Cetoleic Acid Composition

Abstract

The present invention relates to concentrated LCMUFA compositions comprising cetoleic acid. Additionally, the present invention relates to methods for providing concentrated compositions comprising cetoleic acid and uses of such compositions.

Description

[Technical Field]

[0001] The present invention relates to LCMUFA compositions, particularly concentrated LCMUFA compositions containing cetoleic acid. Additionally, the present invention relates to methods for providing compositions containing cetoleic acid, and various uses of the LCMUFA compositions. [Background technology]

[0002] Numerous scientific studies support the important role of omega-3 fatty acids in improving heart disease risk factors, reducing inflammation, and promoting brain health during pregnancy and early life. Omega-3 compounds have a wide range of proven positive effects on human health, making them a big business. Back in the 1960s, a multinational epidemiological study by Keys et al. (Acta Med Scand Suppl. 1966, 460, 1-392) found that long-chain monounsaturated fatty acids (LCMUFAs) derived from fish such as mackerel and herring also had positive health effects, particularly in relation to coronary heart disease. This research sparked public interest in monounsaturated fatty acids and promoted the Mediterranean diet, which is still considered one of the healthiest diets, and is rich in monounsaturated fatty acids, such as oleic acid from olive oil.

[0003] The human body contains many different types of fats, which help store energy, provide insulation, protect vital organs, and perform important structural functions. Some lipids have important biological activities, meaning they trigger specific responses in the body. Dietary fats consumed by humans can be separated into two categories: saturated and unsaturated. The latter are generally considered "healthy fats" because they help increase levels of HDL (good cholesterol) and reduce unhealthy triglycerides. Unsaturated fats can be divided into two main types: monounsaturated fats (MUFAs) and polyunsaturated fats (PUFAs). Long-chain fatty acids are those with carbon chains longer than 18 (C20-C22), and fish oils are rich in these lipids. LCPUFAs include the omega-3 fatty acids EPA and DHA, which are important for health but are produced in small amounts in the human body. Numerous studies have demonstrated the health benefits of EPA and DHA, including reduced inflammation, a lower risk of Alzheimer's disease, and reduced effects of cardiovascular disease. Therefore, dietary intake of EPA and DHA is important for maintaining healthy levels of these fatty acids.

[0004] Fish and marine life are rich sources of EPA and DHA, but in addition, certain fish also contain high levels of LCMUFAs. Compared to EPA and DHA, knowledge about the health benefits of marine LCMUFAs is limited. Cetoleic acid is an omega-11 marine lipid found in higher concentrations in North Atlantic fish such as herring, mackerel, and tobis than in South American fish such as anchovies and sardines, which are more rich in EPA and DHA.

[0005] LCMUFAs, including cetoleic acid, can be utilized as an energy source by mitochondrial β-oxidation and peroxisomal β-oxidation, as described by Bremer et al., J Lipid Res, Feb 1982, 23(2), 43-56. Adipose tissue is the primary storage site for triglycerides, and studies of feeding cetoleic acid-rich oils have shown high absorption of cetoleic acid in adipose tissue, suggesting that cetoleic acid is stored in adipose tissue and may exert important effects in this tissue. Early studies (see Yang et al., Lipids Health Dis, Nov 22, 2016, 15(1), 201; and Yang et al., Mol Nutr Food Res 2016, 60(10), 2208-2218) have shown that cetoleic acid-rich fish oil reduces systemic inflammation and the degree of atherosclerosis in mice. Other positive health effects associated with reduced inflammation and in preventing the development of metabolic dysfunction associated with metabolic syndrome and obesity have also been noted.

[0006] Cell culture studies and human clinical studies support the hypothesis that cetoleic acid-rich fish oils promote the conversion of alpha-linolenic acid to EPA, as disclosed by Ostbye et al., BR, J Nutr. Oct 14 2019, 122(7), 755-768.

[0007] Research in the late 1990s demonstrated that cetolein-rich oils, along with EPA and DHA, may have various activities in the body. Supplementation with EPA and DHA leads to beneficial plasma lipid effects not seen with cetolein-rich oils. In other words, the health benefits of these two fatty acids may extend to various areas of metabolic health. Some evidence supports this. In vitro studies using hepatocytes have demonstrated that EPA and DHA reduce cholesterol and triglyceride content in hepatocytes, but this reduction is even greater when the cells are co-administered with cetolein-rich oil (see Yoshinaga et al., J Oleo Sci. May 1 2021, 70(5), 731-736). While the effects of omega-3 fatty acids are primarily on triglycerides and HDL, cetolein-rich oils affect non-HDL cholesterol and insulin sensitivity (see Yang et al., J Agric Food Chem, Jul 13, 2011, 59(13), 7482-9). This complementary effect is interesting for providing comprehensive metabolic protection. Indeed, data from animal models suggest a beneficial combined effect in metabolic syndrome, as provided by Yang et al., Lipids Health Dis, Dec 1, 2015, 14, 155, whereby oils rich in EPA / DHA and cetoleic acid exhibit combined and complementary actions in metabolic syndrome, including a combined effect on reducing plasma non-HDL cholesterol, improving hyperinsulinemia, liver fat, and plasma lipid levels.

[0008] As with the LCPUFAs EPA and DHA, levels of omega-11 LCMUFAs such as cetoleic acid are dependent on dietary intake as they cannot be synthesized in the body.

[0009] EP2682116 to Nippon Suisan Kaisha describes a drug for use in improving metabolic syndrome, the drug containing MUFAs but little LCPUFAs.

[0010] LCMUFA, including cetoleic acid, exists in natural oils and is a valuable resource.However, MUFA typically exists in the mixture with various other fatty acids.Therefore, there is a need for a composition that contains LCMUFA, and a method for separating MUFA from other fatty acids, in order to provide MUFA composition. Summary of the Invention

[0011] The present invention provides compositions comprising long chain monounsaturated fatty acids (LCMUFAs), particularly cetoleic acid, methods for providing such compositions, and uses thereof.

[0012] In one aspect, the present invention relates to an LCMUFA composition comprising C22:1 n11 (cetoleic acid) and C20:5 n3 (EPA), the concentration of C22:1 n11 is at least 16.0 wt.%; The EPA concentration is at least 3 wt.%.

[0013] In the compositions of the present invention, the DHA concentration is low. In one embodiment, the DHA concentration is up to 4.0 wt.% of the composition. In one embodiment, the EPA:DHA ratio is at least 4:1.

[0014] In one embodiment, the present invention relates to an enriched LCMUFA composition, wherein the concentration of C22:1 n11 is at least 39 wt.%.

[0015] In a second aspect, the present invention relates to a method for preparing a composition of LCMUFA, the method comprising: i) mixing a natural oil with a lipase to obtain a mixture containing LCPUFAs in glyceride form and LCMUFAs in ethyl ester form; ii) distilling the mixture to obtain a first fraction containing an LCMUFA composition and a second fraction containing an LCPUFA composition.

[0016] In one embodiment, the method is for preparing the LCMUFA composition of the first aspect. The method allows for the separation of LCMUFAs such as cetoleic acid and gondoic acid from EPA and DHA.

[0017] In a further aspect, the present invention relates to an LCMUFA composition according to the first aspect for use in a therapeutic or health indication, and use in a feed or food product. [Brief explanation of the drawings]

[0018] [Figure 1]

[0033] Results are provided for a study using a wound healing model (Example 4) showing the area of ​​the wound 14 hours after treatment of the wound and cells, with the cells being supplemented with LCMUFA concentrate or DHA, respectively. [Figure 2] 1 shows melanin concentrations (μg melanin / μg cellular DNA) in human fibroblasts (n=4) after oxidative stress from the wound healing model of Example 4. [Figure 3] 1 shows the relative expression of inflammation-related genes (log2) in human fibroblasts under oxidative stress from the wound healing model of Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0019] Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial departure from what is commonly understood in the art.

[0020] In the present invention, "monounsaturated fatty acids" or "MUFA" refer to fatty acids with one double bond, while "polyunsaturated fatty acids" or "PUFA" refer to fatty acids with four or more double bonds. MUFAs and PUFAs with long-chain fatty acids of 20 or more carbon atoms are referred to as LCMUFAs or LCPUFAs. MUFAs with 20 carbon atoms are called eicosenoic acids in the IUPAC nomenclature system, and are also called cis-icosa-9-enoic acid (n-11, common name: gadoleic acid), cis-icosa-11-enoic acid (n-9, common name: gondoic acid), etc. MUFAs with 22 carbon atoms are called docosenoic acids in the IUPAC nomenclature system, and are also called cis-docosa-11-enoic acid (n-11, common name: cetoleic acid), cis-docosa-13-enoic acid (n-9, common name: erucic acid), etc.

[0021] In the present invention, the following nomenclature is used: Gondic acid, C20:1 n9; Gadoleic acid, C20:1 n11; erucic acid, C22:1 n9; cetoleic acid, C22:1 n11; eicosapentaenoic acid (EPA), C20:5 n3; Docosahexaenoic acid (DHA), C22:6 n3.

[0022] The present invention relates to a composition containing both LCMUFA and LCPUFA obtained from natural oils, and a method for providing such a composition. The present invention provides an LCMUFA composition, particularly a concentrated LCMUFA composition containing cetoleic acid. The present invention also provides a method for producing a composition containing a high concentration of cetoleic acid from natural oils by separating the natural oils from other lipids.

