments, the
processed oil further
comprises linoleic acid, wherein a ratio of oleic acid to linoleic acid in the
processed oil is
between about 0.01 and about 5.
[00335] In an aspect of the ninth, tenth or eleventh embodiments, the
processed oil further
comprises linoleic acid, wherein a ratio of oleic acid to linoleic acid in the
processed oil is
between about 1 and about 4.
[00336] In an aspect of the ninth, tenth or eleventh embodiments, the
processed oil further
comprises linoleic acid, wherein a ratio of oleic acid to linoleic acid in the
processed oil is
between about 3 and about 4.
[00337] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, at
least a portion of said
oleic acid is present in the form of oleic acid monoglyceride (MOG).
[00338] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, at
least a portion of said
oleic acid is present in the form of oleic acid monoglyceride (MOG), and at
least 50% by weight
1-oley1 monoglyceride out of the total amount of MOG.
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661
PCT/US2020/014182
[00339] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, at
least a portion of said
oleic acid is present in the form of oleic acid monoglyceride (MOG), and at
least 60% by weight
1-oley1 monoglyceride out of the total amount of MOG.
[00340] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, at
least a portion of said
oleic acid is present in the form of oleic acid monoglyceride (MOG), and at
least 70% by weight
1-oley1 monoglyceride out of the total amount of MOG.
[00341] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, at
least a portion of said
oleic acid is present in the form of oleic acid monoglyceride (MOG), and at
least 80% by weight
1-oley1 monoglyceride out of the total amount of MOG.
[00342] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, the
processed oil
comprises less than 10% by weight triacylglycerides (TAGs) based on the total
weight of the
processed oil.
[00343] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, the
processed oil
comprises greater than 50% by weight monoacylglyceride (MAGs) based on the
total weight of
the processed oil.
[00344] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, the
processed oil
comprises greater than 60% by weight monoacylglyceride (MAGs) based on the
total weight of
the processed oil.
[00345] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, the
processed oil
comprises greater than 70% by weight monoacylglyceride (MAGs) based on the
total weight of
the processed oil.
[00346] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, the
processed oil
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
66
comprises greater than 80% by weight monoacylglyceride (MAGs) based on the
total weight of
the processed oil.
[00347] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, the
processed oil
comprises greater than 90% by weight monoacylglyceride (MAGs) based on the
total weight of
the processed oil.
[00348] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, the
processed oil is
derived from an oil source.
[00349] In an aspect of the preceding aspect, the processed oil comprises
non-oil
ingredients derived from and naturally present in the oil source such that the
non-oil ingredients
are not added to the processed oil.
[00350] In an aspect of the preceding aspect, the non-oil ingredients are
selected from
antioxidants, vitamins, and mixtures thereof.
[00351] In an aspect of the preceding aspect, said antioxidant is
tocopherol.
[00352] In an aspect of the preceding aspect, said tocopherol is selected
from a-
tocopherol, 13-tocopherol, 6-tocopherol, y-tocopherol, a-tocotrienol, 13-
tocotrienol, 6-tocotrienol,
and y-tocotrienol.
[00353] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, the
processed oil is
substantially free of monochloropropandiol (MCPD).
[00354] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, the
processed oil has less
than 0.10 mg/kg monochloropropandiol (MCPD).
[00355] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, the oil
source is from an
origin selected from a plant, an animal, algae or fish.
[00356] In an aspect of the preceding aspect, said oil source is of plant
origin.
[00357] In an aspect of the preceding aspect, said oil source is selected
from the group
consisting of safflower oil, grape oil, Silybum marianum oil, hemp oil,
sunflower oil, wheat germ
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
67
oil, pumpkin seed oil, sesame oil, rice bran oil, almond oil, rapeseed oil,
peanut oil, olive oil, and
coconut oil.
[00358] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, said
processed oil
comprises a MAG content of from about 50% to about 95% by weight based on the
total weight
of the processed oil.
[00359] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, said
processed oil
comprises a TAG content from about 5% to about 0.5% by weight based on the
total weight of
the processed oil.
[00360] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, said
processed oil has a
fatty acid profile that is substantially the same as a fatty acid profile of
the pre-processed oil from
which the processed oil was produced.
