WO2018156596A1 - Purification of dha containing oils - Google Patents

Abstract

The various examples presented herein are directed to an oil composition comprising at least one carotenoid in an amount greater than 50 mg/kg by weight of the oil composition; a docosahexaenoic acid (DHA) content greater than about 25% of the total weight of fatty acids present in the oil composition; and less than 80 ppb of trans-2-pentanal (t-2-P), less than 30 ppb of hexanal, less than 15 ppb heptanal, or less 1500 ppb of dimethyldisulfide (DMDS).

Description

PURIFICATION OF DHA CONTAINING OILS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application

Serial No. 62/462,015, filed February 22, 2017, entitled "IMPROVED REFINING OF MICROBIAL OILS" which application is hereby incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0002] Oils containing long chain polyunsaturated fatty acids LC-PUFAs can be difficult to purify. These oils are highly susceptible to oxidation and will easily contain oxidation by products that give the oil off flavors and off aromas. Typical vegetable oil refining is used to purify these oils. For example, algal-produced crude docosahexaenoic acid (DHA)-containing oil is typically degummed to remove phosphatides and is subsequently refined to remove free fatty acids. Such oils are then bleached and deodorized resulting in the production of the final, refined, bleached and deodorized oil (RBD oil). But because of the high temperature and duration of these processing conditions, it is only possible to remove oxidation by- products to a certain level as they can also be created during these procedures. In addition, the processing steps to create RBD oils remove salutary components, including, but not limited to tocopherols, sterols, and carotenoids, including β-carotene, and canthaxanthin.

DETAILED DESCRIPTION OF THE INVENTION

[0003] The oil compositions of the various embodiments described herein are not traditional RBD oils to which salutary components (e.g., carotenoids) have been added back in. Instead, the oil compositions of the various embodiments described herein are derived from crude DHA-containing oil that have been mildly refined. The result is an oil composition that contains i) significant amounts of salutary components that are typically only found in crude DHA-containing oils and in minimal/negligible amounts in RBD oil, but ii) also contains negligible amounts of undesired odor-causing components generally found in crude DHA-containing oil. The salutary components include, but are not limited to, carotenoids such as xanthophylls (e.g., canthaxanthin,) and carotenes (e.g., β-carotene). Salutary components like carotenoids have either been removed in the RBD oils or destroyed/degraded in the RBD process. Undesired odor-causing components include, but are not limited to, aldehydes (e.g., trans-2-pentenal (t-2-P), hexanal), ketones (eg., 2- methylcyclopentanone (2-MCP)) and sulfur compounds (e.g., dimethyldisulfide, also known as DMDS).

[0004] The oil compositions of the various embodiments described herein can be highly colored, due to the novel processing utilized herein. As such, the oil compositions of the various embodiments described herein contain significant amounts of salutary components, including carotenoids, which give such oils their color and higher nutritional value relative to RBD oils. And since those of ordinary skill in the art generally seek RBD oils for human nutrition that have little to no color, the oil compositions of the various embodiments described herein are not an intuitive product. In addition, oil compositions of the various embodiments described herein can be more stable than RBD oils lacking added, external antioxidants without the need to add excessive amounts of such antioxidants. While not wishing to be bound by any specific theory, it is believed that the oil

compositions of the various embodiments described herein are more stable than RBD oils, which may require increased amounts of external antioxidants, because components such as carotenoids which act as built-in antioxidants have been removed.

[0005] Some embodiments of the present disclosure are therefore directed to oil compositions comprising at least one carotenoid in an amount greater than about 50 mg/kg by weight of the oil composition; a DHA content greater than about 25% of the total weight of fatty acids present in the oil composition; and less than about 80 ppb of t-2-P, less than about 30 ppb of hexanal, less than about 100 ppb 2-MCP, or less than about 1500 ppb of DMDS.

[0006] As used herein, the term "carotenoid" generally refers to tetraterpenoids produced by plants and algae, as well as several bacteria and fungi. Carotenoids can be produced from fats and other basic organic metabolic building blocks by all these organisms. Carotenoids is a general term used to refer to over 600 known carotenoids. Carotenoids can be split into two classes, xanthophylls and carotenes. Examples of carotenoids include, but are not limited to, β-carotene, canthaxanthin, astaxanthin, lycopersene (7,8,l l,12,15,7',8',H',12',15'-decahydro-Y,Y-carotene), phytofluene, hexahydrolycopene (15-cis-7,8,l l,12,7',8'-hexahydro-Y,Y-carotene), torulene (3',4'- didehydro- ,Y"Carotene), a-zeacarotene (7',8'-dihydro-s,Y-carotene), alloxanthin, cynthiaxanthin, pectenoxanthin, cryptomonaxanthin ((3R,3'R)-7,8,7',8'-tetradehydro- , - carotene-3,3'-diol), crustaxanthin ( ,-carotene-3,4,3',4'-tetrol), gazaniaxanthin ((3R)-5'-cis- ,Y-caroten-3-ol), OH-chlorobactene ( ,2'-dihydro-f,Y-caroten-l'-ol), loroxanthin (β,ε- carotene-3,19,3'-triol, lutein ((3R,3'R,6'R)- ,s-carotene-3,3'-diol), lycoxanthin (γ,γ-caroten- 16-ol), rhodopin (l,2-dihydro-Y,Y-caroten-l-ol), rhodopinol (also known as warmingol/13- cz^'5-l,2-dihydro-Y,Y-carotene-l,20-diol), saproxanthin (3',4'-didehydro- ,2'-dihydro- ,Y- carotene-3,l'-diol), and zeaxanthin.

[0007] The oil compositions of the various embodiments described herein comprise at least one carotenoid in an amount greater than about 50 mg/kg, greater than about 55 mg/kg, greater than about 60 mg/kg, greater than about 65 mg/kg, greater than about 70 mg/kg, greater than about 75 mg/kg, greater than about 80 mg/kg, greater than about 85 mg/kg, greater than about 90 mg/kg, greater than about 95 mg/kg, greater than about 100 mg/kg; about 50 mg/kg to about 100 mg/kg, about 60 mg/kg to about 80 mg/kg, about 70 mg/kg to about 90 mg/kg, or about 80 mg/kg to about 100 mg/kg. It should be understood that the amount of the at least one carotenoid is the sum total of all carotenoids present in the oil compositions of the various embodiments described herein. Some organisms may even produce oils with much more than 100 mg/kg of carotenoids and are also within the scope of the present invention.

