KR20200019111A - Purification of DHA-Containing Oils - Google Patents

Abstract

The various examples shown herein include at least one carotenoid in an amount greater than 50 mg / kg by weight of the oil composition; Docosahexaenoic acid (DHA) content of greater than about 25% of the total weight of fatty acids present in the oil composition; And an oil composition comprising less than 80 ppb trans-2-pentenal (t-2-P), less than 30 ppb hexanal, less than 15 ppb heptane, or less than 1500 ppb dimethyldisulfide (DMDS). will be.

Description

Purification of DHA-Containing Oils

Cross Reference to Related Application

This application claims the priority of US Provisional Patent Application Serial No. 62 / 462,015, filed February 22, 2017, entitled "IMPROVED REFINING OF MICROBIAL OILS," which is incorporated by reference herein in its entirety. Included.

Oils containing long chain polyunsaturated fatty acids LC-PUFAs can be difficult to purify. These oils will be highly sensitive to oxidation and will readily contain oxidation by products that are oily with flavor and aroma. Typical vegetable oil refining is used to purify these oils. For example, seaweed-produced crude docosahexaenoic acid (DHA) -containing oils are typically degumming to remove phospholipids and subsequently purified to remove free fatty acids. Such oils are then bleached and deodorized resulting in the production of final, purified, bleached and deodorized oils (RBD oils). However, due to the high temperatures and duration of these processing conditions, it is only possible to remove oxidation by-products to a certain level during which they can also be produced. In addition, the processing steps to produce the RBD oil remove beneficial ingredients including but not limited to tocopherol, sterols, and carotenoids, including β-carotene, and canthaxanthin.

The oil compositions of the various embodiments described herein are not traditional RBD oils with added beneficial ingredients (eg, carotenoids) again. Instead, the oil compositions of the various embodiments described herein are derived from slightly purified crude DHA-containing oils. The result is that i) contains significant amounts of beneficial components, typically found only in crude DHA-containing oils, and in a minimal / negligible amount in RBD oils, but ii) also in general in crude DHA-containing oils. It is an oil composition containing an undesired odor-induced component in a negligible amount found. Beneficial components include, but are not limited to, carotenoids such as xanthophyll (eg, canthaxanthin) and carotene (eg, β-carotene). Beneficial constituents such as carotenoids are also removed in the RBD oil or destroyed / decomposed in the RBD process. Undesired malodor-causing components include aldehydes (eg trans-2-pentenal (t-2-P), hexanal), ketones (eg 2-methylcyclopentanone (2-MCP) ) And sulfur compounds (eg, dimethyldisulfide, also known as DMDS).

The oil compositions of the various embodiments described herein can be highly colored due to the novel processing utilized herein. Thus, the oil compositions of the various embodiments described herein contain a significant amount of beneficial ingredients, including carotenoids, which provide such oils with higher nutritional value than their color and RBD oils. Because those skilled in the art generally seek RBD oils for human nutrition with little or no color, the oil compositions of the various embodiments described herein are not easy to understand products. In addition, the oil compositions of the various embodiments described herein may be more stable than RBD oils lacking external antioxidants, without the need to add excessive amounts of such antioxidants. Without being bound by any particular theory, the oil compositions of the various embodiments described herein may require increased amounts of external antioxidants, as components that act as built-in antioxidants, such as carotenoids, are removed. It is believed to be more stable than RBD oil.

Some embodiments of the invention therefore comprise more than about 50 mg / kg by weight of the oil composition; Greater than about 25% of the total weight of fatty acids present in the DHA content oil composition; And at least one carotenoid in an amount of DM-2- less than about 80 ppb, t-2-P, less than about 30 ppb hexanal, less than about 100 ppb 2-MCP, or less than about 1500 ppb. .

As used herein, the term "carotenoid" generally refers to tetraterpenoids produced by plants and algae, as well as by some bacteria and fungi. Carotenoids can be prepared from fats and other basic organic metabolic building blocks by all these organisms. Carotenoids are general terms used to refer to more than 600 known carotenoids. Carotenoids can be divided into two classes, xanthophyll and carotene. Examples of carotenoids include β-carotene, canthaxanthin, astaxanthin, lycopperin (7,8,11,12,15,7 ', 8', 11 ', 12', 15'-decahydro-γγ, γ-carotene), phytofluene, hexahydrolycopene (15-cis-7,8,11,12,7 ', 8'-hexahydro-γ, γ-carotene), toluene (3', 4'- Didihydro-β, γ-carotene), α-zeacarotene (7 ', 8'-dihydro-ε, γ-carotene), alloxanthin, cynthiaxanthin, pectenoxanthin, cryptomonaxanthin ((3R, 3 'R) -7,8,7', 8'-tetradihydro-β, β-carotene-3,3'-diol), crustaxanthin (β, -carotene-3,4,3 ', 4 '-Tetrol), gazaniaxanthin ((3R) -5'-cis-β, γ-carotene-3-ol), OH-chlorobacten (1', 2'-dihydro-f, γ-carotene- 1'-ol), lorozanthin (β, ε-carotene-3,19,3'-triol, lutein ((3R, 3'R-6'R) -β, ε-carotene-3,3'- Diol), lycoxanthin (γ, γ-carotene-16-ol), rhodopin (1,2-dihydro-γ, γ-carotene l-ol), rhodopol (also warming / 13- cis- 1 As, 2-dihydro-γ, γ-carotene-1,20-diol Known), including tin sapeu Lausanne (3 ', 4'-Di hydro-1', 2'-dihydro -β, γ- carotene -3,1'- diol) and zeaxanthin, but are not limited to.

