Isolation of Omega-7 fatty acid ethyl esters from natural oils
10314871 ยท 2019-06-11
Assignee
Inventors
- Sandip Shinde (Gilbert, AZ, US)
- Stephen Ventre (Gilbert, AZ, US)
- Jennifer Lloyd-Randolfi (Chandler, AZ, US)
- Michael Lamont (Gilbert, AZ, US)
Cpc classification
C12P7/6458
CHEMISTRY; METALLURGY
International classification
C12P7/64
CHEMISTRY; METALLURGY
C11C3/00
CHEMISTRY; METALLURGY
Abstract
Methods of producing high purity palmitoleic acid esters from natural oils are disclosed. The natural oils may comprise plant oil, nut oil, microalgae oil, and fish oil. The methods of processing the natural oil comprise transesterification with ethanol as the reacting solvent to produce ethyl esters. Methods of producing a high purity fraction of Omega-3, 6, & 9 fatty acid esters from natural oils are also disclosed. The high purity fatty acid esters may be used in nutrition, cosmetic, and nutraceutical products.
Claims
1. A method of producing high purity palmitoleic acid ethyl esters, comprising: a. Providing a natural oil; b. Transesterifying the natural oil with a catalyst and ethanol as a reacting solvent in a reaction comprising at least one stage to produce ethyl esters; c. Distilling the ethyl esters to produce a first fraction comprising C18:1n9, C20:4n6, and C20:5n3 ethyl esters, and a second fraction comprising C16:0 and C16:1n7 ethyl esters; and d. Separating the C16:0 and C16:1n7 ethyl esters of the second fraction to produce a third fraction of C16:0 ethyl esters and a fourth fraction comprising at least 50% w/w C16:1n7 ethyl esters.
2. The method of claim 1, wherein the natural oil consists of a natural oil comprising at least 5% C16:1n7 fatty acid.
3. The method of claim 2, wherein the natural oil comprises oil derived from at least one source selected from the group consisting of macadamia nut oil, menhaden oil, and anchovy oil.
4. The method of claim 1, wherein the catalyst of the transesterifying step comprises at least one catalyst selected from the group consisting of: NaOH, KOH, sodium ethoxide, HCl, BF.sub.3, H.sub.3PO.sub.4, an organic sulfonic acid, sulphuric acid, a metal oxide, MgAl hydrolalcite, and an immobilized enzyme.
5. The method of claim 1, wherein the molar ratio of ethanol in the transesterifying step is in the range of 1:3 to 1:15.
6. The method of claim 5, wherein the molar ratio of ethanol in the transesterifying step is in the range of 1:6 to 1:8.
7. The method of claim 1, wherein the transesterifying step comprises two stages of transesterification reactions.
8. The method of claim 7, wherein the two stages of transesterification reactions use different catalysts.
9. The method of claim 1, wherein the transesterifying step has a yield in the range of 70-90%.
10. The method of claim 1, wherein the transesterifying step is performed at a temperature in the range of 75-85? C.
11. The method of claim 1, wherein the distillation step comprises at least one selected from the group consisting of one pass vacuum distillation, two pass vacuum distillation, fractional distillation, and simple molecular distillation.
12. The method of claim 1, wherein the separation step comprises at least one selected from the group consisting of urea complexation, crystallization, filtration, extraction, and adsorption chromatography.
13. The method of claim 1, wherein the fourth fraction comprises at least 80% w/w C16:1n7 ethyl esters.
14. The method of claim 3, wherein the second fraction derived from natural macadamia nut oil comprises less than 20% w/w C16:0 ethyl esters.
15. The method of claim 14, wherein the fourth fraction derived from natural macadamia nut oil further comprises less than 10% w/w C16:0 ethyl esters.
16. The method of claim 3, wherein the second fraction derived from natural macadamia nut oil comprises less than 1% w/w C18:1n9 ethyl esters.
17. The method of claim 16, wherein the fourth fraction derived from natural macadamia nut oil further comprises less than 5% w/w C18:1n9 ethyl esters.