[0023] Concentrated compositions are provided that may provide complementary benefits from EPA and cetoleic acid with other fatty acids. Applicant has also investigated evidence supporting marine-derived LCMUFAs, particularly omega-11 cetoleic acid (C22:1 n-11) and omega-9 gondoic acid (C20:1 n-9), to provide new compositions, for example, for the marine lipid ingredient market or for human health.

[0024] Thus, in one aspect, the present invention provides an LCMUFA composition comprising C22:1 n11 (cetoleic acid) and C20:5 n3 (EPA), the concentration of C22:1 n11 is at least 16 wt.%; The EPA concentration is at least 3 wt.%.

[0025] The compositions of the present invention have a low concentration of DHA and therefore a high ratio of either EPA:DHA and cetoleic acid:DHA, hi one embodiment, the DHA concentration is up to 4 wt.% of the composition.

[0026] The MUFA compositions of the present invention containing high concentrations of cetoleic acid are obtained from natural starting oils containing at least both cetoleic acid and EPA. The starting oil, also referred to as feedstock, which can be either crude or refined oil used as the starting oil to provide the compositions of the present invention, typically contains at least 9 wt.% cetoleic acid and at least 3 wt.% EPA, more preferably at least 15 wt.% cetoleic acid and at least 7 wt.% EPA, or at least 5 wt.% EPA. Applicant has discovered that it is possible to obtain commercially useful amounts of compositions containing both health-beneficial LCMUFAs and LCPUFAs from the same starting oil. Disclosed herein is a process for separating individual fractions of specific LCMUFAs from specific LCPUFAs. Accordingly, the present invention also relates to methods for producing compositions containing high concentrations of LCMUFAs, particularly cetoleic acid, derived from natural oils, and such compositions are disclosed and claimed. As shown below, a method has been identified that allows for the separation of MUFAC22:1 n11 (cetoleic acid) from other fatty acids, including the polyunsaturated fatty acid C22:6 n3 (DHA) of the same length.

[0027] The raw material used to provide the composition of the present invention is a natural oil from a marine source, preferably fish oil, such as fish oil, squid oil, krill oil, or algae oil. In particular, the raw material is North Atlantic fish oil, such as herring, mackerel, capelin, cod, pollock, or tobis. The raw material can also be obtained from other fish species containing cetoleic acid, such as North Pacific fish species Alaska pollock or Pacific saury. Most preferably, the raw material is oil derived from herring or mackerel, most preferably herring. These North Atlantic oils are suitable due to their abundant availability, high cetoleic acid concentration, and EPA:DHA ratio. Furthermore, North Atlantic fish oil is rich in gondoic acid, c20:1n9, which is another reason for using such oil to provide the composition of the present invention. Therefore, in one embodiment, the MUFA composition provided by the present invention is a fish oil MUFA composition. Furthermore, in one embodiment, the fatty acids of the starting oil for preparing the composition are in triglyceride form. Therefore, providing the MUFA composition of the present invention also includes valuable use of resources, some of which have previously been little used. It is particularly advantageous to be able to preserve and utilize both MUFA and PUFA from MUFA-rich oils. Until now, North Atlantic oils have typically been considered a second choice for EPA and DHA production because of their low EPA and DHA production levels compared to oils such as anchovies and sardines. Rather than using typical EPA / DHA sources such as anchovy oil, or using MUFA-rich oils for EPA / DHA production but without using MUFA components, the present invention utilizes oils with high MUFA content to provide useful MUFA compositions and further provides a method for recovering and utilizing PUFAs.

[0028] In some embodiments, the compositions of the present invention comprise 16.0-60.0 wt.% cetoleic acid, such as 18.0-50.0 wt.% cetoleic acid, more preferably 20.0-50.0 wt.% cetoleic acid, even more preferably 39.0-50.0 wt.% cetoleic acid, preferably about 40-45 wt.% cetoleic acid. In one embodiment, the concentration of cetoleic acid is at least 39 wt.%. Concentration of cetrenic acid can be achieved, for example, as disclosed below.

[0029] In some embodiments, the composition contains LCMUFA in an amount of at least 20.0 wt.%, such as at least 30.0%. More preferably, the concentrated compositions of the present invention contain LCMUFA in an amount of at least 47 wt.%, preferably at least 50 wt.%, such as at least 60 wt.%. In one embodiment, the composition contains LCMUFA in an amount ranging from 47 to 80 wt.%, such as 50 to 76 wt.%, preferably in an amount ranging from 60 to 65 wt.%. LCMUFA includes the C20 fatty acid gondoic acid, and may further contain, in addition to cetoleic acid, gadoleic acid and the C22:1 fatty acid erucic acid. Thus, the provided compositions are enriched in LCMUFA, i.e., C20:1 and 22:1, and the concentration of LCMUFA is significantly higher in the claimed compositions than in the starting oil used for preparation. However, the ratio of C22:1 to C20:1 is similar to that found in the starting oil (natural oil). In one embodiment, the weight ratio of C22:1 to C20:1 is 1.5:1 to 2.6:1, such as about 1.75:1. More specifically, the weight ratio of C22:1n11:C20:1n9 is about 1.75:1. In one embodiment, the weight ratio of C22:1n11:C20:1n9 is 1.5:1 to 2.6:1.

[0030] In some embodiments, the concentration of gondoic acid, C20:1 n9, ranges from 9 to 28 wt.%, such as 15 to 28 wt.%, e.g., 18 to 28 wt.%. More preferably, the concentration of C20:1 n9 ranges from 18 to 26 wt.%. Such high concentrations of gondoic acid, combined with high concentrations of cetoleic acid, are obtained from fish oils, particularly from North Atlantic fish, unlike typical Pacific fish species such as Alaska pollock and Pacific saury. Oils from Pacific fish species typically contain more gadoleic acid, C20:1 n11, than gondoic acid (C20:1 n9). Concentrated compositions and high concentrations can be obtained by the methods disclosed below, particularly when the methods include multiple rounds of concentration.

[0031] In some embodiments, the amount of gadoleic acid, C20:1 n11, is particularly low in the compositions of the present invention, being up to 2.0 wt.%, preferably 1.0-2.0 wt.%.

[0032] In some embodiments, the amount of erucic acid, C22:1 n9, is particularly low in the compositions of the present invention, being up to 3.0 wt.%, preferably 2.0-3.0 wt.%.

[0033] The EPA concentration in the composition is at least 3.0 wt.%, preferably at least 5.0 wt.%. In some embodiments, the concentration is 5.0-11.0 wt.%. In some embodiments, the EPA concentration is about the same as or slightly lower than the starting oil (feedstock). In an exemplary embodiment, for example, when mackerel oil or herring oil, which has an EPA concentration of about 8%, is used as the starting oil, the composition of the present invention contains about 5-8% EPA. In another embodiment, the EPA concentration is even higher, such as up to 40 wt.% of the composition. Thus, the present invention provides compositions comprising an abundant amount of cetoleic acid and an EPA concentration of 5-40 wt.%, such as 20-40 wt.%, as disclosed. To obtain an EPA-rich MUFA composition, an EPA concentrate can be added to the MUFA composition of the present invention.

[0034] Thus, in one embodiment, the EPA-enriched LCMUFA composition of the present invention comprises C22:1 n11 (cetoleic acid) and C20:5 n3 (EPA); the concentration of C22:1 n11 is at least 16.0 wt.%, such as 16-60 wt.%, e.g., 30-50 wt.%, of the composition; The EPA concentration ranges from 20 to 40 wt.% of the composition, such as from 20 to 30 wt.% of the composition. In one embodiment, DHA may be present at a concentration of up to 4.0 wt.% of the composition. Additionally, the EPA-enriched MUFA composition preferably further comprises gondoic acid, C20:1 n9, at a concentration of 9-28 wt.% of the composition.

[0035] In a preferred embodiment, the compositions of the present invention contain only small amounts of DHA. The applicant surprisingly managed to separate LCMUFA, particularly C22:1 MUFA, i.e., cetoleic acid and erucic acid, from these polyunsaturated C22 fatty acids. Thus, the provided MUFA compositions of the present invention contain significantly less DHA than the starting oil, and thus have a higher EPA:DHA ratio. The MUFA compositions of the present invention typically have a cetoleic acid:DHA ratio about 10 times higher than the natural starting oil. Mackerel crude oil typically contains about 11-12 wt.% DHA, while herring stock typically contains about 9-10 wt.% DHA. However, the MUFA compositions of the present invention contain little DHA, but have a high concentration of cetoleic acid, a fatty acid of the same length. In one embodiment, the LCMUFA compositions of the present invention have a cetoleic:DHA ratio of at least 10:1, such as at least 15:1, more preferably at least 20:1.

[0036] Furthermore, the MUFA compositions of the present invention have a high EPA:DHA ratio. In one embodiment, the LCMUFA composition has an EPA:DHA ratio of 4.0:1 to 16.0:1, more preferably 4.0:1 to 8.0:1. In compositions of the present invention with a high amount of EPA, such as EP-enriched MUFA compositions, the DHA content is generally low, and the EPA:DHA ratio can be as high as 50:1. Thus, in one embodiment, the EPA:DHA ratio is 4:1 to 50:1. Thus, the concentration of EPA is at least four times higher than the concentration of DHA in the MUFA composition. However, this ratio decreases as the composition becomes more cetoleic acid-enriched, for example, by concentration by distillation.