The oil compositions of the various embodiments described herein have 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, about 75 mg / kg. greater than 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; In an amount of 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 At least one carotenoid. It is to be understood that the amount of at least one carotenoid is the sum of all carotenoids present in the oil compositions of the various embodiments described herein. Some organisms can even produce oils with much more than 100 mg / kg carotenoids and are also within the scope of the present invention.

Otherwise, the carotenoid content could be expressed as the total, or individual percentage, of the remaining carotenoids in the oil after the reduction in volatile components. In some embodiments more than 50, 60, 70, 80, or 90% of the total, or individual, carotenoids present in the crude oil are retained after the volatile component is reduced.

In some embodiments, the oil compositions of the various embodiments described herein comprise at least 2, at least 3, at least 4 or at least 5 carotenoids. In some embodiments, oil compositions of various embodiments described herein include at least β-carotene, and canthaxanthin. In some embodiments, oil compositions of the various embodiments described herein are at least in amounts of about 50 mg / kg to about 60 mg / kg β-carotene and about 20 mg / kg to about 30 mg / kg canthaxanthin β-carotene and canthaxanthin.

The oil compositions of the various embodiments described herein comprise greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50% of the total weight of fatty acids present in the oil composition. , Greater than about 55%; It also has about 30% to about 50%, or about 25% to about 60%, or 25% to about 70%, or about 40% to about 50% docosahexaenoic acid (DHA) content. In some embodiments, the DHA is substantially in the form of triglycerides. The DHA content of the oil can be readily determined by well-known methods in the art. For example the AOCS methods Ce-2b-11 and Ce1i-07 and Ce-1b-89 have been utilized herein.

The oil compositions of various embodiments described herein are relatively low in odor-inducing components such as trans-2-pentenal (t-2-P), heptanal, DMDS, and 2-methylcyclopentanone (2-MCP). It may have a content.

For example, the oil compositions of various embodiments described herein can be 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 a t-2-P content of about 2.5 ppb to about 10 ppb. Various embodiments may also have levels that are below the detection limit or below the limit of quantitation of the methods utilized herein.

Oil compositions of various embodiments described herein include 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, about 2.5 ppb; About 2.5 ppb to less than about 20 ppb; Or from about 2.5 ppb to about 10 ppb of hexanal content. Various embodiments may also have levels that are below the detection limit or below the limit of quantitation of the methods utilized herein.

The oil compositions of various embodiments described herein include less than about 2000 ppb, less than 1500 ppb, less than 1000 ppb, less than about 500 ppb, about 400, less than about 200 ppb, less than about 100 ppb, less than about 90 ppb, about 80 less than about 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 from about 2.5 ppb to about 10 ppb. Various embodiments may also have levels that are below the detection limit or below the limit of quantitation of the methods utilized herein.

In some embodiments, the oil compositions of the various embodiments described herein comprise about 2000 ppb, less than 1500 ppb, less than 1000 ppb, less than about 500 ppb, about 400, less than about 200 ppb, less than about 100 ppb, about Less than 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, about Less than 2.5 ppb; About 2.5 ppb to about 20 ppb; Or from about 2.5 ppb to about 10 ppb. Various embodiments may also have levels that are below the detection limit or below the limit of quantitation of the methods utilized herein.

The oil compositions of various embodiments described herein include about 1 wt.%, Less than about 0.5 wt.%, Less than about 0.2 wt.%, Less than about 0.1 wt.%; From about 0.01 wt.% To less than about 0.5 wt.%; Between about 0.01 wt.% And about 0.2 wt.%; Or from about 0.01 wt.% To about 0.03 wt.% Total volatiles content. As used herein, “total volatility” refers to the sum of the following weight percentages: hexanal, t-2-P, DMDS, and 2-MCP.

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

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

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

The oil compositions of the various embodiments described herein can be prepared using methods derived from oils isolated from biomass and known in the art. Such methods include separating oil from biomass including marine microorganisms. 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 preparation and separation of oil compositions of the various embodiments described herein include microalgae and chromogenic algae, as described in the filed PCT Application No. WO94 / 28913, which is like Included by reference as fully described in PCT Application No. WO94 / 008467; US Patent No. 5,908,622; US Patent No. 5,688,500; US Patent No. 5,518,918; US Patent No. 5,340,742; US Patent No. 5,340,594; US Patent No. 8,163,515; US Patent No. 9,023,616; Reference is also made to European application numbers 512997 and 669809, both of which are incorporated by reference as if fully set forth herein.

Any organism known to prepare a DHA- containing oil is, for example, a eukaryotic cis-isopropyl Cri (isochrysis) is Ivana (gaibana) and thraustochytrids (e.g., marine bacteria in the kit seukijo Solarium (schizochytrium)), for example Solarium seukijo Kit ( schizochytrium) species and teurawooseu talkie including yttrium (thraustochytrium) Ow reum (aureum), but not limited to, can be used in the fermentation of the oil described herein. See, for example , 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 will recognize that the production of DHA from marine organisms through fermentation has been described extensively in the literature since the late 1980s. Numerous manufacturing organisms and methods are well known. A specific example of useful organisms including: teurawooseu talkie yttrium (thraustochytrium) species ATCC 26185, and seukijo kit Solarium (schizochytrium) species ATCC 20888, but is not limited thereto.