18. The method of claim 3, wherein the second fraction derived from natural macadamia nut oil further comprises at least 70% w/w of C16:1n7 and C18:1n9 ethyl esters combined.
19. The method of claim 1, further comprising including the C16:1n7 ethyl esters in a product selected from the group consisting of a nutrition product, cosmetic product, and nutraceutical product.
20. The method of claim 11, wherein the distillation step is performed at a pressure in the range of 0.3-5 mm Hg and a temperature in the range of 140-280? C.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1) In order to describe the manner in which the above-recited and other advantages and features of the invention can be obtained, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. Understanding that these drawings depict only typical embodiments of the invention and are not therefore to be considered to be limiting of its scope, the invention will be described and explained with additional specificity and detail through the use of the accompanying drawings in which:
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DETAILED DESCRIPTION
(10) A number of nutrition, cosmetic, and nutraceutical products may be produced from oils comprising Omega-3, 6, 7 & 9 fatty acids. In some embodiments, the oil may comprise a natural oil comprising plant oil, nut oil, microalgae oil, or fish oil derived from plants or animals that have not been genetically or transgenically modified, and has not been previously purified to remove certain constituents (e.g., targeted fatty acids). In some embodiments, the oil may be derived from plant oil, nut oil, microalgae oil, or fish oil derived from plants or animals that have been genetically or transgenically modified, may be previously purified to remove certain constituents (e.g., targeted fatty acids), may be synthesized, or combinations thereof. In some embodiments, the oil may comprise at least 5% C16:1n7 fatty acid. In some embodiments, the oil may comprise 5-30% C16:1n7 fatty acid. In some embodiments, the oil may comprise 5-25% C16:1n7 fatty acid. In some embodiments, the oil may comprise 5-10% C16:1n7 fatty acid. In some embodiments, the oil may comprise 10-20% C16:1n7 fatty acid. In some embodiments, the oil may comprise 20-30% C16:1n7 fatty acid. In some embodiments, the oil may be high in C16:1n7 fatty acid (Omega-7) and comprise plant oil (e.g., sea buckthorn), nut oil (e.g., macadamia), microalgae oil (e.g., Nannochloropsis, Nitzschia, Thalasossira, Phaeodactylum), or fish oil (e.g., menhaden, anchovy). In some embodiments, the oil may be processed in a multiple step process to produce high purity Omega-3, 6, 7 and/or 9 oil products. The targeted end product may influence the method steps and inputs in the oil processing methods.
(11) In some embodiments, processing of the oil may comprise at least one transesterification step. In some embodiments, processing of the oil may comprise a distillation step comprising at least one distillation method selected from the group consisting of simple molecular distillation, short path distillation, one pass vacuum distillation, two pass vacuum distillation, and fractional distillation. In some embodiments, processing of the oil may comprise a separation step comprising at least one separation method selected from the group consisting of urea complexation, crystallization, filtration, extraction, and adsorption chromatography. In some embodiments, processing of the oil may comprise combinations of the steps described throughout the specification.
(12) Using the methods and 1 oils described throughout the specification, the inventors surprisingly found that purity levels of Omega-7 esters could be attained that were not previously achievable, particularly purity levels of Omega-7 esters could be attained from natural oils. In some embodiments, processing of the oil may result in a high purity Omega-7 product with a concentration of palmitoleic acid (C16:1n7) esters of at least 30% w/w. In some embodiments, processing of the oil may result in a high purity Omega-7 product with a concentration of palmitoleic acid (C16:1n7) esters of at least 50% w/w. In some embodiments, processing of the oil may result in a high purity Omega-7 product with a concentration of palmitoleic acid (C16:1n7) esters of at least 70% w/w. In some embodiments, processing of the oil may result in a high purity Omega-7 product with a concentration of palmitoleic acid (C16:1n7) esters of at least 80% w/w. In some embodiments, processing of the oil may result in a high purity Omega-7 product with a concentration of palmitoleic acid (C16:1n7) esters of 30-90% w/w. In some embodiments, processing of the oil may result in a high purity Omega-7 product with a concentration of palmitoleic acid (C16:1n7) esters of 30-50% w/w. In some embodiments, processing of the oil may result in a high purity Omega-7 product with a concentration of palmitoleic acid (C16:1n7) esters of 50-70% w/w. In some embodiments, processing of the oil may result in a high purity Omega-7 product with a concentration of palmitoleic acid (C16:1n7) esters of 70-90% w/w.