[0037] Thus, the concentration of DHA is surprisingly low in the provided MUFA compositions. In some embodiments, the DHA concentration of the MUFA compositions is up to 4.0 wt.%, such as in the range of 0.5-3.5 wt.%, i.e., significantly lower than that of suitable marine-source oils. Compositions that are free of DHA, i.e., compositions with a DHA concentration of 0 wt.%, are within the scope of the claimed compositions, i.e., having a DHA concentration of 0.0-4.0 wt.%. For example, a low DHA content may be desirable in compositions for cardiovascular health, such as treating heart disease to avoid adverse effects on blood parameters such as TG, LDL, HDL, and total cholesterol. Furthermore, because the compositions of the present invention contain high concentrations of MUFA and little DHA PUFA, the fatty acids of the compositions of the present invention are less susceptible to oxidation than compositions containing higher concentrations of PUFA. Therefore, the compositions of the present invention have a pleasant taste and aroma and can be included in foods.

[0038] A further reason for providing the claimed composition is that the applicant wishes to offer a product containing cetoleic acid and small amounts of DHA and EPA to give consumers choice and thus control their lipid intake. Consumers are likely already taking omega-3 supplements, and from a commercial perspective, replacing traditional high-concentration omega-3s, which dominate the fish oil market, is undesirable. Furthermore, alternative oils, such as those rich in cetoleic acid, do not provide the levels of EPA and DHA sold in omega-3 concentrates. This leaves a dilemma: a consumer could take one capsule for omega-3 and one capsule for cetoleic acid, but risk doubling their EPA and DHA intake, which is regulated by the FDA to a maximum of 3g. Therefore, consumers risk consuming too much omega-3 if they take additional cetoleic acid supplements. Therefore, a cetoleic acid supplement with low EPA-DHA is needed. Furthermore, EPA and DHA are known to lower plasma levels of triglycerides (TG) and produce modest changes in total cholesterol. While EPA causes a slight decrease in total cholesterol levels, DHA shows a slight increase, primarily due to an increase in HDL cholesterol. As demonstrated by Sarbolouki et al., 2013, Singapore Med J., 54(7):387-90, EPA supplementation can improve insulin sensitivity by reducing fasting blood glucose, HbA1c, and homeostatic model assessment of insulin resistance. Thus, EPA shows a negative correlation with plasma levels of triglycerides (TG) and insulin. However, EPA has little or no effect on total cholesterol. The primary claim for LCMUFA cardiovascular protection lies in its ability to lower LDL levels without lowering HDL levels. Long-term LCMUFA supplementation (Yang, 2015) suggested that LCMUFA significantly reduced plasma levels of insulin and total cholesterol, while having little or no effect on lowering plasma TG. Thus, LCMUFA shows a negative correlation with plasma levels of total cholesterol and insulin.However, LCMUFAs are neutral in terms of lowering plasma triglycerides.

[0039] Thus, LCMUFA and EPA appear to have complementary effects on plasma lipids and insulin. Therefore, the positive effects of LCMUFA (cetoleic acid) support the hypothesis that LCMUFA may have beneficial effects in reducing the risk of cardiovascular disease. LCMUFA intake may also have a positive impact on the treatment of type 2 diabetes by reducing non-HDL cholesterol and plasma insulin. Many believe that omega-3 cardiovascular health is due to the effects of EPA. This is demonstrated by the success of the drugs ethyl icosapentate (Vascepa) (REDUCE-IT trial), which contains 99% EPA, and Epadel (Jellis trial), which contains over 98% EPA. One objective of this invention is to provide a high concentration of cetoleic acid with low levels of EPA and DHA, allowing consumers to easily make an informed choice. However, because EPA is believed to be more important for cardiovascular health, the product is biased toward EPA over DHA.

[0040] Due to the complementary effects of LCMUFA and EPA, the applicant is also interested in providing a clinical product containing concentrated cetoleic acid and EPA. The low amount of DHA provides an option to make such a product by mixing a cetoleic acid concentrate with an EPA concentrate. However, if the LCMUFA fraction is already rich in DHA, the production of such a composition becomes more difficult. The disclosed method efficiently separates cetoleic acid from DHA, significantly increasing the concentration of cetoleic acid while optionally maintaining a low level of DHA.

[0041] In some embodiments, the combined concentration of EPA and DHA ranges from 3.0 to 12.0 wt.%, such as 5.0 to 12.0 wt.%, more preferably 6.0 to 12.0 wt.%, of the composition. In embodiments where the composition is enriched in EPA, the combined concentration of EPA and DHA can be significantly higher.

[0042] In addition to cetoleic acid and EPA, and preferably gondoic acid, the MUFA composition of the present invention may further comprise a variety of other fatty acids, including short- and medium-chain (C18 or less), long-chain (C20-22), or even very long-chain fatty acids (C22 or greater), typically present in the feedstock from which the MUFA composition is derived. These fatty acids may be saturated, monounsaturated, or polyunsaturated fatty acids. In some embodiments, the MUFA composition comprises short-chain fatty acids. However, of course, the higher the concentration of the MUFA composition in cetoleic acid, the lower the concentration of other fatty acids, including shorter fatty acids. Preferably, the content of short-chain saturated fatty acids is kept low, particularly in highly concentrated cetoleic compositions of the present invention. In some embodiments, the concentration of C12:1-C16:1 fatty acids is a maximum of 2.0% or less. In some embodiments, the MUFA composition comprises C18 fatty acids, such as: For example, in the range of 0.5–2.7 wt.% C18:0; For example, in the range of 5.0–16.0 wt.% C18:1; For example, in the range of 0.5-2.5 wt.% C18:2; For example, in the range of 0.5-2 wt.% C18:3; For example, C18:4 in the range of 0.5-1.5 wt.%.

[0043] In one exemplary embodiment, the concentrated LCMUFA composition of the invention comprises C22:1 n11 (cetoleic acid) and C20:5 n3 (EPA); the concentration of C22:1 n11 is at least 39 wt.%; The concentration of EPA is at least 5.0 wt.%.

[0044] Additionally, in certain embodiments, the concentrated MUFA compositions of the present invention have one or more of the following properties: the concentration of C22:1 n11 is at least 39 wt.%; the weight ratio of C22:1 n11:C20:1 n9 is from 1.5:1 to 2.6:1; The concentration of EPA is at least 5.0 wt.%; The maximum concentration of DHA is 4.0 wt.% or less. The concentration of C20:1 n11 (gadrain) is 2.0 wt.% or less at most. The concentration of C12:1-C16:1 fatty acids is up to a maximum of 2.0%. In a preferred embodiment, the enriched MUFA composition meets all of the above characteristics.

[0045] In the applicant's analysis, gas chromatography is used, and GC area % provides information about the fatty acid distribution of the analyzed composition. The provided fatty acid composition preferably consists of close to 100% fatty acids, and therefore there is only a negligible difference between the unit "GC area %" used in the analysis and examples and "weight %" (wt.%) used throughout this specification and claims.

[0046] In certain embodiments, the concentrated MUFA compositions of the present invention comprise the following fatty acids within the ranges listed in Table A:

[0047] TIFF0007819412000001.tif145130

[0048] In a further aspect, the present invention provides a method for preparing a composition of C20-22 LCMUFAs, e.g., according to the first aspect, the method comprising the step of selectively enzymatically separating LCPUFAs from LC-MUFAs. The method comprises the step of selective interesterification using a lipase. In one embodiment, the method comprises the step of enzymatically separating LCPUFAs from LCMUFAs by selectively cleaving LCMUFAs from triglycerides.

[0049] The method of the present invention allows the MUFA and PUFA of the same length to be separated from each other under mild conditions.More specifically, MUFA C22:1n11 (cetoleic acid) is separated from PUFA C22:6n3 (DHA).Therefore, this method allows the fatty acids that have similar boiling points and are therefore difficult to separate to be separated from each other selectively.Other methods can separate C20 fatty acids from C22 fatty acids, but for example, when C22:1 is concentrated from C20 fatty acids, DHA (C22:6) is typically concentrated together with C22:1 fatty acids.

[0050] Thus, in one embodiment, the present invention provides a method for preparing a composition of LCMUFAs, the method comprising: i) mixing a natural oil containing LCMUFA and LCPUFA with a lipase to obtain a mixture containing LCPUFA in the form of glycerides and LCMUFA in the form of ethyl esters; ii) distilling the mixture of step i) to obtain the LCMUFA composition as one fraction and the LCPUFA composition as another fraction.

[0051] The present invention further provides a method for separating LCMUFAs from LCPUFAs, the method comprising: i) mixing a natural oil with a lipase to obtain a mixture containing LCPUFAs in glyceride form and LCMUFAs in ethyl ester form; ii) distilling the mixture of step i) to obtain a first fraction containing an LCMUFA composition and a second fraction containing an LCPUFA composition.

[0052] In these methods, the enzymatic reaction occurs on triglycerides, which is beneficial. Because DHA fatty acids tend to be located in the central position of triglycerides, fatty acids located at positions 1 and 3, such as LCMUFA, can be selectively cleaved using 1,3-specific lipases. Thus, DHA is left on the glycerol as DHA monoglyceride (MAG-DHA). Therefore, both region selectivity and fatty acid selectivity are achieved.