[00361] In an aspect of the preceding aspect, said fatty acid profile
comprises oleic acid,
linoleic acid and linolenic acid
[00362] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, the
amount of oleic acid,
linoleic acid and linolenic acid in the processed oil is within about 10% of
the amount of oleic
acid, linoleic acid and linolenic acid, respectively, in the pre-processed oil
from which the
processed oil was produced.
[00363] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, the
amount of oleic acid,
linoleic acid and linolenic acid in the processed oil is within about 1% of
the amount of oleic
acid, linoleic acid and linolenic acid, respectively, in the pre-processed oil
from which the
processed oil was produced.
[00364] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, the
processed oil
comprises three times the amount of MAGs relative to the amount of TAGs in the
pre-processed
oil from which the processed oil was produced.
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
68
[00365] In an aspect of the eighth, ninth, tenth or eleventh embodiments
and any of the
foregoing aspects of the eighth, ninth, tenth or eleventh embodiments, the
processed oil
comprises oleic acid in the form of oleic acid monoglyceride in an amount that
is about the same
as the amount of oleic acid in the pre-processed oil from which the processed
oil was produced.
[00366] In a twelfth embodiment, a processed oil comprises oleic acid and
linoleic acid
and having a total fatty acid content, wherein said linoleic acid is present
in an amount from
about 10% to about 90% by weight out of the total fatty acid content of the
processed oil, and
wherein the processed oil comprises greater than 50% by weight
monoacylglyceride (MAGs)
based on the total weight of the processed oil.
[00367] In an aspect of the twelfth embodiment, said linoleic acid is
present in an amount
from about 20% to about 90% by weight out of the total fatty acid content of
the processed oil.
[00368] In another aspect of the twelfth embodiment, said linoleic acid is
present in an
amount from about 10% to about 25% by weight out of the total fatty acid
content of the
processed oil.
[00369] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the processed oil further comprises linolenic acid,
wherein said linolenic
acid is present in an amount from about 0.01% to about 2% by weight out of the
total fatty acid
content of the processed oil.
[00370] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, a ratio of oleic acid to linoleic acid in the processed
oil is between about
0.01 and about 5.
[00371] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, a ratio of oleic acid to linoleic acid in the processed
oil is between about 1
and about 4.
[00372] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, a ratio of oleic acid to linoleic acid in the processed
oil is between about 3
and about 4.
[00373] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, a ratio of oleic acid to linolenic acid in the processed
oil is between about 1
and about 100.
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
69
[00374] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, a ratio of oleic acid to linolenic acid in the processed
oil is between about
and about 100.
[00375] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, a ratio of oleic acid to linolenic acid in the processed
oil is between about
10 and about 30.
[00376] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, wherein at least a portion of said oleic acid is present
in the form of oleic
acid monoglyceride (MOG).
[00377] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the MOG in the processed oil comprises at least 50% by
weight 1-oley1
monoglyceride out of the total amount of MOG.
[00378] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the MOG in the processed oil comprises at least 60% by
weight 1-oley1
monoglyceride out of the total amount of MOG.
[00379] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the MOG in the processed oil comprises at least 70% by
weight 1-oley1
monoglyceride out of the total amount of MOG.
[00380] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the MOG in the processed oil comprises at least 80% by
weight 1-oley1
monoglyceride out of the total amount of MOG.
[00381] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the processed oil comprises less than 10% by weight
triacylglycerides
(TAGs) based on the total weight of the processed oil.
[00382] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the processed oil comprises greater than 50% by weight
monoacylglyceride
(MAGs) based on the total weight of the processed oil.
[00383] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the processed oil comprises greater than 60% by weight
monoacylglyceride
(MAGs) based on the total weight of the processed oil.
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
[00384] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the processed oil comprises greater than 70% by weight
monoacylglyceride
(MAGs) based on the total weight of the processed oil.
[00385] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the processed oil comprises greater than 80% by weight
monoacylglyceride
(MAGs) based on the total weight of the processed oil.
[00386] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the processed oil comprises greater than 90% by weight
monoacylglyceride
(MAGs) based on the total weight of the processed oil.
[00387] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the processed oil is processed oil derived from an oil
source.
[00388] In an aspect of the preceding aspect, the processed oil comprises
non-oil
ingredients derived from and naturally present in the oil source such that the
non-oil ingredients
are not added to the processed oil.
