NZ750465B2 - A two-step fractionation method for winterizing oil - Google Patents
A two-step fractionation method for winterizing oil Download PDFInfo
- Publication number
- NZ750465B2 NZ750465B2 NZ750465A NZ75046517A NZ750465B2 NZ 750465 B2 NZ750465 B2 NZ 750465B2 NZ 750465 A NZ750465 A NZ 750465A NZ 75046517 A NZ75046517 A NZ 75046517A NZ 750465 B2 NZ750465 B2 NZ 750465B2
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- NZ
- New Zealand
- Prior art keywords
- oil
- temperature
- period
- time
- reducing
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- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229960002079 calcium pantothenate Drugs 0.000 description 1
- NVTRPRFAWJGJAJ-UHFFFAOYSA-L calcium;2-[2-[carboxylatomethyl(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetate Chemical compound [Ca+2].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O NVTRPRFAWJGJAJ-UHFFFAOYSA-L 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000001413 cellular Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 150000003841 chloride salts Chemical class 0.000 description 1
- UWHZIFQPPBDJPM-FPLPWBNLSA-N cis-vaccenic acid Chemical compound CCCCCC\C=C/CCCCCCCCCC(O)=O UWHZIFQPPBDJPM-FPLPWBNLSA-N 0.000 description 1
- 125000003346 cobalamin group Chemical group 0.000 description 1
- 239000000306 component Substances 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- 238000009295 crossflow filtration Methods 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000004059 degradation Effects 0.000 description 1
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- 239000003974 emollient agent Substances 0.000 description 1
- 235000015897 energy drink Nutrition 0.000 description 1
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- 235000019414 erythritol Nutrition 0.000 description 1
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- 239000000284 extract Substances 0.000 description 1
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 1
- 230000002349 favourable Effects 0.000 description 1
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- 239000012467 final product Substances 0.000 description 1
- 239000004467 fishmeal Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 235000013350 formula milk Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 150000002314 glycerols Chemical class 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000000102 heterotrophic Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
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- 239000000845 maltitol Substances 0.000 description 1
- 229940035436 maltitol Drugs 0.000 description 1
- 235000010449 maltitol Nutrition 0.000 description 1
- RXVWSYJTUUKTEA-CGQAXDJHSA-N maltotriose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O)O[C@H](CO)[C@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 RXVWSYJTUUKTEA-CGQAXDJHSA-N 0.000 description 1
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- 239000008267 milk Substances 0.000 description 1
- 230000001333 moisturizer Effects 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- LPUQAYUQRXPFSQ-UHFFFAOYSA-M monosodium glutamate Chemical compound [Na+].[O-]C(=O)C(N)CCC(O)=O LPUQAYUQRXPFSQ-UHFFFAOYSA-M 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L na2so4 Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 229940053662 nickel sulfate Drugs 0.000 description 1
- 229910000363 nickel(II) sulfate Inorganic materials 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 235000020660 omega-3 fatty acid Nutrition 0.000 description 1
- 239000006014 omega-3 oil Substances 0.000 description 1
- 230000003287 optical Effects 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000000243 photosynthetic Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 235000021395 porridge Nutrition 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
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- 238000002360 preparation method Methods 0.000 description 1
- 230000002335 preservative Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 description 1
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 description 1
- 239000011764 pyridoxine hydrochloride Substances 0.000 description 1
- 235000021067 refined food Nutrition 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 235000019192 riboflavin Nutrition 0.000 description 1
- 239000002151 riboflavin Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- 235000015393 sodium molybdate Nutrition 0.000 description 1
- 229910001948 sodium oxide Inorganic materials 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 235000011496 sports drink Nutrition 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 235000013616 tea Nutrition 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 1
- 239000011747 thiamine hydrochloride Substances 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 230000000699 topical Effects 0.000 description 1
- 235000019798 tripotassium phosphate Nutrition 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000004034 viscosity adjusting agent Substances 0.000 description 1
- 235000019163 vitamin B12 Nutrition 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 235000021307 wheat Nutrition 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- HFHDHCJBZVLPGP-RWMJIURBSA-N α-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO HFHDHCJBZVLPGP-RWMJIURBSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B3/00—Refining fats or fatty oils
- C11B3/006—Refining fats or fatty oils by extraction
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B7/00—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils
- C11B7/0075—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils by differences of melting or solidifying points
Abstract
Provided herein are methods for winterizing microbial oils. In prior art winterizing methods involving dry fractionation a large amount of solids are removed, resulting in a poor liquid oil yield. The method of the invention allows for higher liquid oil yield by stepwise temperature adjustment and solid removal without loss of liquid fraction of the oil. The method as claimed involves heating the oil to a first temperature and maintaining the oil at the first temperature for a first period of time; reducing the first temperature of the oil after the first period of time to a second temperature over a second period of time, wherein reducing the first temperature produces a first solid fraction and first liquid fraction of the oil; removing the first solid fraction from the oil; reducing the second temperature of the first liquid fraction of the oil over a third period of time to a third temperature, wherein reducing the second temperature of the oil produces a second solid fraction and second liquid fraction of the oil; removing the second solid fraction from the oil; and recovering the second liquid fraction of the oil; wherein the oil is a microbial oil; and wherein the method is performed in the absence of solvent. The recovered liquid fraction may be utilized to produce biofuel or in pharmaceuticals, nutraceuticals, food supplements, animal feed additives and cosmetics. solid removal without loss of liquid fraction of the oil. The method as claimed involves heating the oil to a first temperature and maintaining the oil at the first temperature for a first period of time; reducing the first temperature of the oil after the first period of time to a second temperature over a second period of time, wherein reducing the first temperature produces a first solid fraction and first liquid fraction of the oil; removing the first solid fraction from the oil; reducing the second temperature of the first liquid fraction of the oil over a third period of time to a third temperature, wherein reducing the second temperature of the oil produces a second solid fraction and second liquid fraction of the oil; removing the second solid fraction from the oil; and recovering the second liquid fraction of the oil; wherein the oil is a microbial oil; and wherein the method is performed in the absence of solvent. The recovered liquid fraction may be utilized to produce biofuel or in pharmaceuticals, nutraceuticals, food supplements, animal feed additives and cosmetics.