[0053] In an alternative embodiment that also involves transesterification using a lipase, the present invention provides a method for separating LCMUFAs from LCPUFAs, the method comprising: converting fatty acids of natural oils into ethyl esters; distilling the ethyl esters to obtain a fraction of LCMUFAs and LCPUFAs in ethyl ester form; and mixing the ethyl esters with glycerol and lipase to obtain a glyceride mixture containing LCMUFA, with the DHA remaining as the ethyl ester.

[0054] In the method of the present invention, as disclosed above with respect to the composition of the present invention, the starting material is a natural oil containing at least both cetoleic acid (LCMUFA) and EPA (LCPUFA). The starting oil, also referred to as feedstock, can be either a crude oil or a refined oil used as the starting oil to provide the composition of the present invention, but typically contains at least 9 wt.% cetoleic acid and at least 3 wt.% EPA, more preferably at least 15 wt.% cetoleic acid and at least 7 wt.% EPA, for example. Thus, the natural oil for use in the method can be a feedstock oil, such as the feedstock oil typically produced in fish meal production. Alternatively, the natural oil can be a refined oil, for example, an oil from which contaminants have been removed, such as by bleaching, deacidification, peeling, or supercritical extraction. The natural oil is from a marine source, such as fish oil, squid oil, krill oil, or algae oil, preferably from fish oil. In particular, the oil is a North Atlantic fish oil such as herring, mackerel, capelin, or tobis. Most preferably, the oil is oil from herring or mackerel, most preferably from herring. The starting fatty acids are preferably in the form of triglycerides.

[0055] In step i) of the method, enzymatic separation is carried out by using lipase. Lipases are a family of enzymes that catalyze the hydrolysis of fats. Some lipases exhibit a wide substrate range, including esters of cholesterol, phospholipids, and fat-soluble vitamins. In the method of the present invention, at least one lipase is used as an esterification catalyst to cleave fatty acid chains from the triglyceride backbone. As is known, lipases are well suited for use as catalysts in processes involving highly unstable n-3 polyunsaturated fatty acids, such as EPA and DHA, which occur in marine oils. This is due to their ability to operate at low temperatures, neutral pH, and mild action, which helps minimize undesirable side reactions such as cis-trans isomerization, double bond migration, polymerization, and oxidation. Thus, the use of lipases for the hydrolysis of fatty acids in marine oils has already been well documented. However, the applicant unexpectedly discovered that certain lipases can be used to selectively separate MUFAs from PUFAs.

[0056] In step i), lipase is contacted with fatty acids from natural oil to convert LCMUFA into LCMUFA esters. LCMUFA are converted from triglyceride form to esters, while LCPUFA remain as glycerides. This results in a mixture containing LCPUFA in glyceride form and LCMUFA in ethyl ester form. The PUFAs in the mixture typically comprise a mixture of monoglycerides, diglycerides, and triglycerides. In one embodiment, ethanol is used as the substrate, and the MUFAs in the mixture from step i) are in the form of MUFA ethyl esters. A solvent is usually not required, but may be used in some cases, such as when the oil contains an unusually high amount of stearin.

[0057] The reaction conditions, including temperature, pressure, and reaction time, are selected based on the usual operating conditions used when converting triglycerides to ethyl esters using the same enzyme. The reaction with lipase is typically carried out for a period of time, such as 1 to 48 hours, e.g., 20 to 30 hours, e.g., about 24 hours. Typically, a temperature in the range of 25 to 90°C is suitable, and a pressure of 1 to 50 mbar, if necessary, is suitable.

[0058] In most cases, a suitable amount of lipase will be about 10% (w / w) or less of the oil.

[0059] The method of the present invention utilizes a lipase that catalyzes the esterification of MUFAs from triglycerides. Suitable lipases are preferably immobilized, although non-immobilized enzymes may also function, although recovery after use is expected to be more difficult. In one embodiment, the lipase is a 1,3-specific lipase. Examples of useful lipases include Rhizomucor miehei lipase (formerly Mucor miehei lipase), Aspergillus niger lipase, Thermomyces lanuginosus lipase, Candida antarctica lipase, Candida rugosa lipase (formerly Candida cylindracea lipase), Geotrichum candidum lipase, Penicillium roguefortii lipase, Rhizopus delemar lipase, and Rhizopus oryzae lipase. More preferably, the lipase is selected from the group of Rhizomucor miehei lipase, Thermomyces lanuginosus lipase, Candida antarctica lipase, and Candida rugosa lipase.

[0060] Once the reaction in step i) is complete, the material may be cooled and filtered prior to step ii).

[0061] In step ii), the mixture of step i) is distilled to separate the LCPUFA glycerides of the mixture of step i) from their LCMUFA ethyl esters to obtain an LCMUFA composition as a first fraction and an LCPUFA composition as a second fraction. In one embodiment, the distillation is a single column distillation, for example a single column short path distillation.

[0062] The obtained LCMUFA composition has a higher concentration of LCMUFA, especially cetoleic acid, than natural starting oil.In addition, the obtained LCMUFA composition has a very low content of DHA, because this C22 PUFA is separated from C22 MUFA and is found in LCPUFA fraction (PUFA stream).Similarly, the LCPUFA composition has a higher concentration of LCPUFA than starting oil.

[0063] The method of the present invention allows MUFA and PUFA of the same length to be separated from each other under mild conditions. More specifically, MUFA C22:1 n11 (cetoleic acid) is separated from PUFA C22:6 n3 (DHA). The first fraction rich in cetoleic acid obtained from the method can be further concentrated to prepare a more concentrated MUFA composition, or can be used as is. The separated EPA-DHA-rich fraction (PUFA stream) can also be recovered, for example for commercial use. Thus, in a further aspect, the present invention provides a method for producing a MUFA composition according to the first aspect.

[0064] To illustrate what can be achieved by this method, starting from mackerel oil containing approximately 16 wt.% cetoleic acid, 8% EPA, and 12% DHA, the method of the present invention can produce an LCMUFA composition containing approximately 21 wt.% cetoleic acid without further concentration. In the LCMUFA stream obtained from enzymatic separation and a single distillation (steps i and ii), the resulting EPA concentration is typically approximately 6%, while the DHA concentration is only 1.3%. Thus, using the method of the present invention, the cetoleic acid concentration of the LCMUFA composition is significantly increased while the DHA concentration is significantly decreased. Applicant has discovered that the average change in the concentration of cetoleic acid from the feedstock oil is approximately 2% when using the method of the present invention. Thus, the present invention provides a method for producing a composition of C20-22 LC-MUFA. The resulting MUFA composition may be as disclosed in the first aspect, containing C22:1 n11 (cetoleic acid) and C20:5 n3 (EPA), the concentration of C22:1 n11 is at least 16 wt.%; The EPA concentration is at least 3 wt.%.

[0065] In one embodiment, the concentration of DHA in the resulting composition is up to 4.0 wt.% of the composition. In one embodiment, the EPA:DHA ratio is at least 4.0. Further embodiments of the resulting composition are as disclosed in the first aspect.

[0066] Similarly, the method of the present invention separates LCMUFAs from LCPUFAs, but also provides a method for producing enriched compositions of LCPUFAs. Starting with North Atlantic fish oil, such as mackerel oil, the PUFA stream from the disclosed method (steps i and ii) can typically contain about 9-12 wt.% EPA and 13-15% DHA. Thus, the method provides an efficient procedure for separating MUFAs from PUFAs and providing more enriched compositions thereof.

[0067] The LCMUFA composition obtained from the enzymatic separation and single distillation (the "LCMUFA stream") has been found to be valuable, for example, as an energy source, for use in food, feed, or as a source of more highly concentrated LCMUFA compositions. In one embodiment, the LCMUFA composition is used in aquaculture, for example, as part of fish feed. Further uses of the LCMUFA compositions of the present invention, including uses in therapeutic or health indications, are disclosed below.

[0068] The LCPUFA composition obtained from the enzymatic separation and single distillation ("LCPUFA stream") has been found to be valuable, for example, as an energy source, for use in animal feed, or as a source of more highly concentrated LCPUFA compositions. This could be a valuable alternative source of EPA and DHA to anchovy oil from sustainable fish sources. These EPA and DHA can then be further processed into various concentrates of EPA and DHA, which can be used, for example, in dietary supplements or pharmaceuticals.

[0069] The LCMUFA composition and LCPUFA composition obtained from step ii) can each be further concentrated to form a more concentrated composition. Thus, in one embodiment, the first fraction composition of LCMUFA (from step ii) is further concentrated to provide a more concentrated LCMUFA composition, i.e., a more concentrated composition containing a higher concentration of cetoleic acid. As an example, the isolated mackerel oil disclosed above contains approximately 21 wt.% cetoleic acid and can be further concentrated after steps i) and ii) to increase the amount of cetoleic acid. Thus, as disclosed in the first aspect, a composition containing at least 39.0 wt.% cetoleic acid can be achieved. As shown in Examples 2 and 3, a composition containing approximately 43-45 wt.% cetoleic acid can be obtained from mackerel oil or herring oil. Such further concentration to form an LCMUFA-enriched composition can be achieved using processes such as distillation, extraction, enzymatic treatment, chromatography, and / or other fractionation methods known to those skilled in the art. Such further concentration is preferably carried out by one or more distillations, such as high-quality molecular distillation / short-path distillation procedures. Thus, the method optionally further comprises step iii) of concentrating the desired PUFAs or MUFAs in each of the first and second fractions. Such concentration can be achieved using processes such as distillation, extraction, enzyme treatment, chromatography, etc., and is preferably achieved by distillation, for example, molecular distillation (short-path distillation). Thus, in one embodiment, the method optionally comprises a further step of concentrating LCMUFAs, particularly cetoleic acid, for example, by distillation. Thus, such optional further distillation (one or more) is a step that produces a higher concentration of cetoleic acid while still reducing the content of DHA.