[00389] In an aspect of the preceding aspect, the non-oil ingredients are
selected from
antioxidants, vitamins, and mixtures thereof.
[00390] In an aspect of the preceding aspect, said antioxidant is
tocopherol.
[00391] In an aspect of the preceding aspect, said tocopherol is selected
from a-
tocopherol, 13-tocopherol, 6-tocopherol, y-tocopherol, a-tocotrienol, 13-
tocotrienol, 6-tocotrienol,
and y-tocotrienol.
[00392] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the processed oil is substantially free of
monochloropropandiol (MCPD)
[00393] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the processed oil has less than 0.10 mg/kg
monochloropropandiol (MCPD).
[00394] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, said oil source is from an origin selected from a plant,
an animal, algae or
fish.
[00395] In an aspect of the preceding aspect, the oil source is of plant
origin.
[00396] In an aspect of the preceding aspect, said oil source is selected
from the group
consisting of safflower oil, grape oil, Silybum marianum oil, hemp oil,
sunflower oil, wheat germ
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
71
oil, pumpkin seed oil, sesame oil, rice bran oil, almond oil, rapeseed oil,
peanut oil, olive oil, and
coconut oil.
[00397] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, said processed oil comprises a MAG content of from about
50% to about
95% by weight based on the total weight of the processed oil.
[00398] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, said processed oil comprises a TAG content from about 5%
to about 0.5%
by weight based on the total weight of the processed oil
[00399] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, said processed oil has a fatty acid profile that is
substantially the same as a
fatty acid profile of the pre-processed oil from which the processed oil was
produced.
[00400] In an aspect of the preceding aspect, said fatty acid profile
comprises oleic acid,
linoleic acid and linolenic acid.
[00401] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the amount of oleic acid, linoleic acid and linolenic acid
in the processed
oil is within about 10% of the amount of oleic acid, linoleic acid and
linolenic acid, respectively,
in the pre-processed oil from which the processed oil was produced.
[00402] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the amount of oleic acid, linoleic acid and linolenic acid
in the processed
oil is within about 1% of the amount of oleic acid, linoleic acid and
linolenic acid, respectively,
in the pre-processed oil from which the processed oil was produced.
[00403] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the processed oil comprises about the same amount of MAGs
relative to the
amount of TAGs in the pre-processed oil from which the processed oil was
produced.
[00404] In an aspect of the twelfth embodiment or any of the foregoing
aspects of the
twelfth embodiment, the processed oil comprises oleic acid in the form of
oleic acid
monoglyceride in an amount that is about the same as the amount of oleic acid
in the pre-
processed oil from which the processed oil was produced.
[00405] In a thirteenth embodiment, a processed oil has a fatty acid
profile comprising
oleic acid, linoleic acid and linolenic acid, wherein the amount of oleic
acid, linoleic acid and
linolenic acid in the processed oil is within about 10% of the amount of oleic
acid, linoleic acid
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
72
and linolenic acid, respectively, in the pre-processed oil from which the
processed oil was
produced, and wherein the processed oil comprises greater than 50% by weight
monoacylglyceride (MAGs) based on the total weight of the processed oil.
[00406] In a fourteenth embodiment, a processed oil has a fatty acid
profile comprising
oleic acid, linoleic acid and linolenic acid, wherein the amount of oleic
acid, linoleic acid and
linoleic acid in the processed oil is within about 1% of the amount of oleic
acid, linoleic acid and
linolenic acid, respectively, in the pre-processed oil from which the
processed oil was produced,
and wherein the processed oil comprises greater than 50% by weight
monoacylglyceride
(MAGs) based on the total weight of the processed oil.
[00407] In a fifteenth embodiment, a method for promoting glucose
homeostasis in a
subject in need thereof, comprises: administering to said subject a
composition comprising a
processed oil.
[00408] In a sixteenth embodiment, a method for treating type II diabetes
in a subject in
need thereof, comprises: administering to said subject a composition
comprising a processed oil.
[00409] In a seventeenth embodiment, a method for promoting glucose
homeostasis in a
subject in need thereof, comprises: administering to said subject a
composition comprising a
processed oil comprising oleic acid monoglyceride (MOG), wherein at least 50%
by weight of
said MOG is 1-oley1 monoglyceride.