(13) In some embodiments, processing of the oil may result in an Omega-7 & 9 product with a combined concentration of C16:1n7 and C18:1n9 esters of at least 40% w/w. In some embodiments, processing of the oil may result in an Omega-7 & 9 product with a combined concentration of C16:1n7 and C18:1n9 esters of at least 50% w/w. In some embodiments, processing of the oil may result in an Omega-7 & 9 product with a combined concentration of C16:1n7 and C18:1n9 esters of at least 70% w/w. In some embodiments, processing of the oil may result in an Omega-7 & 9 product with a combined concentration of C16:1n7 and C18:1n9 esters of at least 80% w/w. In some embodiments, processing of the oil may result in an Omega-7 & 9 product with a combined concentration of C16:1n7 and C18:1n9 esters of 30-90% w/w. In some embodiments, processing of the oil may result in an Omega-7 & 9 product with a combined concentration of C16:1n7 and C18:1n9 esters of 30-50% w/w. In some embodiments, processing of the oil may result in an Omega-7 & 9 product with a combined concentration of C16:1n7 and C18:1n9 esters of 50-70% w/w. In some embodiments, processing of the oil may result in an Omega-7 & 9 product with a combined concentration of C16:1n7 and C18:1n9 esters of 70-90% w/w.
(14) In some embodiments, processing of the oil may result in a concentration of C16:0 esters of less than 50% w/w. In some embodiments, processing of the oil may result in a concentration of C16:0 esters of less than 40% w/w. In some embodiments, processing of the oil may result in a concentration of C16:0 esters of less than 30% w/w. In some embodiments, processing of the oil may result in a concentration of C16:0 esters of less than 20% w/w. In some embodiments, processing of the oil may result in a concentration of C16:0 esters of less than 10% w/w. In some embodiments, processing of the oil may result in a concentration of C16:0 esters ranging from 5-50% w/w. In some embodiments, processing of the oil may result in a concentration of C16:0 esters ranging from 5-40% w/w. In some embodiments, processing of the oil may result in a concentration of C16:0 esters ranging from 5-30% w/w. In some embodiments, processing of the oil may result in a concentration of C16:0 esters ranging from 5-20% w/w. In some embodiments, processing of the oil may result in a concentration of C16:0 esters ranging from 5-10% w/w.
(15) In some embodiments, processing of the oil may result in a concentration of C18:1n9 esters of less than 30% w/w. In some embodiments, processing of the oil may result in a concentration of C18:1n9 esters of less than 20% w/w. In some embodiments, processing of the oil may result in a concentration of C18:1n9 esters of less than 10% w/w. In some embodiments, processing of the oil may result in a concentration of C18:1n9 esters of less than 5% w/w. In some embodiments, processing of the oil may result in a concentration of C18:1n9 esters of less than 1% w/w. In some embodiments, processing of the oil may result in a concentration of C18:1n9 esters ranging from 0.5-30% w/w. In some embodiments, processing of the oil may result in a concentration of C18:1n9 esters ranging from 0.5-20% w/w. In some embodiments, processing of the oil may result in a concentration of C18:1n9 esters ranging from 0.5-10% w/w. In some embodiments, processing of the oil may result in a concentration of C18:1n9 esters ranging from 0.5-5% w/w. In some embodiments, processing of the oil may result in a concentration of C18:1n9 esters ranging from 0.5-1% w/w.