Description
A TWO-STEP FRACTIONATION METHOD FOR WINTERIZING OIL
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority to U.S. Provisional Application No. 62/364,367, filed
July 20, 2016, which is incorporated by reference herein in its entirety.
BACKGROUND
Polyunsaturated fatty acids (PUFA), more specifically the omega-3 fatty acids, which
include docosahexaenoic acid (DHA), provide numerous health benefits. With the
development of biotechnology, these fatty acids can be produced efficiently by
microorganisms as an alternative source to fish. Microbial lipids, however, do not always
have the physical properties required for handling and are prone to phase separation. A
typical process for removal of solids from microbial lipids by controlled crystallization
involves solvents if crystallizing a desired fraction or dry fractionation by winterizing or
pressing. However, solvents are expensive and impact process safety, and dry fractionation
methods result in a large amount of solids removed, thereby, resulting in a poor liquid oil
yield. Typical methods for obtaining liquid oils from solid fat with the desired composition
of fatty acids are problematic for large scale production.
BRIEF SUMMARY
Provided herein are methods for winterizing microbial oil. The methods include
heating the oil to a first temperature and maintaining the oil at the first temperature for a first
period of time; reducing the first temperature of the oil after the first period of time to a
second temperature over a second period of time, wherein reducing the first temperature
produces a first solid fraction and first liquid fraction of the oil; removing the first solid
fraction from the oil; reducing the second temperature of the first liquid fraction of the oil
over a third period of time to a third temperature, wherein reducing the second temperature of
the oil produces a second solid fraction and second liquid fraction of the oil; removing the
second solid fraction from the oil; and recovering the second liquid fraction of the oil. The
method is carried out in the absence of solvent to result in an optimized winterized oil having
desired physical properties and composition of fatty acids.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph showing the crystal grown in oil as a function of temperature.
Figure 2 is a bar graph showing the fatty acid profile of crude oil and its fractions
during 3-stage solventless winterization. The chart contains fatty acid components with a
content of 0.1% or higher in the oil. *Fatty acid component whose percentage is noted on the
chart.
16492629_1 (GHMatters) P110597.NZ
Figure 3 is a graph showing the temperature change over time of a 45 mL oil sample
during ambient cooling.
DETAILED DESCRIPTION
Normally after oil is extracted, the oil becomes cloudy at ambient conditions due to its
complex fatty acid composition. A refining process called winterization is generally required
to remove saturated components that contribute to the cloudiness. Since algal oils crystallize
and solidify within a small temperature window (less than 10°C), solvents are generally used
to offset the increasing viscosity of the oil mixture to achieve the desired separation of solids
from the liquid oil. When solvents are absent, it leads to either low yield of the liquid fraction
or completely inseparable oil. However, for optimized food safety, alternatives to the use of
solvents are preferred. To date, such alternatives have failed to provide a commercially
viable option due to separation challenges. The challenge of such treatments of algal oil, for
example, lies in the large amount of solid fraction that traps liquid oil hampering separation.
The present methods, in contrast, provide a physical fractionation process that produce clear
microbial oil in its natural form (without degradation of triglycerides) at ambient temperature
through stepwise temperature adjustment and control to achieve separation. As described
herein, the fractionation process is divided into stages, solids are removed promptly and
efficiently without removing too much liquid oil. Improved access to and recovery of the
liquid fraction enhances total yield. Thus, the winterized liquid oil produced by the herein
provided methods optionally has a high DHA content.
Provided herein is a method for winterizing microbial oil comprising the steps of
heating a microbial oil to a first temperature and maintaining the oil at the first temperature
for a first period of time, reducing the first temperature of the oil after the first period of time
to a second temperature over a second period of time, wherein reducing the first temperature
produces a solid fraction and liquid fraction of the oil, removing the first solid fraction from
the oil; reducing the second temperature of the first liquid fraction of the oil over a third
period of time to a third temperature, wherein reducing the second temperature of the oil
produces a second solid fraction and second liquid fraction of the oil, and wherein the oil is
not heated between the steps of reducing the first temperature and reducing the second
temperature; removing the second solid fraction and recovering the second liquid fraction of
the oil thereby obtaining winterized oil. The method is carried out in the absence of solvents.
Also provided herein is a high-yield solventless winterization method involving at
least a two-stage dry fractionation process that refines crude oils made by microorganisms
into clear oils that flow at room temperature. This process is a temperature-controlled
winterization of the crude oil, during which solid fractions are removed at least twice. The
16492629_1 (GHMatters) P110597.NZ
first fraction removal is conducted soon after crystallization occurs, which can be determined
by the oil’s optical density. The resulting liquid fraction continues the winterization process
until crystals appear at a lower temperature. The crystals are then removed at the targeted
temperature. The fractionation process uses no organic solvents. The two-stage process
provides a high yield and elevated DHA content comparable to solvent-assisted winterization
and much higher yield than one-stage dry fractionation. For example, the two-stage process
increases the DHA content in the final oil product. The provided methods for winterizing oil
include the steps of providing an oil; heating the oil to a first temperature and maintaining the
oil at the first temperature for a first period of time; reducing the first temperature of the oil
after the first period of time to a second temperature over a second period of time, wherein
reducing the first temperature produces a first solid fraction and first liquid fraction of the oil;
removing the first solid fraction from the oil; reducing the second temperature of the first
liquid fraction of the oil over a third period of time to a third temperature, wherein reducing
the second temperature of the oil produces a second solid fraction and second liquid fraction
of the oil; removing the second solid fraction from the oil; and recovering the second liquid
fraction of the oil. The second liquid fraction comprises the winterized oil. The method is
carried out in the absence of solvents. Optionally, the oil is filtered prior to heating the oil to
the first temperature to remove impurities. Optionally, a filter aid, such as diatomaceous
earth, is added to the oil.