After the fermentation is complete, the obtained cells can be dried to a suitable moisture content (eg, about 4% moisture content). Suitable non-polar solvents (e.g., pentane, hexane, etc.) are then added to the dried biomass in a suitable container (e.g. glass bottle) and at a suitable time and temperature (e.g. at 25 ° C). 2 hours). Filtration of the biomass The solvent in the filtrate can then be removed by any suitable means (eg, rotary evaporator) to produce crude DHA-containing oil. Otherwise, the crude oil can be separated through solvent free processes that are well known in the art. See, eg , US 9,745,538 or US 9,745,539; Both are incorporated herein by reference.

It is well understood by those skilled in the art that the levels of DHA, carotenoids, and various volatile compounds and impurities present in the crude oil are elements of the specific manufacturing parameters used in fermentation, separation, and refining. The starting value of these various components will thus vary from lots of crude oil.

The oil compositions of various embodiments described herein are derived from oils isolated from biomass, such as crude DHA-containing oils. The crude DHA-containing oil is then slightly purified to obtain oil compositions of the various embodiments described herein. The oil compositions of the various embodiments described herein are not traditional RBD oils with added beneficial ingredients (eg, carotenoids) again. Instead, the oil compositions of the various embodiments described herein are derived from slightly purified crude DHA-containing oils.

As used herein, the term "slightly purified" generally means that the crude DHA-containing oil is not purified to the extent that an RBD oil is obtained. Instead, i) retains significant amounts of beneficial components, typically found only in crude DHA-containing oils and in a minimal / negligible amount in RBD oils, and ii) generally found in crude DHA-containing oils. Substantially eliminating undesired malodor or flavor causing components; The crude DHA-containing oil is purified only to the extent necessary to obtain the oil composition.

Pretreatment

Crude oils isolated from natural sources sometimes contain "gum" consisting primarily of phospholipids, and also contain free fatty acids, sterols, sterol esters, trace metals, trace carbohydrates and proteins, and solid particles. In some cases, the oil also contains antioxidants such as tocopherol and carotenoids. Removing phospholipids from the oil prevents the formation of gum deposits in the refining process and prevents the development of flavor and color release during storage. Otherwise, the crude oil may contain small particles that aggregate and create problems in subsequent processing. While optional for the purposes described herein, pretreatment steps such as degumming or filtration can be used to avoid problems in further processing.

Water degumming is generally used to remove phospholipids and other water soluble components from oils. A common process involves treating crude oil (eg crude DHA-containing oil) with 250-2000 ppm phosphoric acid or citric acid at 60-90 ° C. with stirring. Water (eg 1-5%) is then added to the crude oiled acid at 60-75 ° C. with agitation to help hydrate the phospholipids in the crude oil. The oil is then mixed gently for another 15-60 minutes. Together with some splash entrained oils, it forms an aqueous phase consisting of an emulsion of hydrated phospholipids and other water-soluble compounds. The two phases are separated from each other by precipitation and decantation, or by centrifugation to obtain a stream of acid degumming oil and a stream of wet gum. The neutralization step using an aqueous solution of base can be used to remove residual acid, as well as to remove the oil of the soluble salt in one or more extraction steps with deionized water.

In some embodiments the crude DHA-containing oil is optionally degumming prior to mild deodorization using a suitable aqueous acid such as phosphoric acid or citric acid. In another embodiment the crude DHA-containing oil is initially washed with water prior to mild deodorization. The oils of the various embodiments described herein, however, are not bleached under traditional conditions for processing vegetable oils.

In addition, crude oil may be optionally filtered prior to mild deodorization to remove particulate matter. Filtration by the general method is well known. As part of the integration process, it may be carried out in batch mode or continuously. Filter aids (including but not limited to cellulose, diatomaceous earth, commercially available clays, or silica gels) or other processing aids may be used to remove unwanted particles or dissolved material. These auxiliaries can additionally be utilized to increase the ease and effectiveness of filtration. Filtration can be performed at elevated temperatures and oil can be contacted with the filter aid for an extended time to assist in the absorption or adhesion of particles onto the filter aid.

Deodorization

Briefly, mild deodorization removes volatile compounds that cause "flavor and malodor release" but the process can also remove free fatty acids, and some tocopherols and sterols. The process is often performed under vacuum to help remove certain volatile compounds and to protect the oil from oxidation. Steam, nitrogen or another inert gas can be used as the stripping agent.

The process is fully defined by temperature, time, and pressure. When performed on a commercial scale, it may include a multi-step process including defoaming of the oil, multi-step heating, deodorization-deoxidation, and multi-step cooling. If defoaming is carried out, it can be achieved in a separate vessel connected to a vacuum system (eg 30-50 mm Hg) or even at a lower pressure in the deodorant. Injection steam (or any other inert gas) can be used to improve defoaming.

The process can be carried out batchwise, in a semi-continuous system, or in a continuous system. Stripping efficiency may be better in continuous systems that generally have columns filled with structured packages that provide a high surface area. Backflow contact of the stripping agent and the oil on the structured package can provide efficient stripping in a short contact time. Various configurations of the apparatus can be used (horizontal or vertical vessels, tray-type columns, packed columns or thin film / wiped film evaporators / distillers).

The oil compositions of the various embodiments described herein are obtained by treating the crude DHA-containing oil under a mild deodorization process utilizing a suitable device as described herein.

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

Process for the preparation of purified PUFA oil comprising passing crude PUFA oil through a packed column: wherein the column is heated between 140 and 200 ° C and steam is fed into the column in countercurrent to the flow of crude PUFA oil. And the purified oil is collected from the column.