[0070] In one embodiment, step iii) comprises one or more rounds of distillation, with the fraction richest in cetoleic acid being distilled again in each round. Such a process can yield a composition comprising at least 39 wt.% cetoleic acid, such as 39.0-60.0 wt.% cetoleic acid, as disclosed in the first aspect. By methods of the present invention, including multiple distillations after enzymatic separation, for example, cetoleic acid can be further purified and recovered at a concentration of more than 50.0 wt.%, such as more than 60.0 wt.%, more than 70.0 wt.%, or even more than 80 wt.%.

[0071] In one embodiment, the present invention provides a method for producing an enriched composition of C20-22 LC-MUFAs, the enriched composition comprising: Contains C22:1 n11 (cetoleic acid) and C20:5 n3 (EPA), the concentration of C22:1 n11 is at least 39 wt.%; The concentration of EPA is at least 5.0 wt.

[0072] By using the enzymatic method of the present invention, it has been surprisingly discovered that it is possible to prepare a composition with a high concentration of cetoleic acid, a desired range between cetoleic acid and gondoic acid, and a desired EPA concentration. More specifically, this method allows the preparation of a composition with a low DHA concentration, which is separated from MUFA of the same length. In particular, it is possible to provide a composition containing a high concentration of 20:1 and 22:1 MUFA, and an EPA concentration of more than 5% combined with a very low DHA concentration, for example, in the range of 0.5-3.5%. This combination cannot be prepared by distillation alone, or by other methods such as chromatography (as shown in the following examples).

[0073] The preparation method may further include a step of removing or reducing toxic components. The purified and concentrated compositions of the present invention further have very low amounts of undesirable contaminants. For example, the amount of oligomeric and polymeric by-products, including oxidation products, is reduced from that in the starting oil. Furthermore, the low level of DHA in the composition reduces the tendency to oxidize, which is beneficial to the stability of the composition. Preferably, such oxidation products are at most 1.0% by weight of the fatty acid composition, more preferably at most 1.5% by weight, such as at most 0.5% by weight. More specifically, the amounts of environmental contaminants such as benzo(a)pyrene (BAP) and polycyclic aromatic hydrocarbons (PAH) are low in the compositions of the present invention. In one embodiment, the fatty acid mixture of the composition contains less than 2 pg / kg of benzo(a)pyrene (BAP). In another embodiment, the composition preferably contains less than 10 pg / kg of polycyclic aromatic hydrocarbons (4PAH). 4PAH is defined as the sum of benzo(a)anthracene, chrysene, benzo(b)fluoranthene, and benzo(a)pyrene. Two main methods are used to measure oxidation in oils: measuring the peroxide value and the p-anisidine value.

[0074] The fatty acids of the composition, both MUFA and PUFA, and other fatty acids of the composition, can be in various forms. In one embodiment, the fatty acids of the composition are in a form selected from the group consisting of free fatty acids, fatty acid salts, monoglycerides, diglycerides, triglycerides, esters such as ethyl esters, wax esters, O-acetylated ω-hydroxy fatty acids (OAHFA), cholesteryl esters, ceramides, phospholipids, and sphingomyelin, alone or in combination. Alternatively, the fatty acids may be in any form that can be absorbed in the digestive tract or absorbed on the body surface after topical application. Preferably, the fatty acids are in the form of free fatty acids, fatty acid salts, ethyl esters, or glycerides. In one embodiment, the LCMUFA and LCPUFA are independently selected from the group consisting of free fatty acids, fatty acid esters, and monoglycerides, diglycerides, or triglycerides. In a preferred embodiment, the MUFA of the MUFA composition of the present invention is in the form of ethyl esters. If desired, the form of the fatty acid can be converted to another form. For example, those skilled in the art know how to convert, for example, ethyl esters to free fatty acids or glycerides, and the production method may include such steps.

[0075] When referring to the weight % of fatty acid in the mixture, any of the above-mentioned most broadly defined fatty acid forms can be used as the calculation basis.Furthermore, the fatty acid of the composition provided in any of the above-mentioned forms is preferably not combined with other active ingredients.Therefore, the fatty acid mixture of the composition is a pure, unreacted, high-concentration MUFA and PUFA mixture.However, fatty acid end groups can be changed from their original, for example, from glyceride to ester or free fatty acid, or vice versa.

[0076] The LCMUFA compositions of the present invention are useful in a range of different applications. The disclosed LCMUFA compositions, according to a first aspect, have been found to be valuable, for example, as an energy source, for use in foods, supplements, therapeutic applications, or as a source of more highly concentrated LCMUFA compositions. In particular, the MUFA compositions of the present invention obtained from enzymatic separation and single distillation, the first fraction containing, for example, about 10-25% cetoleic acid, may be commercially interesting, for example, for use in the food or feed industry.

[0077] In one aspect, the present invention provides the use of a MUFA composition disclosed in the first aspect, comprising C22:1 n11 (cetoleic acid) and C20:5 n3 (EPA), in a food or feed, the concentration of C22:1 n11 is at least 16 wt.%; The EPA concentration is at least 3 wt.%. Further embodiments of the composition are as described in relation to the first aspect.

[0078] In one exemplary embodiment, the MUFA composition of the present invention is included in feed or food compositions that usually contain a certain amount of unsaturated fatty acids.Because the claimed composition is highly stable and resistant to oxidation, the claimed MUFA composition can be beneficially replaced with the oils used in food or feed that are more susceptible to oxidation.Another argument for using the MUFA composition in food, feed, supplements or nutritional supplements is that cetoleic acid can act as a catalyst for the conversion of ALA to EPA and DHA.

[0079] In another exemplary embodiment, the MUFA compositions of the present invention are included in feed for use in aquaculture, such as, for example, fish feed compositions, e.g., feed pellets for farmed fish. In particular, the compositions are used in therapeutic feed for fish, e.g., farmed salmon. In one example, this is for the preventative or therapeutic treatment of heart health.

[0080] In some embodiments, the present invention relates to methods and compositions for the treatment and alleviation of diseases or for use in health indicators. As discussed in the Background section, research, particularly by Yang et al., has supported the beneficial effects of cetoleic acid on health, for example, when combined with EPA. The concentrated MUFA compositions of the present invention, which contain high concentrations of cetoleic acid as disclosed in the first aspect, may provide one or more of the following health benefits: reduced inflammation, improved TG and cholesterol, increased fat burning, reduced liver steatosis, improved insulin sensitivity, and improved metabolic syndrome. As suggested in Example 6, the compositions of the present invention may be used to positively affect metabolic syndrome. This may affect parameters related to diabetes (glucose tolerance, insulin sensitivity, intramuscular glucose uptake), diabetic neuropathy, particularly allodynia (temperature sensitivity) and metabolic syndrome (liver fat content), and cardiovascular health (blood pressure).

[0081] In some embodiments, the composition is a dietary supplement, supplemental composition, or pharmaceutical composition, and the composition is used to treat, e.g., prevent, reduce, and / or alleviate the effects, symptoms, etc., of at least one health problem in a subject in need thereof. The MUFA composition is administered to the subject. In one embodiment, the composition is used to maintain good health, such as maintaining a healthy cardiovascular system / heart health. In at least some embodiments of the present invention, the composition does not contain an additional active agent. In this embodiment, the composition can be used for pharmaceutical treatment of a subject, such as a subject diagnosed with reduced MUFA levels.

[0082] In one embodiment, the compositions of the present invention are used for human health as supplements, i.e., in the form of foods, nutritional supplements, or dietary supplements, pharmaceuticals, medical foods, or foods for special medical purposes. In this latter group, the compositions may be selected from the group consisting of enteral nutrients for special medical uses, foods for specified health uses, foods for special medical purposes (FSMP), foods for special nutritional uses (FSDU), medical nutrition, and medical foods. Such compositions are particularly suitable for subjects with deficiencies in specific nutrients, such as cetoleic acid. The compositions are suitable for the nutritional management of subjects with unique nutritional needs. Such compositions are typically administered to subjects under the supervision of a physician. The compositions contain relevant MUFAs to increase or correct MUFA levels in blood or specific tissues, such as in subjects diagnosed with reduced capacity for MUFA synthesis and / or a reduced omega-3 index. The compositions and methods of the present invention are capable of correcting nutritional deficiencies in such subject populations. In one exemplary use, the compositions are for maintaining a healthy cardiovascular system / heart health. In particular, the compositions of the present invention are used as dietary supplements for cardiovascular health.

