Nutritional oil with reduced mosH content and method for its preparation

Abstract

The present disclosure describes nutritional oils comprising LC-PUFAs and having a MOSH content of less than or equal to 7 ppm, and methods of making nutritional oils.

Description

Technical Field

[0001] This disclosure relates to microbial oil and nutrient oil compositions having reduced levels of mineral oil saturated hydrocarbons (MOSH). This disclosure also relates to a method for preparing microbial oil and nutrient oil compositions having reduced levels of MOSH. Background Technology

[0002] Mineral oils comprise complex mixtures of hydrocarbons with varying structures and chain lengths. Mineral oil hydrocarbons (MOHs) include mineral oil saturated hydrocarbons (MOSHs) and mineral oil aromatic hydrocarbons (MOAHs). For example, MOSHs can include alkanes (open-chain, saturated, optionally branched hydrocarbons) and cycloalkanes (cyclic, saturated, optionally highly alkylated hydrocarbons). For example, MOAHs can include monocyclic or polycyclic aromatic, optionally highly alkylated hydrocarbons.

[0003] Molecular oxygen (MOO) is a known food contaminant, particularly concerning in infant nutrition products. While MOOAH is considered more toxic than MOSH, both are subjects of ongoing evaluation to determine their long-term effects on human health. MOH food contamination can originate from a variety of sources, including packaging materials (e.g., plastics, adhesives, paper products, inks, etc.), manufacturing equipment (e.g., lubricants, detergents, etc.), and environmental pollution.

[0004] Docosahexaenoic acid (DHA) and arachidonic acid (ARA) are long-chain polyunsaturated fatty acids (LC-PUFAs), defined as polyunsaturated fatty acids (PUFAs) with 20 to 22 carbon atoms, which can influence infant growth and development. DHA is the most abundant omega-3 fatty acid in the brain and retina. ARA is the most abundant omega-6 fatty acid in the brain. It is believed that providing DHA and ARA together in effective amounts during infancy has a positive impact on children's development. DHA is believed to support brain and cognitive development, psychological adaptability and problem-solving abilities, visual development, and attention and information processing. ARA is believed to play a role in supporting the development of a healthy immune system, bone formation, and blood flow and vascular development.

[0005] Because DHA and ARA are believed to have positive effects on infant growth and development, these nutritional oils are often included in infant nutrition products. However, DHA and ARA products are also susceptible to contamination from MOSH and MOAH from packaging, manufacturing equipment, etc. Therefore, it is desirable to minimize the contamination of beneficial nutritional oils (e.g., oils containing LC-PUFA) with potentially harmful MOHs (including MOSH and MOAH). Summary of the Invention

[0006] This disclosure describes a nutritional oil containing arachidonic acid (ARA) at a content greater than or equal to 300 mg / g based on the total weight of the nutritional oil, free fatty acids at a content greater than or equal to 0.1 wt% based on the total weight of the nutritional oil, and MOSH at a content less than 7 ppm based on the total weight of the nutritional oil.

[0007] This disclosure further describes a nutritional oil comprising ARA at a content greater than or equal to 300 mg / g based on the total weight of the nutritional oil, free fatty acids at less than 0.1 wt% based on the total weight of the nutritional oil, and MOSH at less than or equal to 3 ppm based on the total weight of the nutritional oil.

[0008] This disclosure further describes a nutrient oil mixture comprising a first nutrient oil containing ARA mixed with a second nutrient oil containing DHA, wherein the nutrient oil mixture contains ARA and DHA in a weight ratio of 0.5 to 3 ARA / DHA, and wherein the nutrient oil mixture contains MOSH at a content of less than or equal to 4 ppm.

[0009] This disclosure also describes a method for preparing a nutritional oil, comprising mixing an oil containing LC-PUFA with an organic solvent, removing at least a portion of the organic solvent from the LC-PUFA-containing oil to obtain a crude nutritional oil, and recovering at least a portion of the organic solvent removed from the LC-PUFA-containing oil using a recycled solvent, wherein the recycled solvent contains less than 50 wt% mineral oil based on the total weight of the recycled solvent. Detailed Implementation

[0010] While the following detailed description contains numerous specific details for illustrative purposes, those skilled in the art will understand that many variations and modifications can be made to these details, and such variations and modifications are considered to be included herein. Therefore, the following description of embodiments does not lose any generality, nor does it impose any limitation on any of the appended claims. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0011] As used in this written description, the singular forms “a,” “an,” and “the” include explicit support for plural referents unless the context clearly indicates otherwise. Thus, for example, references to “fatty acid” or “the fatty acid” can include a variety of such fatty acids.

[0012] Unless otherwise stated, all numerical parameters should be understood to be prefixed and modified by the term “about” in all cases, where the numerical parameter has the inherent variability peculiar to the underlying measurement technique used to determine the value of the parameter.

[0013] Concentration, quantity, and other numerical data may be expressed or presented in range format herein. It should be understood that such range format is used merely for convenience and brevity, and therefore should be flexibly interpreted to include not only the values ​​explicitly stated as range limits, but also all individual values ​​or subranges covered within that range, as if each value and subrange were explicitly stated. For example, the numerical range “1 to 5” should be interpreted to include not only the explicitly stated values ​​of 1 to 5, but also the individual values ​​and subranges within the indicated range. Thus, the numerical range includes individual values ​​such as 1, 2, 3, 4, and 5, as well as subranges such as 1 to 3, 2 to 4, 3 to 5, etc. The same principle applies to ranges that state only one value as a minimum or maximum value. Furthermore, this interpretation should apply regardless of the breadth of the range or the characteristics described.

[0014] Throughout this specification, the term "embodiment" refers to a specific feature, component, or characteristic described in connection with that embodiment being included in at least one implementation. Therefore, the phrases "in an embodiment" or "in one embodiment" appearing in different places throughout this specification do not necessarily refer to the same implementation.

[0015] Oil source The methods described herein, as well as the microbial oils or nutrient oil compositions described herein, can be carried out or obtained through a suitable biomanufacturing process employing one or more suitable plants or microorganisms. For example, microbial oils or nutrient oils can be produced and obtained from plants, plant seeds, microalgae, fungi (including yeasts), bacteria, and / or protozoa.

[0016] In some specific embodiments, the nutrient oil may be extracted from or otherwise obtained from plants that have been genetically modified to produce at least one LC-PUFA or to increase the production of at least one LC-PUFA, such as coconut oil, corn oil, cottonseed oil, olive oil, palm oil, peanut oil, rapeseed oil (canola oil), safflower oil, sesame oil, soybean oil, sunflower oil, linseed oil, etc., containing LC-PUFAs. Other embodiments and descriptions can be found in U.S. Patent Publications 2015 / 0152450, 2013 / 0101723, 2013 / 0150599, 2010 / 0313309, 2008 / 0050505, and 2007 / 0220634, each of which is incorporated herein by reference. Plants may be cultivated and oils extracted from plants or their seeds using methods known in the art.

[0017] In some specific embodiments, the microbial oil described herein may be produced by fungi or primarily by fungi. In other specific embodiments, the microbial oil described herein may be produced by fungi of the order Mortierellales or primarily by them.

[0018] In some specific embodiments, the microbial oil described herein can be derived from *Morchella* genus (… Mortierella The microbial oil described herein may be produced or primarily produced by fungi including *Mortierella alpina* and *Mortierella vinifera*. In some embodiments, the microbial oil described herein may be produced by fungi including *Mortierella alpina* and *Mortierella vinifera*. Mortierella vinacea ), Mosqualis multiceps ( Mortierella polycephala ), Long-sporeed molluscum ( Mortierella elongata Fungi that produce or are primarily produced by or in combination with them.

[0019] In some specific embodiments, the microbial oil described herein may be produced by or primarily by microalgae. In some embodiments, the microalgae may be or include dinoflagellates, such as *Cryptodinium kurstii* (…). Crypthecodinium cohnii ).

[0020] In some other specific embodiments, the microbial oil described herein may be produced by or primarily by Thraustochytrid, which is classified as a microalga in this disclosure.

[0021] For the purposes of this disclosure, strains described as *Thraustochytriales* include organisms taxonomically belonging to the order *Thraustochytriales*. In some further embodiments, *Thraustochytriales* may include organisms taxonomically belonging to the family *Thraustochytriidae* or *Thraustochytriaceae*. In even further embodiments, *Thraustochytriales* may include organisms taxonomically belonging to the genus *Thraustochytriales*. Thraustochytrium ), Wuken's Chrystridium ( Ulkenia ), genus Schizochytrium ( Schizochytrium ), genus Chrystridium ( Japonochytrium ), Acinetochytrium ( Aplanochytrium ), genus Arachycetes ( Althornia ), genus *Erythritolium* Elina ), genus *Citrineus* ( Aurantiochytrium ), genus *Chytridum* ( Oblongichytrium ), Stachytrium ( Botryochytrium ), genus *Paritichophyton* ( Parietichytrium ), genus *Cypripedium* ( Sicyoidochytrium ) organisms, or combinations thereof.

[0022] In some further embodiments, the microbial oil described herein may be produced by microorganisms of species from the genera *Cypripedium*, *Wukenia*, *Schizochytrium*, *Japonicus*, and / or *Acinetochytrium*, or microorganisms derived from such species, or at least primarily produced therefrom.

[0023] Other microbial species that can be used to produce microbial oils are available in U.S. Patent No. 8486267, which is incorporated herein by reference.

[0024] Cultivation and Fermentation The microorganisms described herein can be cultured in large-scale industrial bioreactors. These microorganisms can be cultured under conditions that increase the production of biomass and / or target compounds (e.g., total lipid content, total LC-PUFA content, etc.). For example, the production of desired lipids can be enhanced by culturing cells according to methods involving altering one or more culture conditions to obtain larger quantities of the desired polyunsaturated fatty acids (PUFAs), or particularly long-chain PUFAs (LC-PUFAs) with a carbon chain length of 20 to 22 carbon atoms.

[0025] More specifically, LC-PUFAs can typically be produced by culturing microorganisms in a two-stage process, which includes a growth phase of increasing cellular biomass followed by a production phase of biomass-derived lipids. The lipid components can be considered microbial oils and can be separated from the biomass for use as nutrient oils or as components of nutrient oil compositions. The specific growth medium and culture conditions employed will depend on the specific microorganisms being cultured.

[0026] Typically, microorganisms can be cultured to achieve a biomass concentration of at least 20 g / L at the end of the growth phase. In some further embodiments, microorganisms can be cultured to achieve a biomass concentration of at least 40 g / L, at least 60 g / L, at least 80 g / L, at least 90 g / L, or at least 100 g / L at the end of the growth phase. The biomass concentration can be determined based on the mass of washed (e.g., to remove residual salts, carbohydrates, etc.) and lyophilized biomass obtained per unit volume of fermentation medium.

[0027] Microorganisms can typically be cultured to achieve a lipid content (or fat or oil content) of at least 20 wt.% based on the total stem cell weight of the microbial biomass at the end of the production phase. In some further embodiments, microorganisms can be cultured to achieve a lipid content (or fat or oil content) of at least 30 wt.%, at least 40 wt.%, at least 50 wt.%, at least 55 wt.%, at least 60 wt.%, or at least 65 wt.% based on the total stem cell weight of the biomass. The stem cell weight is based on the mass of the biomass after washing (e.g., to remove residual salts, carbohydrates, etc.) and lyophilization.

[0028] In some embodiments, the volume of the culture medium may be at least 2 liters, at least 10 liters, at least 50 liters, at least 100 liters, at least 200 liters, at least 200 liters, at least 1000 liters, at least 10,000 liters, at least 20,000 liters, at least 50,000 liters, at least 100,000 liters, at least 150,000 liters, at least 200,000 liters, or at least 250,000 liters. In some embodiments, the volume of the culture medium is 2 liters to 300,000 liters, 10 liters to 200,000 liters or 250,000 liters, 50 liters to 100,000 liters or 150,000 liters, 100 liters to 50,000 liters, 500 liters to 20,000 liters, or 1,000 liters to 10,000 liters.

[0029] Microbial cells can be cultured for a period of 1 to 60 days. In some embodiments, the culture period for microbial cells can be 1 to 14 days, 2 to 13 days, 3 to 12 days, 4 to 11 days, 5 to 10 days, 6 to 9 days, or 7 to 8 days.

[0030] In some embodiments, the culture may include aerated shaking culture, shaking culture, static culture, batch culture, semi-continuous culture, continuous culture, rolling batch culture, wave culture, etc. In some other embodiments, conventional stirred fermenters, bubble column fermenters (batch or continuous), wave fermenters, etc., may be used for culture. Various methods can be used to aerate the culture, such as shaking, mixing, bubbling, etc.

[0031] Other methods and details relating to cultivation and fermentation can be found in U.S. Patent Nos. 8,669,090, 9,045,785, 9,453,172 and 10,435,725 and U.S. Patent Publication No. 2021 / 0171991, each of which is incorporated herein by reference.

[0032] Pasteurization Optionally, the biomass produced during fermentation can be pasteurized to kill cells and inactivate unwanted substances present in the biomass. For example, the biomass can be pasteurized to inactivate substances that may cause degradation of desired lipids or other compounds. Pasteurization can be performed on the fermentation medium or by separating the biomass from the fermentation medium for pasteurization. Pasteurization can be carried out by heating the biomass and / or the fermentation medium to a temperature sufficient to kill cells and / or inactivate certain unwanted substances.

[0033] In some embodiments, pasteurization may be performed as described in U.S. Patent Publication No. 2005 / 0220958, which is incorporated herein by reference.

[0034] In some other embodiments, pasteurization can be performed at a temperature of 50°C to 90°C for a period of time. In other embodiments, pasteurization can be performed at a temperature of 50°C to 70°C, 60°C to 80°C, or 70°C to 90°C for a period of time.

[0035] Pasteurization time can typically be less than or equal to 120 minutes. In some embodiments, pasteurization time can be from 30 minutes to 120 minutes. In some specific embodiments, pasteurization time can be from 30 minutes to 90 minutes, 45 minutes to 105 minutes, or 60 minutes to 120 minutes. In other embodiments, pasteurization time can be from 45 minutes to 75 minutes or 55 minutes to 90 minutes.

[0036] Pasteurization can be performed using a variety of suitable methods, including indirect and / or direct heating methods. In some embodiments, pasteurization can be performed using indirect heating methods, such as through a heat transfer jacket or plate. In some embodiments, pasteurization can be performed using direct heating methods, such as direct steam injection.

[0037] Harvest Alternatively, biomass can be harvested using a variety of conventional methods. As a non-limiting example, centrifugation (e.g., solid-jet centrifugation), filtration (e.g., cross-flow filtration), and / or other suitable harvesting methods can be used to collect biomass. In some embodiments, a precipitant (e.g., sodium phosphate or calcium chloride) can also be used to accelerate the collection of biomass.

[0038] In some embodiments, the biomass may also be washed with water. In some embodiments, the biomass may be concentrated to achieve the desired solids content. Other methods and details relating to the harvesting of biomass can also be found in U.S. Patent No. 5,130,242, U.S. Patent Publication No. 2002 / 0001833 and International Patent Publication No. WO1997 / 037032, each of which is incorporated herein by reference.

[0039] hydrolysis Optionally, cell hydrolysis (i.e., disruption of biomass cells) can be performed using a variety of methods. In some embodiments, cell hydrolysis can be performed using chemical, enzymatic, and / or mechanical methods.

[0040] Chemical hydrolysis can be carried out in various ways, such as by acid hydrolysis, alkaline hydrolysis, detergent hydrolysis, etc. In some embodiments, chemical hydrolysis can be performed by adding an acid to the biomass mixture. Typically, acid hydrolysis may include washing the biomass with water. The acid may be added to the washed biomass mixture. In some embodiments, the biomass is not dried before the acid is added. In other embodiments, the biomass is dried before the acid is added. Non-limiting examples of acids that can be used for acid hydrolysis may include sulfuric acid, hydrochloric acid, phosphoric acid, hydrobromic acid, nitric acid, perchloric acid, or other strong acids.

[0041] An amount of acid sufficient to hydrolyze the cells can be added to the biomass mixture for an incubation period sufficient to hydrolyze the cells. In some embodiments, the acid can be added to the biomass mixture in an amount sufficient to achieve a final acid concentration of 100 mM to 200 mM. In some other embodiments, the acid can be added to the biomass mixture in an amount sufficient to achieve a final acid concentration of 100 mM to 140 mM, 120 mM to 160 mM, 140 mM to 180 mM, or 160 mM to 200 mM.

[0042] The incubation period for hydrolyzed cells is also affected by the temperature at which acid hydrolysis is performed. In some embodiments, acid hydrolysis can be carried out at temperatures ranging from 30°C to 200°C. In some further embodiments, acid hydrolysis can be carried out at temperatures ranging from 30°C to 80°C, 50°C to 100°C, 75°C to 125°C, 100°C to 150°C, 125°C to 175°C, or 150°C to 200°C.

[0043] The incubation period can be a sufficient time interval to hydrolyze the cells at the stated acid concentration and temperature. For example, incubating the mixture at a higher temperature can result in hydrolysis proceeding at a faster rate (i.e., requiring a shorter hydrolysis time). Similarly, in some embodiments, incubating the mixture at a higher acid concentration can result in hydrolysis proceeding at a faster rate.

[0044] In some embodiments, enzymatic hydrolysis can be used to hydrolyze cells in biomass. For example, the biomass can be contacted with one or more enzymes under conditions that induce cell destruction. In some embodiments, the enzyme can be a protease. A non-limiting example of a protease that can be used for the enzymatic hydrolysis of biomass cells is ALCALASE 2.4L FG (Novozymes; Franklinton, North Carolina). In some embodiments, the biomass can be washed prior to enzymatic hydrolysis.

[0045] Microorganisms can typically ferment and float in aqueous media. The fermentation medium can be gravity-sedimented in a fermenter and can be decanted or otherwise removed to provide the desired concentration of biomass. Alternatively, the fermentation medium can be concentrated by centrifugation to provide the desired concentration of biomass. In some embodiments, the biomass can be contacted with one or more enzymes while the microorganisms are in the fermentation medium. The optimal temperature, time, pH, and enzyme concentration depend on the specific enzyme used. Enzymatic hydrolysis can be carried out with or without surfactants.