Optionally, the winterized oil is clear at room temperature. As used herein, the term
clear or clear oil refers to an oil that is transparent (i.e., not cloudy), which allows light to
pass through the oil. The term clear is not intended to imply that the oil must be free of color
as an oil that is clear may also have a color, i.e., orange or yellow.
Optionally, the winterized oil comprises one or more polyunsaturated fatty acids (e.g.,
docosahexaenoic acid (DHA). The total lipids in the oil comprise, for example, 40% or more
DHA. Optionally, the total lipids in the oil comprise 35 to 45% DHA.
In the provided methods, the first temperature is, optionally, above the melting point
of the oil. As used herein, the term melting point refers to the temperature at which the oil
becomes clear. The oil is in a liquid state at or above the melting point. Optionally, the first
temperature is above the melting point, for example, from about 25°C to 65°C, from about
40°C to 65°C, or any temperature within these ranges. These temperatures can be determined
by known methods including those established by the American Oil Chemistry Society
(AOCS) and American Society of Testing and Materials (ASTM), which establishes
specifications for determining the melting, cloud and pour points of fluids such as lipids and
oils. For example, the melting point can be determined using AOCS Official Method Cc 1-
16492629_1 (GHMatters) P110597.NZ
, cloud point can be determined using AOCS Official Method Cc 6-25, and pour point can
be determined using ASTM Official Method D97.
The oil is maintained at the first temperature for a selected period of time. Optionally,
the oil is maintained at the first temperature for 1 to 60 minutes or more. Optionally, the oil
is maintained at the first temperature for at least about 5 minutes.
In the provided methods, the first temperature is reduced over the second period of
time to a second temperature. Optionally, the first temperature is reduced by 0.5 to 2 degrees
per hour over the second period of time to reach the second temperature. The temperature
can be reduced by 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2
degrees per hour over the second period of time. The second period of time is selected, for
example, from 1 to 10 hours, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours, or for any period of time in
between. Optionally, the temperature is reduced by 1 degree per hour over the second period
of time.
The oil can be agitated during the second period of time by stirring, mixing, blending,
shaking, vibrating, or a combination thereof. Optionally, the oil is mixed during the second
period of time at a mixing speed of 50 to 200 rpm or any amount in between 50 and 200 rpm.
In the provided methods, the second temperature is at or near the cloud point of the
oil. As used herein, the term cloud point refers to the temperature of the oil at which the oil
begins to crystalize. One of skill in the art recognizes or knows how at measure and assess
the cloud point of an oil. For example, the cloud point can be routinely determined by the
cloud point test, e.g. AOCS Official Method Cc 6-25. Optionally, the second temperature is
between about 10°C to about 20°C, between about 20°C to about 30°C, or any value within
these ranges.
Optionally, the oil is maintained at the second temperature for about 1 to 30 minutes
or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29 or 30 minutes. For example, the oil is maintained at the second temperature for 5 to 20
minutes. Optionally, the second temperature is reduced by about 0.5 to 2 degrees per hour
over the third period of time to the third temperature. For example, the temperature is
reduced by 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 degrees per
hour over the third period of time of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours.
The third temperature is optionally about room temperature. Optionally, the third
temperature is about 3-5°C or about 4°C.
Optionally, the provided methods further comprise reducing the third temperature of
the second liquid fraction of the oil over a fourth period of time to a fourth temperature,
wherein reducing the third temperature of the oil produces a third solid fraction and third
16492629_1 (GHMatters) P110597.NZ
liquid fraction of the oil. Optionally, the method further comprises removing the third solid
fraction of the oil. Optionally, the method further comprises recovering the third liquid
fraction of the oil (i.e., the winterized oil). Optionally, the fourth temperature is about room
temperature. Optionally, the fourth temperature is about 3-5°C or about 4°C.
In the provided methods, the solid fractions of the oil can be removed by any one or
more means including, but not limited to, filtration and centrifugation.
Optionally, the oil to be winterized comprises triglycerides. More specifically, the oil
can comprise alpha linolenic acid, arachidonic acid, docosahexanenoic acid,
docosapentaenoic acid, eicosapentaenoic acid, gamma-linolenic acid, linoleic acid, linolenic
acid, or any combination thereof. Optionally, the oil to be winterized comprises triglycerides.
Optionally, the oil comprises fatty acids selected from the group consisting of palmitic acid
(C16:0), myristic acid (C14:0), palmitoleic acid (C16:1(n-7)), cis-vaccenic acid (C18:1(n-7)),
docosapentaenoic acid (C22:5(n-6)), docosahexaenoic acid (C22:6(n-3)), and any
combination thereof.
Oil that is processed using the provided methods is microbial oil, derived from a
population of microorganisms, e.g., oil-producing algae, fungi, bacteria and protists.