[0008] Alternatively, carotenoid content could be represented as a percentage of total, or individual, carotenoids remaining in the oil after reduction of the volatile components. In some embodiments greater than 50, 60, 70, 80, or 90% of the total, or individual, carotenoids present in the crude oil are retained after the volatile components has been reduced.

[0009] In some embodiments, the oil compositions of the various embodiments described herein comprise at least two, at least three, at least four or at least five carotenoids. In some embodiments, the oil compositions of the various embodiments described herein comprise at least β-carotene, and canthaxanthin. In some embodiments, the oil compositions of the various embodiments described herein comprise at least β- carotene and canthaxanthin in an amount of from about 50 mg/kg to about 60 mg/kg β- carotene and about 20 mg/kg to about 30 mg/kg canthaxanthin.

[0010] The oil compositions of the various embodiments described herein also have a Docosahexaenoic acid (DHA) content greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%; about 30% to about 50%, or about 25% to about 60%, or 25% to about 70%, or about 40% to about 50% of the total weight of fatty acids present in the oil composition. In some embodiments, the DHA is substantially in the form of a triglyceride. DHA content of oils can be easily determined by well-known methods in the art. For example AOCS methods Ce-2b-l l and Celi-07 and Ce-lb-89 were utilized herein.

[0011] The oil compositions of the various embodiments described herein can have a relatively low content of odor-causing components such as trans-2-pentenal (t-2-P), heptanal, DMDS, and 2-methylcyclopentanone (2-MCP).

[0012] For example, the oil compositions of the various embodiments described herein can have a t-2-P content of less than about 100 ppb, less than about 90 ppb, less than about 80 ppb, less than about 70 ppb, less than about 60 ppb, less than about 50 ppb, less than about 40 ppb, less than about 30 ppb, less than about 20 ppb, less than about 10 ppb, less than about 5 ppb, less than about 2.5 ppb; about 2.5 ppb to about 20 ppb; or about 2.5 ppb to about 10 ppb of t-2-P. Various embodiments may also have levels that are below the limit of detection or below the limit of quantification of the methods utilized herein.

[0013] The oil compositions of the various embodiments described herein can have a hexanal content of less than about 30 ppb, less than about 50 ppb, less than about 40 ppb, less than about 30 ppb, less than about 20 ppb, less than about 10 ppb, less than about 5 ppb, less than about 2.5 ppb; about 2.5 ppb to about 20 ppb; or about 2.5 ppb to about 10 ppb of hexanal. Various embodiments may also have levels that are below the limit of detection or below the limit of quantification of the methods utilized herein.

[0014] The oil compositions of the various embodiments described herein can have a DMDS content of less than about 2000 ppb, less than 1500 ppb, less than 1000 ppb, less than about 500 ppb, less than about 400, less than about 200 ppb, less than about 100 ppb, less than about 90 ppb, less than about 80 ppb, less than about 70 ppb, less than about 60 ppb, less than about 50 ppb, less than about 40 ppb, less than about 30 ppb, less than about 20 ppb, less than about 10 ppb, less than about 5 ppb, less than about 2.5 ppb; about 2.5 ppb to about 20 ppb; or about 2.5 ppb to about 10 ppb of DMDS. Various embodiments may also have levels that are below the limit of detection or below the limit of quantification of the methods utilized herein.

[0015] In some embodiments, the oil compositions of the various embodiments described herein can have a 2-MCP content of less than about 2000 ppb, less than 1500 ppb, less than 1000 ppb, less than about 500 ppb, less than about 400, less than about 200 ppb, less than about 100 ppb, less than about 90 ppb, less than about 80 ppb, less than about 70 ppb, less than about 60 ppb, less than about 50 ppb, less than about 40 ppb, less than about 30 ppb, less than about 20 ppb, less than about 10 ppb, less than about 5 ppb, less than about 2.5 ppb; about 2.5 ppb to about 20 ppb; or about 2.5 ppb to about 10 ppb of 2-MCP. Various embodiments may also have levels that are below the limit of detection or below the limit of quantification of the methods utilized herein.

[0016] The oil compositions of the various embodiments described herein can have a total volatiles content of less than about 1 wt.%, less than about 0.5 wt.%, less than about 0.2 wt.%, less than about 0.1 wt.%; about 0.01 wt.% to about 0.5 wt.%; about 0.01 wt.% to about 0.2 wt.%; or about 0.01 wt.% to about 0.03 wt.%. "Total Volatiles" and used herein means the sum in weight percent of the following: hexanal, t-2-P, DMDS, and 2-MCP.

[0017] In some embodiments, the oil compositions of the various embodiments described herein can have a combination of one or more or all of the values described herein of DHA, carotenoid content, t-2-P content, hexanal content, DMDS content, and 2-MCP content. In some embodiments, therefore, the oil compositions of the various embodiments described herein can have a Total Volatiles content of about 0.01 wt.% to about 0.5 wt.%; a DHA content of about 25% to about 60%; a sum total of all carotenoids present in the oil compositions of the various embodiments described herein of about 70 mg/kg to about 90 mg/kg; a t-2-P content of less than about 2.5 ppb; a hexanal content of less than about 2.5 ppb; a DMDS content of less than about 2.5 ppb; or a 2-MCP content of less than about 2.5 ppb to about 3 ppb.

[0018] In some embodiments, the oil compositions of the various embodiments described herein can have a combination of one or more or all of the values described herein of DHA, carotenoid content, t-2-P content, hexanal content, DMDS content, and 2-MCP content. In some embodiments, therefore, the oil compositions of the various embodiments described herein can have a DHA content of about 25% to about 50%; a sum total of all carotenoids present in the oil compositions of the various embodiments described herein of greater than about 50 mg/kg; a t-2-P content of less than about 2.5 ppb; a hexanal content of less than about 2.5 ppb; a DMDS content of less than about 2.5 ppb; or a 2-MCP content of less than about 2.5 ppb.