[0046] Other hydrolysis methods may also be used. Non-limiting embodiments may include bead milling, ultrasonic treatment, rapid decompression, high-shear mechanical methods, homogenization, ultrasound, Freund's crusher, cold pressing, osmotic shock, heating, drying, pressure oscillation, solvent extraction, expression of autolysing genes, etc., or combinations thereof. In some embodiments, a combination of chemical, enzymatic and / or mechanical methods may be used sequentially or simultaneously to hydrolyze biomass cells.

[0047] Other methods or details regarding hydrolysis can be found in U.S. Patent Nos. 9,408,404 and U.S. Patent Publications Nos. 2022 / 0145211 and 2022 / 0154098, each of which is incorporated herein by reference.

[0048] extract Lipids can be extracted from plant or microbial biomass in a variety of ways. As used herein, the term "lipid" includes phospholipids, free fatty acids, fatty acid esters, acylglycerols (e.g., triacylglycerols, diacylglycerols, monoacylglycerols), lysophospholipids, soaps, phospholipids, sterols, sterol esters, carotenoids, lutein, hydrocarbons, etc. Lipid-rich substances extracted from biomass are generally referred to as LC-PUFA-containing oils or crude oils.

[0049] Depending on the extraction technique used, different types or components of lipids can be extracted. In some embodiments, lipids can be isolated from plants or microorganisms using standard techniques without further purification or refining. In some embodiments, lipids can be isolated using physical and / or mechanical extraction methods, such as, but not limited to, extraction by homogenization or compression.

[0050] In other embodiments, solvent extraction can be used to extract lipids. A variety of organic solvents can be used when solvent extraction is employed. Non-limiting embodiments may include hexane, isopropanol, methylene chloride, dodecane, methanol, ethylated oil, supercritical carbon dioxide, etc., or combinations thereof. Polar lipids (e.g., phospholipids) are typically extracted with polar solvents (e.g., chloroform / methanol). Non-polar lipids (e.g., triglycerides) are typically extracted with non-polar solvents (e.g., hexane). In some specific embodiments, when solvent extraction is used, the extraction solvent may be or include hexane.

[0051] In solvent extraction, organic solvents and biomass can be mixed for a duration suitable for lipid extraction from biomass. For example, organic solvents and biomass can be mixed for 10 minutes to 2 hours or longer. Subsequently, the lipids can be separated from the remaining components of the mixture by centrifugation, filtration, or a combination thereof.

[0052] In other embodiments, a reduced amount of organic solvent can be used for extraction compared to the amount typically used for extracting lipids from whole dried biomass. For example, the biomass-to-organic solvent ratio required for extracting lipids from whole dried biomass is typically 1:4 or higher. Therefore, a reduced amount of organic solvent can provide a biomass-to-organic solvent ratio of less than 1:4.

[0053] Optionally, edible oils or biofuels can be used for lipid extraction. In some embodiments, the edible oil may be a vegetable oil, such as coconut oil, palm oil, canola oil, sunflower oil, soybean oil, corn oil, olive oil, safflower oil, palm kernel oil, cottonseed oil, its alkylated derivatives, etc., or combinations thereof. In some other embodiments, the edible oil may be a microbial oil, alkylated derivatives, etc., or combinations thereof. As used herein, “biofuel” means any fuel, fuel additive, aromatic and / or aliphatic compound derived from a biomass feedstock. In some embodiments, biofuels may be derived from plant or microbial sources. Non-limiting examples of biofuel sources may include microalgae, corn, switchgrass, sugarcane, sugar beet, rapeseed, soybean, etc. As used herein, the term organic solvent does not include biofuels as defined herein, nor does it include edible oils.

[0054] Optionally, in the case of extracting lipids from biomass using edible oils and / or biofuels, the edible oils and / or biofuels are not completely removed from the lipid extract, or in some cases are substantially not removed. Subsequent fractionation of the extracted oil, where the added oil or biofuel remains only in one of the oil fractions, is not considered a removal of the edible oil or biofuel from the lipid extract. In some embodiments, after recovery, the crude oil can be combined with other edible oils or biofuels to be used as or incorporated into one or more nutrient oil compositions described herein. Edible oils or biofuels can be added during the extraction step to oils containing long-chain polyunsaturated fatty acids (LC-PUFAs) as a substitute or supplement to the combination with LC-PUFA-containing oils after the recovery process. In some cases, adding edible oils or biofuels during the extraction step can facilitate the separation of LC-PUFA-containing oils from the biomass.

[0055] In conventional methods for separating LC-PUFA-containing oils from biomass using organic solvent extraction, the organic solvent is removed from the LC-PUFA-containing oil after recovery, although trace amounts may remain. In the methods described herein, where edible oil or biofuel is used to assist in the extraction of LC-PUFA-containing oils, optionally, at least 80 wt.% of the edible oil or biofuel added during the extraction step may remain in the extracted LC-PUFA-containing oil and may be incorporated into the final nutrient oil composition. In other embodiments, at least 85 wt.%, at least 90 wt.%, at least 95 wt.%, at least 98 wt.%, or 100 wt.% of the edible oil or biofuel used in the extraction step may remain in the extracted LC-PUFA-containing oil and may be incorporated into the final nutrient oil composition.

[0056] Alternatively, mechanical methods can be used to extract the LC-PUFA-containing oil. For example, hydrolyzed biomass can be centrifuged to separate the LC-PUFA-containing oil from other components. In some embodiments, the LC-PUFA-containing oil may be contained in the upper layer of the centrifuged material and can be separated from the residual material by suction or decantation.

[0057] Refining Nutrient oils or microbial oils isolated from other components of plant or microbial biomass mixtures can be referred to as crude nutrient oils, which may include LC-PUFAs produced by plants and / or microorganisms, as well as various other components extracted together with the LC-PUFAs, such as sterols, other fatty acids, residual edible oils / biofuels used in the extraction process, etc. In some embodiments, the nutrient oil compositions described herein may be or include crude nutrient oils extracted from biomass.

[0058] Optionally, the crude nutrient oil may undergo further refining processes to produce refined oil or final oil. In some embodiments, the nutrient oil compositions described herein may be or include refined nutrient oil or final nutrient oil. In some specific embodiments, the refined nutrient oil or final nutrient oil may be refined microbial oil or final microbial oil.

[0059] The refining of crude vegetable oils can include any of the standard vegetable oil refining steps, such as degumming, neutralization / alkali refining, decolorization, deodorization, winterization / dewaxing, etc. During refining, various impurities or minor components can be removed, such as, but not limited to, free fatty acids, water, impurities, colorants, phospholipids, minerals, carotenoids, sterols, tocopherols / tocotrienols, waxes, residual cell debris, residual solvents, etc.

[0060] More specifically, degumming can be performed as needed to remove phospholipids / colloids from crude oil. For example, degumming can also help minimize refining losses and oil decomposition. In some embodiments, degumming may include aqueous degumming and / or acid degumming.

[0061] Neutralization typically occurs after degumming to remove free fatty acids. Neutralization usually involves saponifying the free fatty acids using an alkaline solution and extracting the resulting soap with an aqueous phase that can be easily separated from the oil. Neutralization can also be used to remove phospholipids, oxidized components, metal ions, colorants (e.g., pigments), insoluble impurities, etc., from oils.

[0062] Decolorization typically occurs after neutralization and can be used to remove colorants (e.g., pigments) from oil. Decolorization can be performed by adding bleaching clay or other suitable adsorbents to adsorb the colorants present in the oil. The bleaching clay or other adsorbents can then be removed by filtration.

[0063] Winterization, or dewaxing, typically occurs after decolorization and can be used to remove wax or saturated fatty acids from oils. The presence of saturated fatty acids causes oils to appear cloudy or hazy. Winterization is usually performed by cooling the oil to precipitate the saturated fatty acids. The precipitated solids can then be filtered out and removed from the oil.

[0064] Deodorization is typically performed last in the entire oil refining process to ensure the cleanliness of the feedstock. This process usually involves high-temperature steam stripping of the oil under vacuum to help remove unwanted volatile compounds, odors, hydroperoxides, and other contaminants from the oil.

[0065] Other methods and details relating to the refining of crude oil can be found in U.S. Patent Nos. 7,419,596 and 11,672,258, each of which is incorporated herein by reference.

[0066] Minimize mineral oil hydrocarbons Mineral oil hydrocarbons (MOHs) can be introduced into nutrient oil compositions in a variety of ways. As mentioned earlier, MOHs can be introduced into nutrient oil compositions through packaging materials, manufacturing equipment, environmental sources, etc. Therefore, adjustments to any of these factors can be considered to minimize MOH contamination.

[0067] In some specific embodiments, MOH reduction can be minimized by addressing the sources of MOH contamination during the manufacturing process. However, MOH can be an effective component in various aspects of the manufacturing process. For example, mineral oil-based systems are often used to absorb and recover evaporated extraction solvents for subsequent reuse.

[0068] Specifically, oils containing LC-PUFA can be extracted from plant or microbial biomass using organic solvents. Organic solvents can be removed from the LC-PUFA-containing oil (typically by evaporation), and solvent recovery systems can be used to capture and reuse them. More specifically, the exhaust gas leaving the solvent recovery system contains solvent vapors and can be introduced into a mineral oil-based absorber system to absorb residual solvents from the exhaust gas before it is released into the atmosphere. The exhaust gas can be introduced into the mineral oil-based system, causing it to rise through a solid packing material to maximize the oil / vapor contact surface area. The mineral oil is introduced into the mineral oil-based system in a countercurrent manner with the exhaust gas. Therefore, the organic solvents carried by the exhaust gas can be absorbed by the mineral oil.

[0069] After absorbing the organic solvent, the mineral oil can be heated with steam under vacuum to strip and condense the organic solvent for later reuse. The stripped mineral oil can then be cooled and recycled back into the mineral oil-based system for reuse. However, trace amounts of mineral oil saturated hydrocarbons (MOSH) and / or mineral oil aromatics (MOAH) can contaminate the organic extraction solvent, for example, through aerosolization. These trace amounts of MOSH and MOAH can subsequently transfer to the oil containing LC-PUFA during extraction, rather than remaining in the extraction solvent, resulting in undesirable oil contamination. The amount of MOSH and / or MOAH contamination can depend on a variety of factors, such as operating rate, vapor pressure, temperature, cleanliness of the packed phase, state of the extraction solvent, and state of the mineral oil.

[0070] Therefore, this disclosure describes a method for minimizing MOSH and / or MOAH in nutrient oils by employing alternative but effective recycled solvents. More specifically, the method may include replacing at least a portion of the mineral oil in a mineral oil-based system. Thus, used organic solvents can be recovered and MOH contamination of the nutrient oil can be minimized using a recycled solvent containing less than 50 wt% mineral oil based on the total weight of the recycled solvent. More specifically, the method may include combining an LC-PUFA-containing oil with an organic solvent, removing at least a portion of the organic solvent from the LC-PUFA-containing oil to obtain a crude nutrient oil, and recovering at least a portion of the organic solvent using a recycled solvent containing less than 50 wt% mineral oil based on the total weight of the recycled solvent.

[0071] Oils containing LC-PUFAs can be any LC-PUFA-containing oil combined with an organic solvent (e.g., for extraction). In some embodiments, oils containing LC-PUFAs may include eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA), or combinations thereof. In some specific embodiments, oils containing LC-PUFAs may include EPA. In some specific embodiments, oils containing LC-PUFAs may include DHA. In some specific embodiments, oils containing LC-PUFAs may include ARA.

[0072] Various organic solvents can be combined with oils containing LC-PUFA. Non-limiting embodiments may include hexane, isopropanol, methylene chloride, dodecane, methanol, ethanol, acetone, ethylated oil, supercritical carbon dioxide, etc., or combinations thereof. In some specific embodiments, the organic solvent may include hexane. In some other specific embodiments, the organic solvent may include isopropanol. In some further specific embodiments, the organic solvent may include ethanol and / or methanol.

[0073] In some embodiments, the organic solvent may include at least a portion of recovered organic solvent, which was previously mixed with an oil containing LC-PUFA and subsequently recovered using a solvent recovery method. In some embodiments, the organic solvent may contain more than 40 wt%, more than 50 wt%, more than 60 wt%, or more than 70 wt% of recovered organic solvent.

[0074] The recovered organic solvent may typically contain less than 100 ppm of MOSH. In some other embodiments, the recovered organic solvent may contain less than 50 ppm of MOSH, less than 40 ppm of MOSH, less than 30 ppm of MOSH, less than 20 ppm of MOSH, less than 10 ppm of MOSH, less than 5 ppm of MOSH, or less than 1 ppm of MOSH.

[0075] Furthermore, the recovered organic solvent may typically contain less than 100 ppm of MOAH. In some other embodiments, the recovered organic solvent may contain less than 50 ppm of MOAH, less than 40 ppm of MOAH, less than 30 ppm of MOAH, less than 20 ppm of MOAH, less than 10 ppm of MOAH, less than 5 ppm of MOAH, or less than 1 ppm of MOAH.

[0076] MOSH and MOAH can be detected and measured using any suitable analytical method. As a non-limiting example, gas chromatography (GC) with flame ionization detection (FID) can be used to analyze MOSH and MOAH.

[0077] Mixing organic solvents and / or recycled organic solvents with LC-PUFA-containing oils is generally used for the extraction of LC-PUFA-containing oils from biomass (e.g., plant biomass or microbial biomass). In specific embodiments, organic solvents and / or recycled organic solvents can be mixed with LC-PUFA-containing oils to extract LC-PUFA-containing oils from plant biomass. In some other embodiments, organic solvents and / or recycled organic solvents can be mixed with LC-PUFA-containing oils to extract LC-PUFA-containing oils from microbial biomass.

[0078] Organic solvents and / or recovered organic solvents can be removed from oils containing LC-PUFAs to obtain crude nutrient oils. Crude nutrient oils, after removal, typically contain less than 5 wt% organic solvents. In some further embodiments, the crude nutrient oil may contain less than 3 wt%, less than 1 wt%, less than 0.5 wt%, less than 0.1 wt%, less than 0.05 wt%, less than 0.01 wt%, less than 0.005 wt%, or less than 0.001 wt% organic solvents. Therefore, organic solvents can be at least partially removed from oils containing LC-PUFAs. In some further embodiments, organic solvents can be substantially removed from oils containing LC-PUFAs.

[0079] To prevent organic solvents from being released into the atmosphere, solvent recovery systems can be used to recover and reuse them. Furthermore, solvent recovery systems typically employ a recovered solvent to absorb the organic solvent. Typically, the recovered solvent contains less than 50 wt% mineral oil based on its total weight. In some other embodiments, the recovered solvent contains less than 40 wt%, less than 30 wt%, less than 20 wt%, less than 10 wt%, less than 5 wt%, or less than 1 wt% mineral oil based on its total weight. This minimizes the contribution of the recovered solvent to the MOSH and / or MOAH levels of the crude nutrient oil.

[0080] In other embodiments, the recovered solvent may include edible oils, such as coconut oil, palm oil, canola oil, sunflower oil, soybean oil, corn oil, olive oil, safflower oil, palm kernel oil, cottonseed oil, medium-chain triglycerides, their alkylated derivatives, etc., or combinations thereof. In further embodiments, the recovered solvent may include biofuels. In some specific embodiments, the recovered solvent may include medium-chain triglycerides. In some specific embodiments, the recovered solvent may include canola oil. In some specific embodiments, the recovered solvent may include sunflower oil.

[0081] Organic solvents can be removed from oils containing LC-PUFA to provide crude nutrient oil. In some embodiments, the nutrient oil may be crude nutrient oil. In some further embodiments, the crude nutrient oil may be further processed to obtain refined nutrient oil or final nutrient oil.

[0082] In some other embodiments, minimization of MOSH and / or MOAH in the nutrient oil can be achieved or further achieved by replacing at least a portion of the mineral oil used elsewhere in the manufacturing process (e.g., as an equipment lubricant) with alternative equipment materials (which may be the same as or different from the recycled solvent). In practice, the equipment material typically contains less than 50 wt% mineral oil based on its total weight. In some other embodiments, the equipment material contains less than 40 wt%, less than 30 wt%, less than 20 wt%, less than 10 wt%, less than 5 wt%, or less than 1 wt% mineral oil based on its total weight. In some specific embodiments, the equipment material is an equipment lubricant.

[0083] In other embodiments, the device material may be the same as the recycled solvent. In some alternative embodiments, the device material may be a different material from the recycled solvent. In some embodiments, the device material may include edible oils, such as coconut oil, palm oil, canola oil, sunflower oil, soybean oil, corn oil, olive oil, safflower oil, palm kernel oil, cottonseed oil, medium-chain triglycerides, their alkylated derivatives, etc., or combinations thereof. In further embodiments, the device material may include biofuels. In some specific embodiments, the device material may include medium-chain triglycerides. In some specific embodiments, the device material may include canola oil. In some specific embodiments, the recycled solvent may include sunflower oil.

[0084] Composition This disclosure describes various nutrient oil compositions. In some embodiments, the nutrient oil may be a crude nutrient oil. In some embodiments, the nutrient oil may be a refined nutrient oil or a final nutrient oil.

[0085] In some embodiments, the nutrient oil may be a crude nutrient oil and may contain more than 0.1 wt% free fatty acids based on the total weight of the nutrient oil. In further embodiments, the crude nutrient oil may contain more than 0.15 wt%, more than 0.2 wt%, more than 0.25 wt%, more than 0.3 wt%, more than 0.35 wt%, more than 0.4 wt%, more than 0.45 wt%, more than 0.5 wt% or more than 0.55 wt% free fatty acids based on the total weight of the nutrient oil. In some other embodiments, the nutrient oil may contain less than 0.5 wt%, less than 0.45 wt%, less than 0.4 wt%, less than 0.35 wt%, less than 0.3 wt%, less than 0.25 wt%, less than 0.2 wt%, less than 0.15 wt%, less than 0.1 wt% or less than 0.05 wt% free fatty acids based on the total weight of the nutrient oil. In some specific embodiments, the nutritional oil may be a refined nutritional oil or a final nutritional oil, and may contain less than 0.1 wt% of free fatty acids based on the total weight of the nutritional oil.

[0086] The content of free fatty acids can be determined using any suitable analytical method. In some non-limiting embodiments, the content of free fatty acids can be determined using AOCS Ca 5a-40 or a similar method.