Optionally, the oil is a plant seed oil. The population of microorganisms is optionally
selected from the genus Oblongichytrium, Aurantiochytrium Thraustochytrium, and
Schizochytrium or any mixture thereof. Optionally, the microorganism is Thraustochytrids of
the order Thraustochytriales, more specifically Thraustochytriales of the genus
Thraustochytrium. Exemplary microorganisms include Thraustochytriales as described in
U.S. Patent Nos. 5,340,594 and 5,340,742, which are incorporated herein by reference in
their entireties. The microorganism can be a Thraustochytrium species, such as the
Thraustochytrium species deposited as ATCC Accession No. PTA-6245 (i.e., ONC-T18), as
described in U.S. Patent No. 8,163,515, which is incorporated by reference herein in its
entirety.
Microalgae are acknowledged in the field to represent a diverse group of organisms.
For the purpose of this document, the term microalgae is used to describe unicellular
microorganisms derived from aquatic and/or terrestrial environments (some cyanobacteria are
terrestrial/soil dwelling). Aquatic environments extend from oceanic environments to
freshwater lakes and rivers, and also include brackish environments such as estuaries and
river mouths. Microalgae can be photosynthetic; optionally, microalgae are heterotrophic.
Microalgae can be of eukaryotic nature or of a prokaryotic nature. Microalgae can be non-
motile or motile.
16492629_1 (GHMatters) P110597.NZ
The term thraustochytrid, as used herein, refers to any member of the order
Thraustochytriales, which includes the family Thraustochytriaceae. Strains described as
thraustochytrids include the following organisms: Order: Thraustochytriales; Family:
Thraustochytriaceae; Genera: Thraustochytrium (Species: sp., arudimentale, aureum,
benthicola, globosum, kinnei, motivum, multirudimentale, pachydermum, proliferum, roseum,
striatum), Ulkenia (Species: sp., amoeboidea, kerguelensis, minuta, profunda, radiata,
sailens, sarkariana, schizochytrops, visurgensis, yorkensis), Schizochytrium (Species: sp.,
aggregatum, limnaceum, mangrovei, minutum, octosporuni), Japonochytrium (Species: sp.,
marinum), Aplanochytrium (Species: sp., haliotidis, kerguelensis, profunda, stocchino ?),
Althornia (Species: sp., crouchii), or Elina (Species: sp., marisalba, sinorifica). Species
described within Ulkenia are considered to be members of the genus Thraustochytrium.
Strains described as being within the genus Thrautochytrium may share traits in common
with and also be described as falling within the genus Schizochytrium. For example, in some
taxonomic classifications ONC-T18 may be considered within the genus Thrautochytrium,
while in other classifications it may be described as within the genus Schizochytrium because
it comprises traits indicative of both genera.
The provided methods include or can be used in conjunction with additional steps for
culturing microorganisms according to methods known in the art and obtaining the oil
therefrom. For example, a Thraustochytrid, e.g., a Thraustochytrium sp., can be cultivated
according to methods described in U.S. Patent Publications 2009/0117194 or 2012/0244584,
which are herein incorporated by reference in their entireties for each step of the methods or
compositions used therein.
To isolate oil from microorganisms, the microorganisms are grown in a growth
medium (also known as culture medium). Any of a variety of media are suitable for use in
culturing the microorganisms described herein. Optionally, the medium supplies various
nutritional components, including a carbon source and a nitrogen source, for the
microorganism. Medium for Thraustochytrid culture can include any of a variety of carbon
sources. Examples of carbon sources include fatty acids (e.g., oleic acid), lipids, glycerols,
triglycerols, carbohydrates, polyols, amino sugars, and any kind of biomass or waste stream.
Carbohydrates include, but are not limited to, glucose, cellulose, hemicellulose, fructose,
dextrose, xylose, lactulose, galactose, maltotriose, maltose, lactose, glycogen, gelatin, starch
(corn or wheat), acetate, m-inositol (e.g., derived from corn steep liquor), galacturonic acid
(e.g., derived from pectin), L-fucose (e.g., derived from galactose), gentiobiose, glucosamine,
alpha-D-glucosephosphate (e.g., derived from glucose), cellobiose, dextrin, alpha-
cyclodextrin (e.g., derived from starch), and sucrose (e.g., from molasses). Polyols include,
16492629_1 (GHMatters) P110597.NZ
but are not limited to, maltitol, erythritol, and adonitol. Amino sugars include, but are not
limited to, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, and N-acetyl-beta-D-
mannosamine.
Optionally, the microorganisms provided herein are cultivated under conditions that
increase biomass and/or production of a compound of interest (e.g., oil or total fatty acid
(TFA) content). Thraustochytrids, for example, are typically cultured in saline or salt-
containing medium. The culture medium optionally includes NaCl or natural or artificial sea
salt and/or artificial seawater.
Thraustochytrids can be cultured, for example, in medium having a salt concentration
from about 0.5 g/L to about 50.0 g/L, from about 0.5 g/L to about 35 g/L, or from about 18
g/L to about 35 g/L. Optionally, the Thraustochytrids described herein can be grown in low
salt conditions (e.g., salt concentrations from about 0.5 g/L to about 20 g/L or from about 0.5
g/L to about 15 g/L).
Alternatively, the culture medium for Thraustochytrids, for example, can include non-
chloride-containing sodium salts as a source of sodium, with or without NaCl. Examples of
non-chloride sodium salts suitable for use in accordance with the present methods include,
but are not limited to, soda ash (a mixture of sodium carbonate and sodium oxide), sodium
carbonate, sodium bicarbonate, sodium sulfate, and mixtures thereof. See, e.g., U.S. Pat.