[00410] In an eighteenth embodiment, a method for treating diabetes in a
subject in need
thereof, comprises: administering to said subject a composition comprising a
processed oil
comprising oleic acid monoglyceride (MOG), wherein at least 50% by weight of
said MOG is 1-
oleyl monoglyceride.
[00411] In an aspect of the seventeenth or eighteenth embodiment, at least
60% by weight
of said MOG is 1-oley1 monoglyceride.
[00412] In another aspect of the seventeenth or eighteenth embodiment, at
least 70% by
weight of said MOG is 1-oley1 monoglyceride.
[00413] In another aspect of the seventeenth or eighteenth embodiment, at
least 80% by
weight of said MOG is 1-oley1 monoglyceride.
[00414] In an aspect of the fifteenth, sixteenth, seventeenth or eighteenth
embodiment or
any of the foregoing aspects of the seventeenth or eighteenth embodiment, said
processed oil is a
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
73
processed oil according to any one of the fifth through fourteenth embodiments
and any of the
foregoing aspects thereof.
[00415] In an aspect of the fifteenth, sixteenth, seventeenth or eighteenth
embodiment or
any of the foregoing aspects of the fifteenth, sixteenth, seventeenth or
eighteenth embodiment,
the composition is a food product comprising the processed oil of a food
product of any of the
first, second or third embodiment and any of the foregoing aspects thereof.
[00416] In an aspect of the fifteenth or seventeenth embodiment or any of
the foregoing
aspects of the fifteenth or seventeenth embodiment, said subject is suffering
from a condition
that affects glucose homeostasis.
[00417] In an aspect of the preceding aspect, the condition is insulin
resistance or type II
diabetes.
[00418] In an aspect of the fifth, sixth or seventh embodiment or any of
the foregoing
aspects of the fifth, sixth or seventh embodiment, said MOG contributes
between about 10% and
about 75% by weight of the total fatty acid content of the processed oil.
[00419] In an aspect of the fifth, sixth or seventh embodiment or any of
the foregoing
aspects of the fifth, sixth or seventh embodiment, said MOG contributes
between about 20% and
about 75% by weight of the total fatty acid content of the processed oil.
[00420] In an aspect of the fifth, sixth or seventh embodiment or any of
the foregoing
aspects of the fifth, sixth or seventh embodiment, said MOG contributes
between about 30% and
about 75% by weight of the total fatty acid content of the processed oil.
[00421] In an aspect of the fifth, sixth or seventh embodiment or any of
the foregoing
aspects of the fifth, sixth or seventh embodiment, said MOG contributes
between about 40% and
about 75% by weight of the total fatty acid content of the processed oil.
[00422] In an aspect of the fifth, sixth or seventh embodiment or any of
the foregoing
aspects of the fifth, sixth or seventh embodiment, said MOG contributes
between about 50% and
about 75% by weight of the total fatty acid content of the processed oil.
[00423] In an aspect of the fifth, sixth or seventh embodiment or any of
the foregoing
aspects of the fifth, sixth or seventh embodiment, said MOG contributes
between about 60% and
about 75% by weight of the total fatty acid content of the processed oil.
[00424] In an aspect of the eighth, ninth, tenth, eleventh, twelfth,
thirteenth or fourteenth
embodiment or any of the foregoing aspects of the eighth, ninth, tenth,
eleventh, twelfth,
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661
PCT/US2020/014182
74
thirteenth or fourteenth embodiment, said processed oil comprises oleic acid
monoglyceride
(MOG) in an amount that contributes 5% to about 75% by weight of the fatty
acid content of the
processed oil.