[0019] In some embodiments, the oil compositions of the various embodiments described herein can have a combination of one or more or all of the values described herein of DHA, carotenoid content, t-2-P content, hexanal content, DMDS content, and 2-MCP content. In some embodiments, therefore, the oil compositions of the various embodiments described herein can have a DHA content of about 25% to about 60%; a sum total of all carotenoids present in the oil compositions of greater than about 50 mg/kg; a t-2-P content below the limit of detection or quantification; a hexanal content below the limit of detection or quantification; a DMDS content below the limit of detection or quantification; or a 2- MCP content below the limit of detection or quantification.

[0020] The oil compositions of the various embodiments described herein are derived from an oil isolated from a biomass and can be produced using methods known in the art. Such methods include isolating the oils from a biomass comprising a marine microorganism. Examples of suitable marine microorganisms include, but are not limited to, at least one of algae, bacteria, fungi, and protists. Some specific examples of marine microorganisms useful for the production and isolation of the oil compositions of the various embodiments described herein include microalgae and chromophytic algae as described in Published PCT Appl. No. W094/28913, which is incorporated by reference as if fully set forth herein. See also Published PCT Appl. No. WO94/008467; U.S. Patent No. 5,908,622; U.S. Patent No. 5,688,500; U.S. Patent No. 5,518,918; U.S. Patent No.

5,340,742; U.S. Patent No. 5,340,594; U.S. Patent No. 8,163,515; U.S. Patent

No.9,023,616; Published European Appl. No. 512997 and 669809, all of which are incorporated by reference as if fully set forth herein.

[0021] Any organism known to produce DHA-containing oils can be used in the fermentation of oils described herein including but not limited to, eukaryotes such as Isochrysis gaibana and thraustochytrids (e.g., a marine microorganism of genus

Schizochytrium ) such as Schizochytrium sp. and Thraustochytrium aureum. See, e.g. , Structured and Modified Lipids 376 (Frank D. Gunstone ed., Marcel Dekker Inc. 2001), which is incorporated by reference as if fully set forth herein. The skilled person would appreciate that production of DHA from marine organisms through fermentation has been be extensively described in the literature since the late 1980s. Numerous production organisms and methods are well known. Specific examples of useful organisms include but are not limited to the following Thraustochytrium sp ATCC 26185, and Schizochytrium sp. ATCC 20888.

[0022] After fermentation is complete the harvested cells can be dried to a suitable moisture content (e.g., about a 4% moisture content). A suitable non-polar solvent (e.g., pentane, hexanes, and the like) can then be added to the dried biomass in a suitable container (e.g., a glass kettle) and stirred for a suitable time and temperature (e.g., 2 hours at 25 °C). Following filtration of the biomass, the solvent in the filtrate can be removed by any suitable means (e.g., rotary evaporator) to produce crude DHA-containing oil.

Alternatively, the crude oil may be isolated through a solvent free processes well known in the art. See, eg., US 9,745,538 or US 9,745,539; both of which are hereby incorporated by reference.

[0023] It is well understood to those in the art that the levels of DHA, carotenoids, and level of various volatile compounds and impurities present in a crude oil will be a factor of the particular production parameters used in the fermentation, isolation, and refining. So the starting values of these various components will vary from lot to lot of crude oil.

[0024] The oil compositions of the various embodiments described herein are derived from an oil isolated from a biomass, such as a crude DHA-containing oil. A crude DHA-containing oil is then mildly refined to obtain the oil compositions of the various embodiments described herein. The oil compositions of the various embodiments described herein are not traditional RBD oils to which salutary components (e.g., carotenoids) have been added back in. Instead, the oil compositions of the various embodiments described herein are derived from crude DHA-containing oil that have been mildly refined.

[0025] As used herein, the term "mildly refined" generally means that a crude

DHA-containing oil is not refined to the extent that one would obtain an RBD oil. Instead, the crude DHA-containing oil is refined only to the extent necessary to obtain an oil composition that; i) retains significant amounts of salutary components that are typically only found in crude DHA-containing oils and in minimal/negligible amounts in RBD oil, and ii) has undesired odor or flavor causing components generally found in crude DHA- containing oil substantially removed.

Pretreatment

[0026] Crude oils isolated from natural sources sometimes contain "gums," primarily consisting of phospholipids, and also contain free fatty acids, sterols, sterol esters, trace metals, traces of carbohydrates and proteins, and solid particles. In some cases, the oils also contain antioxidants, such as tocopherols and carotenoids. Removing

phospholipids from the oil prevents the formation of gum deposits further down in the refining process and prevents development off flavors and color during storage.

Alternatively, the crude oil may contain small particles that aggregate and create problems in later processing. While optional for the purposes described herein, pretreatment steps such as degumming or filtration can be used to avoid issues in further processing. [0027] Water degumming is commonly used to remove phospholipids and other water soluble components from the oil. A general process includes treating crude oil (e.g., crude DHA-containing oil) with 250-2000 ppm of phosphoric acid or citric acid at 60-90°C with stirring. Water (e.g., 1-5%) is then added to the acid treated crude oil at 60-75°C with stirring to aid in the hydration of the phospholipids in the crude oil. The oil is then gently mixed for another 15-60 min. An aqueous phase is formed consisting of an emulsion of hydrated phospholipids and other water-soluble compounds, along with some entrained oil. The two phases are separated from each other by settling and decantation, or by

centrifugation, yielding a stream of acid degummed oil and a stream of wet gums. A neutralization step with an aqueous solution of base can be used to remove residual acid, as well as one or more extraction steps with deionized water to rid the oil of soluble salts.

[0028] In some embodiments the crude DHA-containing oil is optionally degummed prior to mild deodorization using a suitable aqueous acid, such as phosphoric acid or citric acid. In other embodiments the crude DHA-containing oil is initially washed with water prior to mild deodorization. The oils of the various embodiments described herein, however, are at no point bleached under traditional conditions to process vegetable oil.

[0029] Additionally, the crude oil may optionally be filtered to remove particulate matter prior to mild deodorization. Filtration by common methods is well known. It may be performed in a batch manner or continuously as part of an integrated process. A filtration aid (including but not limited to cellulose, diatomaceous earth, commercially available clays, or silica gel) or other processing aid may be used to remove unwanted particles or dissolved materials. These aids may be additionally utilized to increase the ease and effectiveness of filtration. Filtration may be performed at an elevated temperature and the oil may be contacted with the filter aid for an extended period of time to aid in the adsorption or adherence of particles onto the filter aid.