[0083] Applicant is also investigating the potential beneficial role of the MUFA compositions of the present invention in skin health, which is likely to be an important area of ​​research in the future. Skin lacking the ability to produce MUFAs is characterized by underdeveloped sebaceous glands, which normally prevent skin dryness. This suggests the importance of MUFAs in skin biology. In one embodiment, the compositions are used to treat skin, such as contributing to skin barrier function, maintaining a healthy skin appearance, preventing wrinkles or red spots, and protecting against the harmful effects of the sun's ultraviolet rays. Skin and hair-related diseases and conditions that can be treated with the compositions for use in the present invention include at least the following: dry and wrinkled skin, skin inflammation, oxidation, or sensitivity, protecting against the harmful effects of the sun's ultraviolet rays (i.e., preventative treatment), improving wound healing capabilities, adverse effects on hair follicles, and reduced hair health, including the risk of hair loss. Examples of skin diseases and conditions that typically cause skin irritation / oxidation and can benefit from treatment with the composition for use are, for example, eczema, psoriasis, dermatitis, acne, and rosacea (papulopustular rosacea).In one embodiment, the MUFA composition of the present invention is included in skin preparations such as cosmetics.By using the composition or method of the present invention, the fatty acid composition of tissues such as skin can be normalized, for example, by supplementing abnormal sebum fatty acid composition.Such use or method can be considered as therapeutic or non-therapeutic.

[0084] See Examples 4 and 5 below. The effects of the compositions of the present invention on skin, such as human fibroblasts, can be seen as improving the cells' ability to regenerate after injury (wound healing), affecting the production of melanin (skin pigment), or reducing the expression of genes involved in inflammation (Example 4). In one embodiment, use of the composition results in an improvement in the degree and severity of eczema, such as atopic dermatitis (Example 5). Use of the composition can indicate changes in molecular markers, such as lipid mediators, inflammatory markers, and blood lipids. More specifically, use of the composition can positively affect one or more of the following: ceramide / lipid composition in the skin, inflammatory signaling molecules in the skin, erythrocyte omega-3 index, erythrocyte cetoleic acid, cholesterol / blood lipid profile, transepidermal water loss (TEWL), plasma hydroxylated omega-3 fatty acids, the association between omega-3 index and Eczema Area and Severity Index (EASI) parameters, and the association between erythrocyte cetoleic acid content and EASI parameters.

[0085] Thus, in one embodiment, the present invention provides a composition of the first aspect for use as a dietary supplement, food supplement, food additive, or cosmetic.

[0086] The compositions according to the present invention, such as supplements, can be delivered or administered in any suitable form, including, but not limited to, oral delivery, transdermal delivery, or mucosal delivery, including eye drops. The compositions of the present invention can be formulated, along with other ingredients, in the form of an oral delivery vehicle, such as a capsule, preferably a gelatin capsule, a liquid, an emulsion, a tablet, or a powder, to include an excipient and / or carrier acceptable for oral consumption.

[0087] Thus, the present invention provides a MUFA composition as disclosed in the first aspect, comprising C22:1 n11 (cetoleic acid) and C20:5 n3 (EPA), for use in a treatment or health indication; the concentration of C22:1 n11 is at least 16 wt.%; The EPA concentration is at least 3 wt.%.

[0088] Similarly, the present invention provides a method of treatment comprising administering to a subject a MUFA composition as disclosed in the first aspect, comprising C22:1 n11 (cetoleic acid) and C20:5 n3 (EPA); the concentration of C22:1 n11 is at least 16 wt.%; The EPA concentration is at least 3 wt.%.

[0089] In some embodiments, the composition for use comprises: an enriched LCMUFA composition comprising C22:1 n11 (cetoleic acid) and C20:5 n3 (EPA); the concentration of C22:1 n11 is at least 39 wt.%; The concentration of EPA is at least 5.0 wt.

[0090] Thus, the present invention provides a composition according to the first aspect for use as a supplement, a medicine, a medical food, or a food for special medical purposes, or a food for the skin.

[0091] The MUFA composition for use is as detailed in the first aspect. In particular, the composition for use in therapy or the composition for use in a treatment method is used to reduce inflammation, improve TG and cholesterol, increase fat burning, reduce fatty liver, or improve insulin sensitivity or metabolic syndrome, which are indicators of the combined benefits of cetoleic acid and EPA. Furthermore, the composition of the present invention can be used for one or more of the following: reducing triacylglycerides, increasing HDL cholesterol, lowering blood pressure, reducing inflammation, and acting as a precursor to specialized proresolving mediators (SPMs). Furthermore, the composition of the present invention can be used for one or more of the effects of cetoleic acid: reducing non-HDL cholesterol, reducing hyperinsulinemia, regulating glucose, and reducing inflammation. Based on the research of Yang et al. in 2016, the MUFA composition of the present invention was developed with specific therapeutic effects in mind. First, in reducing the risk of cardiovascular disease, and second, as an alternative means of providing omega-3 levels to humans, which provides benefits in areas such as chronic inflammation, cognition, and healthy aging. Cell culture studies and human clinical studies by Ostbye et al., BR, J Nutr. Oct 14, 2019, 122(7), 755-768 support the hypothesis that cetoleic acid-rich fish oil promotes the conversion of alpha-linolenic acid to EPA, supporting the use of cetoleic acid-rich oils in the claimed composition as a related source of EPA and DHA. In one embodiment, the composition is used to treat one or more of metabolic syndrome, fatty liver disease (NAFLD, NASH), diabetes, prediabetes, and cardiovascular disease.

[0092] In a further aspect, the present invention provides a composite formulation. Such a formulation may be a combination of the MUFA composition as disclosed in the first aspect with other marine oils. As an alternative, the MUFA composition of the present invention is combined with, for example, anchovy oil or a fraction thereof to further concentrate the composition with PUFAs and provide a formulation useful for various types of applications.

[0093] The present invention is not intended to be limited to the embodiments and examples shown. While various embodiments of the present disclosure have been described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications and variations to the embodiments described herein, as well as variations and substitutions of the embodiments, will be apparent to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments described herein can be employed in implementing the present disclosure.

[0094] It should be understood that any embodiment of the present disclosure may be optionally combined with any one or more of the other embodiments described herein.

[0095] It should be understood that embodiments disclosed for one aspect also apply to other aspects of the invention, for example, embodiments disclosed for compositions also apply to aspects relating to methods for production and compositions for use.

[0096] It should be understood that each component, compound, composition, or parameter disclosed herein is construed as being disclosed for use alone or in combination with one or more of all other components, compounds, compositions, or parameters disclosed herein. Furthermore, each amount / value or amount / value range for each component, compound, or parameter disclosed herein should also be construed as being disclosed in combination with each amount / value or amount / value range for any other component(s), compound(s), or parameter(s) disclosed herein; and thus, any combination of amount / values ​​or amount / value ranges for two or more components, compounds, or parameters disclosed herein should also be understood to be disclosed in combination with each other for purposes of this description. Any feature described herein, and any combination of such features, is within the scope of the present invention, unless the features are mutually inconsistent.

[0097] It should be understood that each lower limit of each range disclosed herein is interpreted as a disclosure in combination with each upper limit of each range disclosed herein for the same component, compound, or parameter. Thus, a disclosure of two ranges should be interpreted as a disclosure of four ranges derived by combining each lower limit with each upper limit of each range. A disclosure of three ranges should be interpreted as a disclosure of nine ranges derived by combining each lower limit with each upper limit of each range, and so on. Furthermore, a specific amount / value of a component, compound, or parameter disclosed in the description or examples should be interpreted as a disclosure of either a lower or upper limit of a range, and thus can be combined with any other lower or upper limit or range or specific amount / value of the same component, compound, or parameter disclosed elsewhere in this application to form a range of that component, compound, or parameter. [Example]

[0098] Example 1. Comparison - Two-Stage Distillation vs. Use of a Lipase Process According to the Invention The starting oil in this example was mackerel oil that had been deacidified (to remove free fatty acids) and had organic contaminants removed by single-column short-path distillation. Refining is optional and can be done in a variety of ways, such as pre-bleaching, employing supercritical extraction, membrane filtration, etc. The same comments apply to other examples.

[0099] For comparison, batch 1 was prepared by chemically ethylating fish oil (starting oil) using 2% sodium ethoxide / ethanol, with quantitative yields of ethyl esters (>95%). Workup was traditional, with glycerol removed after evaporation of excess ethanol, followed by washing of the ethyl esters with water and citric acid. The area was 0.06 m. 2 The ester was distilled using a VTA short-path distillation pilot plant (VK 83-6) consisting of two columns with a column diameter of 0.285 m and a height of 0.285 m. The flow rate was adjusted to about 4 kg / h, and the temperature was adjusted to 115°C in the first column, while it was adjusted to 165°C in the second column. The pressure was maintained at 100°C during the distillation. -3 The distillation was adjusted to remove mainly short chain fatty acids (C14-C16) and maintain the content of long chain fatty acids (Table 1).

[0100] Batch 2 was produced by selective interesterification according to the present invention using a 1,3-position specific lipase with a stoichiometric amount of ethanol. The lipase was 2% (w / w) of the oil. Once the target conversion was reached (which varied), the lipase was removed by filtration. Excess ethanol was removed and the ethyl esters were recovered by single column stripping using the same VTA pilot plant as above. The temperature of the stripping column was 165°C and the pressure was 10 -3 The pressure was less than mbar.

[0101] Results from both batches are shown in Table 1 below. It is noted that the compositions of gondoic and cetoleic acids were fairly similar in both batches, 12-13% (121-123 mg / g) and 20-21% (199-203 mg / g), respectively. However, significant differences were observed in the amounts of EPA and DHA between the batches. In Batch 1, the total EPA+DHA was 26%, and the EPA / DHA ratio was 0.68, a similar ratio to that found in the starting oil. In Batch 2, using lipase, the total EPA+DHA was only 7%, and the EPA / DHA ratio was 4.4, a more than six-fold increase over the starting oil. The cetoleic acid / DHA ratio was 16.04, a more than 11-fold increase over the starting oil.