[00425] In an aspect of the eighth, ninth, tenth, eleventh, twelfth,
thirteenth or fourteenth
embodiment or any of the foregoing aspects of the eighth, ninth, tenth,
eleventh, twelfth,
thirteenth or fourteenth embodiment, said processed oil comprises oleic acid
monoglyceride
(MOG) in an amount that contributes between about 10% and about 75% by weight
of the total
fatty acid content of the processed oil
[00426] In an aspect of the eighth, ninth, tenth, eleventh, twelfth,
thirteenth or fourteenth
embodiment or any of the foregoing aspects of the eighth, ninth, tenth,
eleventh, twelfth,
thirteenth or fourteenth embodiment, said processed oil comprises oleic acid
monoglyceride
(MOG) in an amount that contributes between about 20% and about 75% by weight
of the total
fatty acid content of the processed oil
[00427] In an aspect of the eighth, ninth, tenth, eleventh, twelfth,
thirteenth or fourteenth
embodiment or any of the foregoing aspects of the eighth, ninth, tenth,
eleventh, twelfth,
thirteenth or fourteenth embodiment, said processed oil comprises oleic acid
monoglyceride
(MOG) in an amount that contributes between about 30% and about 75% by weight
of the total
fatty acid content of the processed oil
[00428] In an aspect of the eighth, ninth, tenth, eleventh, twelfth,
thirteenth or fourteenth
embodiment or any of the foregoing aspects of the eighth, ninth, tenth,
eleventh, twelfth,
thirteenth or fourteenth embodiment, said processed oil comprises oleic acid
monoglyceride
(MOG) in an amount that contributes between about 40% and about 75% by weight
of the total
fatty acid content of the processed oil
[00429] In an aspect of the eighth, ninth, tenth, eleventh, twelfth,
thirteenth or fourteenth
embodiment or any of the foregoing aspects of the eighth, ninth, tenth,
eleventh, twelfth,
thirteenth or fourteenth embodiment, said processed oil comprises oleic acid
monoglyceride
(MOG) in an amount that contributes between about 50% and about 75% by weight
of the total
fatty acid content of the processed oil.
[00430] In an aspect of the eighth, ninth, tenth, eleventh, twelfth,
thirteenth or fourteenth
embodiment or any of the foregoing aspects of the eighth, ninth, tenth,
eleventh, twelfth,
thirteenth or fourteenth embodiment, said processed oil comprises oleic acid
monoglyceride
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661 PCT/US2020/014182
(MOG) in an amount that contributes between about 60% and about 75% by weight
of the total
fatty acid content of the processed oil
[00431] In a nineteenth embodiment, a processed oil has a fatty acid
profile wherein the
fatty acid profile of the processed oil is substantially the same as the pre-
processed oil from
which the processed oil was produced, and wherein the processed oil comprises
greater than 50%
by weight monoacylglyceride (MAGs) based on the total weight of the processed
oil.
[00432] In a twentieth embodiment, a food product comprises the processed
oil of any one
of the fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth,
thirteenth or fourteenth
embodiment or any of the foregoing aspects of the fifth, sixth, seventh,
eighth, ninth, tenth,
eleventh, twelfth, thirteenth or fourteenth embodiments.
[00433] In a twenty-first embodiment, a processed oil comprises greater
than 50% by
weight monoacylglyceride (MAGs) based on the total weight of the processed oil
and having a
total fatty acid content.
[00434] In a twenty-second embodiment, a processed oil comprises greater
than 60% by
weight monoacylglyceride (MAGs) based on the total weight of the processed oil
and having a
total fatty acid content.
[00435] In a twenty-third embodiment, a processed oil comprises greater
than 70% by
weight monoacylglyceride (MAGs) based on the total weight of the processed oil
and having a
total fatty acid content.
[00436] In a twenty-fourth embodiment, a processed oil comprises greater
than 80% by
weight monoacylglyceride (MAGs) based on the total weight of the processed oil
and having a
total fatty acid content.
[00437] In a twenty-fifth embodiment, a processed oil comprises greater
than 90% by
weight monoacylglyceride (MAGs) based on the total weight of the processed oil
and having a
total fatty acid content.
[00438] In an aspect of the twenty-first, twenty-second, twenty-third,
twenty-fourth or
twenty-fifth embodiment, the processed oil further comprises oleic acid in
amount of from about
5% to about 75% by weight out of the total fatty acid content of the processed
oil.
[00439] In another aspect of the twenty-first, twenty-second, twenty-third,
twenty-fourth
or twenty-fifth embodiment, the processed oil further comprises oleic acid in
amount of from
about 10% to about 75% by weight out of the total fatty acid content of the
processed oil.
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661
PCT/US2020/014182
76
[00440] In another aspect of the twenty-first, twenty-second, twenty-third,
twenty-fourth
or twenty-fifth embodiment, the processed oil further comprises oleic acid in
amount of from
about 20% to about 75% by weight out of the total fatty acid content of the
processed oil.
[00441] In another aspect of the twenty-first, twenty-second, twenty-third,
twenty-fourth
or twenty-fifth embodiment, the processed oil further comprises oleic acid in
amount of from
about 30% to about 75% by weight out of the total fatty acid content of the
processed oil.