Oil composition

Ultimately, the oil compositions of the various embodiments described herein, derived from oils isolated from biomass by the weak refining process described herein, have a total volatile content, DHA, carotenoid content, t-2-P content. One or more or all of the values described herein in the hexanal content, DMDS content, and 2-MCP content. In some embodiments, the slightly purified oil isolated from the biomass, therefore, has a total volatiles content of about 0.01 wt.% To about 0.5 wt.%; DHA content of about 25% to about 60%; Total carotenoids content in slightly refined oil isolated from biomass, greater than about 70 mg / kg; T-2-P content of about 0.5 ppb to about 3 ppb; Hexanal content of about 0.5 ppb to about 3 ppb; Heptanal content of about 0.5 ppb to about 3 ppb; 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.

As mentioned herein, known DHA-containing oils used in human nutrition are fully purified using typical vegetable oil techniques to produce highly refined RBD oils. Refining removes volatile compounds, metals, and pigments. However, the process removes ingredients that are beneficial to human nutrition, such as carotenoids, as well as creating the need for high levels of antioxidants to preserve the oil. The oil compositions of the various embodiments described herein, derived from oils separated from biomass by the weak refining process described herein, have a low content of malodor-causing compounds and at the same time retain beneficial ingredients such as carotenoids. The presence of carotenoids can be attributed to antioxidants (e.g., γ tocopherol (vitamin E), tocotrienol, ascorbic acid (vitamin C), retinol (vitamin A), tertiary butylhydroquinone (TBHQ), butylated hydroxyanisole ( Although BHA), and butylated hydroxytoluene (BHT)) can be added to the oil compositions of the various embodiments described herein, this leads to more nutritious or stable products without the need for additional antioxidants.

The compositions described herein can be used in food, dietary supplement, and feed applications. A further embodiment of the invention is a food product comprising the oils described herein. The food may be delivered in any manner intended for infants or adults and known to those skilled in the art. Alternatively, the compositions described herein may be formulated for consumption as a dietary supplement in the form of a liquid, powder, or capsule.

Values expressed in range form not only include explicitly stated numbers such as range limitations, but also all individual numbers or sub-ranges within which the respective values and sub-ranges are contained within such ranges as explicitly stated. Should be interpreted in a flexible manner, including: For example, the range of "about 0.1% to about 5%" or "about 0.1% to 5%", as well as about 0.1% to about 5%, as well as individual values ( eg , 1%, 2%, 3%, and 4%) and sub-ranges ( eg , 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated ranges. The state "about X to Y" has the same meaning as "about X to about Y," unless otherwise indicated. Likewise, state "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z," unless otherwise indicated.

In this document, the terms "a," "an," or "the" are used to include one or more unless the context clearly dictates otherwise. The term "or" is used to denote "or" which is not exclusive unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation, unless defined otherwise. Any use of section headings is intended to assist in reading the document and should not be construed as limiting. In addition, information related to section headings may occur within or outside of that particular section. In addition, all publications, patents, and patent documents mentioned in this document are incorporated by reference in their entirety herein, even if incorporated individually by reference. In the case of inconsistent usage between this document and the document incorporated by reference, the usage incorporated by reference should be considered as a supplement to the usage of this document; For inconsistencies that cannot be resolved, the content of this document is controlled.

In the methods described herein, the steps may be performed in any order without departing from the principles of the present invention, except when the transient or operational order is explicitly stated. In addition, the specified steps may be performed simultaneously unless stated to be performed separately. For example, the claimed step of performing X and the claimed step of performing Y may be performed simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

The term "about," as used herein, may enable specified limits of a specified value or range of degrees of variability within a value or range, eg, within 10%, within 5%, or within 1%. . In addition, all ranges mentioned herein may otherwise be read without a water meter "about." Thus, if a range is specified as "about X to about Y", we specifically consider the exact range of "X to Y."

The term "substantially" as used herein, refers to at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9 Majority, or most, such as%, 99.99%, or at least about 99.999% or more.

Example

The invention may be better understood by reference to the following examples, which are provided by way of illustration. The invention is not limited by the examples given herein.

Methods for the production and processing of DHA oils from microalgae and pigmented algae are well known in the art for over 30 years as described above. Otherwise, crude DHA-containing seaweed oil can be purchased from Cargill Incorporated. Additional commercial suppliers of crude seaweed oils are also known to those skilled in the art.

Example  One:

material

Crude with citric acid DHA -Oil refining

The 1 L jacketed flask with 4-port headpiece was equipped with a direct drive overhead stirrer and the heated circulation bath was set to 60 ° C. The vessel was kept under nitrogen for the duration of the experiment. The flask was filled with 750 mL of crude algae oil. The mechanical stirrer was turned on and set to 100 RPM. The oil was equilibrated at 60 ° C. for 30 minutes. 500 ppm citric acid solution was prepared in degassed, deionized water. 37.5 mL (5% of the amount of seaweed oil) of 500 ppm citric acid solution was added to the vessel. The material was stirred at 60 ° C. for 30 minutes. The stirrer was turned off and the aqueous layer was separated from the seaweed 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 at 60 ° C. for 10 minutes. The stirrer was turned off and the aqueous layer was separated from the seaweed 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 at 60 ° C. for 10 minutes. The stirrer was turned off and the aqueous layer was separated from the algal oil and removed. Seaweed oil was transferred to a 250 mL centrifuge bottle under nitrogen. The oil was centrifuged at 30 ° C. and 3000 RPM for 10 minutes and separated from the remaining aqueous layer. The oil was transferred to a glass storage vessel, headspace flushed with nitrogen and stored in the freezer at -9 ° C.