(16) Transesterification
(17) Synthesis of ethyl esters is known in the art for the enrichment of Omega-3 fatty acids, and the inventors found that such synthesis may also be adapted for application to Omega-6, 7 & 9 fatty acids. Transesterification of Omega-7 rich oil may be performed using a variety of different catalysts and conditions. In some embodiments, the transesterification may be performed enzymatically, with an acid or base catalyst, a heterogeneous catalyst, or combinations thereof. In some embodiments, the base catalyst may comprise NaOH, KOH, sodium ethoxide, and combinations thereof. In some embodiments, the acid catalyst may comprise HCl, BF.sub.3, H.sub.3PO.sub.4, organic sulfonic acids, and combinations thereof. In some embodiments, the heterogeneous catalyst may comprise metal oxide (e.g., CaO, MgO, SrO, ZnO, La.sub.2O.sub.3), MgAl hydrolalcite, and combinations thereof. In some embodiments, the enzymatic method may comprise immobilized enzyme-based catalysts, such as those commercially available from TransBiodiesel Ltd. (P.O. Box 437, Shfar-Am 20200 Israel).
(18) In some embodiments, the catalyst may preferably comprise at least one catalyst selected from the group consisting of sulphuric acid and sodium ethoxide. In some embodiments, the transesterification method may use methanol as a reacting solvent and produce fatty acid methyl esters (FAMEs). In some embodiments, the transesterification method may use ethanol as a reacting solvent and produce fatty acid ethyl esters (FAEEs). In some embodiments, the molar ratio of the reacting solvent may range from 1:3 to 1:15. In some embodiments, the molar ratio of the reacting solvent may range from 1:4 to 1:14. In some embodiments, the molar ratio of the reacting solvent may range from 1:5 to 1:13. In some embodiments, the molar ratio of the reacting solvent may range from 1:5 to 1:12. In some embodiments, the molar ratio of the reacting solvent may range from 1:7 to 1:10. In some embodiments, the molar ratio of the reacting solvent may range from 1:6 to 1:8. In some embodiments, the molar ratio of the reacting solvent may range from 1:9 to 1:11.
(19) In some embodiments, the temperature of the transesterification step may comprise a temperature in the range of 70-90? C. In some embodiments, the temperature of the transesterification step may comprise a temperature in the range of 75-85? C. In some embodiments, the temperature of the transesterification step may comprise a temperature in the range of 76-84? C. In some embodiments, the temperature of the transesterification step may comprise a temperature in the range of 77-83? C. In some embodiments, the temperature of the transesterification step may comprise a temperature in the range of 78-82? C. In some embodiments, the temperature of the transesterification step may comprise a temperature in the range of 79-81? C.
(20) In some embodiments, the yield of the transesterification step may comprise at least 50%. In some embodiments, the yield of the transesterification step may comprise 50-90%. In some embodiments, the yield of the transesterification step may comprise 60-80%. In some embodiments, the yield of the transesterification step may comprise 70-90%. In some embodiments, the yield of the transesterification step may comprise 75-90%. In some embodiments, the yield of the transesterification step may comprise 80-90%. In some embodiments, the yield of the transesterification step may comprise 75-85%.
(21) Purification of Esters
(22) Purification of esters may be performed with adsorption chromatography, fractional or simple molecular distillation, low-temperature crystallization (winterization), supercritical fluid extraction, solvent extraction, and urea complexation. Suitable solvents for extraction include, but are not limited to ethanol, methanol, and hexane. With the help of proper absorbents, adsorption chromatography may be used to separate the esters based on the number of carbons as well as the degree of unsaturation. In some embodiments, such absorbents may comprise AgBF.sub.4/SBA15.HPSiOEtIM.PF.sub.6, Silver resin Chromatography, Ag+ immobilized by ionic liquid precursor molecules covalently bonded to silica gel, and mesoporous SBA-15.
(23) Molecular distillation is known in the art to be useful for the separation, purification and concentration of thermolabile substances with low vapor pressure. Molecular distillation techniques may be used as simple or short path distillation, especially when purifying crude esters such as but not limited to microalgae oil FAEEs. Fractional distillation also provides a method of producing higher purity FAEEs from oils, such as but not limited to nut oil FAEE (e.g., macadamia) or fish oil FAEE (e.g., menhaden, anchovy).