[00442] In another aspect of the twenty-first, twenty-second, twenty-third,
twenty-fourth
or twenty-fifth embodiment, the processed oil further comprises oleic acid in
amount of from
about 40% to about 75% by weight out of the total fatty acid content of the
processed oil.
[00443] In another aspect of the twenty-first, twenty-second, twenty-third,
twenty-fourth
or twenty-fifth embodiment, the processed oil further comprises oleic acid in
amount of from
about 50% to about 75% by weight out of the total fatty acid content of the
processed oil.
[00444] In another aspect of the twenty-first, twenty-second, twenty-third,
twenty-fourth
or twenty-fifth embodiment, the processed oil further comprises oleic acid in
amount of from
about 60% to about 75% by weight out of the total fatty acid content of the
processed oil.
[00445] In an aspect of the twenty-first, twenty-second, twenty-third,
twenty-fourth or
twenty-fifth embodiment or any of the foregoing aspects of the twenty-first,
twenty-second,
twenty-third, twenty-fourth or twenty-fifth embodiment, the processed oil
further comprises
linoleic acid in an amount of from about 1.5% to about 90% by weight out of
the total fatty acid
content of the processed oil.
[00446] In an aspect of the twenty-first, twenty-second, twenty-third,
twenty-fourth or
twenty-fifth embodiment or any of the foregoing aspects of the twenty-first,
twenty-second,
twenty-third, twenty-fourth or twenty-fifth embodiment, the processed oil
further comprises
linoleic acid in an amount of from about 10% to about 25% by weight out of the
total fatty acid
content of the processed oil.
[00447] In an aspect of the twenty-first, twenty-second, twenty-third,
twenty-fourth or
twenty-fifth embodiment or any of the foregoing aspects of the twenty-first,
twenty-second,
twenty-third, twenty-fourth or twenty-fifth embodiment, the processed oil
further comprises
linolenic acid in an amount of from about 0.1% to about 2% by weight out of
the total fatty acid
content of the processed oil.
SUBSTITUTE SHEET (RULE 26)
CA 03127093 2021-07-16
WO 2020/150661
PCT/US2020/014182
77
[00448] In
another aspect of the twenty-first, twenty-second, twenty-third, twenty-fourth
or twenty-fifth embodiment, the processed oil further comprises oleic acid and
linoleic acid,
wherein a ratio of oleic acid to linoleic acid is between about 0.01 and 5.
[00449] In
another aspect of the twenty-first, twenty-second, twenty-third, twenty-fourth
or twenty-fifth embodiment, the processed oil further comprises oleic acid and
linoleic acid,
wherein a ratio of oleic acid to linoleic acid is between about 1 and 4.
[00450] In
another aspect of the twenty-first, twenty-second, twenty-third, twenty-fourth
or twenty-fifth embodiment, the processed oil further comprises oleic acid and
linoleic acid,
wherein a ratio of oleic acid to linoleic acid is between about 3 and 4.
[00451] In
another aspect of the twenty-first, twenty-second, twenty-third, twenty-fourth
or twenty-fifth embodiment, the processed oil further comprises oleic acid and
linolenic acid,
wherein a ratio of oleic acid to linolenic acid is between about 1 and 100.
[00452] In
another aspect of the twenty-first, twenty-second, twenty-third, twenty-fourth
or twenty-fifth embodiment, the processed oil further comprises oleic acid and
linolenic acid,
wherein a ratio of oleic acid to linolenic acid is between about 10 and 100.
[00453] In
another aspect of the twenty-first, twenty-second, twenty-third, twenty-fourth
or twenty-fifth embodiment, the processed oil further comprises oleic acid and
linolenic acid,
wherein a ratio of oleic acid to linolenic acid is between about 10 and 30.
[00454] The
foregoing description of specific embodiments of the present disclosure has
been presented for purpose of illustration and description. The exemplary
embodiments were
chosen and described in order to best explain the principles of the disclosure
and its practical
application, to thereby enable others skilled in the art to best utilize the
subject matter and
various embodiments with various modifications are suited to the particular
use contemplated.
Different features and disclosures of the various embodiments within the
present disclosure may
be combined within the scope of the present disclosure.
SUBSTITUTE SHEET (RULE 26)