Crude without citric acid DHA -Oil refining

The 1 L jacketed flask with 4-port headpiece was equipped with a direct drive overhead stirrer and the heated circulation bath was set to 60 ° C. The vessel was kept under nitrogen for the duration of the refining step. The flask was filled with 750 mL of crude algae oil. The mechanical stirrer was turned on and set to 100 rpm. The oil was 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 at 60 ° C. for 10 minutes. The stirrer was turned off and the aqueous layer was separated from the seaweed oil. The aqueous layer was removed. Seaweed oil was removed and placed under nitrogen in a 250 mL centrifuge bottle. The oil was centrifuged at 30 ° C. and 3000 RPM for 10 minutes and separated from the remaining aqueous layer. The oil was transferred to a glass storage vessel, headspace flushed with nitrogen and stored in the freezer at -9 ° C.

Deodorization

(i) degassing apparatus with variable speed drive mechanism (wetted part 316 stainless steel), glass inner condenser, 2000 mL glass graduation feeder and stopper; (ii) a rotatable, multiple receiver flask comprising four 200 mL receiver flasks; (iii) 500 mL distillate receiver flask, glass cold trap, 316 stainless steel wiper retainer and carbon wiper blade; (iv) a metal band heater set (part no. 40782-G1) having a high temperature “J” thermocouple assembly and an insulating jacket to maintain the desired temperature; (v) a silicone rubber heater and thermocouple (part number 40645) and an insulating jacket (part number 40669) to maintain the temperature of the feed material; (vi) an Edwards mechanical rotary vacuum pump / oil diffusion pump system to provide a vacuum during experimental operation; (vii) Pirani-type gauges ranging from 0.001 Torr to 1000 Torr; (viii) a digital RPM indicator for providing constant monitoring of variable speed drive; And (ix) Pope Scientific Inc. with Cole-Palmer chillers to maintain the temperature of the internal condenser. 2 "wipe (wiped) -film evaporator (part number 40450-01).

The feed and degassing assembly was filled with purified crude DHA-containing oil (purified with or without citric acid). The entire system was kept under nitrogen for the duration of operation. The feed assembly was maintained at 45 ° C. Pure nitrogen was used to degas the feed material for 1 hour. The internal condenser of the wiped-film evaporator was kept at 2 ° C. over the cooler for all deodorization operations. The variable speed drive mechanism of the evaporator was set at a constant speed of 400 RPM. The feed rate for the deodorant was 175 mL / hr throughout the experimental run. Deodorization temperature and pressure were varied to assess the deodorization conditions while maintaining the carotenoid content. Samples were obtained throughout the operation at the desired conditions. Samples were transferred to storage vessels, headspace flushed with nitrogen and stored frozen until analyzed.

Process conditions for refining and deodorization are shown in Table 1 below:

The slightly purified DHA-containing oil (Samples 1A-1F) had the features shown in Table 2.

The slightly purified 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.

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

** a β- carotene and Khan Tarzan tin, respectively, the specified method EN 12823-2: 2000 and according to the Roche index n _o 2264, was determined by an external laboratory, Eurofins

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

Example  2:

Refining vessel-Pretreatment of the crude oil was carried out with various process aids using the refining vessel. Different combinations of clay, silica, and cellulose were tested. The mixer attached on top allowed mixing / stirring of the contents. Supplier: HAB Heiland Apparatebau (Germany). Form: vertical cylindrical, conical bottom with viewing window. Volume: 40 L (inside) with 5.68 L (jacket). Materials of Construction: Stainless Steel. Equipped with heating jacket, stirrer with variable speed (propeller type, two wings), baffle plate, spray nozzle for returning oil back from the filtration loop.

Candle Filter-Perform filtration of oil from the refining vessel using a candle filter with a multifilament cloth. When the cake is laminated to the cloth and the oil downstream of the filter is transparent (visually observed from the see-through window), the transfer operation to the batch deodorant begins. Supplier: Amafilter group Lochem BV (Netherlands)-Filtration group. Form: vertical cylindrical. Volume: 8 L. Filtration area: 0.05 m ² . Construction material: stainless steel with Viton (gasket); Polyethylene (filter cloth).

Abrasive Filter—Place the abrasive filter downstream of the candle filter as the oil is transferred from the refining vessel to the batch deodorant. This filter removes any particles not held by the candle filter. Supplier: Amafilter group Lochem BV (Netherlands)-Filtration group. Particle Retention: 1 micron. Filtration area: 0.09 m ² . Materials of Construction: Stainless steel with Viton gasket. Bag Filter (Polypropylene Needle Felt from EATON (Belgium))

Batch Deodorant-Intended container for performing a batch deodorant operation. The vapor (or nitrogen) can be continuously injected through the oil, typically applying a reduced pressure of ˜2 mbar-a. Supplier: HAB Heiland Apparatebau (Germany). Form: vertical cylindrical, flat bottom. Volume: 31 L (internal); 5.5 L (jacket). Materials of Construction: Stainless Steel. Equipped with heating jacket, jet steam loop, and sight glass.

 Packed Column Deodorant-Packed column deodorant consists of structuring a packed bed that can accommodate different types of packages and heights. Raschig Superpack RSP 250X was used as packaging material. The oil is heated in an in-line electric heater and sprayed onto the packing bed at the top of the column to ensure good oil distribution and intimate contact with the stripping agent. Stripping steam (or nitrogen) is introduced to the bottom of the column, countercurrent to the flow of oil. Designed by Cargill and manufactured by VGM (Netherlands). Form: vertical cylindrical. Structured packaging: Raschig Superpack RSP 250X; Diameter: 255 mm; Height: 708 mm. Capacity (oil process): 15-25 kg / h. Materials of Construction: Stainless Steel. Viewing window near oil distribution nozzle. At the bottom of the packed column deodorant was placed an outlet vessel with a cooling jacket.