[0102] The results presented in the table further demonstrate that when lipase was used, the amount of short-chain fatty acids (C14-C16) was approximately 27% (w / w), compared to 7% when distillation alone was used. This is a major advantage of the intermediate: the significantly lower boiling points of these short-chain fatty acids allow for further concentration by distillation, which is reflected in the overall oil yield. This example demonstrates that by utilizing a 1,3-position-specific lipase characterized by specific fatty acid selectivity, a similar LCMUFA composition can be achieved compared to distillation, but with much less EPA + DHA compared to EPA, and even less DHA than expected. Those skilled in the art will recognize that separation of fatty acids or their derivatives can be achieved either by their polarity (degree of unsaturation) or boiling point. Those skilled in the art will understand that separating LCMUFAs such as cetoleic acid and gondoic acid from EPA and DHA is not easy, and the results achieved by this example are surprising and novel.

[0103] [Table 1]

[0104] Example 2. Concentration of cetoleic acid by distillation with or without lipase This example demonstrates how surprisingly effective the ethyl esters made by selective ethylation according to the present invention were in concentrating cetoleic acid compared to non-selective ethylation. Two batches made in Example 1 were further distilled using the same pilot plant as in Example 1. Fractions were collected and analyzed by GC to estimate mg / g and % area of ​​major fatty acids. The results are shown in Table 2 below.

[0105] When concentrating batch 2 and batch 1 from Example 1, fractions A (selective / lipase) and C (non-selective / chemical) were produced with comparable oil yields, i.e., 45% vs. 51%, respectively. The sum of 20:1 + 22:1 was similar in both cases, 47% and 42%, respectively. Significant differences between the methods were noted in terms of both the EPA + DHA and cetoleic acid / DHA ratios: 11% and 12.23 for fraction A, compared with 31% and 1.37 for fraction C. Second, fraction A was still significantly richer in short-chain fatty acids (C14-C16 and C18) than fraction C, supporting further enrichment of cetoleic acid. Therefore, batch 2 (lipase) was further concentrated, resulting in fraction B with an overall yield of 31%. This fraction had a similar amount of short-chain fatty acids to that found in C (9.8% vs. 13.9%). However, fraction B was much richer in cetoleic acid than that found in fraction C, with 44% cetoleic acid EE (423 mg / g as EE) in fraction B compared to only 27% cetoleic acid (265 mg / g as EE) in fraction C for the non-selective process. Only 12% EPA + DHA was still retained in fraction B, and the cetoleic acid / DHA ratio was high, i.e., 13.08, which was 9.5 times higher than that found in fraction C and 9.6 times higher than that found in the starting oil.

[0106] It is further noted from Table 2 that the yields of cetoleic acid in Fractions B and C were substantial, 83% and 87%, respectively, despite Fraction B containing approximately 1.6-fold higher amounts of this fatty acid. This demonstrates that the lipase process of the present invention is more efficient at producing a high concentration of cetoleic acid while still maintaining low amounts of EPA + DHA and a high cetoleic / DHA ratio. The cetoleic / cetoleic ratio remains similar to that found in the starting oil.

[0107] Those skilled in the art will recognize that comparative fraction C can be further distilled until the cetoleic acid content is similar to that found in fraction B. However, this will lead to a significant decrease in the yield of oil and cetoleic acid. Further distillation of C will lead to a proportional increase in the content of DHA and cetoleic acid, because the boiling points of these fatty acid esters are similar. Therefore, this cetoleic acid concentrate will be rich in cetoleic acid and DHA, and the cetoleic acid / DHA ratio will be low. Therefore, this example demonstrates that the use of 1,3-position specific lipases characterized by specific fatty acid selectivity is surprisingly more effective in concentrating cetoleic acid than distillation.

[0108] [Table 2]

[0109] Example 3. Enrichment of cetoleic acid by using menhaden oil and 1,3-position specific lipase Refined herring oil was subjected to selective interesterification using a 1,3-position specific lipase and stoichiometric amounts of ethanol and lipase. Once the target conversion was reached (variable), the lipase was removed by filtration. Excess ethanol was removed, and the ethyl esters were recovered by single column stripping at 165°C, designated as Fraction A. The distillate was further distilled using the same VTA two-column short-path distillation plant used in Example 1. The pressure was maintained at 10°C under the distillation process. -3The pressure was less than mbar and the column temperature was 100-140° C. during the two-stage distillation (concentration of cetoleic acid). The results can be seen in Table 3 below.

[0110] After distillation of fraction A, an oil containing 44% cetoleic acid (434 mg / g EE) was collected, which contained only 10% EPA + DHA and an EPA / DHA ratio of 4.72, 5.2 times higher than that found in refined herring oil. The cetoleic acid / DHA ratio also increased significantly from 2.22 in the starting oil to 26.49 in the 44% cetoleic acid concentrate, an increase of approximately 12 times. The ratio of gondoic acid to cetoleic acid is similar to that found in the starting oil. Therefore, the process maintains this ratio while concentrating cetoleic acid. Therefore, the method of the present invention can be used with a variety of fish oils rich in cetoleic acid.

[0111] [Table 3]

[0112] Example 4. Study using human fibroblasts to determine the effect of LCMUFA concentrate on skin health, including wound healing Various fish oils contain varying amounts of omega-3 fatty acids and other less well-known fatty acids. North Atlantic fish oils contain lower levels of EPA and DHA than South American fish oils, but are richer in long-chain monounsaturated fatty acids (LCMUFAs), particularly C22:n1-11 (cetoleic acid), which are unique to marine oils. Compared to EPA and DHA, research on the absorption, tissue incorporation, and health benefits of marine LCMUFA supplementation is lacking. Early research (Huang et al., Cosmetic and Therapeutic Applications of Fish Oil's Fatty Acids on the Skin. Mar. Drugs 2018, 16, 256) has shown that fatty acids from fish oil taken as an oral supplement can improve skin barrier function, reduce UV-induced inflammation, reduce unwanted pigmentation, and improve wound healing. Some studies suggest positive effects of fish oil and omega-3s on skin disorders, but meta-analyses have produced conflicting results. These contradictory results may be explained by the complex composition and composition of fish oil; furthermore, the quality of the oil may vary depending on the fish species used and the manufacturing process. Both the LC-MUFA and omega-3 content of fish oil are important factors when determining the health benefits of fish oil. Studies conducted by the applicant and NOFIMA in rats and salmon have shown that for both species, the highest concentrations of the fatty acid C22:1n-11 (cetoleic acid) are found in the skin.

[0113] To test the potential health benefits of LCMUFA in skin, a preliminary cell study was conducted using human fibroblasts.

[0114] The main objective of the study was to determine whether the addition of LCMUFA concentrate (cetoleic acid concentrate) or DHA to cell culture media 1) affecting the ability of cells to regenerate after injury (the so-called "wound healing model"); 2) Affects the production of melanin (skin pigment), 3) To demonstrate whether it affects the expression of genes involved in inflammation after oxidative stress induction.

[0115] The LCMUFA concentrate used in the study was a North Atlantic fish oil concentrate prepared from mackerel oil and contained the following, given as GC area %: Cetoleic acid, C22:1: 48.74%; DHA, C22:6:n3:3.96%; C20:1:18.84%.

[0116] In the test material used in this study, the concentration of EPA, C20:5 n3 was 0.80 GC area %.

[0117] Wound healing model: Treatment groups included the addition of DHA or LCMUFA concentrate to the cell culture medium, respectively. Controls included cells receiving culture medium alone without fatty acids. The effect on wound closure 14 hours after wound initiation was measured.

[0118] The results are shown in the figures. In Figure 1, the Y-axis shows the scratch area % and the X-axis provides bars for the control, DHA, and LCMUFA concentrates. The initial wound area was defined as 100%, and then the wound opening was measured 14 hours later as a percentage of the original wound. The reduction rates were approximately 60% for LC-MUFA and 50% for DHA, respectively. Therefore, a trend toward improved wound healing was observed in the LCMUFA test group.

[0119] Menadione was added to the cell culture medium to induce oxidative stress. Melanin production was reduced in both LCMUFA- and DHA-treated cells, see Figure 2, which shows melanin levels after oxidative stress.

[0120] Expression of inflammation-related genes showed no statistically significant differences between treatment groups, although there was a trend toward lower expression of MMP2, ATF6B, and TNF-α genes in the LCMUFA group compared to controls; see Figure 3. DHA-treated cells showed no such trend; data not shown.

[0121] Conclusion: Preliminary data from cell studies suggest a positive effect of LCMUFA concentrate on skin health, particularly on unwanted hyperpigmentation due to a reduction in melanin synthesis.

[0122] Example 5. Randomized, double-blind nutritional study to determine the effects of concentrated cetoleic acid fish oil on atopic dermatitis The primary objective of this study is to determine whether 2 g of the investigational drug "EPAX Cetoleic 30" taken daily changes the extent and severity of mild-to-moderate atopic dermatitis, as assessed by the Eczema Area and Severity Index (EASI), in patients with mild-to-moderate atopic dermatitis. The secondary objective is to determine whether 2 g of EPAX Cetoleic 30 taken daily is associated with changes in molecular markers (lipid mediators, inflammatory markers, and blood lipids) in patients with mild-to-moderate atopic dermatitis, compared with placebo.