Nos. 5,340,742 and 6,607,900, the entire contents of each of which are incorporated by
reference herein. A significant portion of the total sodium, for example, can be supplied by
non-chloride salts such that less than about 100%, 75%, 50%, or 25% of the total sodium in
culture medium is supplied by sodium chloride.
Media for Thraustochytrid cultures can include any of a variety of nitrogen sources.
Exemplary nitrogen sources include ammonium solutions (e.g., NH in H O), ammonium or
amine salts (e.g., (NH ) SO , (NH ) PO , NH NO , NH OOCH CH (NH Ac)), peptone,
4 2 4 4 3 4 4 3 4 2 3 4
tryptone, yeast extract, malt extract, fish meal, sodium glutamate, soy extract, casamino acids
and distiller grains. Concentrations of nitrogen sources in suitable medium typically range
between and including about 1 g/L and about 25 g/L.
The medium optionally includes a phosphate, such as potassium phosphate or sodium-
phosphate. Inorganic salts and trace nutrients in medium can include ammonium sulfate,
sodium bicarbonate, sodium orthovanadate, potassium chromate, sodium molybdate, selenous
acid, nickel sulfate, copper sulfate, zinc sulfate, cobalt chloride, iron chloride, manganese
chloride calcium chloride, and EDTA. Vitamins such as pyridoxine hydrochloride, thiamine
hydrochloride, calcium pantothenate, p-aminobenzoic acid, riboflavin, nicotinic acid, biotin,
folic acid and vitamin B12 can be included.
16492629_1 (GHMatters) P110597.NZ
The pH of the medium can be adjusted to between and including 3.0 and 10.0 using
acid or base, where appropriate, and/or using the nitrogen source. Optionally, the medium is
sterilized.
Generally a medium used for culture of a microorganism is a liquid medium.
However, the medium used for culture of a microorganism can be a solid medium. In
addition to carbon and nitrogen sources as discussed herein, a solid medium can contain one
or more components (e.g., agar or agarose) that provide structural support and/or allow the
medium to be in solid form.
The resulting biomass can be pasteurized to inactivate undesirable substances present
in the biomass. For example, the biomass can be pasteurized to inactivate compound
degrading substances, such as degradative enzymes. The biomass can be present in the
fermentation medium or isolated from the fermentation medium for the pasteurization step.
The pasteurization step can be performed by heating the biomass and/or fermentation
medium to an elevated temperature. For example, the biomass and/or fermentation medium
can be heated to a temperature from about 50°C to about 140°C (e.g., from about 55°C to
about 90°C or from about 65°C to about 80°C). Optionally, the biomass and/or fermentation
medium can be heated from about 30 minutes to about 120 minutes (e.g., from about 45
minutes to about 90 minutes, or from about 55 minutes to about 75 minutes). The
pasteurization can be performed using a suitable heating means, such as, for example, by
direct steam injection.
The biomass can be harvested according to a variety of methods, including those
currently known to one skilled in the art. For example, the biomass can be collected from the
fermentation medium using, for example, centrifugation (e.g., with a solid-ejecting
centrifuge) and/or filtration (e.g., cross-flow filtration). Optionally, the harvesting step
includes use of a precipitation agent for the accelerated collection of cellular biomass (e.g.,
sodium phosphate or calcium chloride).
The biomass is optionally washed with water. The biomass can be concentrated up to
about 30% solids. For example, the biomass can be concentrated to about 1% to about 20%
solids, from about 5% to about 20%, from about 7.5% to about 15% solids, or to any
percentage within the recited ranges.
Prior to winterization, the oil or polyunsaturated fatty acids are obtained or extracted
from the biomass or microorganisms using one or more of a variety of methods, including
those currently known to one of skill in the art. For example, methods of isolating oil or
polyunsaturated fatty acids are described in U.S. Patent No. 8,163,515, which is incorporated
by reference herein in its entirety. Alternatively, the oil or polyunsaturated fatty acids are
16492629_1 (GHMatters) P110597.NZ
isolated as described in U.S. Publication No. 2015-0176042, which is incorporated by
reference herein in its entirety. Optionally, the one or more polyunsaturated fatty acids are
selected from the group consisting of alpha linolenic acid, arachidonic acid,
docosahexanenoic acid, docosapentaenoic acid, eicosapentaenoic acid, gamma-linolenic acid,
linoleic acid, linolenic acid, and any combination thereof.
Winterized oil or derivatives thereof (e.g., polyunsaturated fatty acids (PUFAs) and
other lipids) can be utilized in any of a variety of applications exploiting their biological,
nutritional, or chemical properties. Thus, the winterized oil or derivatives thereof can be used
to produce biofuel. Optionally, the oil is used in pharmaceuticals, nutraceuticals, food
supplements, animal feed additives, cosmetics, and the like.
Optionally, the liquid fractions of oil or the solid fractions of oil produced according
to the methods described herein can be incorporated into a final product (e.g., a food or feed
supplement, an infant formula, a pharmaceutical, a fuel, and the like). Optionally, the solid
fractions are incorporated into animal feed. Optionally, the liquid fractions are incorporated
into a food supplement, e.g., a nutritional or dietary supplement such as a vitamin. Suitable
food or feed supplements into which the lipids can be incorporated include beverages such as
milk, water, sports drinks, energy drinks, teas, and juices; confections such as candies, jellies,
and biscuits; fat-containing foods and beverages such as dairy products; processed food
products such as soft rice (or porridge); infant formulae; breakfast cereals; or the like.