Deodorization

[0030] Briefly, mild deodorization removes volatile compounds that are responsible for "off flavors and odors," but the process may also remove free fatty acids, and some tocopherols and sterols. The process is often performed under vacuum to aid in the removal of specific volatile compounds, and to protect the oil from oxidation. Steam, nitrogen or another inert gas can be used as a stripping agent.

[0031] The process is fully defined by temperature, time, and pressure. When performed on a commercial scale, it can include a multi-step process including de-aeration, multi-stage heating, deodorization-deacidification, and multi-stage cooling of the oil. If de- aeration is performed, it can be accomplished in a separate vessel connected to a vacuum system (e.g., 30-50 mm Hg), or at even lower pressures in the deodorizer. Sparge steam (or any other inert gas) may be used to improve de-aeration.

[0032] The process can be performed in a batch wise, in a semi-continuous system, or in a continuous system. Stripping efficiency can be better in a continuous system, which generally has a column filled with structured packing that provides a high surface area. Counter-current contact of the oil with a stripping agent over the structured packing can provide efficient stripping in a short contact time. Various configurations of apparatus can be used (horizontal or vertical vessels, tray-type columns, packed columns or thin film/wiped film evaporators/distillators).

[0033] The oil compositions of the various embodiments described herein are obtained by treating a crude DHA-containing oil under a mild deodorization process that utilizes a suitable apparatus, as described herein.

[0034] A further aspect of the present invention is the use of a packed column for the purification of a PUFA containing oil. Deodorization columns of this type are known in the art and used in the physical refining of vegetable oils.

[0035] a process for the production of a purified PUFA oil comprising passing a crude PUFA oil though a packed column: wherein the column is heated to between 140 and 200 degrees C and steam in fed into the column counter currently to the flow of crude PUFA oil, and the purified oil is collected from the column.

Oil Compositions

[0036] Ultimately, the oil compositions of the various embodiments described herein, which are derived from an oil isolated from a biomass by the mild refining process described herein, is one that has a combination of one or more or all of the values described herein of Total Volatiles content, DHA, carotenoid content, t-2-P content, hexanal content, DMDS content, and 2-MCP content. In some embodiments, therefore, the mildly refined oil isolated from a biomass can have a Total Volatiles content of about 0.01 wt.% to about 0.5 wt.%; a DHA content of about 25% to about 60%; a total carotenoids content in the mildly refined oil isolated from a biomass of greater than about 70 mg/kg; a t-2-P content of about 0.5 ppb to about 3 ppb; a hexanal content of about 0.5 ppb to about 3 ppb; a heptanal content of about 0.5 ppb to about 3 ppb; a DMDS content of about 0.5 ppb to about 3 ppb; and a 2-MCP content of about 0.5 ppb to about 3 ppb. [0037] As mentioned herein, known DHA-containing oils used for human nutrition have been fully refined using typical vegetable oil techniques to create a highly purified RBD oil. Refining removes volatile compounds, metals, and pigments. But the process not only removes components that are beneficial to human nutrition, such as carotenoids, but also creates a need for high levels of antioxidants to preserve the oils. The oil compositions of the various embodiments described herein, which are derived from an oil isolated from a biomass by the mild refining process described herein, have a low content of odor causing compounds and, at the same time retain salutary components such as carotenoids. The presence of carotenoids leads to a more nutritional or stable product, without the need to use as much additional antioxidants— even though antioxidants (e.g., gamma tocopherol (vitamin E), tocotrienol, ascorbic acid (vitamin C), retinol (vitamin A), tertiary

butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), and butylated

hydroxytoluene (BHT)) can be added to the oil compositions of the various embodiments described herein.

[0038] Compositions described herein can be used in food, dietary supplement, and feed applications. A further embodiment of the invention is a food product comprising an oil described herein. The food maybe a intended for an infant or adult and delivered in any manner known to one of skill in the art. Alternatively, the compositions described herein could be formulated for consumption as a dietary supplement in liquid, powder, or capsule form.

[0039] Values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not just about 0.1 % to about 5%, but also the individual values (e.g. , 1%, 2%, 3%, and 4%) and the sub-ranges (e.g. , 0.1 % to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement "about X to Y" has the same meaning as "about X to about Y," unless indicated otherwise. Likewise, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z," unless indicated otherwise.

[0040] In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading may occur within or outside of that particular section.

Furthermore, all publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0041] In the methods described herein, the steps can be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Furthermore, specified steps can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed step of doing X and a claimed step of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0042] The term "about" as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range. In addition, all ranges mentioned herein can alternatively be read without the quantifier "about". So if a range is stated as "from about X to about Y", the inventor are specifically contemplating the exact range of "from X to Y".

[0043] The term "substantially" as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more.

Examples

[0044] The present invention can be better understood by reference to the following examples which are offered by way of illustration. The present invention is not limited to the examples given herein.

[0045] Methods for the production and processing of DHA oils from microalgae and chromophyitic algae have been well known in the art for over 30 years as described above. Alternatively, crude DHA containing algal oil can be purchased from Cargill Incorporated. Additional commercial suppliers of crude algal oil are also known to those skilled in the art. Example 1:

Materials

Crude DHA-Containing Oil Refining With Citric Acid

[0046] A 1 L jacketed flask equipped with a four-port headpiece was fitted with a direct drive overhead stirrer and a heated circulating bath set at 60°C. The vessel was kept under nitrogen for the duration of the experiment. The flask was charged with 750 mL of crude algal oil. The mechanical stirrer was turned on and set to 100 RPM. The oil equilibrated at 60°C for 30 minutes. A 500 ppm citric acid solution was prepared in degassed, deionized water. 37.5 mL (5% relative to the amount of algal oil) of the 500 ppm citric acid solution was added to the vessel. The material was stirred at 60°C for 30 min. The stirrer was turned off and the aqueous layer allowed to separate from the algal oil. After 15 minutes the aqueous layer was removed and 37.5 mL of degassed, deionized water was added to the reaction flask. The material was stirred for 10 min at 60°C. The stirrer was turned off and the aqueous layer allowed to separate from the algal oil. The aqueous layer was removed and another 37.5 mL of degassed, deionized water was added to the reaction flask. The material was stirred for 10 min at 60°C. The stirrer was turned off and the aqueous layer allowed to separate from the algal oil and removed. The algal oil was transferred under nitrogen to 250 mL centrifuge bottles. The oil was centrifuged at 30°C and 3000 RPM for 10 min and separated from the remaining aqueous layer. The oil was transferred to a glass storage container, the headspace flushed with nitrogen, and stored in a freezer at -9°C.