[0087] The nutritional oils described herein typically have a MOSH content of 7 ppm or less. In some other embodiments, the nutritional oils described herein may contain 5 ppm or less, or 3 ppm or less, of MOSH. In some specific embodiments, the nutritional oil compositions described herein may contain MOSH at a content of 7 ppm or less, 6 ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2.5 ppm or less, 2 ppm or less, 1.5 ppm or less, 1 ppm or less, or 0.5 ppm or less based on the total weight of the nutritional oil. In some other embodiments, the nutritional oil may contain MOSH at a content of 0.1 ppm to 7 ppm based on the total weight of the nutritional oil. In some further embodiments, the nutritional oil may contain MOSH at a content of 0.1 ppm to 2 ppm, 0.5 ppm to 3 ppm, 1 ppm to 4 ppm, or 1.5 ppm to 5 ppm based on the total weight of the nutritional oil.

[0088] In some specific embodiments, the nutritional oil described herein may contain MOAH at a content of less than or equal to 5 ppm, less than or equal to 4 ppm, less than or equal to 3 ppm, less than or equal to 2 ppm, less than or equal to 1.5 ppm, less than or equal to 1 ppm, or less than or equal to 0.5 ppm based on the total weight of the nutritional oil. In some other embodiments, the nutritional oil described herein may contain MOAH at a content of 0.001 ppm to 2 ppm or 0.01 to 5 ppm based on the total weight of the nutritional oil.

[0089] MOSH and MOAH can be detected and measured using any suitable analytical method. As a non-limiting example, MOSH and MOAH can be separated using solid-phase extraction, followed by analysis using gas chromatography with flame ionization detection. MOSH and MOAH can also be analyzed using DIN EN 16995: 2017-08 or similar methods.

[0090] The nutritional oils described herein may contain a variety of long-chain polyunsaturated fatty acids (LC-PUFAs). In some embodiments, the nutritional oils described herein may contain arachidonic acid (ARA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), or a combination thereof. In some embodiments, the nutritional oils described herein may contain ARA. In some embodiments, the nutritional oils described herein may contain DHA. In some embodiments, the nutritional oils described herein may contain EPA. In some further embodiments, the nutritional oils described herein may contain ARA, DHA, and EPA.

[0091] It should be noted that nutritional oils typically contain a significant amount of acylglycerols, such as triglycerides, diglycerides, and monoglycerides. In some embodiments, the nutritional oil may contain triglycerides in an amount greater than or equal to 50 wt%, greater than or equal to 55 wt%, greater than or equal to 60 wt%, greater than or equal to 65 wt%, greater than or equal to 70 wt%, greater than or equal to 75 wt%, greater than or equal to 80 wt%, greater than or equal to 85 wt%, greater than or equal to 90 wt%, or greater than or equal to 95 wt% based on the total weight of the nutritional oil.

[0092] The triglyceride content in nutritional oils can be measured using any suitable analytical method. In a non-limiting embodiment, the following method can be used: 1 H-NMR was used to measure triglyceride content.

[0093] More specifically, fatty acids (such as PUFAs) can bind to the glycerol backbone at one of three positions to form acylglycerols. The two terminal hydroxyl groups along the glycerol backbone are called the sn-1 and sn-3 positions. The central hydroxyl group is called the sn-2 position. Typically, PUFAs bind to the glycerol backbone to form acylglycerols. Therefore, when referring to any particular PUFA, it should be understood that such references include both PUFAs bound to the glycerol backbone to form acylglycerols and free PUFAs, unless otherwise stated.

[0094] In some embodiments, the nutritional oil described herein may contain LC-PUFA at a content greater than or equal to 300 mg / g based on the total weight of the nutritional oil. In some other embodiments, the nutritional oil may contain LC-PUFA at a content greater than or equal to 350 mg / g, greater than or equal to 400 mg / g, greater than or equal to 450 mg / g, greater than or equal to 500 mg / g, or greater than or equal to 550 mg / g based on the total weight of the nutritional oil. In some further embodiments, LC-PUFA may be present in the nutritional oil in amounts of 300 mg / g to 400 mg / g, 350 mg / g to 450 mg / g, 400 mg / g to 500 mg / g, 450 mg / g to 550 mg / g, 500 mg / g to 600 mg / g, or 550 mg / g to 650 mg / g based on the total weight of the nutritional oil.

[0095] The amount of LC-PUFA or other fatty acids in nutritional oils can be determined using any suitable analytical method. In some non-limiting embodiments, fatty acids (e.g., LC-PUFA) can be quantified against a reference standard using AOCS Ce 1b-89 or a similar method.

[0096] In some specific embodiments, the nutritional oil may contain ARA. In some further embodiments, the nutritional oil may contain ARA at a content greater than or equal to 300 mg / g based on the total weight of the nutritional oil. In some other embodiments, the nutritional oil may contain ARA at a content greater than or equal to 350 mg / g, greater than or equal to 400 mg / g, greater than or equal to 450 mg / g, greater than or equal to 500 mg / g, or greater than or equal to 550 mg / g based on the total weight of the nutritional oil. In still other embodiments, the nutritional oil may contain ARA at a content between 300 mg / g and 400 mg / g, 350 mg / g and 450 mg / g, 400 mg / g and 500 mg / g, 450 mg / g and 550 mg / g, or 500 mg / g and 600 mg / g based on the total weight of the nutritional oil.

[0097] In some specific embodiments, the nutritional oil may contain dihedral-gamma-linolenic acid (DGLA) (20:3 n-6). In some further embodiments, the nutritional oil may contain DGLA at a concentration of ≥10 mg / g based on the total weight of the nutritional oil. In some other embodiments, the nutritional oil may contain DGLA at a concentration of ≥15 mg / g, ≥20 mg / g, ≥25 mg / g, or ≥30 mg / g based on the total weight of the nutritional oil. In still other embodiments, the nutritional oil may contain DGLA at a concentration of 10 mg / g to 20 mg / g, 15 mg / g to 25 mg / g, 20 mg / g to 30 mg / g, or 25 mg / g to 35 mg / g based on the total weight of the nutritional oil.

[0098] In some specific embodiments, the nutritional oil may contain gamma-linolenic acid (GLA) (18:3 n-6). In some further embodiments, the nutritional oil may contain GLA at a concentration of ≥10 mg / g based on the total weight of the nutritional oil. In some other embodiments, the nutritional oil may contain GLA at a concentration of ≥15 mg / g, ≥20 mg / g, ≥25 mg / g, ≥30 mg / g, or ≥35 mg / g based on the total weight of the nutritional oil. In still other embodiments, the nutritional oil may contain GLA at a concentration of 10 mg / g to 20 mg / g, 15 mg / g to 25 mg / g, 20 mg / g to 30 mg / g, 25 mg / g to 35 mg / g, or 30 mg / g to 40 mg / g based on the total weight of the nutritional oil.

[0099] In some embodiments, the nutrient oil may contain at least 30 mg / g of palmitic acid based on the total weight of the nutrient oil. In other embodiments, the nutrient oil may contain at least 40 mg / g, at least 50 mg / g, at least 60 mg / g, at least 70 mg / g, at least 80 mg / g, or at least 90 mg / g of palmitic acid based on the total weight of the nutrient oil. In some further embodiments, the nutrient oil may contain palmitic acid from 50 mg / g to 200 mg / g, 60 mg / g to 150 mg / g, or 70 mg / g to 130 mg / g based on the total weight of the nutrient oil.

[0100] In some embodiments, the nutrient oil may also contain additives. Non-limiting embodiments of additives may include antioxidants, antimicrobial agents, flavoring agents, odor masking agents, edible oils and / or biofuels, or combinations thereof.

[0101] Various added antioxidants may be included in the nutritional oils described herein. In some embodiments, antioxidants may be or include natural antioxidants such as tocopherols, phospholipids, ascorbic acid / ascorbyl palmitate, phytic acid, phenolic acids, citric acid, rosemary extract, etc., or combinations thereof. In some other embodiments, antioxidants may be or include synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate, tert-butylhydroquinone (TBHQ), etc., or combinations thereof. In some specific embodiments, the antioxidants in the oil composition may be or include tocopherols, ascorbyl palmitate, rosemary extract, or combinations thereof.

[0102] In some specific embodiments, the nutritional oil described herein may contain an antioxidant at a concentration of 50 ppm or more based on the total weight of the nutritional oil. In other embodiments, the nutritional oil described herein may contain an antioxidant at a concentration of 75 ppm or more, 100 ppm or more, 125 ppm or more, or 150 ppm or more based on the total weight of the nutritional oil. In some further embodiments, the nutritional oil may contain an antioxidant at a concentration of 300 ppm or more, 500 ppm or more, 700 ppm or more, 900 ppm or more, 1200 ppm or more, 1500 ppm or more, 1800 ppm or more, 2000 ppm or more, 2200 ppm or more, 2400 ppm or more, 2600 ppm or more, 2800 ppm or more, or 3000 ppm or more based on the total weight of the nutritional oil. In some other embodiments, the nutritional oil may contain antioxidants at concentrations of 10,000 ppm, 7,500 ppm, 5,000 ppm, 4,000 ppm, 3,000 ppm, or 2,500 ppm based on the total weight of the nutritional oil. In other embodiments, the nutritional oil may contain antioxidants at concentrations of 500 ppm, 400 ppm, 300 ppm, or 200 ppm based on the total weight of the nutritional oil.

[0103] In some embodiments, the nutritional oil may contain low levels of 3-monochloropropane-1,2-diol (3-MCPD) and 2,3-epoxy-1-propanol (glycidyl). When 3-MCPD and / or glycidyl is mentioned, unless otherwise stated, such mentions also include references to their respective esters. In some embodiments, the nutritional oil may contain less than 1 ppm of 3-MCPD based on the total weight of the nutritional oil. In some other embodiments, the nutritional oil may contain less than 0.5 ppm, less than 0.4 ppm, less than 0.3 ppm, less than 0.2 ppm, less than 0.15 ppm, or less than 0.1 ppm of 3-MCPD based on the total weight of the nutritional oil.

[0104] In some embodiments, the nutritional oil may contain less than 0.5 ppm of glycidyl ether based on the total weight of the nutritional oil. In some other embodiments, the nutritional oil may contain less than 0.2 ppm, less than 0.1 ppm, less than 0.05 ppm, less than 0.04 ppm, less than 0.03 ppm, or less than 0.02 ppm of glycidyl ether based on the total weight of the nutritional oil.

[0105] The amounts of 3-MCPD and glycidol can be determined using any suitable analytical method. In some non-limiting embodiments, GC-MS / MS can be used to determine the content of 3-MCPD and glycidol.

[0106] Nutritional oils can typically have low anisidine values ​​(AV). In some embodiments, nutritional oils can have an AV of less than 20. In other embodiments, nutritional oils can have an AV of less than 15, less than 12, less than 10, less than 8, or less than 5. Anisidine values ​​can typically be determined using AOCS Cd 18-90.

[0107] Nutritional oils can also typically have low peroxide values ​​(PV). In some embodiments, nutritional oils may have a PV of less than 10 meq / kg based on the total weight of the nutritional oil. In other embodiments, nutritional oils may have a PV of less than 8 meq / kg, less than 5 meq / kg, less than 3 meq / kg, less than 2 meq / kg, or less than 1 meq / kg based on the total weight of the nutritional oil. Peroxide value can typically be determined using AOCS Cd 8-53.

[0108] The nutritional oils described herein can be formulated into a variety of nutritional or pharmaceutical products to utilize their biological or nutritional properties. Non-limiting examples of potential applications may include pharmaceuticals, food supplements, animal feed additives, cosmetics, etc.

[0109] The nutritional oils described herein can be incorporated into a variety of foods or feed supplements. For example, nutritional oils can be included in various beverages, such as milk, sports drinks, energy drinks, tea, and fruit juices. Nutritional oils can also be incorporated into confectionery (e.g., jelly and biscuits), dairy products, processed foods (e.g., soft rice or porridge), infant formula, breakfast cereals, and many other foods. Other non-limiting embodiments may include pet food, such as cat food, dog food, poultry feed, and fish feed (including aquaculture).

[0110] In another embodiment, the nutritional oil may be incorporated into dietary supplements, such as multivitamins, omega fatty acid supplements, or other suitable dietary supplements.

[0111] In some embodiments, nutritional oils may be incorporated into the pharmaceutical composition. The pharmaceutical composition may be formulated with a pharmaceutically acceptable carrier and administered to a subject in a suitable dosage form. In some embodiments, the pharmaceutical composition may be formulated as a liquid dosage form, such as a suspension, elixir, oil, gel, paste, etc. In some other embodiments, the pharmaceutical composition may be formulated as a solid dosage form, such as a powder, tablet, capsule, gummies, lozenges, etc.

[0112] In addition to those specifically mentioned in this article, nutritional oils can be used for a variety of other nutritional and / or pharmaceutical applications in humans and animals.

[0113] Nutritional oil mixture In some embodiments, the nutrient oils described herein may be mixed with other nutrient oils to form a nutrient oil mixture. In some embodiments, the mixture may include two microbial oils. In some embodiments, the mixture may include microbial oil and fish oil. In some embodiments, the mixture may include microbial oil and vegetable oil. In some embodiments, the mixture may include vegetable oil and fish oil. In some embodiments, the mixture may include two vegetable oils.

[0114] In some specific embodiments, the nutritional oil mixture may include a first nutritional oil containing ARA mixed with a second nutritional oil containing DHA. The first and second nutritional oils may be mixed together in various proportions to form nutritional oil mixtures having various ARA / DHA ratios. In some embodiments, the nutritional oil mixture may contain ARA and DHA at a weight ratio of 0.1 to 5 ARA / DHA. In other embodiments, the nutritional oil mixture may contain ARA and DHA at a weight ratio of 0.5 to 3. In still other embodiments, the nutritional oil mixture may contain ARA and DHA at a weight ratio of 0.1 to 1, 0.5 to 1.5, 1 to 2, 1.5 to 2.5, 2 to 3, or 2.5 to 3.5 ARA / DHA.

[0115] In some specific embodiments, the first nutrient oil containing ARA can be produced by fungi of the order Mortierellales. In some specific embodiments, the second nutrient oil containing DHA can be produced by microalgae of the order Thraustochytriales (e.g., microorganisms of the genera *Thraustochytrium* or *Schizochytrium*). In some specific embodiments, the second nutrient oil containing DHA can be produced by dinoflagellates (e.g., *Cryptodinium kurstii*). Crypthecodinium cohnii ))produce.

[0116] The nutrient oil mixtures described herein typically contain less than or equal to 6 ppm of MOSH based on the total weight of the nutrient oil mixture. In some other embodiments, the nutrient oil mixtures described herein may contain less than or equal to 5 ppm of MOSH, or less than or equal to 3 ppm of MOSH based on the total weight of the nutrient oil mixture. In some specific embodiments, the nutrient oil mixtures described herein may contain MOSH at levels of less than or equal to 6 ppm, less than or equal to 5 ppm, less than or equal to 4 ppm, less than or equal to 3 ppm, less than or equal to 2.5 ppm, less than or equal to 2 ppm, less than or equal to 1.5 ppm, less than or equal to 1 ppm, or less than or equal to 0.5 ppm based on the total weight of the nutrient oil mixture. In some other embodiments, the nutrient oil mixtures may contain MOSH at levels ranging from 0.1 ppm to 6 ppm based on the total weight of the nutrient oil mixture. In some further embodiments, the nutrient oil may contain MOSH at a concentration of 0.1 ppm to 2 ppm, 0.5 ppm to 3 ppm, 1 ppm to 4 ppm, or 1.5 ppm to 5 ppm based on the total weight of the nutrient oil mixture.

[0117] In some specific embodiments, the nutrient oil mixture described herein may contain MOAH at a concentration of less than or equal to 5 ppm, less than or equal to 4 ppm, less than or equal to 3 ppm, less than or equal to 2 ppm, less than or equal to 1.5 ppm, less than or equal to 1 ppm, or less than or equal to 0.5 ppm based on the total weight of the nutrient oil mixture. In some other embodiments, the nutrient oil mixture described herein may contain MOAH at a concentration of 0.001 ppm to 2 ppm or 0.01 to 5 ppm based on the total weight of the nutrient oil mixture.

[0118] MOSH and MOAH can be detected and measured using any suitable analytical method. As a non-limiting example, MOSH and MOAH can be separated using solid-phase extraction, followed by analysis using gas chromatography with flame ionization detection. MOSH and MOAH can also be analyzed using DIN EN 16995: 2017-08 or similar methods.

[0119] In some further embodiments, the nutrient oil mixture may contain ARA at a content greater than or equal to 100 mg / g based on the total weight of the nutrient oil mixture. In some other embodiments, the nutrient oil mixture may contain ARA at a content greater than or equal to 150 mg / g, greater than or equal to 200 mg / g, greater than or equal to 250 mg / g, greater than or equal to 300 mg / g, or greater than or equal to 350 mg / g based on the total weight of the nutrient oil mixture. In still other embodiments, the nutrient oil mixture may contain ARA at a content of 100 mg / g to 200 mg / g, 150 mg / g to 250 mg / g, 200 mg / g to 300 mg / g, 250 mg / g to 350 mg / g, or 300 mg / g to 400 mg / g based on the total weight of the nutrient oil mixture.

[0120] In some further embodiments, the nutrient oil mixture may contain DHA at a concentration of 100 mg / g or greater based on the total weight of the nutrient oil mixture. In some other embodiments, the nutrient oil mixture may contain DHA at a concentration of 150 mg / g, 200 mg / g, 250 mg / g, 300 mg / g, or 350 mg / g or greater based on the total weight of the nutrient oil mixture. In still other embodiments, the nutrient oil mixture may contain DHA at a concentration of 100 mg / g to 200 mg / g, 150 mg / g to 250 mg / g, 200 mg / g to 300 mg / g, 250 mg / g to 350 mg / g, or 300 mg / g to 400 mg / g based on the total weight of the nutrient oil mixture.

[0121] In some embodiments, the nutrient oil mixture may also contain additives. Non-limiting embodiments of additives may include antioxidants, antimicrobial agents, flavoring agents, odor masking agents, edible oils and / or biofuels, or combinations thereof.