Optionally, one or more of the winterized oils or compounds therein (e.g., PUFAs)
can be incorporated into a nutraceutical or pharmaceutical product or a cosmetic. Examples
of such a nutraceuticals or pharmaceuticals include various types of tablets, capsules,
drinkable agents, etc. Optionally, the nutraceutical or pharmaceutical is suitable for topical
application, e.g., as a lotion or ointment. Dosage forms can include, for example, capsules,
oils, granula, granula subtilae, pulveres, tabellae, pilulae, trochisci, or the like.
The winterized oil or lipids portions thereof produced according to the methods
described herein can be incorporated into products as described herein in combination with
any of a variety of other agents. For instance, such compounds can be combined with one or
more binders or fillers, chelating agents, pigments, salts, surfactants, moisturizers, viscosity
modifiers, thickeners, emollients, fragrances, preservatives, etc., or any combination thereof.
All ranges as recited herein include each and every value or fractional value within
the range and are inclusive of their end points.
Disclosed are materials, compositions, and components that can be used for, can be
used in conjunction with, can be used in preparation for, or are products of the disclosed
methods and compositions. These and other materials are disclosed herein, and it is
16492629_1 (GHMatters) P110597.NZ
understood that when combinations, subsets, interactions, groups, etc. of these materials are
disclosed that while specific reference of each various individual and collective combinations
and permutations of these compounds may not be explicitly disclosed, each is specifically
contemplated and described herein. For example, if a method is disclosed and discussed and
a number of modifications that can be made to a number of molecules including the method
are discussed, each and every combination and permutation of the method, and the
modifications that are possible are specifically contemplated unless specifically indicated to
the contrary. Likewise, any subset or combination of these is also specifically contemplated
and disclosed. This concept applies to all aspects of this disclosure including, but not limited
to, steps in methods using the disclosed compositions. Thus, if there are a variety of
additional steps that can be performed, it is understood that each of these additional steps can
be performed with any specific method steps or combination of method steps of the disclosed
methods, and that each such combination or subset of combinations is specifically
contemplated and should be considered disclosed.
Publications cited herein and the material for which they are cited are hereby
specifically incorporated by reference in their entireties.
The examples below are intended to further illustrate certain aspects of the methods
and compositions described herein, and are not intended to limit the scope of the claims.
Examples
Example 1. Solvent winterization
All experimental oil was obtained from standard cultivation of ONC-T18 on glucose
and subsequent enzymatic hydrolysis. Solvent winterization uses organic solvents, e.g.
hexane, acetone, to assist oil fractionation. In this experiment, oil samples (duplicates of 20 g
oil each) were dissolved in hexane with solvent to oil ratios of 2:1, 1:1 and 0.5:1. To obtain
clear oil at room temperature, i.e., 20 C, fractionation temperature was lowered to 10 C and
kept there overnight. Liquid oil fractions were recovered by centrifugation (4600rpm×20min)
and removal of solvent by evaporation at ambient conditions. Yield was calculated based on
the weight of liquid fraction over the total weight of starting oil. DHA content was analyzed
based on FAME analysis by gas chromatography (Table 1).
16492629_1 (GHMatters) P110597.NZ
Table 1. Results of Solvent Winterization
Solvent to oil ratio Yield of liquid fraction (%) DHA in liquid fraction
(%)*
2:1 91.6+1.4 41.8
1:1 83.8+0.7 42.5
0.5:1 83.0+0.6 42.6
*DHA content in starting oil is 40.9%
Example 2. One-stage solventless winterization.
The same oil was used in this and subsequent experiments to compare with the
result of above solvent winterization. Twenty 20g of oil were melted at 50°C for 30 minutes
to eliminate its thermal history. It was then cooled to 1°C above its cloud point (i.e., 26.4°C)
and kept cooling slowly at a controlled rate at 1°C/h until 20°C was reached. The sample was
kept at 20°C overnight. Mixing was achieved by using a stir plate and a speed set to 60 rpm.
The liquid oil fraction was recovered by vacuum filtration through Whatman® No. 1 filter
paper (Maidstone, United Kingdom). Experiment was conducted with duplicate samples.
51.8% oil was recovered with a final DHA content of 43.3% (Table 2).
Example 3. Two-stage solventless winterization
The melted oil was cooled from 50°C to 30°C and further to 26.3°C at a fixed rate of
1°C/h. The temperature was maintained at 26.3°C for 12 minutes before saturates were
removed by vacuum filtration. Thus obtained liquid fraction was subjected to a second stage
of cooling at 1°C/h until it reached 20°C. As the two-stage solventless winterization separates
the oil fractions at lower crystal contentrations (Figure 1), it avoids high viscosity and big oil
loss. A yield of 82.9% was achieved with DHA content at 43.0% (Table 2).
Table 2. Solventless Winterization.
Dry fractionation Yield of liquid fraction DHA in liquid fraction
(%) (%)
One-stage 51.8 ± 0.7 43.3
Two-stage 82.9 ± 2.5 43.0
DHA content in starting oil is 40.9%.
Example 4. Two-stage winterization at a higher cooling rate
16492629_1 (GHMatters) P110597.NZ
The experiment was carried out as in Example 3 except for using a higher cooling rate
of 1.5 C/min. Saturates were separated from the liquid fraction by vacuum filtration. It
resulted in a recovery yield of 65.1%, higher than that obtained in a one-stage solventless
winterization (i.e., 51.8%), but lower than that in a two-stage solventless winterization (i.e.,
82.9%), indicating a slower cooling rate is favorable to efficient phase separation although a
faster cooling rate shortens the process greatly. The DHA content in final oil was 41.8%.
Example 5. Two-stage winterization at a high cooling rate followed by centrifugal
concentration.