Crude DHA-Containing Oil Refining Without Citric Acid

[0047] A 1 L jacketed flask equipped with a four-port headpiece was fitted with a direct drive overhead stirrer and a heated circulating bath set at 60°C. The vessel was kept under nitrogen for the duration of the refining step. The flask was charged with 750 mL of crude algal oil. The mechanical stirrer was turned on and set to 100 rpm. The oil equilibrated at 60°C for 30 minutes and 37.5 mL of degassed, deionized water was added to the reaction flask. The material was stirred for 10 min at 60°C. The stirrer was turned off and the aqueous layer allowed to separate from the algal oil. The aqueous layer was removed. The algal oil was removed and placed under nitrogen in 250 mL centrifuge bottles. The oil was centrifuged at 30°C and 3000 RPM for 10 min and separated from the remaining aqueous layer. The oil was transferred to a glass storage container, the headspace flushed with nitrogen, and stored in a freezer at -9°C.

Deodorization

[0048] A Pope Scientific Inc. 2" wiped- film evaporator (part No. 40450-01) complete with (i) a variable speed drive mechanism (wetted parts 316 stainless steel), glass internal condenser, 2000 mL glass graduated feed and degasser with stopper; (ii) a rotatable, multiple receiver flask including four 200 mL receiver flasks; (iii) a 500 mL distillate receiver flask, glass cold trap, 316 stainless steel wiper retainer and carbon wiper blades; (iv) a metal band heater set (part No. 40782-G1) with a high temperature "J" thermocouple assembly and insulating jacket maintained the desired temperatures; (v) a silicone rubber heater and thermocouple (part No. 40645) and insulating jacket (part No. 40669) for maintaining temperature of the feed material; (vi) an Edward's mechanical rotary vacuum pump/oil diffusion pump system for providing vacuum during experimental runs; (vii) Pirani-type gauge with a range of 0.001 Torr to 1000 Torr; (viii) a digital RPM indicator for providing constant monitoring of the variable speed drive; and (ix) a Cole-Palmer chiller for maintaining the temperature of the internal condenser.

[0049] The feed and degasser assembly was charged with refined crude DHA- containing oil (refined with or without citric acid). The entire system was kept under nitrogen for the duration of the run. The feed assembly was held at 45 °C. Pure nitrogen was used to degas the feed material for one hour. The internal condenser of the wiped-film evaporator was maintained at 2°C via the chiller for all deodorization operations. The variable speed drive mechanism of the evaporator was set at a constant speed of 400 RPM. Feed rate to the deodorizer was 175 mL/hr throughout the experimental runs. The deodorization temperature and pressure was varied in order to evaluate deodorization conditions while retaining the carotenoid content. Samples were obtained throughout the run at the desired conditions. The samples were transferred to storage containers, the headspace flushed with nitrogen, and stored frozen until subjected to analyses.

[0050] The process conditions for refining and deodorization are shown in Table 1 : Table 1

[0051] Mildly refined DHA-containing oil (samples 1A-1F) had the properties shown in Table 2. Mildly refined DHA-containing oil (samples 1A-1F) had the profiles shown in Table 3, in terms of t-2-P content, hexanal content, DMDS content, and 2-MCP content.

Table 2

*Total Carotenoid content measured according to British Standard 684-2.20: 1977

_** -Carotene and Canthaxanthin were measured by an external lab, Eurofins, according to stated methods EN 12823-2:2000 and Roche Index n₀ 2264 respectively Table 3

t-2-P is trans-2-pentenal; DMDS is dimethyl disulfide;

2-MCP is 2-methylcyclopentanone

Example 2:

[0052] Refining Vessel - The refining vessel was used to carry out pretreatment of the crude oil with various process aids. Different combinations of clay, silica, and cellulose were tested. A mixer attached to the top allowed mixing/agitation of the contents.

Supplier: HAB Heiland Apparatebau (Germany). Shape: vertical cylindrical, conical bottom, with a sight glass. Volume: 40 L (internal) with a 5.68 L (jacket). Material of construction: Stainless steel. Equipped with a heating jacket, stirrer with variable speed (propeller type, 2 wings), baffle plates, spray nozzle for returning oil back from the filtration loop.

[0053] Candle Filter - A candle filter with a multifilament cloth is used to perform the filtration of the oil from the refining vessel. A cake is built-up on the cloth and when the oil downstream of the filter is clear (visually observed from a sight glass), the transfer operation to the batch deodorizer is started. Supplier: Amafilter group Lochem B.V. (The Netherlands) - Filtration Group. Shape: vertical cylindrical. Volume: 8 L. Filtration area: 0.05 m². Material of construction: Stainless steel with Viton (gaskets); Polyethylene (filter cloth).

[0054] Polishing Filter- A polishing filter is located downstream of the candle filter when the oil is transferred from the refining vessel to the batch deodorizer. This filter removes any particles that were not retained by the candle filter. Supplier: Amafilter group Lochem B.V. (The Netherlands) - Filtration Group. Particle retention: 1 micron. Filtration area: 0.09 m². Material of construction: Stainless steel with Viton gaskets. Bag filter (Polypropylene needle felt from EATON (Belgium))

[0055] Batch Deodorizer - Vessel intended for performing batch deodorization operations. Steam (or nitrogen) can be continuously sparged through the oil, usually while applying a reduced pressure of ~2 mbar-a. Supplier: HAB Heiland Apparatebau (Germany). Shape: vertical cylindrical, flat bottom. Volume: 31 L (internal); 5.5 L (jacket). Material of construction: Stainless steel. Equipped with a heating jacket, sparge steam ring, and a sight glass.