[0122] Various added antioxidants may be included in the nutritional oil mixtures described herein. In some embodiments, antioxidants may be or include natural antioxidants such as tocopherols, phospholipids, ascorbic acid / ascorbyl palmitate, phytic acid, phenolic acids, citric acid, rosemary extract, etc., or combinations thereof. In some other embodiments, antioxidants may be or include synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate, tert-butylhydroquinone (TBHQ), etc., or combinations thereof. In some specific embodiments, the antioxidants in the oil composition may be or include tocopherols, ascorbyl palmitate, rosemary extract, or combinations thereof.

[0123] In some specific embodiments, the nutrient oil mixture described herein may contain an antioxidant at a concentration of 50 ppm or more based on the total weight of the nutrient oil mixture. In other embodiments, the nutrient oil mixture described herein may contain an antioxidant at a concentration of 75 ppm or more, 100 ppm or more, 125 ppm or more, or 150 ppm or more based on the total weight of the nutrient oil mixture. In some further embodiments, the nutrient oil mixture may contain an antioxidant at a concentration of 300 ppm or more, 500 ppm or more, 700 ppm or more, 900 ppm or more, 1200 ppm or more, 1500 ppm or more, 1800 ppm or more, 2000 ppm or more, 2200 ppm or more, 2400 ppm or more, 2600 ppm or more, 2800 ppm or more, or 3000 ppm or more based on the total weight of the nutrient oil mixture. In some other embodiments, the nutrient oil mixture may contain antioxidants at concentrations of 10,000 ppm, 7,500 ppm, 5,000 ppm, 4,000 ppm, 3,000 ppm, or 2,500 ppm based on the total weight of the nutrient oil mixture. In other embodiments, the nutrient oil mixture may contain antioxidants at concentrations of 500 ppm, 400 ppm, 300 ppm, or 200 ppm based on the total weight of the nutrient oil mixture.

[0124] In some embodiments, the nutrient oil mixture may contain low levels of 3-monochloropropane-1,2-diol (3-MCPD) and 2,3-epoxy-1-propanol (glycidyl). When 3-MCPD and / or glycidyl is mentioned, unless otherwise stated, such mentions also include references to their respective esters. In some embodiments, the nutrient oil mixture may contain less than 1 ppm of 3-MCPD based on the total weight of the nutrient oil mixture. In some other embodiments, the nutrient oil mixture may contain less than 0.5 ppm, less than 0.4 ppm, less than 0.3 ppm, less than 0.2 ppm, less than 0.15 ppm, or less than 0.1 ppm of 3-MCPD based on the total weight of the nutrient oil mixture.

[0125] In some embodiments, the nutritional oil mixture may contain less than 0.5 ppm of glycidyl ether based on the total weight of the nutritional oil mixture. In some other embodiments, the nutritional oil mixture may contain less than 0.2 ppm, less than 0.1 ppm, less than 0.05 ppm, less than 0.04 ppm, less than 0.03 ppm, or less than 0.02 ppm of glycidyl ether based on the total weight of the nutritional oil mixture.

[0126] The amounts of 3-MCPD and glycidol can be determined using any suitable analytical method. In some non-limiting embodiments, GC-MS / MS can be used to determine the content of 3-MCPD and glycidol.

[0127] Nutrient oil blends can typically have low anisidine values ​​(AV). In some embodiments, the nutrient oil blend can have an AV of less than 20. In other embodiments, the nutrient oil can have an AV of less than 15, less than 12, less than 10, less than 8, or less than 5. Anisidine values ​​can typically be determined using AOCS Cd 18-90.

[0128] Nutritional oils can also typically have low peroxide values ​​(PV). In some embodiments, the nutritional oil mixture may have a PV of less than 10 meq / kg based on the total weight of the nutritional oil mixture. In other embodiments, the nutritional oil mixture may have a PV of less than 8 meq / kg, less than 5 meq / kg, less than 3 meq / kg, less than 2 meq / kg, or less than 1 meq / kg based on the total weight of the nutritional oil mixture. Peroxide value can typically be determined using AOCS Cd 8-53.

[0129] The nutritional oil mixtures described herein can be formulated into a variety of nutritional or pharmaceutical products to utilize their biological or nutritional properties. Non-limiting examples of potential applications may include pharmaceuticals, food supplements, animal feed additives, cosmetics, etc.

[0130] The nutritional oil mixture described herein can be incorporated into a variety of food or feed supplements. For example, the nutritional oil mixture can be included in various beverages, such as milk, sports drinks, energy drinks, tea, and fruit juice. The nutritional oil mixture can also be incorporated into confectionery (e.g., jellies and biscuits), dairy products, processed foods (e.g., soft rice or porridge), infant formula, breakfast cereals, and many other foods. Other non-limiting embodiments may include pet food, such as cat food, dog food, poultry feed, and fish feed (including aquaculture).

[0131] In another embodiment, the nutritional oil mixture may be incorporated into dietary supplements, such as multivitamins, omega fatty acid supplements, or other suitable dietary supplements.

[0132] In some embodiments, the nutritional oil mixture may be incorporated into the pharmaceutical composition. The pharmaceutical composition may be formulated with a pharmaceutically acceptable carrier and administered to a subject in a suitable dosage form. In some embodiments, the pharmaceutical composition may be formulated as a liquid dosage form, such as a suspension, elixir, oil, gel, paste, etc. In some other embodiments, the pharmaceutical composition may be formulated as a solid dosage form, such as a powder, tablet, capsule, gummies, lozenges, etc.

[0133] In addition to those specifically mentioned in this article, nutritional oil blends can be used for a variety of other nutritional and / or pharmaceutical applications in humans and animals.

[0134] Exemplary Implementation This document discloses a variety of compositions and methods. To aid in understanding these compositions and methods, several non-limiting exemplary embodiments are provided in the following numbered clauses for illustrative purposes only. Unless the context clearly indicates otherwise, the following exemplary embodiments can be combined in any suitable combination.

[0135] Clause 1. A nutritional oil comprising: Based on an ARA content greater than or equal to 300 mg / g based on the total weight of the nutritional oil; Based on the content of free fatty acids greater than or equal to 0.1 wt% by total weight of the nutritional oil; and MOSH content is less than 7 ppm based on the total weight of the nutritional oil.

[0136] Clause 2. The nutritional oil as described in Clause 1, which contains ARA at a content of 350 mg / g or greater, optionally 400 mg / g, optionally 450 mg / g, optionally 500 mg / g, or optionally 550 mg / g based on the total weight of the nutritional oil.

[0137] Clause 3. The nutritional oil as described in any of the preceding clauses contains ARA at a content of 400 mg / g or more based on the total weight of the nutritional oil.

[0138] Clause 4. The nutritional oil as described in any of the preceding clauses contains ARA at a content greater than or equal to 450 mg / g based on the total weight of the nutritional oil.

[0139] Clause 5. The nutritional oil as described in any of the preceding clauses contains ARA at a content greater than or equal to 500 mg / g based on the total weight of the nutritional oil.

[0140] Clause 6. The nutritional oil as described in any of the preceding clauses contains ARA at a content greater than or equal to 550 mg / g based on the total weight of the nutritional oil.

[0141] Clause 7. The nutritional oil as described in any of the preceding clauses, comprising ARA in a content of 300 mg / g to 400 mg / g, optionally 350 mg / g to 450 mg / g, optionally 400 mg / g to 500 mg / g, optionally 450 mg / g to 550 mg / g, or optionally 500 mg / g to 600 mg / g based on the total weight of the nutritional oil.

[0142] Clause 8. The nutritional oil as described in any one of Clauses 1 to 7, which contains ARA at a content of 350 mg / g to 450 mg / g based on the total weight of the nutritional oil.

[0143] Clause 9. The nutritional oil as described in any one of Clauses 1 to 7, which contains ARA at a content of 400 mg / g to 500 mg / g based on the total weight of the nutritional oil.

[0144] Clause 10. The nutritional oil as described in any one of Clauses 1 to 7, comprising ARA at a content of 450 mg / g to 550 mg / g based on the total weight of the nutritional oil.

[0145] Clause 11. The nutritional oil as described in any one of Clauses 1 to 7, comprising ARA at a content of 500 mg / g to 600 mg / g based on the total weight of the nutritional oil.

[0146] Clause 12. The nutritional oil as described in any of the preceding clauses contains MOSH at a concentration of less than or equal to 6 ppm, optionally less than or equal to 5 ppm, optionally less than or equal to 4 ppm, or optionally less than or equal to 3.5 ppm based on the total weight of the nutritional oil.

[0147] Clause 13. The nutritional oil as described in any of the preceding clauses contains less than or equal to 5 ppm of MOSH based on the total weight of the nutritional oil.

[0148] Clause 14. The nutritional oil as described in any of the preceding clauses contains less than or equal to 4 ppm of MOSH based on the total weight of the nutritional oil.

[0149] Clause 15. The nutritional oil as described in any of the preceding clauses contains MOSH at a concentration of less than or equal to 3.5 ppm based on the total weight of the nutritional oil.

[0150] Clause 16. The nutritional oil as described in any of the preceding clauses contains MOSH at a concentration of 0.5 ppm to less than 7 ppm based on the total weight of the nutritional oil.

[0151] Clause 17. The nutritional oil as described in any of the preceding clauses contains MOSH in an amount of 2 ppm to less than 7 ppm.

[0152] Clause 18. The nutritional oil as described in any of the preceding clauses, comprising MOAH at a concentration of less than or equal to 5 ppm, optionally less than or equal to 4 ppm, optionally less than or equal to 3 ppm, optionally less than or equal to 2 ppm, optionally less than or equal to 1.5 ppm, or optionally less than or equal to 1 ppm based on the total weight of the nutritional oil.

[0153] Clause 19. The nutrient oil as described in any of the preceding clauses contains MOAH at a concentration of less than or equal to 4 ppm based on the total weight of the nutrient oil.

[0154] Clause 20. The nutrient oil as described in any of the preceding clauses contains MOAH at a concentration of less than or equal to 3 ppm based on the total weight of the nutrient oil.

[0155] Clause 21. The nutrient oil as described in any of the preceding clauses contains MOAH at a concentration of less than or equal to 2 ppm based on the total weight of the nutrient oil.

[0156] Clause 22. The nutrient oil as described in any of the preceding clauses contains MOAH at a concentration of less than or equal to 1.5 ppm based on the total weight of the nutrient oil.

[0157] Clause 23. The nutrient oil as described in any of the preceding clauses contains MOAH at a content of less than or equal to 1 ppm based on the total weight of the nutrient oil.

[0158] Clause 24. The nutritional oil as described in any of the preceding clauses further contains added antioxidants.

[0159] Clause 25. Nutritional oils as described in Clause 24, wherein the added antioxidants include tocopherol.

[0160] Clause 26. The nutritional oil as described in any of the preceding clauses contains 50 wt% or more, 55 wt% or more, 60 wt% or more, 65 wt% or more, 70 wt% or more, 75 wt% or more, 80 wt% or more, or 85 wt% or more based on the total weight of the nutritional oil.

[0161] Clause 27. The nutritional oil as described in any of the preceding clauses, comprising 50 wt% or more, preferably 55 wt% or more, preferably 60 wt% or more, preferably 65 wt% or more, more preferably 70 wt% or more, more preferably 75 wt% or more, more preferably 80 wt% or more, or more preferably 85 wt% or more, based on the total weight of the nutritional oil.

[0162] Clause 28. The nutritional oil as described in any of the preceding clauses contains 3-MCPD in a concentration of less than or equal to 0.2 ppm, less than or equal to 0.15 ppm, or less than or equal to 0.1 ppm based on the total nutritional oil content.

[0163] Clause 29. The nutritional oil as described in any of the preceding clauses, comprising 3-MCPD in a content of less than or equal to 0.2 ppm, preferably less than or equal to 0.15 ppm, or more preferably less than or equal to 0.1 ppm based on the total nutritional oil content.

[0164] Clause 30. The nutritional oil as described in any of the preceding clauses contains glycidyl glycerin in amounts of less than or equal to 0.1 ppm, less than or equal to 0.05 ppm, less than or equal to 0.04 ppm, less than or equal to 0.03 ppm, or less than or equal to 0.02 ppm based on the total amount of nutritional oil.

[0165] Clause 31. The nutritional oil as described in any of the preceding clauses, comprising glycidyl glycerin in a content of less than or equal to 0.1 ppm, preferably less than or equal to 0.05 ppm, preferably less than or equal to 0.04 ppm, more preferably less than or equal to 0.03 ppm, or more preferably less than or equal to 0.02 ppm based on the total amount of nutritional oil.

[0166] Clause 32. The nutrient oil as described in any of the preceding clauses, wherein the anisidine value of the nutrient oil is less than 20, less than 15, less than 12, less than 10 or less than 8.

[0167] Clause 33. The nutritional oil as described in any of the preceding clauses, wherein the anisidine value of the nutritional oil is less than 20, preferably less than 15, preferably less than 12, more preferably less than 10, or more preferably less than 8.

[0168] Clause 34. The nutrient oil as described in any of the preceding clauses, wherein the nutrient oil has a peroxide value of less than or equal to 10 meq / kg, less than or equal to 8 meq / kg, less than or equal to 5 meq / kg, less than or equal to 3 meq / kg, less than or equal to 2 meq / kg, or less than or equal to 1 meq / kg based on the total weight of the nutrient oil.

[0169] Clause 35. The nutritional oil as described in any of the preceding clauses, wherein the nutritional oil has a peroxide value of less than or equal to 10 meq / kg, preferably less than or equal to 8 meq / kg, preferably less than or equal to 5 meq / kg, preferably less than or equal to 3 meq / kg, more preferably less than or equal to 2 meq / kg, or more preferably less than or equal to 1 meq / kg based on the total weight of the nutritional oil.

[0170] Clause 36. The nutrient oil as described in any of the preceding clauses, wherein the nutrient oil is a microbial oil.

[0171] Clause 37. Nutrient oil as described in Clause 36, wherein the microbial oil is produced by *Morchella alpina* (… Mortierella alpina )produce.

[0172] Clause 38. A nutritional oil comprising: Based on an ARA content greater than or equal to 300 mg / g based on the total weight of the nutritional oil; Based on a free fatty acid content of less than 0.1 wt% based on the total weight of the nutritional oil; and MOSH content is based on a total weight of less than or equal to 3 ppm based on the total weight of the nutrient oil.

[0173] Clause 39. Nutritional oil as described in Clause 38, which contains ARA at a content of 350 mg / g or greater, optionally 400 mg / g, optionally 450 mg / g, or optionally 500 mg / g based on the total weight of the nutritional oil.

[0174] Clause 40. The nutritional oil as described in any one of Clauses 38 to 39, which contains ARA at a content of greater than or equal to 400 mg / g based on the total weight of the nutritional oil.

[0175] Clause 41. The nutritional oil as described in any one of Clauses 38 to 40, which contains ARA at a content greater than or equal to 450 mg / g based on the total weight of the nutritional oil.

[0176] Clause 42. The nutritional oil as described in any one of Clauses 38 to 41, which contains ARA at a content of greater than or equal to 500 mg / g based on the total weight of the nutritional oil.

[0177] Clause 43. The nutritional oil as described in any one of Clauses 38 to 42, which contains ARA at a content of greater than or equal to 550 mg / g based on the total weight of the nutritional oil.

[0178] Clause 44. The nutritional oil as described in any one of Clauses 38 to 43, comprising ARA in a content of 300 mg / g to 400 mg / g, optionally 350 mg / g to 450 mg / g, or optionally 400 mg / g to 500 mg / g based on the total weight of the nutritional oil.

[0179] Clause 45. The nutritional oil as described in any one of Clauses 38 to 44, which contains ARA at a content of 350 mg / g to 450 mg / g based on the total weight of the nutritional oil.

[0180] Clause 46. The nutritional oil as described in any one of Clauses 38 to 44, which contains ARA at a content of 400 mg / g to 500 mg / g based on the total weight of the nutritional oil.

[0181] Clause 47. The nutritional oil as described in any one of Clauses 38 to 44, which contains ARA at a content of 450 mg / g to 550 mg / g based on the total weight of the nutritional oil.

[0182] Clause 48. The nutritional oil as described in any one of Clauses 38 to 44, which contains ARA at a content of 500 mg / g to 600 mg / g based on the total weight of the nutritional oil.

[0183] Clause 49. The nutritional oil as described in any one of Clauses 38 to 48, comprising MOSH at a concentration of less than or equal to 2.5 ppm, optionally less than or equal to 2 ppm, optionally less than or equal to 1.5 ppm, or optionally less than or equal to 1 ppm based on the total weight of the nutritional oil.

[0184] Clause 50. The nutrient oil as described in any one of Clauses 38 to 49 contains less than or equal to 2 ppm of MOSH based on the total weight of the nutrient oil.

[0185] Clause 51. The nutrient oil as described in any one of Clauses 38 to 50, which contains less than or equal to 1.5 ppm of MOSH based on the total weight of the nutrient oil.

[0186] Clause 52. The nutrient oil as described in any one of Clauses 38 to 51, which contains less than or equal to 1 ppm of MOSH based on the total weight of the nutrient oil.

[0187] Clause 53. The nutrient oil as described in any one of Clauses 38 to 52, comprising 0.5 ppm to 2 ppm of MOSH based on the total weight of the nutrient oil.

[0188] Clause 54. The nutritional oil as described in any one of Clauses 38 to 53 contains 1 ppm to 3 ppm of MOSH.

[0189] Clause 55. The nutrient oil as described in any one of Clauses 38 to 54, comprising MOAH at a concentration of less than or equal to 5 ppm, optionally less than or equal to 4 ppm, optionally less than or equal to 3 ppm, optionally less than or equal to 2 ppm, optionally less than or equal to 1.5 ppm, or optionally less than or equal to 1 ppm based on the total weight of the nutrient oil.

[0190] Clause 56. The nutrient oil as described in any one of Clauses 38 to 55, which contains MOAH at a content of less than or equal to 4 ppm based on the total weight of the nutrient oil.

[0191] Clause 57. The nutrient oil as described in any one of Clauses 38 to 56, which contains MOAH at a content of less than or equal to 3 ppm based on the total weight of the nutrient oil.

[0192] Clause 58. The nutrient oil as described in any one of Clauses 38 to 57, which contains less than or equal to 2 ppm of MOAH based on the total weight of the nutrient oil.