Final Product Abrasive Filter—The second abrasive filter is placed downstream of the overall process and used after batch deodorant or after packaged column deodorant prior to collecting samples for analysis. Supplier: Amafilter group Lochem BV (Netherlands)-Filtration group. Particle Retention: 1 micron. Filtration area: 0.09 m ² . Materials of Construction: Stainless steel with Viton gasket. Bag Filter (Polypropylene Needle Felt from EATON (Belgium))

Processing aids: silica gel-JKC-5 and JKC-7 (FIT, Netherlands); Bleached clay-Tonsil 772FF (Clariant Iberia, Spain); Cellulose-Filtracel Active 112 (JRS, Germany).

A drum of crude seaweed oil (190 kg) was obtained from Cargill Incorporated frozen at -20 ° C. The oil was melted using a thermal blanket around the crude oil drum. To minimize contact with air when moving the crude oil for the experiment, the head space was vacuumed, nitrogen was continuously injected through the oil, and the positive pressure of nitrogen was maintained in the drum.

To collect the sample, an aluminum bottle pre-flushed with nitrogen and containing an inner plastic cap was used. Samples were collected while flushing the bottles with nitrogen while keeping headspace to a minimum and stored at -30 ° C.

Example  2A

Crude oil (43 Kg) was transferred from the drum into the refining vessel and heated to 80 ⁰ C. About 0.5 L of heated crude algal oil was removed from the refining vessel under nitrogen into a 1 L flask, both with respect to the initial crude oil mass, 1.0 wt% silica (JKC-5) and 0.03 wt% cellulose (Filtracel). Active 112) was added to the flask. Nitrogen was bubbled through the oil in the flask to minimize oil-to-air contact. Desalted water (0.4 wt% relative to the initial oil mass) was added to the slurry in the flask. The slurry was then emptied into a refining vessel under nitrogen. The contents in the scouring vessel were stirred at 270 rpm for 20 minutes at atmospheric pressure and then for 5 minutes at <2 mbar-a.

The oil containing silica and cellulose was circulated through a filtration loop (via a candle filter) until it was transparently seen through the viewing window. It was then passed through the abrasive filter and transferred to a storage container, which was used as a feed tank for packed column deodorant. The oil in the storage vessel was maintained at 40 ° C. with slight nitrogen pressure.

The oil filtered from the storage vessel was fed to the packed column deodorant at a flow rate of 20 kg / h. The column inlet nozzle upstream in-line electric heater heated the oil to a temperature of 190 ° C. The vacuum in the column was kept constant at ˜2.0 mbar-a. Stripping steam (2.0 wt% with respect to oil flow rate) was used in countercurrent mode to facilitate removal of volatile compounds. Only oil samples for analysis were collected when steady state was reached, as observed by constant flow and temperature. Deodorized oil was collected in a collection vessel cooled to ˜50 ^O C and sampled through a polishing filter. The collected sample is 2A.

Example  2B

Crude seaweed oil (30 Kg) was transferred from the drum into a refining vessel. The crude oil was then heated to 80 ° C. Approximately 2 L of hot crude seaweed oil was injected into a 5 L plastic bucket flushed with nitrogen. While maintaining a nitrogen atmosphere, 0.5 wt% clay (Tonsil 772FF) and 0.03 wt% cellulose (Filtracel Active 112) were added to the oil in the plastic buckets with stirring, both with respect to the initial amount of crude oil. The slurry was then transferred to a 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 minutes at atmospheric pressure, and then at 150 mbar-a for 15 minutes and at <2 mbar-a for an additional 5 minutes. Since filtration of clay is very difficult, more cellulose (2 wt% relative to the initial crude oil mass) was added to the refining vessel to enable faster filtration rates in the candle filter. Then, when sent to the batch deodorant, the oil was passed through the candle filter and then through the abrasive filter until it was transparent through the viewing window.

The clay-treated oil was then heated to a deodorization temperature of 150 ° C. in a batch deodorant and the pressure was lowered between 2.4-2.9 mbar-a. Injection steam (2.0 wt% per hour, relative to the mass of oil in the deodorant) was fed when the oil reached 100 ° C. After 3 hours the heat was shut off and the oil was cooled to 60 ° C. to collect samples for analysis through the final polishing filter. The collected sample is 2B.

Example  2C

Crude seaweed oil (40 Kg) was transferred from the drum into the refining vessel and heated to 80 ° C. The oil / cellulose slurry was prepared in a 5 L plastic bucket, using 2.0 wt% cellulose (Filtracel Active 112), with respect to 2 L crude hot algae oil and initial crude oil mass, as previously described. Nitrogen was injected 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 minutes and then reduced to 30-60 rpm before starting filtration. When transferred to the batch deodorant, the cellulose treated oil was filtered through the candle filter and then through the abrasive filter until it was visually clear from the viewing window.