[0056] Packed Column Deodorizer - The packed column deodorizer consists of a structured packed bed which can accommodate different types of packing and heights. Raschig Superpack RSP 250X was used as the packing material. Oil is heated up in an inline electric heater and sprayed onto the packing bed at the top of the column to guarantee good oil distribution and intimate contact with the stripping agent. Stripping steam (or nitrogen) is introduced at the bottom of the column, counter current to the flow of the oil. Designed by Cargill and manufactured by VGM (The Netherlands). Shape: vertical cylindrical. Structured packing: Raschig Superpack RSP 250X; diameter: 255 mm; max height: 708 mm. Capacity (oil process): 15 - 25 kg/h. Material of construction: Stainless steel. Sight glass near the oil distribution nozzle. An outlet vessel with a cooling jacket is located at the bottom of the packed column deodorizer.

[0057] Final product polishing filter - A second polishing filter is located downstream of the entire process and is used after the batch deodorizer or after the packed column deodorizer, prior to collecting samples for analysis. Supplier: Amafilter group Lochem B.V. (The Netherlands) - Filtration Group. Particle retention: 1 micron. Filtration area: 0.09 m². Material of construction: Stainless steel with Viton gaskets. Bag filter (Polypropylene needle felt from EATON (Belgium))

[0058] Processing aids: Silica gel - JKC-5 and JKC-7 (FIT, The Netherlands);

Bleaching clay - Tonsil 772FF (Clariant Iberia, Spain); Cellulose - Filtracel Active 112 (JRS, Germany).

[0059] A drum of crude Algal oil (190 kg) was obtained from Cargill Incorporated that had been stored frozen at -20°C. The oil was melted using a thermal blanket around the crude oil drum. To minimize contact with air when transferring the crude oil for the experiments, the head space was evacuated and nitrogen sparged continuously through the oil, and a positive pressure of nitrogen maintained in the drum.

[0060] Aluminum bottles pre-flushed with nitrogen and containing an inner plastic cap were used for collecting samples. Samples were collected while flushing the bottles with nitrogen while headspace was kept to a minimum, and stored at -30°C.

Example 2A

[0061] Crude oil (43 Kg) was transferred from the drum into the refining vessel and heated up to 80°C. About 0.5 L of heated crude algae oil was removed from the refining vessel into a 1 L flask under nitrogen, and 1.0 wt% silica (JKC-5) and 0.03 wt% cellulose (Filtracel Active 112), both relative to the initial crude oil mass, was added to the flask. Nitrogen was bubbled through the oil in the flask to minimize contact of air with the oil. Demineralized water (0.4 wt% relative to the initial oil mass) was added to the slurry in the flask. The slurry was then emptied into the refining vessel under nitrogen. The contents in the refining vessel were stirred at 270 rpm for 20 min at atmospheric pressure, and then for 5 min at <2 mbar-a.

[0062] The oil containing silica and cellulose was circulated through the filtration loop (via candle filter) until it was observed to be clear through the sight glass. It was then passed also through a polishing filter and transferred to a storage vessel, which was used as the feed tank for the packed column deodorizer. The oil in the storage vessel was maintained at 40°C with a slight nitrogen pressure.

[0063] The filtered oil from the storage vessel was fed at a flow rate of 20 kg/h to the packed column deodorizer. An inline electric heater upstream the column inlet nozzle heated the oil to a temperature of 190°C. The vacuum in the column was maintained constant at -2.0 mbar-a. Stripping steam (2.0 wt% relative to the oil flow rate) was used in a counter-current mode to facilitate removal of volatile compounds. Oil samples for analysis were collected only when a steady state was reached, as observed by a constant flow and temperature. The deodorized oil was collected in the collection vessel that was cooled to ~50°C and sampled out through a polishing filter. The sample collected is 2A.

Example 2B

[0064] Crude algal oil (30 Kg) was transferred from the drum into the refining vessel. The crude oil was then heated to 80°C. Approximately 2 L of the hot crude algal oil was poured into a 5 L plastic bucket that was flushed with nitrogen. While maintaining a nitrogen atmosphere, 0.5 wt% clay (Tonsil 772FF) and 0.03 wt% cellulose (Filtracel Active 112), both relative to the initial amount of crude oil, was added to the oil in the plastic bucket, with agitation. The slurry was then transferred to the refining vessel under nitrogen. Water (0.5 wt% relative to the initial crude oil mass) was added to the slurry. The mixture in the refining vessel was stirred at 270 rpm for 5 min at atmospheric pressure, and then for 15 min at 150 mbar-a, and a further 5 min at <2 mbar-a. Since filtration of the clay was difficult, more cellulose (2 wt% relative to the initial crude oil mass) was added to the refining vessel to enable a faster filtration rate in the candle filter. The oil was then passed through the candle filter until it was clear as viewed through the sight glass, and then through the polishing filter, when being sent to the batch deodorizer.

[0065] The clay-treated oil was then heated up to the deodorization temperature of

150°C in the batch deodorizer, and the pressure lowered to between 2.4-2.9 mbar-a. Sparge steam (2.0 wt% per hour, relative to the mass of oil in the deodorizer) was supplied once the oil reached 100°C. The heat was shut off after 3 hours and the oil cooled to 60°C before collecting samples for analysis via the final polishing filter. The sample collected is 2B.

Example 2C

[0066] Crude algal oil (40 Kg) was transferred from the drum into the Refining vessel and heated up to 80°C. An oil/cellulose slurry was prepared in a 5 L plastic bucket as previously described, using 2 L crude hot algae oil and 2.0 wt% cellulose (Filtracel Active 112), relative to the initial crude oil mass. Nitrogen was sparged through the oil in the plastic bucket to minimize contact with air. After transferring this slurry to the refining vessel, the mixture in the refining vessel was stirred at 270 rpm for 5 min, and then reduced to 30-60 rpm before commencing filtration. The cellulose treated oil was filtered through the candle filter until visually clear from the sight glass, and then through the polishing filter, when being transferred to the batch deodorizer.