[0193] Clause 59. The nutrient oil as described in any one of Clauses 38 to 58, which contains MOAH at a content of less than or equal to 1.5 ppm based on the total weight of the nutrient oil.

[0194] Clause 60. The nutrient oil as described in any one of Clauses 38 to 59, which contains MOAH at a content of less than or equal to 1 ppm based on the total weight of the nutrient oil.

[0195] Clause 61. The nutritional oil as described in any one of Clauses 38 to 60 further contains added antioxidants.

[0196] Clause 62. Nutritional oils as described in Clause 61, wherein the added antioxidants include tocopherol.

[0197] Clause 63. The nutritional oil as described in any one of Clauses 38 to 62, comprising 60 wt% or more, 65 wt% or more, 70 wt% or more, 75 wt% or more, 80 wt% or more, 85 wt% or more, 90 wt% or more, or 95 wt% or more based on the total weight of the nutritional oil.

[0198] Clause 64. The nutritional oil as described in any one of Clauses 38 to 63, comprising 50 wt% or more, preferably 55 wt% or more, preferably 60 wt% or more, preferably 65 wt% or more, more preferably 70 wt% or more, more preferably 75 wt% or more, more preferably 80 wt% or more, or more preferably 85 wt% or more, based on the total weight of the nutritional oil.

[0199] Clause 65. The nutrient oil as described in any one of Clauses 38 to 64, comprising 3-MCPD in a concentration of less than or equal to 0.2 ppm, less than or equal to 0.15 ppm, or less than or equal to 0.1 ppm based on the total amount of nutrient oil.

[0200] Clause 66. The nutritional oil as described in any one of Clauses 38 to 65, comprising 3-MCPD in a content of less than or equal to 0.2 ppm, preferably less than or equal to 0.15 ppm, or more preferably less than or equal to 0.1 ppm based on the total amount of nutritional oil.

[0201] Clause 67. The nutritional oil as described in any one of Clauses 38 to 66, comprising glycidyl ether in amounts of less than or equal to 0.1 ppm, less than or equal to 0.05 ppm, less than or equal to 0.04 ppm, less than or equal to 0.03 ppm, or less than or equal to 0.02 ppm based on the total amount of nutritional oil.

[0202] Clause 68. The nutritional oil as described in any one of Clauses 38 to 67, comprising glycidyl glycerin in a content of less than or equal to 0.1 ppm, preferably less than or equal to 0.05 ppm, preferably less than or equal to 0.04 ppm, more preferably less than or equal to 0.03 ppm, or more preferably less than or equal to 0.02 ppm based on the total amount of nutritional oil.

[0203] Clause 69. The nutrient oil as described in any one of Clauses 38 to 68, wherein the nutrient oil has anisidine values ​​of less than 20, less than 15, less than 12, less than 10, or less than 8.

[0204] Clause 70. The nutritional oil as described in any one of Clauses 38 to 69, wherein the nutritional oil has anisidine value of less than 20, preferably less than 15, preferably less than 12, more preferably less than 10, or more preferably less than 8.

[0205] Clause 71. The nutrient oil as described in any one of Clauses 38 to 70, wherein the nutrient oil has a peroxide value of less than or equal to 10 meq / kg, less than or equal to 8 meq / kg, less than or equal to 5 meq / kg, less than or equal to 3 meq / kg, less than or equal to 2 meq / kg, or less than or equal to 1 meq / kg based on the total weight of the nutrient oil.

[0206] Clause 72. The nutritional oil as described in any one of Clauses 38 to 71, wherein the nutritional oil has a peroxide value of less than or equal to 10 meq / kg, preferably less than or equal to 8 meq / kg, preferably less than or equal to 5 meq / kg, preferably less than or equal to 3 meq / kg, more preferably less than or equal to 2 meq / kg, or more preferably less than or equal to 1 meq / kg based on the total weight of the nutritional oil.

[0207] Clause 73. The nutrient oil as described in any one of Clauses 38 to 72, wherein the nutrient oil is a microbial oil.

[0208] Clause 74. Nutrient oil as described in Clause 73, wherein the microbial oil is produced by *Morchella alpina*.

[0209] Clause 75. A nutritional oil mixture comprising: A first nutrient oil containing ARA, mixed with a second nutrient oil containing DHA, wherein the nutrient oil mixture contains ARA and DHA in a weight ratio of 0.5 to 3 ARA / DHA, and wherein the nutrient oil mixture contains MOSH at a content of less than or equal to 4 ppm.

[0210] Clause 76. The nutrient oil mixture as described in Clause 75, comprising ARA at a content of 100 mg / g or greater, optionally 150 mg / g, optionally 200 mg / g, optionally 250 mg / g, optionally 300 mg / g, or optionally 350 mg / g based on the total weight of the nutrient oil mixture.

[0211] Clause 77. The nutrient oil mixture as described in any one of Clauses 75 to 76, comprising an ARA content of greater than or equal to 150 mg / g based on the total weight of the nutrient oil mixture.

[0212] Clause 78. The nutrient oil mixture as described in any one of Clauses 75 to 77, comprising an ARA content of greater than or equal to 200 mg / g based on the total weight of the nutrient oil mixture.

[0213] Clause 79. The nutrient oil mixture as described in any one of Clauses 75 to 78, comprising an ARA content of greater than or equal to 250 mg / g based on the total weight of the nutrient oil mixture.

[0214] Clause 80. The nutrient oil mixture as described in any one of Clauses 75 to 79, comprising an ARA content of greater than or equal to 300 mg / g based on the total weight of the nutrient oil mixture.

[0215] Clause 81. The nutrient oil mixture as described in any one of Clauses 75 to 80, comprising an ARA content of greater than or equal to 350 mg / g based on the total weight of the nutrient oil mixture.

[0216] Clause 82. The nutrient oil mixture as described in Clause 75, comprising ARA in a content of 100 mg / g to 200 mg / g, optionally 150 mg / g to 250 mg / g, optionally 200 mg / g to 300 mg / g, optionally 250 mg / g to 350 mg / g, or optionally 300 mg / g to 400 mg / g based on the total weight of the nutrient oil mixture.

[0217] Clause 83. The nutrient oil mixture as described in Clause 75, comprising ARA in a content of 150 mg / g to 250 mg / g based on the total weight of the nutrient oil mixture.

[0218] Clause 84. The nutrient oil mixture as described in Clause 75, comprising ARA at a content of 200 mg / g to 300 mg / g based on the total weight of the nutrient oil mixture.

[0219] Clause 85. The nutrient oil mixture as described in Clause 75, comprising ARA in a content of 250 mg / g to 350 mg / g based on the total weight of the nutrient oil mixture.

[0220] Clause 86. The nutrient oil mixture as described in Clause 75, comprising ARA at a content of 300 mg / g to 400 mg / g based on the total weight of the nutrient oil mixture.

[0221] Clause 87. A nutrient oil mixture as described in any one of Clauses 75 to 86, comprising DHA at a content of 100 mg / g or greater, optionally 150 mg / g, optionally 200 mg / g, optionally 250 mg / g, optionally 300 mg / g, or optionally 350 mg / g based on the total weight of the nutrient oil mixture.

[0222] Clause 88. The nutrient oil mixture as described in any one of Clauses 75 to 87, comprising DHA at a content greater than or equal to 150 mg / g based on the total weight of the nutrient oil mixture.

[0223] Clause 89. The nutrient oil mixture as described in any one of Clauses 75 to 88, comprising DHA at a content greater than or equal to 200 mg / g based on the total weight of the nutrient oil mixture.

[0224] Clause 90. The nutrient oil mixture as described in any one of Clauses 75 to 89, comprising DHA at a content greater than or equal to 250 mg / g based on the total weight of the nutrient oil mixture.

[0225] Clause 91. The nutrient oil mixture as described in any one of Clauses 75 to 90, comprising DHA at a content greater than or equal to 300 mg / g based on the total weight of the nutrient oil mixture.

[0226] Clause 92. The nutrient oil mixture as described in any one of Clauses 75 to 91, comprising DHA at a content greater than or equal to 350 mg / g based on the total weight of the nutrient oil mixture.

[0227] Clause 93. A nutrient oil mixture as described in any one of Clauses 75 to 86, comprising DHA in a content of 100 mg / g to 200 mg / g, optionally 150 mg / g to 250 mg / g, optionally 200 mg / g to 300 mg / g, optionally 250 mg / g to 350 mg / g, or optionally 300 mg / g to 400 mg / g based on the total weight of the nutrient oil mixture.

[0228] Clause 94. The nutrient oil mixture as described in any one of Clauses 75 to 86, comprising DHA in a content of 150 mg / g to 250 mg / g based on the total weight of the nutrient oil mixture.

[0229] Clause 95. The nutrient oil mixture as described in any one of Clauses 75 to 86, comprising DHA in a content of 200 mg / g to 300 mg / g based on the total weight of the nutrient oil mixture.

[0230] Clause 96. The nutrient oil mixture as described in any one of Clauses 75 to 86, comprising DHA in a content of 250 mg / g to 350 mg / g based on the total weight of the nutrient oil mixture.

[0231] Clause 97. The nutrient oil mixture as described in any one of Clauses 75 to 86, comprising DHA in a content of 300 mg / g to 400 mg / g based on the total weight of the nutrient oil mixture.

[0232] Clause 98. A nutritional oil mixture as described in any one of Clauses 75 to 97, comprising ARA and DHA in a weight ratio of 0.5 to 3 ARA / DHA, optionally 0.5 to 1.5 ARA / DHA, optionally 1 to 2 ARA / DHA, optionally 1.5 to 2.5 ARA / DHA, or optionally 2 to 3 ARA / DHA.

[0233] Clause 99. The nutritional oil mixture as described in any one of Clauses 75 to 98, comprising ARA and DHA in a weight ratio of 0.5 to 1.5 ARA / DHA.

[0234] Clause 100. A nutritional oil mixture as described in any one of Clauses 75 to 99, comprising ARA and DHA in a weight ratio of 1 to 2 ARA / DHA.

[0235] Clause 101. A nutritional oil mixture as described in any one of Clauses 75 to 100, comprising ARA and DHA in a weight ratio of 1.5 to 2.5 ARA / DHA.

[0236] Clause 102. The nutritional oil mixture as described in any one of Clauses 75 to 101, comprising ARA and DHA in a weight ratio of 2 to 3 ARA / DHA.

[0237] Clause 103. The nutrient oil mixture as described in any one of Clauses 75 to 102, comprising MOSH at a concentration of less than or equal to 3.5 ppm, optionally less than or equal to 3 ppm, optionally less than or equal to 2.5 ppm, optionally less than or equal to 2 ppm, optionally less than or equal to 1.5 ppm, or optionally less than or equal to 1 ppm based on the total weight of the nutrient oil mixture.

[0238] Clause 104. The nutrient oil mixture as described in any one of Clauses 75 to 103, comprising MOSH at a content of less than or equal to 3 ppm based on the total weight of the nutrient oil mixture.

[0239] Clause 105. The nutrient oil mixture as described in any one of Clauses 75 to 104, comprising MOSH at a content of less than or equal to 2.5 ppm based on the total weight of the nutrient oil mixture.

[0240] Clause 106. The nutrient oil mixture as described in any one of Clauses 75 to 105, comprising MOSH at a content of less than or equal to 2 ppm based on the total weight of the nutrient oil mixture.

[0241] Clause 107. The nutrient oil mixture as described in any one of Clauses 75 to 106, comprising MOSH at a content of less than or equal to 1.5 ppm based on the total weight of the nutrient oil mixture.

[0242] Clause 108. The nutrient oil mixture as described in any one of Clauses 75 to 107, comprising MOSH at a content of less than or equal to 1 ppm based on the total weight of the nutrient oil mixture.

[0243] Clause 109. The nutrient oil mixture as described in any one of Clauses 75 to 108, comprising 0.5 ppm to 3 ppm of MOSH based on the total weight of the nutrient oil mixture.

[0244] Clause 110. The nutrient oil mixture as described in any one of Clauses 75 to 109, comprising MOSH in a content of 1 ppm to 4 ppm based on the total weight of the nutrient oil mixture.

[0245] Clause 111. A nutrient oil mixture as described in any one of Clauses 75 to 110, comprising MOAH at a concentration of less than or equal to 5 ppm, optionally less than or equal to 4 ppm, optionally less than or equal to 3 ppm, optionally less than or equal to 2 ppm, optionally less than or equal to 1.5 ppm, or optionally less than or equal to 1 ppm based on the total weight of the nutrient oil mixture.

[0246] Clause 112. The nutrient oil mixture as described in any one of Clauses 75 to 111, comprising MOAH at a content of less than or equal to 4 ppm based on the total weight of the nutrient oil mixture.

[0247] Clause 113. The nutrient oil mixture as described in any one of Clauses 75 to 112, comprising MOAH at a content of less than or equal to 3 ppm based on the total weight of the nutrient oil mixture.

[0248] Clause 114. The nutrient oil mixture as described in any one of Clauses 75 to 113, comprising MOAH at a content of less than or equal to 2 ppm based on the total weight of the nutrient oil mixture.

[0249] Clause 115. The nutrient oil mixture as described in any one of Clauses 75 to 114, comprising MOAH at a content of less than or equal to 1.5 ppm based on the total weight of the nutrient oil mixture.

[0250] Clause 116. The nutrient oil mixture as described in any one of Clauses 75 to 115, comprising MOAH at a content of less than or equal to 1 ppm based on the total weight of the nutrient oil mixture.

[0251] Clause 117. The nutritional oil mixture as described in any one of Clauses 75 to 116 further comprises added antioxidants.

[0252] Clause 118. A nutritional oil blend as described in Clause 117, wherein the added antioxidant comprises tocopherol.

[0253] Clause 119. The nutritional oil mixture as described in any one of Clauses 75 to 118, comprising 60 wt% or more, 65 wt% or more, 70 wt% or more, 75 wt% or more, 80 wt% or more, 85 wt% or more, 90 wt% or more, or 95 wt% or more based on the total weight of the nutritional oil mixture.

[0254] Clause 120. The nutritional oil mixture as described in any one of Clauses 75 to 119, comprising 60 wt% or more, preferably 65 wt% or more, more preferably 70 wt% or more, more preferably 75 wt% or more, more preferably 80 wt% or more, more preferably 85 wt% or more, more preferably 90 wt% or more, or more preferably 95 wt% or more, based on the total weight of the nutritional oil mixture.

[0255] Clause 121. The nutrient oil mixture as described in any one of Clauses 75 to 120, comprising 3-MCPD in a concentration of less than or equal to 0.2 ppm, less than or equal to 0.15 ppm, or less than or equal to 0.1 ppm based on the total amount of the nutrient oil mixture.

[0256] Clause 122. The nutrient oil mixture as described in any one of Clauses 75 to 121, comprising 3-MCPD in a content of less than or equal to 0.2 ppm, preferably less than or equal to 0.15 ppm, or more preferably less than or equal to 0.1 ppm based on the total amount of the nutrient oil mixture.

[0257] Clause 123. The nutritional oil mixture as described in any one of Clauses 75 to 122, comprising glycidyl glycerol in an amount of less than or equal to 0.1 ppm, less than or equal to 0.05 ppm, less than or equal to 0.04 ppm, less than or equal to 0.03 ppm, or less than or equal to 0.02 ppm based on the total amount of the nutritional oil mixture.

[0258] Clause 124. The nutritional oil mixture as described in any one of Clauses 75 to 123, comprising glycidyl glycerin in a content of less than or equal to 0.1 ppm, preferably less than or equal to 0.05 ppm, preferably less than or equal to 0.04 ppm, more preferably less than or equal to 0.03 ppm, or more preferably less than or equal to 0.02 ppm based on the total amount of the nutritional oil mixture.

[0259] Clause 125. A nutrient oil mixture as described in any one of Clauses 75 to 124, wherein the anisidine value of the nutrient oil mixture is less than 20, less than 15, less than 12, less than 10, or less than 8.

[0260] Clause 126. A nutrient oil mixture as described in any one of Clauses 75 to 125, wherein the anisidine value of the nutrient oil mixture is less than 20, preferably less than 15, preferably less than 12, more preferably less than 10, or more preferably less than 8.

[0261] Clause 127. A nutrient oil mixture as described in any one of Clauses 75 to 126, wherein the nutrient oil mixture has a peroxide value of less than or equal to 10 meq / kg, less than or equal to 8 meq / kg, less than or equal to 5 meq / kg, less than or equal to 3 meq / kg, less than or equal to 2 meq / kg, or less than or equal to 1 meq / kg based on the total weight of the nutrient oil mixture.

[0262] Clause 128. A nutrient oil mixture as described in any one of Clauses 75 to 127, wherein the nutrient oil mixture has a peroxide value of less than or equal to 10 meq / kg, preferably less than or equal to 8 meq / kg, preferably less than or equal to 5 meq / kg, preferably less than or equal to 3 meq / kg, more preferably less than or equal to 2 meq / kg, or more preferably less than or equal to 1 meq / kg based on the total weight of the nutrient oil mixture.

[0263] Clause 129. A nutrient oil mixture as described in any one of Clauses 75 to 128, wherein the nutrient oil mixture comprises microbial oil.

[0264] Clause 130. A nutrient oil mixture as described in Clause 129, wherein the microbial oil contains DHA.

[0265] Clause 131. A nutrient oil mixture as described in Clause 130, wherein the microbial oil is derived from *Cryptodinium kurstii* (…). Crypthecodinium cohnii )produce.

[0266] Clause 132. A nutrient oil mixture as described in Clause 130, wherein the microbial oil is composed of *Cypripedium* (…). Thraustochytrium ) or Schizochytrium ( Schizochytrium Microorganisms that produce .

[0267] Clause 133. A nutrient oil mixture as described in Clause 129, wherein the microbial oil contains ARA.

[0268] Clause 134. The nutrient oil mixture as described in Clause 133, wherein the microbial oil is derived from *Morchella alpina* (… Mortierella alpina ))produce.

[0269] Clause 135. A nutritional oil mixture as described in any one of Clauses 128 to 134, comprising a first microbial oil containing ARA and a second microbial oil containing DHA.