The experiment was carried out as in Example 4, e.g., cooling rate of 1.5 C/min,
except that saturates were separated using Sartorius Vivaspin® 20mL Centrifugal
Concentrators (Littleton, MA) in a centrifuge at 4600 rpm for 20 min. The yield of oil was
improved to 76.3%. The DHA content in final oil was 41.6%.
Example 6. Three-stage solventless winterization
Oil (440g) was melted at 50 C for 30 min to eliminate its thermal history. The
winterization was performed at three stages. In the first stage, the oil was cooled at a rate of
1.5 C/min to its cloud point at 26.4 C . The oil was maintained at 26.4°C for 12 min before
phase separation by vacuum filtration. Such obtained liquid fraction was subjected to a
second stage of cooling at a rate of 2 C/h until it reached 20 C remaining at this temperature
for half an hour. Saturates were then removed by vacuum filtration and the second liquid
fraction was cooled in a third stage of winterization at 2 C/h until it reached 4 C.
The yield and DHA content of each liquid fraction are shown in Table 3. The overall
yield of the three-stage winterization was 60.8%. Winterization improved oil appearance and
flow property. A clear oil at room temperature was obtained after the 2 stage fractionation.
o nd
The oil also flowed after storing at 4 C. It was noted the crystallization in the 2 liquid when
put under a temperature under 20 C differed from that of the crude oil when put under its
cloud point. When the crude oil was cooled, saturates came out and formed a solid layer
below the liquid fraction. It was difficult to blend it into the liquid phase, which caused an oil
loss after a certain period of storage. However, the crystals from the 2 liquid were loosely
packed. They did not settle but were able to be mixed with the liquid fraction and poured out
of the storage jar, which is desirable for storage and reuse. A 3 fractionation made the oil
clear at 4 C with a relatively high yield (i.e., 93.1%). A complete fatty acid profile is listed in
Table 4 and major fatty acid components are shown in Figure 2.
16492629_1 (GHMatters) P110597.NZ
Table 3. Three Stage Solventless Winterization
Fraction Yield (%) DHA (%) Pour Point (°C) Form/Appearance
Crude Oil - 38.5 18 Solid at 20°C
1 Liquid 74.5 40.1 0 Flow at 20°C
2 Liquid 87.6 41.8 -3 Clear at 20°C
3 Liquid 93.1 42.5 -6 Clear at 4°C
Table 4. Fatty acid profiles of crude oil and fractions before and after the three-stage
solventless winterization
Crude oil 1st liquid 2nd liquid 3rd liquid 1st solid 2nd solid 3rd solid
C10:0
0.1 0.1 0.1 0.1 0.1 0.1 0.1
C12:0
1.0 1.0 1.0 0.9 1.0 1.3 1.5
C13:0
0.0 0.0 0.0 0.0 0.1 0.1 0.1
C14:0
13.5 12.8 11.8 11.3 15.7 20.5 17.4
C14:1
0.1 0.2 0.1 0.1 0.1 0.1 0.1
C15:0
0.4 0.4 0.4 0.4 0.5 0.5 0.5
C16:0
26.0 24.5 23.3 23.0 30.3 34.6 26.6
C16:1
.5 5.8 5.9 5.9 5.0 4.4 5.6
C17:0
0.1 0.1 0.1 0.1 0.1 0.1 0.1
C18:0
0.7 0.7 0.6 0.6 0.9 1.0 0.8
C18:1 Ole
0.2 0.2 0.2 0.2 0.2 0.2 0.2
C18:1 Vac
3.5 3.6 3.7 3.6 3.3 3.1 4.2
C18:3n-6
0.1 0.1 0.1 0.1 0.1 0.1 0.1
C18:4
0.2 0.2 0.2 0.2 0.2 0.2 0.2
C20:0
0.1 0.1 0.1 0.1 0.1 0.1 0.1
C20:2 n-6
0.0 0.0 0.0 0.0 0.1 0.0 0.1
C20:3 n-6
0.1 0.1 0.1 0.1 0.1 0.1 0.1
C20:4 n-6
0.3 0.3 0.3 0.3 0.3 0.2 0.3
:4n3
0.4 0.4 0.4 0.4 0.4 0.3 0.4
C20:5 n-3
1.1 1.2 1.2 1.3 1.0 0.8 1.0
C22:0
0.1 0.1 0.1 0.1 0.0 0.0 0.0
C22:4 n-6
0.1 0.1 0.1 0.0 0.0 0.0 0.1
C22:5 n-6
7.5 7.8 8.1 8.1 6.4 5.1 6.5
22:5 n-3
0.3 0.3 0.3 0.2 0.2 0.1 0.3
C24:0
0.0 0.0 0.0 0.2 0.1 0.1 0.0
C22:6 n-3
38.5 40.1 41.8 42.5 33.8 27.0 33.9
Example 7. One-stage winterization.
In this one-step ambient cooling process, oil was first heated to 50°C for thirty
minutes. Then the oil was placed at room temperature (20-21°C) and cooled. The cooling
rate varied as it was not controlled. The temperature drop was fast but gradually slowed down
as can be seen in Figure 3. Samples were stored at room temperature for 24 hours. Separation
16492629_1 (GHMatters) P110597.NZ
was achieved by vacuum filtration (11µm) at room temperature. Examples of yield and
change of DHA content are listed in Table 5.