The cellulose-treated oil was heated to 150 ° C. in a batch deodorant under reduced pressure of 3.2-3.5 mbar-a. Injection steam started when the oil reached 100 ° C. Amount of spray steam of 1.0 wt% per hour (relative to the mass of oil in the deodorant) was used. After 1 hour, the oil was cooled to 100 ° C. while maintaining a nitrogen overpressure of ˜1.1 bar-a in the batch deodorizer before feeding to the packed column deodorant. Oil from the batch deodorant was fed to a packed column deodorant at a rate of 20 kg / hr. The packed column deodorant was operated at a reduced pressure of ˜2.0 mbar-a. The oil entering the packaged column deodorant was heated to 190 ° C using a column inlet nozzle upstream inline heater. Stripping steam (2.0 wt% with respect to oil flow rate) was fed countercurrent to the flow of oil. The process, indicated by a stable flow rate and temperature, cooled and collected the oil exiting the packed column deodorant to <60 ° C. when steady state was reached. The temperature setting for the column inlet nozzle was then changed to 200 ° C. and the sample was collected once again when the process reached a new steady state. The collected samples were labeled with 2C (i) (190 ° C) and 2C (ii) (200 ° C).

Example  2D

Crude seaweed oil (40 Kg) was transferred from the drum into a refining vessel. The oil was heated to 120 ° C. 2 L of hot oil was transferred to a 5 L plastic bucket under an atmosphere of nitrogen, both with 0.5 wt% silica (JKC-7) and 1.0 wt% cellulose (Filtracel Active 112), relative to the initial crude oil mass, Nitrogen was sprayed through the oil and added to the oil in the plastic bucket. The contents were pivoted until the mixture was homogeneous. The slurry was then injected into the refining vessel under nitrogen. The contents in the refining vessel were stirred at 270 rpm for 30 minutes at atmospheric pressure and then for 5 minutes at <2 mbar-a.

The temperature was reduced to 100 ° C. in the refining vessel before filtration in the candle filter was allowed to proceed. After completion of filtration, the oil was transferred to a batch deodorant, which passed through the abrasive filter. The filtered oil in the batch deodorant was kept at 100 ° C. for 30 minutes under reduced pressure (˜3.3 mbar-a) while providing spray vapor (1.5 wt% per hour based on the mass of oil in the deodorant). .

The oil from the batch deodorant was then sent to the packed column deodorant. The column inlet nozzle upstream inline heater heated the oil entering the column to 190 ° C. The oil was fed at a flow rate of 15 kg / hr. A reduced pressure of ˜2.0 mbar-a was maintained in the packed column deodorant. Stripping steam (2.0 wt% with respect to oil flow rate) was provided in countercurrent to the flow of oil. When reaching steady state, samples were collected for analysis through the final polishing filter, as indicated by constant flow and temperature. Sampling was performed at <60 ° C by supplying cooling water to a collection vessel attached to the bottom of the packed column deodorant. The column inlet nozzle upstream temperature was then set to 200 ° C. and samples were collected again after the system reached a new steady state. Samples collected were 3D (i) (190 ° C) and 3D (ii) (200 ° C).

"ND" as used in Table 4 means that the presence of the designated compound was not detected and the detection limit was 2.5 ppb. Thus ND can otherwise be interpreted as <2.5 ppb. "<LOQ" as used in Table 4 means that the presence of the specified compound was not detected above the limit of quantitation. LOQ was 2.5 ppb; Thus, <LOQ could otherwise be interpreted as <2.5 ppb. All samples have a DHA content between 42.5% and 44.5%.

Weak deodorization of the process described above has a dramatic effect on the level of impurities and it will be clear from Table 4 that they are reduced below the detection (ND) or quantitative (LOQ) limit. At the same time, however, the levels of carotenoids are substantially preserved with a retention of 68.2% to 86.9%, which enables the oils to maintain their colored appearance. In contrast, crude oils contain high levels of the volatile compounds described in Tables 3 and 4 and conventionally refined, bleached and deodorized oils (RBD) have retained carotenoids with very little or no.

Volatility Analysis

Dynamic of Seaweed Oil Headspace  sampling.

Slightly purified DHA-containing oil sample is loaded onto an automatic sampler tray. Thermal desorption tubes packed with Tenax TA® (Gerstel, P / N 013741-005-00) are loaded into a Gerstel Thermal Desorption Unit (TDU) tube tray on an automatic sampler. The Gerstel Multi-Purpose Sampler (MPS) moves sample vials into a dynamic headspace (DHS) incubator for 10 minutes at equilibrium at 75 ° C. Samples are shaken at 1000 RPM during this equilibrium. After equilibration, the 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 on the sample and through the TDU trap (13.33 min). The extraction temperature is 75 ° C and the trap is kept at 35 ° C during extraction. After extraction, the TDU tube was moved to the TDU for desorption and volatiles were trapped in the cold-cooled inlet. The TDU temperature program is from 35 ° C (0.5 minutes) to 300 ° C (5 minutes) at 120 ° C / min in non-isolated transfer mode. The inlet temperature program is from -120 ° C (0.2 minutes) to 270 ° C (5 minutes) at 12 ° C / s. The inlet was maintained at the solvent discharge mode setting with a helium column flow (constant flow) set at 1.5 mL / min. The inlet purge time is 1 second and the purge flow is 30 mL / minute. The solvent discharge time is set at 30 seconds, the solvent discharge flow is set at 75 mL / min, and the solvent discharge pressure is set at 12.9 psi. The extracted volatiles are separated and analyzed on a LECO® Pegasus® 4D 2-D Gas Chromatograph-Time Flight Mass Spectrometer (2DGC-TOFMS). 30 m DB®-624 (0.25 mm x 1.4 μm) column (Agilent Technologies, P / N122-1334) in one dimension and 1.5 m BPX®90 (0.25 mm x 0.25 μm) column (SGE Analytical Science, P / N054570) is used in two dimensions. The temperature program for a one-dimensional column ranges from 40 ° C. (2 minutes) to 5 ° C./minute to 120 ° C. and 10 ° C./minute to 250 ° C. (3 minutes). The two-dimensional temperature program is identical to the five degree offset one-dimensional program. The modulator in this method is not used and therefore operates the system in 1D mode. The moving line temperature is 250 ° C. The mass spectrometer scanned a mass range of 25-550 amu scanned at 50 scans / s. The detector voltage was set at a 200V offset to the regulated voltage and the source temperature was 225 ° C.