[0270] Clause 136. A method for preparing a nutritional oil according to any one of Clauses 1 to 74, comprising: i. Mix the oil containing LC-PUFA with an organic solvent; ii. Removing at least a portion of the organic solvent from the oil containing LC-PUFA to obtain a crude nutritional oil; and iii. Recover at least a portion of the organic solvent removed in step ii using a recycled solvent, wherein the recycled solvent contains less than 50 wt% mineral oil based on the total weight of the recycled solvent. The nutritional oil mentioned above has a MOSH content of less than 7 ppm.

[0271] Clause 137. A method for preparing a nutritional oil, comprising: Mix the oil containing LC-PUFA with an organic solvent; Remove at least a portion of the organic solvent from the oil containing LC-PUFA to obtain a crude nutritional oil; and At least a portion of the organic solvent removed in step ii is recovered using a recycled solvent, wherein the recycled solvent contains less than 50 wt% mineral oil based on the total weight of the recycled solvent.

[0272] Clause 138. The method of any one of Clauses 136 to 137, wherein the organic solvent comprises hexane.

[0273] Clause 139. The method of any one of Clauses 136 to 138, wherein the organic solvent comprises at least a portion of the recovered organic solvent previously mixed with a second LC-PUFA-containing oil.

[0274] Clause 140. The method of Clause 139, wherein the recovered organic solvent comprises less than 100 ppm of MOSH, less than 75 ppm of MOSH, less than 50 ppm of MOSH, less than 25 ppm of MOSH, less than 10 ppm of MOSH, less than 5 ppm of MOSH, or less than 1 ppm of MOSH.

[0275] Clause 141. The method of any one of Clauses 139 and 140, wherein the recovered organic solvent comprises less than 100 ppm of MOSH, preferably less than 75 ppm of MOSH, preferably less than 50 ppm of MOSH, preferably less than 25 ppm of MOSH, more preferably less than 10 ppm of MOSH, more preferably less than 5 ppm of MOSH, or more preferably less than 1 ppm of MOSH.

[0276] Clause 142. The method of any one of Clauses 136 to 141, wherein mixing the oil containing LC-PUFA with an organic solvent comprises using said organic solvent to extract microbial oil from microorganisms.

[0277] Clause 143. The method as described in Clause 142, wherein the microorganism belongs to the order Mortierellales.

[0278] Clause 144. The method as described in Clause 143, wherein said microorganism is *Morchella alpina* ( Mortierella alpina ).

[0279] Clause 145. The method described in Clause 142, wherein the microorganism belongs to the order Thraustochytriales.

[0280] Clause 146. The method as described in Clause 145, wherein the microorganism belongs to the genus *Cypripedium* (…). Thraustochytrium ) or Schizochytrium ( Schizochytrium ).

[0281] Clause 147. The method as described in Clause 142, wherein the microorganism is a dinoflagellate.

[0282] Clause 148. The method as described in Clause 147, wherein said microorganism is *Cryptodinium kurstii* (…). Crypthecodinium cohnii ).

[0283] Clause 149. The method of any one of Clauses 136 to 148, wherein the recovered solvent comprises less than 40 wt%, less than 30 wt%, less than 20 wt%, less than 10 wt%, or less than 5 wt% mineral oil based on the total weight of the recovered solvent.

[0284] Clause 150. The method of any one of Clauses 136 to 149, wherein the recovered solvent comprises less than 40 wt%, preferably less than 30 wt%, preferably less than 20 wt%, more preferably less than 10 wt%, or more preferably less than 5 wt% mineral oil based on the total weight of the recovered solvent.

[0285] Clause 151. The method of any one of Clauses 136 to 149, wherein the recovered solvent comprises triglycerides.

[0286] Clause 152. The method of any one of Clauses 136 to 151, wherein the recovered solvent comprises sunflower oil, medium-chain triglycerides, rapeseed oil, or a combination thereof.

[0287] Clause 153. The method of any one of Clauses 136 to 152, wherein the nutrient oil is the crude nutrient oil.

[0288] Clause 154. The method of any one of Clauses 136 to 153, wherein the nutrient oil is a final nutrient oil obtained by further processing the crude nutrient oil to obtain a final nutrient oil.

[0289] Clause 155. The method of any one of Clauses 136 to 154, wherein the nutritional oil contains one or more of ARA, DHA or EPA in an amount greater than or equal to 300 mg / g based on the total amount of nutritional oil.

[0290] Clause 156. The method of any one of Clauses 136 to 155, wherein the nutrient oil contains less than or equal to 6 ppm, optionally less than or equal to 5 ppm, optionally less than or equal to 4 ppm, optionally less than or equal to 3 ppm, optionally less than or equal to 2.5 ppm, optionally less than or equal to 2 ppm, optionally less than or equal to 1.5 ppm, optionally less than or equal to 1 ppm, or optionally less than or equal to 0.5 ppm based on the total weight of the nutrient oil.

[0291] Clause 157. The method of any one of Clauses 136 to 156, wherein the nutrient oil contains less than or equal to 5 ppm of MOSH based on the total weight of the nutrient oil.

[0292] Clause 158. The method of any one of Clauses 136 to 157, wherein the nutrient oil contains less than or equal to 4 ppm of MOSH based on the total weight of the nutrient oil.

[0293] Clause 159. The method of any one of Clauses 136 to 158, wherein the nutrient oil contains MOSH at a content of less than or equal to 3 ppm based on the total weight of the nutrient oil.

[0294] Clause 160. The method of any one of Clauses 136 to 159, wherein the nutrient oil contains MOSH at a content of less than or equal to 2.5 ppm based on the total weight of the nutrient oil.

[0295] Clause 161. The method of any one of Clauses 136 to 160, wherein the nutrient oil contains MOSH at a content of less than or equal to 2 ppm based on the total weight of the nutrient oil.

[0296] Clause 162. The method of any one of Clauses 136 to 161, wherein the nutrient oil contains less than or equal to 1.5 ppm of MOSH based on the total weight of the nutrient oil.

[0297] Clause 163. The method of any one of Clauses 136 to 162, wherein the nutrient oil contains MOSH at a content of less than or equal to 1 ppm based on the total weight of the nutrient oil.

[0298] Clause 164. The method of any one of Clauses 136 to 163, wherein the nutrient oil contains MOSH at a content of less than or equal to 0.5 ppm based on the total weight of the nutrient oil.

[0299] Clause 165. The method of any one of Clauses 136 to 156, wherein the nutrient oil contains MOSH at a content of 0.1 ppm to 2 ppm based on the total weight of the nutrient oil.

[0300] Clause 166. The method of any one of Clauses 136 to 156, wherein the nutrient oil contains MOSH at a content of 0.5 ppm to 3 ppm based on the total weight of the nutrient oil.

[0301] Clause 167. The method of any one of Clauses 136 to 156, wherein the nutrient oil contains MOSH at a content of 1 ppm to 4 ppm based on the total weight of the nutrient oil.

[0302] Clause 168. The method of any one of Clauses 136 to 156, wherein the nutrient oil contains 1.5 ppm to 5 ppm of MOSH based on the total weight of the nutrient oil.

[0303] Clause 169. The method of any one of Clauses 136 to 168, wherein the nutrient oil contains MOAH at a content of less than or equal to 5 ppm, optionally less than or equal to 4 ppm, optionally less than or equal to 3 ppm, optionally less than or equal to 2 ppm, optionally less than or equal to 1.5 ppm, or optionally less than or equal to 1 ppm based on the total weight of the nutrient oil.

[0304] Clause 170. The method of any one of Clauses 136 to 169, wherein the nutrient oil contains MOAH at a content of less than or equal to 4 ppm based on the total weight of the nutrient oil.

[0305] Clause 171. The method of any one of Clauses 136 to 170, wherein the nutrient oil contains MOAH at a content of less than or equal to 3 ppm based on the total weight of the nutrient oil.

[0306] Clause 172. The method of any one of Clauses 136 to 171, wherein the nutrient oil contains MOAH at a content of less than or equal to 2 ppm based on the total weight of the nutrient oil.

[0307] Clause 173. The method of any one of Clauses 136 to 172, wherein the nutrient oil contains MOAH at a content of less than or equal to 1.5 ppm based on the total weight of the nutrient oil.

[0308] Clause 174. The method of any one of Clauses 136 to 173, wherein the nutrient oil contains MOAH at a content of less than or equal to 1 ppm based on the total weight of the nutrient oil.

[0309] Clause 175. The method of any one of Clauses 136 to 174, wherein the nutritional oil comprises, based on the total weight of the nutritional oil, greater than 0.1 wt%, optionally greater than 0.15 wt%, greater than 0.2 wt%, optionally greater than 0.25 wt%, optionally greater than 0.3 wt%, optionally greater than 0.35 wt%, optionally greater than 0.4 wt%, optionally greater than 0.45 wt%, optionally greater than 0.5 wt%, or optionally greater than 0.55 wt% of free fatty acids.

[0310] Clause 176. The method of any one of Clauses 136 to 175, wherein the nutrient oil contains less than 0.1 wt% or less than 0.05 wt% of free fatty acids based on the total weight of the nutrient oil.

[0311] Clause 177. The method of any one of Clauses 136 to 176, wherein the nutritional oil contains less than 0.1 wt% or preferably less than 0.05 wt% of free fatty acids based on the total weight of the nutritional oil.

[0312] Clause 178. The method as described in any one of Clauses 136 to 177 further comprises using equipment materials containing less than or equal to 50 wt% mineral oil based on the total weight of the equipment materials.

[0313] Clause 179. The method described in Clause 178, wherein the equipment material is an equipment lubricant.

[0314] Clause 180. The method of any one of Clauses 178 and 179, wherein the equipment material comprises rapeseed oil, sunflower seed oil, mixed-chain triglycerides, or combinations thereof.

[0315] Clause 181. A nutritional oil produced by any one of Clauses 137 to 180, comprising: Based on an ARA content greater than or equal to 300 mg / g based on the total weight of the nutritional oil; and MOSH is 0.5 ppm to 10 ppm based on the total weight of the nutrient oil.

[0316] Clause 182. The nutritional oil as described in Clause 1, which contains ARA at a content of 350 mg / g or greater, optionally 400 mg / g, optionally 450 mg / g, optionally 500 mg / g, or optionally 550 mg / g based on the total weight of the nutritional oil.

[0317] Clause 183. The nutritional oil as described in any of the preceding clauses contains ARA at a content greater than or equal to 400 mg / g based on the total weight of the nutritional oil.

[0318] Clause 184. The nutritional oil as described in any of the preceding clauses contains ARA at a content greater than or equal to 450 mg / g based on the total weight of the nutritional oil.

[0319] Clause 185. The nutritional oil as described in any of the preceding clauses contains ARA at a content greater than or equal to 500 mg / g based on the total weight of the nutritional oil.

[0320] Clause 186. The nutritional oil as described in any of the preceding clauses contains ARA at a content greater than or equal to 550 mg / g based on the total weight of the nutritional oil.

[0321] Clause 187. The nutritional oil as described in any of the preceding clauses, comprising ARA in a content of 300 mg / g to 400 mg / g, optionally 350 mg / g to 450 mg / g, optionally 400 mg / g to 500 mg / g, optionally 450 mg / g to 550 mg / g, or optionally 500 mg / g to 600 mg / g based on the total weight of the nutritional oil.

[0322] Clause 188. The nutritional oil as described in any one of Clauses 1 to 7, comprising ARA at a content of 350 mg / g to 450 mg / g based on the total weight of the nutritional oil.

[0323] Clause 189. The nutritional oil as described in any one of Clauses 1 to 7, comprising ARA at a content of 400 mg / g to 500 mg / g based on the total weight of the nutritional oil.

[0324] Clause 190. The nutritional oil as described in any one of Clauses 1 to 7, comprising ARA at a content of 450 mg / g to 550 mg / g based on the total weight of the nutritional oil.

[0325] Clause 191. The nutritional oil as described in any one of Clauses 1 to 7, comprising ARA at a content of 500 mg / g to 600 mg / g based on the total weight of the nutritional oil.

[0326] Clause 192. The nutritional oil as described in any one of Clauses 181 to 191, comprising free fatty acids at a content greater than or equal to 0.1 wt% based on the total weight of the nutritional oil.

[0327] Clause 193. The nutritional oil as described in Clause 192, which contains MOSH at a concentration of less than or equal to 7 ppm, optionally less than or equal to 6 ppm, optionally less than or equal to 5 ppm, optionally less than or equal to 4 ppm, or optionally less than or equal to 3.5 ppm based on the total weight of the nutritional oil.

[0328] Clause 194. The nutrient oil as described in any one of Clauses 192 to 193 contains less than or equal to 6 ppm of MOSH based on the total weight of the nutrient oil.

[0329] Clause 195. The nutrient oil as described in any one of Clauses 192 to 194, which contains less than or equal to 5 ppm of MOSH based on the total weight of the nutrient oil.

[0330] Clause 196. The nutrient oil as described in any one of Clauses 192 to 195 contains less than or equal to 4 ppm of MOSH based on the total weight of the nutrient oil.

[0331] Clause 197. The nutrient oil as described in any one of Clauses 192 to 196, which contains less than or equal to 3.5 ppm of MOSH based on the total weight of the nutrient oil.

[0332] Clause 198. The nutrient oil as described in any one of Clauses 192 to 197, which contains MOSH at a content of 0.5 ppm to less than 7 ppm based on the total weight of the nutrient oil.

[0333] Clause 199. The nutritional oil as described in any one of Clauses 192 to 198 contains MOSH in an amount of 2 ppm to less than 7 ppm.

[0334] Clause 200. The nutritional oil as described in any one of Clauses 181 to 191 contains less than 0.1 wt% of free fatty acids based on the total weight of the nutritional oil.

[0335] Clause 201. The nutritional oil as described in Clause 200 contains MOSH in amounts of 0.1 ppm to 5 ppm, optionally 0.1 ppm to 2 ppm, optionally 0.5 ppm to 3 ppm, optionally 1 ppm to 4 ppm, or optionally 1.5 ppm to 5 ppm based on the total weight of the nutritional oil.

[0336] Clause 202. The nutritional oil as described in any one of Clauses 200 to 201 contains 0.1 ppm to 2 ppm of MOSH based on the total weight of the nutritional oil.

[0337] Clause 203. The nutritional oil as described in any one of Clauses 200 to 201, comprising 0.5 ppm to 3 ppm of MOSH based on the total weight of the nutritional oil.

[0338] Clause 204. The nutritional oil as described in any one of Clauses 200 to 201 contains 1 to 4 ppm of MOSH based on the total weight of the nutritional oil.

[0339] Clause 205. The nutrient oil as described in any one of Clauses 200 to 201, comprising 1.5 ppm to 5 ppm of MOSH based on the total weight of the nutrient oil.

[0340] Clause 206. The nutrient oil as described in any one of Clauses 181 to 205, comprising MOAH at a concentration of less than or equal to 5 ppm, optionally less than or equal to 4 ppm, optionally less than or equal to 3 ppm, optionally less than or equal to 2 ppm, optionally less than or equal to 1.5 ppm, or optionally less than or equal to 1 ppm based on the total weight of the nutrient oil.

[0341] Clause 207. The nutrient oil as described in any one of Clauses 181 to 206, which contains MOAH at a content of less than or equal to 4 ppm based on the total weight of the nutrient oil.

[0342] Clause 208. The nutrient oil as described in any one of Clauses 181 to 207, which contains less than or equal to 3 ppm of MOAH based on the total weight of the nutrient oil.

[0343] Clause 209. The nutrient oil as described in any one of Clauses 181 to 208, which contains less than or equal to 2 ppm of MOAH based on the total weight of the nutrient oil.

[0344] Clause 210. The nutrient oil as described in any one of Clauses 181 to 209 contains less than or equal to 1.5 ppm of MOAH based on the total weight of the nutrient oil.

[0345] Clause 211. The nutrient oil as described in any one of Clauses 181 to 210 contains MOAH at a content of less than or equal to 1 ppm based on the total weight of the nutrient oil.

[0346] Clause 212. The nutritional oil as described in any one of Clauses 181 to 211 further contains added antioxidants.

[0347] Clause 213. Nutritional oils as described in Clause 212, wherein the added antioxidants include tocopherol.

[0348] Clause 214. The nutritional oil as described in any one of Clauses 181 to 213, comprising 50 wt% or more, 55 wt% or more, 60 wt% or more, 65 wt% or more, 70 wt% or more, 75 wt% or more, 80 wt% or more, or 85 wt% or more based on the total weight of the nutritional oil.

[0349] Clause 215. The nutritional oil as described in any one of Clauses 181 to 214, comprising 50 wt% or more, preferably 55 wt% or more, preferably 60 wt% or more, preferably 65 wt% or more, more preferably 70 wt% or more, more preferably 75 wt% or more, more preferably 80 wt% or more, or more preferably 85 wt% or more, based on the total weight of the nutritional oil.

[0350] Clause 216. The nutrient oil as described in any one of Clauses 181 to 215, comprising 3-MCPD in a concentration of less than or equal to 0.2 ppm, less than or equal to 0.15 ppm, or less than or equal to 0.1 ppm based on the total amount of nutrient oil.

[0351] Clause 217. The nutritional oil as described in any one of Clauses 181 to 216, comprising 3-MCPD in a content of less than or equal to 0.2 ppm, preferably less than or equal to 0.15 ppm, or more preferably less than or equal to 0.1 ppm based on the total amount of nutritional oil.

[0352] Clause 218. The nutritional oil as described in any one of Clauses 181 to 217, comprising glycidyl glycerin in a concentration of less than or equal to 0.1 ppm, less than or equal to 0.05 ppm, less than or equal to 0.04 ppm, less than or equal to 0.03 ppm, or less than or equal to 0.02 ppm based on the total amount of nutritional oil.

[0353] Clause 219. The nutritional oil as described in any one of Clauses 181 to 218, comprising glycidyl glycerin in a content of less than or equal to 0.1 ppm, preferably less than or equal to 0.05 ppm, preferably less than or equal to 0.04 ppm, more preferably less than or equal to 0.03 ppm, or more preferably less than or equal to 0.02 ppm based on the total amount of nutritional oil.

[0354] Clause 220. The nutrient oil as described in any one of Clauses 181 to 219, wherein the nutrient oil has anisidine values ​​of less than 20, less than 15, less than 12, less than 10, or less than 8.

[0355] Clause 221. The nutritional oil as described in any one of Clauses 181 to 220, wherein the nutritional oil has anisidine value of less than 20, preferably less than 15, preferably less than 12, more preferably less than 10, or more preferably less than 8.