Table 5. One-stage solventless winterization
Sample # Yield (%) DHA in crude oil DHA in winterized oil
(%) (%)
1 88.8 40.5 42.2
2 92.9 37.7 38.5
3 87.2 40.7 42.3
Example 8. Effects of Filter Aid and Filtration on Solventless Winterization
Crude oil was heated to 50°C for 30 minutes before filtration (11µm) to remove visible
impurities. Filtered oil was thus obtained. Both crude and filtered oil were heated to 50°C
again for half hour and cooled at room temperature (20-21°C) for 24 hours. Fractions were
separated by vacuum filtration (11µm) at room temperature. The yields of liquid fraction
were compared but showed no significant difference between using crude and filtered oils
(Table 6). Diatomaceous earth (filter aid) was added to both crude and filtered oil to repeat
the same winterization conditions as above. The result showed that filter aid does not
significantly impact yield (Table 6).
Table 6. Experiments on pre-filtration and using filter aid
Sample # Yield of liquid fraction from winterization conditions as below (%)
From crude oil From filtered oil From crude oil From filtered oil
with filter aid with filter aid
1 93.0 92.0 93.5 93.1
2 80.2 83.7 78.8 82.8
16492629_1 (GHMatters) P110597.NZ
Claims (31)
1. A method for winterizing oil comprising the steps of: (a) heating an oil to a first temperature and maintaining the oil at the first temperature for a first period of time; (b) reducing the first temperature of the oil after the first period of time to a second temperature over a second period of time, wherein reducing the first temperature produces a first solid fraction and first liquid fraction of the oil; (c) removing the first solid fraction from the oil; (d) reducing the second temperature of the first liquid fraction of the oil over a third period of time to a third temperature, wherein reducing the second temperature of the oil produces a second solid fraction and second liquid fraction of the oil, and wherein the oil is not heated between the steps of reducing the first temperature and reducing the second temperature; (e) removing the second solid fraction from the oil; and (f) recovering the second liquid fraction of the oil, wherein the oil is a microbial oil, derived from a population of microorganisms and wherein the method is carried out in the absence of solvents.
2. The method of claim 1, wherein the first temperature is above the melting point of the oil.
3. The method of claim 1 or claim 2, wherein the first temperature is from 25°C to 65°C.
4. The method of claim 3, wherein the first temperature is from 40°C to 65°C.
5. The method of any one of claims 1-4, wherein the oil is maintained at the first temperature for 5 to 60 minutes prior to the reducing step.
6. The method of any one of claims 1-5, wherein the first temperature is reduced by 0.5 to 2 degrees per hour over the second period of time to the second temperature.
7. The method of any one of claims 1-5, wherein the first temperature is reduced by 0.5 to 2 degrees per minute over the second period of time to the second temperature.
8. The method of any one of claims 1-7, wherein the second period of time is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours.
9. The method of any one of claims 1-8, wherein the oil is mixed during the second period of time.
10. The method of claim 9, wherein the mixing comprises a speed of 50 to 200 rpm.
11. The method of any one of claims 1-10, wherein the second temperature is the cloud point of the oil. 16492629_1 (GHMatters) P110597.NZ
12. The method of any one of claims 1-11, wherein the second temperature is between 10°C to 20°C.
13. The method of any one of claims 1-11, wherein the second temperature is between 20°C to 30°C.
14. The method of any one of claims 1-13, wherein the oil is maintained at the second temperature for 5 to 20 minutes.
15. The method of any one of claims 1-14, wherein the second temperature is reduced by 0.5 to 2 degrees per hour over the third period of time to the third temperature.
16. The method of any one of claims 1-14, wherein the second temperature is reduced by 0.5 to 2 degrees per minute over the third period of time to the third temperature.
17. The method of any one of claims 1-16, wherein the third period of time is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours.
18. The method of any one of claims 1-17, wherein the third temperature is room temperature.
19. The method of any one of claims 1-17, wherein the third temperature is 4°C.
20. The method of any one of claims 1-19, wherein the method further comprises reducing the third temperature of the second liquid fraction of the oil over a fourth period of time to a fourth temperature, wherein reducing the third temperature of the oil produces a third solid fraction and third liquid fraction of the oil.
21. The method of claim 20, wherein the method further comprises removing the third solid fraction of the oil.
22. The method of claim 20, wherein the method further comprises recovering the third liquid fraction of the oil.
23. The method of any one of claims 20-22, wherein the fourth temperature is room temperature.
24. The method of any one of claims 20-22, wherein the fourth temperature is 4°C.
25. The method of any one of claims 1-24, wherein 80% or more of the oil is clear at room temperature.
26. The method of any one of claims 1-25, wherein the oil comprises one or more polyunsaturated fatty acids.
27. The method of claim 26, wherein the polyunsaturated fatty acid is docosahexaenoic acid (DHA).
28. The method of claim 27, wherein the oil comprises 40% or more DHA.
29. The method of claim 1, wherein the population of microorganisms is selected from the group consisting of algae, fungi, bacteria and protists. 16492629_1 (GHMatters) P110597.NZ
30. The method of claim 1, wherein the population of microorganisms is selected from the genus Oblongichytrium, Aurantiochytrium Thraustochytrium, and Schizochytrium or any mixture thereof.
31. The method of claim 1, wherein the population of microorganisms is a Thraustochytrium sp. deposited as ATCC Accession No. PTA-6245. 16492629_1 (GHMatters) P110597.NZ 0 50 100 150 200 Time (min) Temperature (°C)
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US201662364367P | 2016-07-20 | 2016-07-20 | |
US62/364,367 | 2016-07-20 | ||
PCT/IB2017/054412 WO2018015926A1 (en) | 2016-07-20 | 2017-07-20 | A two-step fractionation method for winterizing oil. |
Publications (2)
Publication Number | Publication Date |
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NZ750465A NZ750465A (en) | 2020-09-25 |
NZ750465B2 true NZ750465B2 (en) | 2021-01-06 |
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