Determination of Volatile Compounds from Seaweed Oil.

Calibration curves were made using purified, bleached, and deodorized (RBD) seaweed 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 is under the 2.5 ppb quantification limit. The storage calibration oil is added by adding approximately 15 mg of each standard to one 20 mL vial containing 6.5 g of RBD oil, capping and mixing to produce about 2500 ppm stock for each standard. Manufactured. The stock oil was serially diluted to form a 0 ppb to 400 ppb calibration curve. 2 g of each standard were weighed into vials and extracted and analyzed according to the analytical method. The calibration curve was generated by plotting the amount of each compound (x-axis) injected versus the area under the peak for each compound (y-axis). The amount (μg) of each compound monitored in the sample is determined by using a regression equation and calculating x (injected amount). The concentration (ppb) is determined by multiplying the mass of the sample by the amount of compound injected and by multiplying by 1000. Samples with very low levels can be interpreted in one of two ways. The linearity of the calibration curve at very low concentrations is not sufficient to enable quantifying sample dates. Therefore, the limit of quantitation is determined to be 2.5 ppb. Samples with levels below this limit of quantitation (LOQ) can be reported as <2.5 ppb or <LOQ only. Otherwise, the sample may be analyzed and no signal detected, and the sample was considered below the level of detection (LOD). It is also below the level of quantitation but can be reported as <2.5 ppb or non-detection (ND). Volatile monitored were: trans-2-pentenal (t-2-P); Hexanal; Dimethyl disulfide (DMDS); 2-methylcyclopentanone (2-MCP).

In oil Carotenoids  UV- to measure the concentration Vis  Way

The method for measuring total carotenoids according to the present specification 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 vegetable oil Light absorption method for measuring carotene.

The spectrophotometer used in this study is a Genesys 10S UV-Vis spectrometer model (Thermo Scientific). The instrument uses a 1 cm path length cuvette. ACS grade hexane (Fisher) was used as 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 in a 25 ml solution with hexanes using a volumetric flask. The concentration was reported as c (g oil per 100 mL solution). Depending on the concentration of the carotenoids in the oil, the dilution of the oil in the hexanes was adjusted so that the absorbance measured at 445 nm was between 0.1 and 1 absorbance units. The equation for measuring carotenoid concentration is:

Concentration (ppm) = (383 * E) / (L * c)

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

Thus, 0.4793 g of slightly purified DHA-containing oil sample was added to a 25 mL volumetric flask as an example. The flask was filled with hexanes up to the 25 mL mark. The flask was capped and the samples mixed to ensure a homogeneous solution. 1 cm route cuvettes were filled with diluted solution. Hexane was filled with a separate 1 cm path cuvette and used to empty the UV-Vis spectrometer at 445 nm. The oil sample was measured and the absorbance in 0.525 units recorded. The concentration of oil in hexane c is (0.4793 g / 25 ml) * 4 = 1.917 g / 100 ml. The carotene concentration is then: (383 * 0.525) / (1 * 1.917) = 104.9 ppm carotenoids in oil.

The individual carotenoids reported in Table 2 were measured by their external commercial laboratory Eurofins utilizing their internal commercial methods.

Claims (13)

Oil composition comprising:
i) at least one carotenoid oil in an amount greater than 50 mg / kg by weight of the composition;
ii) a docosahexaenoic acid (DHA) content of greater than about 25% of the total weight of fatty acids present in the oil composition; And
iii) less than 80 ppb trans-2-pentenal (t-2-P), less than 30 ppb hexanal, less than 1500 ppb dimethyl disulfide (DMDS), or less than 1500 ppb 2-methylcyclopentanone (2- MCP).
The composition of claim 1, wherein the composition has less than 30 ppb of trans-2-pentenal (t-2-P), less than 10 ppb of hexanal, less than 100 ppb of dimethyl disulfide (DMDS), or less than 100 ppb of 2-methyl. An oil composition comprising cyclopentanone (2-MCP).
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.
The oil composition of claim 1, wherein the composition comprises a DHA content of greater than about 40%.
The 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 dimethyl disulfide (DMDS), or less than 2.5 ppb of 2-methyl. An oil composition comprising cyclopentanone (2-MCP).
The 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 dimethyl disulfide (DMDS), and less than 2.5 ppb of 2-methyl. An oil composition comprising cyclopentanone (2-MCP).
The oil composition of claim 1, wherein the composition is derived from oil separated from biomass.
8. The oil composition of claim 7, wherein the biomass comprises marine microorganisms.
The oil composition of claim 8, wherein the marine microorganism is at least one of algae, bacteria, fungi, and protists.
The method of claim 9, wherein the marine microorganism seukijo kit Solarium (schizochytrium) in the at least one marine microorganism oil composition.
The oil composition of claim 1, wherein the at least one carotenoid comprises β-carotene or canthaxanthin.
An edible product which is a food, feed, or dietary supplement comprising the oil of claim 1.
The product of claim 12 which is a dietary supplement.