[0067] The cellulose-treated oil was heated in the batch deodorizer to 150°C, under a reduced pressure of 3.2-3.5 mbar-a. Sparge steam was started when the oil reached 100°C. A sparge steam amount of 1.0 wt% per hour (relative to the oil mass in the deodorizer) was used. After 1 hour the oil was cooled down to 100°C before being fed to the packed column deodorizer, while a nitrogen overpressure of -1.1 bar-a was maintained in the batch deodorizer. The oil from the batch deodorizer was fed to the packed column deodorizer at the rate of 20 kg/hr. The packed column deodorizer was operated at a reduced pressure of -2.0 mbar-a. An inline heater upstream the column inlet nozzle was used to heat up the oil entering the packed column deodorizer to 190°C. Stripping steam (2.0 wt% relative to the oil flow rate) was supplied counter current to the flow of the oil. When the process reached a steady state, indicated by a stable flow rate and temperature, the oil exiting the packed column deodorizer was cooled to <60°C and collected. The temperature setting for the column inlet nozzle was then changed to 200°C, and once again samples collected once the process reached the new steady state. The collected samples are labelled 2C(i) (190 °C) and 2C(ii) (200 °C).

Example 2D

[0068] Crude algal oil (40 kg) was transferred from the drum into the refining vessel. The oil was heated up to 120°C. Two liters of the hot oil was transferred to a 5 L plastic bucket under an atmosphere of nitrogen and 0.5 wt% silica (JKC-7) and 1.0 wt% cellulose (Filtracel Active 112), both relative to the initial crude oil mass, were added to the oil in the plastic bucket while sparging nitrogen through the oil. The contents were swirled until the mixture became homogeneous. The slurry was then poured into the refining vessel under nitrogen. The contents in the refining vessel were stirred at 270 rpm for 30 min at atmospheric pressure, and then for 5 min at <2 mbar-a.

[0069] The temperature was reduced to 100 °C in the refining vessel before proceeding with filtration in the candle filter. After filtration completion, the oil is transferred to the batch deodorizer, passing through the polishing filter. The filtered oil in the batch deodorizer was kept at 100°C under reduced pressure (-3.3 mbar-a) for 30 minutes, during which sparge steam (1.5 wt% per hour, based on the oil mass in the deodorizer) was provided.

[0070] The oil from the batch deodorizer was then sent to the packed column deodorizer. The inline heater upstream the column inlet nozzle heated the oil entering the column to 190°C. The oil was supplied at a flow rate of 15 kg/hr. A reduced pressure of -2.0 mbar-a was maintained in the packed column deodorizer. Stripping steam (2.0 wt% relative to the oil flow rate) was provided counter-current to the flow of the oil. Once a steady state was reached, as indicated by a constant flow and temperature, samples for analysis were collected through the final polishing filter. Cooling water was supplied to the collection vessel attached to the bottom of the packed column deodorizer, so sampling could be done at <60 °C. The temperature upstream the column inlet nozzle was then set to 200°C, and samples collected again after the system reached the new steady state. The samples collected were 3D(i) (190 °C) and 3D(ii) (200 °C). Table 4

"ND" as used in table 4 means the presence of designated compound was not detected and the limit of detection was 2.5 ppb. So ND could alternatively interpreted as < 2.5 ppb. "<LOQ" as used in table 4 means the presence of designated compound was not detected above the limit of quantification. The LOQ was 2.5 ppb; so <LOQ could alternatively interpreted as < 2.5 ppb. All samples have a DHA content of between 42.5% and 44.5%.

[0071] It is clear from Table 4 that the mild deodorization of the process described above has a dramatic effect on the level of impurities, reducing them below the limit of detection (ND) or quantification (LOQ). At the same time, however, the level of carotenoids is substantially preserved with a retention of 68.2% to 86.9% allowing the oils to maintain their colored appearance. In contrast, the crude oil contains high levels of the volatile compounds described in Tables 3 and 4 and conventionally refined, bleached and deodorized oil (RBD) has very little or no retained carotenoids.

Volatiles Analysis

Dynamic headspace sampling of algal oils.

[0072] Mildly refined DHA-containing oil samples are loaded onto an auto sampler tray. Thermal desorption tubes packed with Tenax TA® (Gerstel, P/N 013741-005-00) are loaded into the Gerstel Thermal Desorption Unit (TDU) tube tray on the auto sampler. The Gerstel Multi-Purpose Sampler (MPS) transfers the sample vial to the dynamic headspace (DHS) incubator for ten minutes of equilibration at 75 °C. The sample is shaken at 1000 RPM during this equilibration. After equilibration, a TDU tube is loaded into the DHS extractor and the sample is purged with helium gas at a flow of 75 mL/min for a total flow of 1 liter of gas flowing over the sample and through the TDU trap (13.33 min). The extraction temperature is 75 °C and the trap is held at 35 °C during extraction. After the extraction, the TDU tube is transferred to the TDU for desorption and the volatiles are trapped in the cryo-cooled inlet. The TDU temperature program is 35°C (0.5 min) to 300°C (5 min) at 120°C/min in splitless transfer mode. The inlet temperature program is -120°C (0.2 min) to 270°C (5 min) at 12°C/s. The inlet was kept in solvent vent mode setting with helium column flow set to 1.5 niL/min (constant flow). The inlet purge time is 1 sec and the purge flow 30 mL/min. The solvent vent time is setting 30 seconds, solvent vent flow setting of 75 mL/min, solvent vent pressure setting of 12.9 psi. The extracted volatiles are separated and analysed on a LECO ® Pegasus ® 4D 2-Dimensional Gas Chromatograph- Time of Flight Mass Spectrometer (2DGC-TOFMS). A 30 m DB™-624 (0.25 mm x 1.4 μιη) column (Agilent Technologies, P/N122-1334) is used in the first dimension and a 1.5 m BPX™90 (0.25 mm x 0.25μιη) column (SGE Analytical Science, P/N054570) is used in the second dimension. The temperature program for the first dimension column is 40°C (2 min) to 120°C at 5°C/min to 250°C (3 min) at 10°C/min. The second dimension temperature program is the same as the 1st dimension program with a 5 degree offset. The modulator is not used in this method thus running the system in ID mode. The transfer line temperature is 250°C. The mass spectrometer scanned a mass range of 25-550 amu scanning at 50 scans/s. The detector voltage was set to 200V offset to the tune voltage and the source temperature was 225 °C.