[0356] Clause 222. The nutrient oil as described in any one of Clauses 181 to 221, wherein the nutrient oil has a peroxide value of less than or equal to 10 meq / kg, less than or equal to 8 meq / kg, less than or equal to 5 meq / kg, less than or equal to 3 meq / kg, less than or equal to 2 meq / kg, or less than or equal to 1 meq / kg based on the total weight of the nutrient oil.

[0357] Clause 223. The nutritional oil as described in any one of Clauses 181 to 222, wherein the nutritional oil has a peroxide value of less than or equal to 10 meq / kg, preferably less than or equal to 8 meq / kg, preferably less than or equal to 5 meq / kg, preferably less than or equal to 3 meq / kg, more preferably less than or equal to 2 meq / kg, or more preferably less than or equal to 1 meq / kg based on the total weight of the nutritional oil.

[0358] Clause 224. The nutrient oil as described in any one of Clauses 181 to 223, wherein the nutrient oil is a microbial oil.

[0359] Clause 225. Nutrient oil as described in Clause 224, wherein the microbial oil is produced by Alpine Glomerella.

[0360] Experimental Example After providing a general description of nutrient oils and related methods, further understanding can be gained by referring to the experimental examples provided below. These examples are provided for illustrative purposes only and are not intended to be limiting.

[0361] Example 1 – Comparative Example (Crude Oil) Several microbial oil samples containing greater than 400 mg / g arachidonic acid were produced using *Morchella alpina*. The manufacturing equipment used in producing the microbial oil samples employed mineral oil as a lubricant. Each microbial oil sample was extracted from biomass using hexane recovered from a previous extraction process, employing a mineral oil-based system. Specifically, the hexane used to extract this microbial oil had previously been used to extract other microbial oils from other biomass samples. The hexane was separated from the previous microbial oil by evaporation and collected for reuse. Specifically, the exhaust gas leaving the solvent recovery system contained evaporated hexane vapor, and residual hexane was absorbed from the exhaust gas using a mineral oil-based system before being released into the atmosphere. The mineral oil-based system contained solid packing material to maximize the oil / vapor contact surface area. The adsorbed solvent was collected and reused for extracting this microbial oil sample.

[0362] The initial crude oil samples contained more than 0.1 wt% free fatty acids. The level of MOSH in the microbial oil was determined by GC-FID analysis of each crude oil sample. More specifically, a glass solid-phase extraction (SPE) column with a PTFE sieve was packed with a lower layer of activated alumina (1.5 g) and an upper layer of silica gel impregnated with 10% silver nitrate (2.5 g). The column was conditioned by washing with several volumes of high-purity hexane. Approximately 1.5 g of each crude oil sample was dissolved separately and quantitatively transferred to the column in two 1 mL aliquots of hexane. The MOSH fraction of the crude oil samples was eluted with 5 mL of hexane added dropwise. An internal standard (n-octadecane) was added to the collected MOSH fraction. The sample was then concentrated to a final volume of approximately 1 mL under a gentle nitrogen flow.

[0363] The MOSH of concentrated samples was analyzed using the AGILENT 7890 GC-FID, which is equipped with an upgraded "multi-mode" injection port, via gas chromatography and flame ionization.

[0364] Chromatographic conditions: Injection parameters Injection volume: 50 μL Injection rate: 80 μL / min.

[0365] Inlet settings Carrier gas: hydrogen Inlet mode: PTV solvent venting Inlet pressure: 6.3143 psi Septum purging flow rate: 3 mL / min.

[0366] Split outlet purge flow rate: 60 mL / min after 4.5 min.

[0367] Solvent purging flow rate: 100 mL / min at 5 psi for 1 min.

[0368] Inlet temperature: 45°C for 1 min, then increased to 370°C at a rate of 600°C / min and held.

[0369] Column box installation Carrier gas flow rate: 2 mL / min H2 (constant flow mode) Temperature: Hold at 50°C for 4 min, then increase to 370°C at a rate of 30°C / min and hold for 5 min.

[0370] Runtime: 19.7 min.

[0371] Flame ionization detector parameters Temperature: 370°C Hydrogen flow rate: 40 mL / min.

[0372] Airflow rate: 450 mL / min.

[0373] Makeup airflow (N2): 20 mL / min (column flow + makeup flow) Maintain the injection port at a temperature close to the boiling point of the sample solvent (hexane) for 1 minute, and simultaneously inject the sample slowly while applying a gas flow to purge the evaporated solvent from the purge port, allowing any MOSH to deposit on the injection port liner wall. After injection, rapidly increase the injection port temperature and direct the gas flow to the GC column to vaporize the analyte and transfer it to the column. Then, increase the column oven temperature to allow any MOSH to elute in a broadband manner.

[0374] Quantification was achieved by comparing the total peak area of ​​the MOSH peak with that of the internal standard peak. System suitability standards were also injected to validate the performance of the chromatographic system. The limit of quantitation for this method was determined to be 7 ppm. The results are shown in Table 1 below: Table 1 – Comparison of MOSH levels in samples (crude oil) Example 2 – Replacing a mineral oil-based system (crude oil) Several microbial oil samples containing greater than 400 mg / g arachidonic acid were produced using *Morchella alpina*. The manufacturing equipment used in producing the microbial oil samples employed mineral oil as a lubricant. Each microbial oil sample was extracted from biomass using hexane recovered from previous extraction processes, employing a sunflower oil-based system. Specifically, the hexane used to extract this microbial oil had previously been used to extract other microbial oils from other biomass samples. The hexane was separated from the previous microbial oils by evaporation and collected for reuse. Specifically, the exhaust gas leaving the solvent recovery system contained evaporated hexane vapor, and residual hexane was absorbed from the exhaust gas using a sunflower oil-based system before being released into the atmosphere. The sunflower oil-based system contained solid packing material to maximize the oil / vapor contact surface area. The adsorbed solvent was collected and reused for extracting this microbial oil sample.

[0375] The initial crude oil samples contained more than 0.1 wt% free fatty acids. Each crude oil sample was analyzed by GC-FID using the same method described in Example 1 to determine the level of MOSH in the microbial oil. The results are shown in Table 2 below: Table 2 – MOSH Levels (Crude Oil) in Embodiments of the Invention Example 3 – Stability of Alternative Solvents for Mineral Oil Scrubber Systems This embodiment aims to simulate a mineral oil-based scrubber system for removing volatile hexane after separation from extracted microbial oil. Mineral oil-based scrubber systems are typically part of a larger hexane solvent recovery unit, reducing volatile organic compound (VOC) emissions. However, when the captured hexane is recovered for further extraction of microbial oil, mineral oil is also a potential source of MOH (including MOSH and MOAH) contamination in the microbial oil. Therefore, this study seeks alternative recovery solvents for mineral oil that may be suitable for use in scrubber systems.

[0376] Three experiments were conducted to simulate a mineral oil scrubber system, but mineral oil was substituted for the following non-limiting examples: 1) high-oleic sunflower oil (HOSO); 2) medium-chain triglycerides (MCT); or 3) canola oil. In each experiment, 300 g of the substituted recycled solvent was introduced into a Parr 4500 series reactor. 2.3 wt% of reconstituted vapor condensate and 1.4 wt% hexane were added to the system. The mixture was sealed and heated to 105°C while mixing and maintaining a target pressure of 20 psi. The reaction was sampled twice daily over a period of 5–9 days.

[0377] Free fatty acids (FFA) in each mixture sample were analyzed as an indicator of hydrolysis over time. Figure 1It can be seen that during the testing period, the FFA content in the HOSO sample increased sharply after approximately 4 days, while in MCT or canola oil it increased only very slowly. HOSO had the highest amount of monounsaturated fatty acids, followed by canola oil and MCT, the latter being the least unsaturated. This data indicates that HOSO decomposes faster than canola oil and MCT, thus requiring more frequent replacement in scrubber systems compared to canola oil and MCT. FFA transferred from recovered hexane to subsequent batches of extracted microbial oil is generally easier to remove from microbial oil than MOSH and MOAH contamination from mineral oil.

[0378] Example 4 – Adsorption capacity of alternative recycled solvents Oil is pumped to the top of the glass column using a Milton Roy positive displacement piston pump. The oil is then allowed to flow downwards along the column wall by gravity and collected in a glass flask. The target oil flow rate is the set volumetric flow rate. A flask containing n-hexane is heated and evaporated, entering from the bottom of the column. A purge gas (nitrogen) is added to prevent hexane condensation and to simulate the non-condensable load typically present in mineral oil scrubbing systems. Qualitative measurements are performed on the nitrogen purge flow. Moisture (volatiles) is measured by infrared analysis to determine the amount of solvent collected.

[0379] As shown in Table 3, the adsorption range of n-hexane for samples 1 and 2 under experimental conditions was 10-14%, with HOSO showing slightly better adsorption performance than mineral oil. These experiments were repeated for samples 3 and 4, with the nitrogen purge gas reduced, resulting in a slight increase in recovery rate to 11-22%. In both experiments, HOSO recovered slightly more n-hexane solvent than mineral oil.

[0380] Table 3 – Initial Adsorption Capacity of HOSO and Mineral Oil In Table 4, nitrogen flow rates were reduced to the lowest possible level for laboratory-scale experiments, and mineral oil was compared with HOSO, MCT oil, and canola oil. Compared to the experiments in Table 3, the total solvent adsorption increased. MCT oil showed the highest recovery rate, while mineral oil showed the lowest. HOSO and canola oil exhibited similar n-hexane adsorption capacities.

[0381] Table 4 – Adsorption capacity of recovered solvent Example 5 – Replacing Mineral Oil-Based Systems (Refined Oils) Antioxidant blends, which may include tocopherols, are typically added to the final oil. In some cases, tocopherols may also contain some MOSH, thus contributing to the total MOSH in the final oil. To assess the contribution of tocopherols to the final oil, two separate tocopherol samples were procured from different suppliers and analyzed to determine the MOSH content of the samples. The MOSH content of the corresponding samples was determined using the following methods.

[0382] Accurately weigh approximately 0.5 g of tocopherol into a glass test tube. Add 3 mL of reagent alcohol (denatured ethanol) and 1 mL of 45% potassium hydroxide (aqueous solution). Cover the sample, place it on a heating block, set to 100°C, and continue for 30 minutes to saponify the lipid fraction. Extract the unsaponifiables with GC-MS grade hexane. Add an internal standard (n-octadecane) for quantification. Wash the hexane layer with deionized water and concentrate under a nitrogen stream.

[0383] An empty glass solid-phase extraction (SPE) column with a PTFE sieve was packed with a lower layer of 1.5 g activated alumina and an upper layer of 2.5 g silica gel (impregnated with 10% silver nitrate). The column was conditioned by washing with 20 mL of GC-MS grade hexane. The concentrated hexane extract from the sample was loaded onto the conditioned column. The MOSH fraction was then eluted with 5 mL of hexane. The hexane layer was concentrated to approximately 1 mL under a gentle nitrogen flow.

[0384] The MOSH content of concentrated fractions was analyzed using an AGILENT 8890 gas chromatograph equipped with a flame ionization detector (GC-FID). The instrument is upgraded with a "multi-mode injector" to allow for larger volume injections and improve sensitivity.

[0385] The blank oil sample was prepared using the same procedure, but the tocopherol sample was omitted to account for any trace MOSH contribution that the reagents might have brought.

[0386] Maintain the injection port at a temperature close to the boiling point of the sample solvent (hexane) for 1 minute, and simultaneously inject the sample slowly while applying a gas flow to purge the evaporated solvent from the purge port, allowing any MOSH to deposit on the injection port liner wall. After injection, rapidly increase the injection port temperature and direct the gas flow to the GC column to vaporize the analyte and transfer it to the column. Then, increase the column oven temperature to allow any MOSH to elute in a broadband manner.

[0387] Chromatographic conditions: Injection parameters Injection volume: 50 μL Injection rate: 80 μL / min.

[0388] Inlet settings Carrier gas: hydrogen Inlet mode: PTV solvent venting Inlet pressure: 6.3143 psi Septum purging flow rate: 3 mL / min.

[0389] Split outlet purge flow rate: 60 mL / min after 4.5 min.

[0390] Solvent purging flow rate: 100 mL / min at 5 psi for 1 min.

[0391] Inlet temperature: 45°C for 1 min, then increased to 370°C at a rate of 600°C / min and held.

[0392] Column box installation Carrier gas flow rate: 2 mL / min H2 (constant flow mode) Temperature: Hold at 50°C for 4 min, then increase to 370°C at a rate of 30°C / min and hold for 5 min.

[0393] Runtime: 19.7 min.

[0394] Flame ionization detector parameters Temperature: 370°C Hydrogen flow rate: 40 mL / min.

[0395] Airflow rate: 450 mL / min.

[0396] Makeup airflow (N2): 20 mL / min (column flow + makeup flow) Maintain the injection port at a temperature close to the boiling point of the sample solvent (hexane) for 1 minute, and simultaneously inject the sample slowly while applying a gas flow to purge the evaporated solvent from the purge port, allowing any MOSH to deposit on the injection port liner wall. After injection, rapidly increase the injection port temperature and direct the gas flow to the GC column to vaporize the analyte and transfer it to the column. Then, increase the column oven temperature to allow any MOSH to elute in a broadband manner.

[0397] Quantification was achieved by comparing the total peak area of ​​the MOSH peak with that of the internal standard peak. System suitability standards were also injected to validate the performance of the chromatographic system. The limit of quantitation for this method was determined to be 2 ppm.

[0398] The analytical results of the two separate tocopherol samples are reported in Table 5 below.

[0399] Table 5 – MOSH content in tocopherol samples As can be seen from Table 5, the samples from one tocopherol supplier had significantly higher MOSH values ​​than those from another supplier.

[0400] Crude microbial oil containing greater than 300 mg / g arachidonic acid was produced using *Morchella alpina* in the same manner as in Example 2. The initial crude oil sample contained greater than 0.1 wt% free fatty acids. The crude oil sample was further refined using degumming, caustic refining, decolorization, winterization, and deodorization (RBWD) processes commonly used in the art.

[0401] An antioxidant mixture was added to the refined oils to help protect them from oxidative degradation. Specifically, two oil samples with the same levels of tocopherol were prepared, but batch V7 from Table 5 was added to one sample and batch K4 from Table 5 was added to the other. Both refined oil samples were then analyzed using the same methods described above for the tocopherol samples to determine the MOSH content. Other data points were determined using the corresponding methods described elsewhere in this document. Various properties of the oil samples are reported in Table 6 below.

[0402] Table 6 – Characteristics of the Final Oil in the Embodiments of the Invention Example 6 – ARA and DHA mixture From Schizochytrium ( Schizochytrium DHA crude oil was prepared using a strain and extracted using an aqueous extraction technique as described in US 10,385,289 B2, which is incorporated herein by reference. The crude oil sample contained greater than 550 mg / g of DHA. The crude oil was further refined using degumming, alkali refining, decolorization, winterization, and deodorization processes commonly used in the oil refining industry. An antioxidant mixture containing tocopherol from batch K4 of Table 5 was added to the final DHA oil. This final DHA oil was then mixed with the ARA final oil tested as Sample 2 in Table 6 at two different ratios (ARA:DHA). The MOSH content of the two mixtures was analyzed using the same method described in Example 5. The results are shown in Table 7 below.

[0403] Table 7 – MOSH content in ARA / DHA mixtures

Claims

1. A nutritional oil comprising: Based on the content of arachidonic acid (ARA) greater than or equal to 300 mg / g based on the total weight of the nutrient oil. Based on the free fatty acid content greater than or equal to 0.1 wt% of the total weight of the nutritional oil; and Based on the total weight of the nutrient oil, the content of mineral oil saturated hydrocarbons (MOSH) is less than 7 ppm.

2. The nutritional oil of claim 1, comprising ARA at a content greater than or equal to 350 mg / g, greater than or equal to 400 mg / g, greater than or equal to 450 mg / g, greater than or equal to 500 mg / g, or greater than or equal to 550 mg / g based on the total weight of the nutritional oil.

3. The nutritional oil as claimed in any of the preceding claims, comprising ARA in a content of 300 mg / g to 400 mg / g, 350 mg / g to 450 mg / g, 400 mg / g to 500 mg / g, 450 mg / g to 550 mg / g, or 500 mg / g to 600 mg / g based on the total weight of the nutritional oil.

4. The nutritional oil as claimed in any of the preceding claims, comprising MOSH at a concentration of less than or equal to 6 ppm, less than or equal to 5 ppm, less than or equal to 4 ppm, or less than or equal to 3.5 ppm based on the total weight of the nutritional oil.

5. The nutritional oil as claimed in any of the preceding claims, comprising MOSH in a content of 0.5 ppm to less than 7 ppm or 2 ppm to less than 7 ppm based on the total weight of the nutritional oil.

6. The nutritional oil as claimed in any of the preceding claims, comprising mineral oil aromatics (MOAHs) in amounts of less than or equal to 5 ppm, less than or equal to 4 ppm, less than or equal to 3 ppm, less than or equal to 2 ppm, less than or equal to 1.5 ppm, or less than or equal to 1 ppm based on the total weight of the nutritional oil.

7. The nutritional oil as described in any of the preceding claims further comprises added antioxidants.

8. The nutritional oil of claim 7, wherein the added antioxidant comprises tocopherol.

9. The nutritional oil as claimed in any of the preceding claims, comprising 50 wt% or more, 55 wt% or more, 60 wt% or more, 65 wt% or more, 70 wt% or more, 75 wt% or more, 80 wt% or more, or 85 wt% or more based on the total weight of the nutritional oil.

10. The nutritional oil as claimed in any of the preceding claims, comprising 3-MCPD in a content of less than or equal to 0.2 ppm, less than or equal to 0.15 ppm, or less than or equal to 0.1 ppm based on the total amount of the nutritional oil.

11. The nutritional oil as claimed in any of the preceding claims, comprising glycidyl glycerin in an amount of less than or equal to 0.1 ppm, less than or equal to 0.05 ppm, less than or equal to 0.04 ppm, less than or equal to 0.03 ppm, or less than or equal to 0.02 ppm based on the total amount of the nutritional oil.