[0123] Investigational drug: North Atlantic fish oil concentrate containing at least 300 mg / g cetoleic acid (EPAX Cetoleic 30). Specifically, the investigational product contains the following fatty acids, given by weight % and GC area %: EPA(C20:5): 5.7wt%, 6.12 area% DHA(C22:6): 1.6wt%, 1.66 area% Gondoic acid (C20:1 n9): 21.6 wt%, 23.16 area% Cetoleic acid (C22:1 n11): 41.5 wt%, 45.46 area%.

[0124] The total LCMUFA weight percent is 67.6% (TG).

[0125] The study drug was prepared according to the method requested. The study drug will be provided in the form of 1 gram capsules. Subjects will take 2 x 1 g capsules daily for 6 months. The placebo will be corn oil.

[0126] The study population was based on volunteers (aged 18-80 years) with mild to moderate atopic dermatitis and an Eczema Area and Severity Index (EASI) of 1.1-21.0. The project used medical records from Norwegian hospitals to pre-screen patients with mild to moderate atopic dermatitis. The study population was block randomized to ensure equal representation of drug-treated and non-treated volunteers in each group.

[0127] The study is double-blind and subjects are assigned to one of two nutritional groups according to a randomization plan, with 30 subjects in each group. Group 1: Placebo: Corn oil capsules Group 2: Study supplement: EPAX Cetoleic 30 capsules

[0128] Inclusion criteria: subjects diagnosed with active atopic dermatitis, with an EASI score of 1.1–21.0, willing to abstain from oil supplements (e.g., omega-3, borage, evening primrose) for one month before the start of the study (wash-out) and during the study period, and willing to take the study supplements for six months.

[0129] Study visits include clinical assessments, blood sample collection, skin sample collection, and subject assessments (questionnaires) at day 1, 6 weeks, 3 months, and 6 months.

[0130] Samples and analysis: Clinical evaluation: Eczema Area and Severity Index (EASI) Patient-reported assessments: Pruritus Numerical Rating Scale (NRS), Dermatology Life Quality Index (DLQI), Patient-Oriented Eczema Measure (POEM), use of topical treatments Skin tape samples (10 consecutive tapes): ceramides, cytokines Skin barrier: Transepithelial water loss (TEWL) Blood samples (red blood cells (RBC) and plasma): Omega-3 Index, hydroxylated omega-3s Blood samples for blood lipids (cholesterol-package) and liver enzymes (ALAT, ASAT, GTT)

[0131] The primary efficacy variables will be changes in clinical and subject-assessed parameters for the assessment of eczema. Secondary efficacy variables were: Changes in skin ceramide / lipid composition Changes in inflammatory signaling molecules in the skin Changes in the omega-3 index of red blood cells Changes in cetoleic acid in erythrocytes Changes in cholesterol / blood lipid profile Changes in transepithelial water loss (TEWL) Changes in plasma hydroxylated omega-3 fatty acids Association between Omega-3 Index and EASI parameters Relationship between cetoleic acid content in RBC and EASI parameters

[0132] At the end of the study, Group 2, subjects receiving the cetoleic acid-enriched capsules, are expected to show a favorable change in at least one of the parameters for the evaluation of eczema compared to Group 1 receiving the placebo.

[0133] Example 6. Animal studies to evaluate effects on metabolic syndrome A study was conducted using ZDSD rats, a rat model of obese diabetic rats that spontaneously develop type 2 diabetes. Rats were fed a diet supplemented with herring oil (containing cetoleic acid) for six weeks to evaluate the effect on metabolic syndrome.

[0134] Feeding diets supplemented with menhaden oil was found to improve insulin sensitivity and blood glucose levels compared to soy controls, parameters that are important targets for diabetes control and metabolic syndrome.

[0135] Future studies are planned using Zucker fa / fa rats, which are naturally prediabetic. This means that the rats will exhibit mild insulin insensitivity and elevated fasting blood glucose levels, but these levels will be lower than those seen in fully diabetic rats. The rats will be divided into a low-cetoleic acid group (menhaden oil), a high-cetoleic acid group (EPAX Cetoleic 30, the same investigational drug used in Example 5), and a soy control group, and will be treated for six weeks. The study will measure diabetes-related parameters (glucose tolerance, insulin sensitivity, and muscle glucose uptake), diabetic neuropathy, particularly allodynia (temperature sensitivity), metabolic syndrome (liver fat content), and cardiovascular health (blood pressure).

Claims

1. A composition enriched in long chain monounsaturated fatty acids (LCMUFA), said composition comprising C22:1 n11 (cetoleic acid) and C20:5 n3 (EPA); the fatty acids of the composition are obtained from natural oils containing cetoleic acid and EPA; The concentration of cetoleic acid is at least 16.0 wt. %; EPA concentration is at least 3 wt. %; Any concentration of DHA up to 4 wt. %; an EPA:DHA ratio of at least 4:1; A composition, wherein the composition comprises a fatty acid in the form of an ethyl ester, a free fatty acid, or a fatty acid salt.

2. 2. The LCMUFA composition of claim 1, obtained from fish oil, in particular from herring, mackerel, capelin, cod, pollock or tobis oil.

3. 10. The LCMUFA composition of claim 1, comprising 16.0 to 60.0 wt. % cetoleic acid.

4. 2. The LCMUFA composition of claim 1, wherein the LCMUFA composition further comprises at least one C20:1 fatty acid, and the weight ratio of C22:1 fatty acid to C20:1 fatty acid is from 1.5:1 to 2.6:

1.

5. 10. The LCMUFA composition of claim 1, comprising EPA at a concentration of 5.0 to 40.0 wt.% of the composition.

6. 2. The LCMUFA composition of claim 1, comprising gondoic acid, C20:1 n9, in the range of 9-28 wt% of the composition.

7. 2. The LCMUFA composition of claim 1, wherein the cetoleic acid:DHA ratio is at least 10:

1.

8. The concentration of cetoleic acid is at least 39 wt. %; 2. The LCMUFA composition of claim 1, wherein the concentration of EPA is at least 5.0 wt.%.

9. 10. The LCMUFA composition of claim 1, wherein the combined concentration of EPA and DHA ranges from 6.0 to 12.0 wt.% of the composition.

10. 2. The LCMUFA composition of claim 1, wherein the cetoleic acid:DHA ratio is at least 15:

1.

11. 1. A method for preparing a composition enriched in long-chain monounsaturated fatty acids (LCMUFA) containing at least 16 wt. % cetoleic acid (C22:1 n11) and long-chain polyunsaturated fatty acids (LCPUFA) from a natural oil containing LCMUFA including cetoleic acid, comprising: i) selectively transesterifying fatty acids of the natural oil by mixing the natural oil with lipase and ethanol to obtain a mixture comprising LCPUFA in glyceride form and LCMUFA in ethyl ester form; ii) distilling the mixture of step i) to separate LCMUFA as one fraction and LCPUFA as another fraction, and recovering the LCMUFA fraction to obtain an LCMUFA-enriched composition.

12. 12. A method according to claim 11 for preparing an LCMUFA-enriched composition according to any one of claims 1 to 10.

13. 12. The method of claim 11 for preparing an LCMUFA-enriched composition, wherein the concentration of cetoleic acid is at least 39 wt.% and the concentration of EPA is at least 5.0 wt.%.

14. 12. The method of claim 11, wherein the natural oil is obtained from herring, mackerel, capelin, cod, pollock, or sea bass.

15. 12. The method of claim 11, wherein the fatty acids of the natural oil are predominantly in the form of triglycerides.

16. The method of claim 11, wherein the lipase is a 1,3 specific lipase.

17. 12. The method of claim 11, wherein the lipase is selected from Rhizomucor miehei lipase, Aspergillus niger lipase, Thermomyces lanuginosus lipase, Candida antarctica lipase, Candida rugosa lipase, Geotrichum candidum lipase, Penicillium roguefortii lipase, Rhizopus delemar lipase, and Rhizopus oryzae lipase.

18. Use of the LCMUFA composition according to any one of claims 1 to 10 in a food, feed, supplement or dietary supplement.

19. 11. The LCMUFA composition according to any one of claims 1 to 10 for use in therapy.

20. 20. The LCMUFA composition for use according to claim 19, for use in the treatment of one or more of metabolic syndrome, insulin sensitivity, fatty liver disease (NAFLD, NASH), diabetes, prediabetes, diabetic neuropathy, high LDL cholesterol, cardiovascular disease and risk factors, and skin diseases or conditions.

21. 20. The LCMUFA composition for use according to claim 19, wherein said use is in skin health or skin treatment.

22. 22. An LCMUFA composition for use according to claim 21, wherein the treatment of the skin contributes to the skin's barrier function, maintaining a healthy appearance of the skin, avoiding wrinkles or red spots, or protecting against the harmful effects on the skin of the sun's ultraviolet rays.

23. 21. The LCMUFA composition for use according to claim 20, wherein the skin disease or condition is selected from the group of eczema, psoriasis, dermatitis, acne, and rosacea (papulopustular rosacea).

24. 20. The LCMUFA composition for use according to claim 19, which is comprised in a pharmaceutical composition, a supplement, i.e. a food, a nutritional or dietary supplement, a medical food or a food for special medical purposes.

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