Quantification of volatiles from algal oils.

[0073] A calibration curve was made using refined, bleached, and deodorized

(RBD) algal oil. High purity standards of trans-2-pentenal, hexanal, dimethyl disulfide, and 2-methylcyclopentanone were purchased from Sigma. The concentration of each standard in the RBD oil falls below the 2.5 ppb quantification limit. A stock calibration oil was prepared by adding approximately 15 mg of each standard to one 20 mL vial containing 6.5 grams of RBD oil which is capped and mixed to create a stock around 2500 part per million for each standard. The stock oil is serially diluted to form a calibration curve from 0 ppb to ~400ppb. Two grams of each standard is weighed into a vial and extracted and analysed according to the analysis method. A calibration curve is generated by plotting the amount of each compound injected (x-axis) versus the area under the peak for each compound (y- axis). The amount of each compound (in μg) being monitored in a sample is determined by using the regression line equation and solving for x (the amount injected). The

concentration (in ppb) is determined by multiplying the amount of compound injected by the mass of the sample and multiplying by 1000. The sample with very low levels can be interpreted in one of two ways. At very low concentrations the linearity of the calibration curve is not sufficient to allow the sample date to be quantified. Therefore, the

quantification limit is determined to be 2.5 ppb. Samples with levels below this limit of quantification (LOQ) can only be reported as <2.5 ppb or <LOQ. Alternatively, a sample could be analysed and no signal detected and the sample considered below the level of detection (LOD). This would also be below the level of quantification but may be reported as <2.5 ppb or non-detected (ND). The volatiles that were monitored are: trans-2-pentenal (t-2-P); hexanal; dimethyl disulfide (DMDS); 2-methylcyclopentanone (2-MCP).

UV-Vis Method to Measure Carotenoid Concentration in Oil

[0074] The method to measure total carotenoids followed herein is British Standard

684-2.20: 1977 (Methods of analysis of Fats and Fatty oils - Part 2: Other methods - Section 2.20: Determination of carotene in vegetable oils), which is a light absorbance method to measure carotene in vegetable oil.

[0075] The spectrophotometer used in this study was a Genesys 10S UV-Vis

Spectrometer model (Thermo Scientific). The instrument uses a 1 cm path length cuvette. ACS grade Hexane (Fisher) was used for the solvent. Absorbance was measured at 445 nm. The oil to be tested was diluted in hexane. Approximately 0.5-1.0 g of oil was diluted to 25 ml solution with hexane using a volumetric flask. The concentration was recorded as c (g oil per 100 mL solution). Depending on the concentration of carotenoids in the oil, the dilution of the oil in hexane was adjusted so the measured absorbance at 445 nm would fall between 0.1 and 1 absorbance units. The formula to measure carotenoid concentration is:

[0076] Concentration (ppm) = (383 * E )/( L * c)

where E is the absorbance measured at 445 nm (after correcting for the blank); L is the path length of the cuvette (in cm); and c is the concentration of the oil (g oil/100 mL solution).

[0077] Thus, as an example 0.4793 g of mildly refined DHA-containing oil samples was added to a 25 mL volumetric flask. The flask was filled to the 25 mL mark with hexane. The flask was capped and the sample was mixed to ensure a homogeneous solution. A 1 cm path cuvette was filled with the diluted solution. Hexane filled a separate 1 cm path cuvette and was used to blank the UV-Vis spectrometer at 445nm. The oil sample was measured and recorded an absorbance of 0.525 units. The concentration of the oil in hexane c, is (0.4793 g/25ml) * 4 = 1.917 g/lOOml. The carotene concentration is then: (383 * 0.525)/(l * 1.917) = 104.9 ppm carotenoids in the oil.

[0078] Individual carotenoids reported in Table 2 were measured by an outside testing laboratory Eurofins utilizing their internal commercial methods.

Claims

CLAIMS What is claimed is:

1. An oil composition comprising:

i) at least one carotenoid in an amount greater than 50 mg/kg by weight of the oil composition;

ii) a docosahexaenoic acid (DHA) content greater than about 25% of the total weight of fatty acids present in the oil composition; and

iii) less than 80 ppb of trans-2-pentenal (t-2-P), less than 30 ppb of hexanal, less than 1500 ppb of dimethyldisulfide (DMDS), or less than 1500 ppb 2-methylcyclopentanone (2- MCP).

2. The oil composition of claim 1, wherein the composition comprises less than 30 ppb of trans-2-pentenal (t-2-P), less than 10 ppb of hexanal, less than 100 ppb of dimethyldisulfide (DMDS), or less than 100 ppb 2-methylcyclopentanone (2-MCP).

3. The oil composition of claim 2, wherein the composition comprises at least one carotenoid in an amount greater than 75 mg/kg by weight of the oil composition.

4. The oil composition of claim 1, wherein the composition comprises a DHA content of greater than about 40%.

5. The oil composition of claim 4, wherein the composition comprises less than 2.5 ppb of trans-2-pentenal (t-2-P), less than 2.5 ppb of hexanal, less than 2.5 ppb of dimethyldisulfide (DMDS), or less than 2.5 ppb 2-methylcyclopentanone (2-MCP).

6. The oil composition of claim 4, wherein the composition comprises less than 2.5 ppb of trans-2-pentenal (t-2-P), less than 2.5 ppb of hexanal, less than 2.5 ppb of dimethyldisulfide (DMDS), and less than 2.5 ppb 2-methylcyclopentanone (2-MCP).

7. The oil composition of claim 1, wherein the composition is derived from an oil isolated from a biomass.

8. The oil composition of claim 7, wherein the biomass comprises a marine microorganism.

9. The oil composition of claim 8, wherein the marine microorganism is at least one of algae, bacteria, fungi, and protists.

10. The oil composition of claim 9, wherein the marine microorganism is at least one marine microorganism of genus Schizochytrium.

11. The oil composition of claim 1, wherein the at least one carotenoid comprises β- carotene or canthaxanthin.

12. An edible product comprising the oil of claim 1 that is a food, feed, or dietary supplement.

13. The product of claim 12 that is a dietary supplement.