12. The nutritional oil as claimed in any of the preceding claims, wherein the anisidine value of the nutritional oil is less than 20, less than 15, less than 12, less than 10 or less than 8.

13. The nutritional oil as claimed in any of the preceding claims, wherein the nutritional oil has a peroxide value of less than or equal to 10 meq / kg, less than or equal to 8 meq / kg, less than or equal to 5 meq / kg, less than or equal to 3 meq / kg, less than or equal to 2 meq / kg, or less than or equal to 1 meq / kg based on the total weight of the nutritional oil.

14. The nutritional oil as claimed in any of the preceding claims, wherein the nutritional oil is a microbial oil.

15. The nutrient oil of claim 14, wherein the microbial oil is derived from *Morchella alpina* (…). Mortierella alpina )produce.

16. A nutritional oil comprising: Based on an ARA content greater than or equal to 300 mg / g based on the total weight of the nutritional oil; Based on the free fatty acids content of less than 0.1 wt% based on the total weight of the nutritional oil; and The MOSH content is less than or equal to 3 ppm based on the total weight of the nutritional oil.

17. The nutritional oil of claim 16, comprising ARA at a content greater than or equal to 350 mg / g, greater than or equal to 400 mg / g, greater than or equal to 450 mg / g, or greater than or equal to 500 mg / g based on the total weight of the nutritional oil.

18. The nutritional oil according to any one of claims 16 to 17, comprising ARA in a content of 300 mg / g to 400 mg / g, 350 mg / g to 450 mg / g, or 400 mg / g to 500 mg / g based on the total weight of the nutritional oil.

19. The nutritional oil according to any one of claims 16 to 18, comprising MOSH in a content of less than or equal to 2.5 ppm, less than or equal to 2 ppm, less than or equal to 1.5 ppm, or less than or equal to 1 ppm based on the total weight of the nutritional oil.

20. The nutritional oil according to any one of claims 16 to 18, comprising 0.1 ppm to 2 ppm or 0.5 ppm to 3 ppm of MOSH based on the total weight of the nutritional oil.

21. The nutritional oil according to any one of claims 16 to 20, comprising MOAH in amounts of less than or equal to 5 ppm, less than or equal to 4 ppm, less than or equal to 3 ppm, less than or equal to 2 ppm, less than or equal to 1.5 ppm, or less than or equal to 1 ppm based on the total weight of the nutritional oil.

22. The nutritional oil according to any one of claims 16 to 21, further comprising added antioxidants.

23. The nutritional oil of claim 22, wherein the added antioxidant comprises tocopherol.

24. The nutritional oil according to any one of claims 16 to 23, comprising 60 wt% or more, 65 wt% or more, 70 wt% or more, 75 wt% or more, 80 wt% or more, 85 wt% or more, 90 wt% or more, or 95 wt% or more based on the total weight of the nutritional oil.

25. The nutritional oil according to any one of claims 16 to 24, comprising 3-MCPD in a content of less than or equal to 0.2 ppm, less than or equal to 0.15 ppm, or less than or equal to 0.1 ppm based on the total amount of the nutritional oil.

26. The nutritional oil according to any one of claims 16 to 25, comprising glycidyl glycerin in a content of less than or equal to 0.1 ppm, less than or equal to 0.05 ppm, less than or equal to 0.04 ppm, less than or equal to 0.03 ppm, or less than or equal to 0.02 ppm based on the total amount of the nutritional oil.

27. The nutritional oil according to any one of claims 16 to 26, wherein the nutritional oil has anisidine value of less than 20, less than 15, less than 12, less than 10 or less than 8.

28. The nutritional oil according to any one of claims 16 to 27, wherein the nutritional oil has a peroxide value of less than or equal to 10 meq / kg, less than or equal to 8 meq / kg, less than or equal to 5 meq / kg, less than or equal to 3 meq / kg, less than or equal to 2 meq / kg, or less than or equal to 1 meq / kg based on the total weight of the nutritional oil.

29. The nutrient oil according to any one of claims 16 to 28, wherein the nutrient oil is a microbial oil.

30. The nutrient oil of claim 29, wherein the microbial oil is produced by *Morchella alpina*.

31. A nutritional oil mixture comprising: A first nutrient oil containing ARA, mixed with a second nutrient oil containing docosahexaenoic acid (DHA), wherein the nutrient oil mixture contains ARA and DHA in a weight ratio of 0.5 to 3 ARA / DHA, and wherein the nutrient oil mixture contains MOSH at a content of less than or equal to 4 ppm.

32. The nutrient oil mixture of claim 31, comprising ARA at a content greater than or equal to 100 mg / g, greater than or equal to 150 mg / g, greater than or equal to 200 mg / g, greater than or equal to 250 mg / g, greater than or equal to 300 mg / g, or greater than or equal to 350 mg / g based on the total weight of the nutrient oil mixture.

33. The nutrient oil mixture according to any one of claims 31 to 32, comprising ARA in a content of 100 mg / g to 200 mg / g, 150 mg / g to 250 mg / g, 200 mg / g to 300 mg / g, 250 mg / g to 350 mg / g, or 300 mg / g to 400 mg / g based on the total weight of the nutrient oil mixture.

34. The nutrient oil mixture according to any one of claims 31 to 33, comprising DHA at a content greater than or equal to 100 mg / g, greater than or equal to 150 mg / g, greater than or equal to 200 mg / g, greater than or equal to 250 mg / g, greater than or equal to 300 mg / g, or greater than or equal to 350 mg / g based on the total weight of the nutrient oil mixture.

35. The nutrient oil mixture according to any one of claims 31 to 34, comprising DHA in a content of 100 mg / g to 200 mg / g, 150 mg / g to 250 mg / g, 200 mg / g to 300 mg / g, 250 mg / g to 350 mg / g, or 300 mg / g to 400 mg / g based on the total weight of the nutrient oil mixture.

36. The nutritional oil mixture according to any one of claims 31 to 35, comprising ARA and DHA in a weight ratio of 0.5 to 1.5 ARA / DHA, 1 to 2 ARA / DHA, 1.5 to 2.5 ARA / DHA, or 2 to 3 ARA / DHA.

37. The nutrient oil mixture of any one of claims 31 to 36, comprising MOSH in a content of less than or equal to 3.5 ppm, less than or equal to 3 ppm, less than or equal to 2.5 ppm, less than or equal to 2 ppm, less than or equal to 1.5 ppm, or less than or equal to 1 ppm based on the total weight of the nutrient oil mixture.

38. The nutrient oil mixture according to any one of claims 31 to 36, comprising MOSH in an amount of 0.1 ppm to 2 ppm, 0.5 ppm to 3 ppm, 1 ppm to 4 ppm, or 1.5 ppm to 5 ppm based on the total weight of the nutrient oil mixture.

39. The nutrient oil mixture of any one of claims 31 to 38, comprising MOAH in a content of less than or equal to 5 ppm, less than or equal to 4 ppm, less than or equal to 3 ppm, less than or equal to 2 ppm, less than or equal to 1.5 ppm, or less than or equal to 1 ppm based on the total weight of the nutrient oil mixture.

40. The nutritional oil mixture of any one of claims 31 to 38, further comprising added antioxidants.

41. The nutritional oil mixture of claim 40, wherein the added antioxidant comprises tocopherol.

42. The nutritional oil mixture according to any one of claims 31 to 41, comprising 60 wt% or more, 65 wt% or more, 70 wt% or more, 75 wt% or more, 80 wt% or more, 85 wt% or more, 90 wt% or more, or 95 wt% or more based on the total weight of the nutritional oil mixture.

43. The nutrient oil mixture of any one of claims 31 to 42, comprising 3-MCPD in a content of less than or equal to 0.2 ppm, less than or equal to 0.15 ppm, or less than or equal to 0.1 ppm based on the total amount of the nutrient oil mixture.

44. The nutritional oil mixture according to any one of claims 31 to 43, comprising glycidyl glycerol in an amount of less than or equal to 0.1 ppm, less than or equal to 0.05 ppm, less than or equal to 0.04 ppm, less than or equal to 0.03 ppm, or less than or equal to 0.02 ppm based on the total amount of the nutritional oil mixture.

45. The nutrient oil mixture of any one of claims 31 to 44, wherein the anisidine value of the nutrient oil mixture is less than 20, less than 15, less than 12, less than 10, or less than 8.

46. ​​The nutrient oil mixture according to any one of claims 31 to 45, wherein the nutrient oil mixture has a peroxide value of less than or equal to 10 meq / kg, less than or equal to 8 meq / kg, less than or equal to 5 meq / kg, less than or equal to 3 meq / kg, less than or equal to 2 meq / kg, or less than or equal to 1 meq / kg based on the total weight of the nutrient oil mixture.

47. The nutrient oil mixture of any one of claims 31 to 46, wherein the nutrient oil mixture comprises microbial oil.

48. The nutrient oil mixture of claim 47, wherein the microbial oil comprises DHA.

49. The nutrient oil mixture of claim 48, wherein the microbial oil is composed of *Cryptodinium kurstii* (…). Crypthecodinium cohnii )produce.

50. The nutrient oil mixture of claim 48, wherein the microbial oil is composed of *Cypripedium* (…). Thraustochytrium ) or Schizochytrium ( Schizochytrium Microorganisms that produce .

51. The nutrient oil mixture of claim 47, wherein the microbial oil comprises ARA.

52. The nutrient oil mixture of claim 51, wherein the microbial oil is produced by *Morchella alpina*.

53. The nutrient oil mixture according to any one of claims 47 to 52, comprising a first microbial oil containing ARA and a second microbial oil containing DHA.

54. A method for preparing a nutritional oil according to any one of claims 1 to 30, comprising: i. Mix the oil containing LC-PUFA with an organic solvent; ii. To remove at least a portion of the organic solvent from the oil containing LC-PUFA to obtain a crude nutritional oil; and iii. Recover at least a portion of the organic solvent removed in step ii using a recycled solvent, wherein the recycled solvent contains less than 50 wt% mineral oil based on the total weight of the recycled solvent. The nutritional oil mentioned above has a MOSH content of less than 7 ppm.

55. A method for preparing a nutritional oil, comprising: i. Mix the oil containing LC-PUFA with an organic solvent; ii. To remove at least a portion of the organic solvent from the oil containing LC-PUFA to obtain a crude nutritional oil; and iii. Recover at least a portion of the organic solvent removed in step ii using a recycled solvent, wherein the recycled solvent contains less than 50 wt% mineral oil based on the total weight of the recycled solvent.

56. The method of any one of claims 54 to 55, wherein the organic solvent comprises hexane.

57. The method of any one of claims 54 to 57, wherein the organic solvent comprises at least a portion of the recovered organic solvent previously mixed with a second LC-PUFA-containing oil.

58. The method of claim 58, wherein the recovered organic solvent comprises less than 100 ppm of MOSH, less than 75 ppm of MOSH, less than 50 ppm of MOSH, less than 25 ppm of MOSH, less than 10 ppm of MOSH, less than 5 ppm of MOSH, or less than 1 ppm of MOSH.

59. The method of any one of claims 54 to 58, wherein mixing the LC-PUFA-containing oil with an organic solvent comprises using the organic solvent to extract microbial oil from microorganisms.

60. The method of claim 59, wherein the microorganism belongs to the order Mortierellales.

61. The method of claim 60, wherein the microorganism is *Morchella alpina*.

62. The method of claim 59, wherein the microorganism belongs to the order Thraustochytriales.

63. The method of claim 62, wherein the microorganism belongs to the genus Schizochytrium or the genus Schizochytrium.

64. The method of claim 59, wherein the microorganism is a dinoflagellate.

65. The method of claim 64, wherein the microorganism is Cryptodinium kurstii.

66. The method of any one of claims 54 to 65, wherein the recovered solvent comprises less than 40 wt%, less than 30 wt%, less than 20 wt%, less than 10 wt%, or less than 5 wt% mineral oil based on the total weight of the recovered solvent.

67. The method of any one of claims 54 to 66, wherein the recovered solvent comprises triglycerides.

68. The method of any one of claims 54 to 67, wherein the recovered solvent comprises sunflower oil, medium-chain triglycerides, rapeseed oil, or a combination thereof.

69. The method of any one of claims 54 to 68, wherein the nutrient oil is a crude nutrient oil.

70. The method of claim 69, wherein the nutritional oil comprises free fatty acids in amounts greater than 0.1 wt%, greater than 0.15 wt%, greater than 0.2 wt%, greater than 0.25 wt%, greater than 0.3 wt%, greater than 0.35 wt%, greater than 0.4 wt%, greater than 0.45 wt%, greater than 0.5 wt%, or greater than 0.55 wt% based on the total weight of the nutritional oil.

71. The method of any one of claims 54 to 68, wherein the nutrient oil is a final nutrient oil obtained by further processing the crude nutrient oil to obtain a final nutrient oil.

72. The method of any one of claims 54 to 71, wherein the nutritional oil comprises free fatty acids in a content of less than 0.1 wt% or less than 0.05 wt% based on the total weight of the nutritional oil.

73. The method of any one of claims 54 to 72, wherein the nutritional oil comprises one or more of ARA, DHA, or EPA in an amount greater than or equal to 300 mg / g based on the total amount of the nutritional oil.

74. The method of any one of claims 54 to 73, wherein the nutrient oil comprises MOSH in amounts of less than or equal to 6 ppm, less than or equal to 5 ppm, less than or equal to 4 ppm, less than or equal to 3 ppm, less than or equal to 2.5 ppm, less than or equal to 2 ppm, less than or equal to 1.5 ppm, or less than or equal to 1 ppm, or less than or equal to 0.5 ppm based on the total weight of the nutrient oil.

75. The method of any one of claims 54 to 73, wherein the nutrient oil comprises MOSH in a content of 0.1 ppm to 2 ppm, 0.5 ppm to 3 ppm, 1 ppm to 4 ppm, or 1.5 ppm to 5 ppm based on the total weight of the nutrient oil.

76. The method of any one of claims 54 to 75, wherein the nutrient oil contains MOAH at a concentration of less than or equal to 5 ppm, less than or equal to 4 ppm, less than or equal to 3 ppm, less than or equal to 2 ppm, less than or equal to 1.5 ppm, or less than or equal to 1 ppm based on the total weight of the nutrient oil.

77. The method of any one of claims 54 to 76, further comprising using equipment material comprising less than or equal to 50 wt% mineral oil based on the total weight of the equipment material.

78. The method of claim 77, wherein the equipment material is an equipment lubricant.

79. The method of any one of claims 77 and 78, wherein the equipment material comprises rapeseed oil, sunflower seed oil, mixed-chain triglycerides, or combinations thereof.

80. A nutritional oil produced by the method of any one of claims 54 to 79, comprising: Based on the ARA content of ≥300 mg / g based on the total weight of the said nutritional oil; and MOSH is 0.5 ppm to 10 ppm based on the total weight of the nutritional oil.

81. The nutritional oil of claim 80, comprising ARA at a content greater than or equal to 350 mg / g, greater than or equal to 400 mg / g, greater than or equal to 450 mg / g, greater than or equal to 500 mg / g, or greater than or equal to 550 mg / g based on the total weight of the nutritional oil.

82. The nutritional oil according to any one of claims 80 to 81, comprising ARA in a content of 300 mg / g to 400 mg / g, 350 mg / g to 450 mg / g, 400 mg / g to 500 mg / g, 450 mg / g to 550 mg / g, or 500 mg / g to 600 mg / g based on the total weight of the nutritional oil.

83. The nutritional oil according to any one of claims 80 to 82, comprising free fatty acids in a content greater than or equal to 0.1 wt% based on the total weight of the nutritional oil.

84. The nutritional oil of claim 83, comprising MOSH in amounts of 0.5 ppm to 2 ppm, 1 ppm to 3 ppm, 2 ppm to 4 ppm, or 3 ppm to 5 ppm, 4 ppm to 6 ppm, or 5 ppm to 7 ppm based on the total weight of the nutritional oil.

85. The nutritional oil according to any one of claims 80 to 82, comprising less than 0.1 wt% of free fatty acids based on the total weight of the nutritional oil.

86. The nutritional oil of claim 85, comprising MOSH in an amount of 0.1 ppm to 2 ppm, 0.5 ppm to 3 ppm, 1 ppm to 4 ppm, or 1.5 ppm to 5 ppm based on the total weight of the nutritional oil.

87. The nutritional oil according to any one of claims 80 to 86, comprising MOAH in amounts of less than or equal to 5 ppm, less than or equal to 4 ppm, less than or equal to 3 ppm, less than or equal to 2 ppm, less than or equal to 1.5 ppm, or less than or equal to 1 ppm based on the total weight of the nutritional oil.

88. The nutritional oil according to any one of claims 80 to 87, further comprising added antioxidants.

89. The nutritional oil of claim 85, wherein the added antioxidant comprises tocopherol.

90. The nutritional oil according to any one of claims 80 to 89, comprising 50 wt% or more, 55 wt% or more, 60 wt% or more, 65 wt% or more, 70 wt% or more, 75 wt% or more, 80 wt% or more, or 85 wt% or more based on the total weight of the nutritional oil.

91. The nutritional oil according to any one of claims 80 to 90, comprising 3-MCPD in a content of less than or equal to 0.2 ppm, less than or equal to 0.15 ppm, or less than or equal to 0.1 ppm based on the total amount of the nutritional oil.

92. The nutritional oil according to any one of claims 80 to 91, comprising glycidyl glycerin in a content of less than or equal to 0.1 ppm, less than or equal to 0.05 ppm, less than or equal to 0.04 ppm, less than or equal to 0.03 ppm, or less than or equal to 0.02 ppm based on the total amount of the nutritional oil.

93. The nutritional oil according to any one of claims 80 to 92, wherein the anisidine value of the nutritional oil is less than 20, less than 15, less than 12, less than 10 or less than 8.

94. The nutritional oil according to any one of claims 80 to 93, wherein the nutritional oil has a peroxide value of less than or equal to 10 meq / kg, less than or equal to 8 meq / kg, less than or equal to 5 meq / kg, less than or equal to 3 meq / kg, less than or equal to 2 meq / kg, or less than or equal to 1 meq / kg based on the total weight of the nutritional oil.

95. The nutrient oil according to any one of claims 80 to 94, wherein the nutrient oil is a microbial oil.

96. The nutrient oil of claim 95, wherein the microbial oil is produced by *Morchella alpina*.

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