(24) In some embodiments, the distillation may comprise one pass or multiple pass vacuum distillation and take place under pressure. In some embodiments, the distillation may take place under pressure ranging from 0.1-10 mm Hg. In some embodiments, the distillation may take place under pressure ranging from 0.2-7.5 mm Hg. In some embodiments, the distillation may take place under pressure ranging from 0.3-5 mm Hg. In some embodiments, the distillation may take place under pressure ranging from 1-4 mm Hg. In some embodiments, the distillation may take place under pressure ranging from 2-3 mm Hg. In some embodiments, multiple passes in a distillation take place at the same pressures. In some embodiments, multiple passes in a distillation take place at different pressures
(25) In some embodiments, the temperature during distillation may range from 140-280? C. In some embodiments, the temperature during distillation may range from 160-270? C. In some embodiments, the temperature during distillation may range from 170-250? C. In some embodiments, the temperature during distillation may range from 180-225? C. In some embodiments, the temperature during distillation may range from 190-210? C. In some embodiments, the distillation may comprise a single pass at a constant temperature. In some embodiments, the distillation may comprise a single pass at varying temperatures. In some embodiments, the distillation may comprise multiple passes at the same temperature. In some embodiments, the distillation may comprise multiple passes at different temperatures.
(26) Low-temperature crystallization (winterization) may also be used to produce high purity esters, and takes advantage of the solubility of the esters in different solvents. In some embodiments, the solvents may comprise: alcohols (e.g., methanol, ethanol), esters (e.g., ethyl acetate), ethers (e.g., diethyl ether), ketones (e.g., acetone), aromatic hydrocarbons (e.g., toluene), hydrocarbons (e.g., hexane), and n-heptane. In some embodiments, low-temperature crystallization may be used to purify the Omega-3 EPA or DHA fatty acids or esters from oil with higher concentrations of EPA or DHA, such as fish or microalgae oil. Urea complexation may also be used to effectively separate the saturated fat from PUFAs. In some embodiments, urea complexation may be used with simple principle straight-chain saturated fatty acids with six carbon atoms confined in the complex. In such six carbon atom embodiments, the presence of double bonds in the fatty acids increases the bulk of the molecule and reduces the likelihood of the molecule's complexation with urea, which helps to achieve the separation.
(27) In some embodiments, the temperature of the urea complexation step may comprise a temperature in the range of 70-90? C. In some embodiments, the temperature of the urea complexation step may comprise a temperature in the range of 75-85? C. In some embodiments, the temperature of the urea complexation step may comprise a temperature in the range of 76-84? C. In some embodiments, the temperature of the urea complexation step may comprise a temperature in the range of 77-83? C. In some embodiments, the temperature of the urea complexation step may comprise a temperature in the range of 78-82? C. In some embodiments, the temperature of the urea complexation step may comprise a temperature in the range of 79-81? C.
Example 1
(28) An experiment was conducted to determine the purity of Omega-7 ethyl esters that could be isolated from natural macadamia nut oil. Natural macadamia nut oil was analyzed and found to contain the fatty acid profile as shown in Table 3.
(29) TABLE-US-00003 TABLE 3 Natural Macadamia Oil Composition Analyte % C14:0 1.1 C16:0 8.3 C16:1n7 22.0 (Omega-7) C18:0 3.1 C18:1n9 53.7 (Omega-9) C18:1n11t 4.2 C18:2 2.3 C20:0 2.8 C20:1 2.6 Total Fatty 100.0 Acids
(30) One non-limiting embodiment of the multi-step process for producing an isolated high purity Omega-7 product from natural nut oil, such as macadamia nut oil, is shown in
(31) Transesterification Step:
(32) Macadamia nut oil (1 kg) was transesterified in a 5 L, 3-neck round bottom flask using 2.5% w/w sodium ethoxide as the catalyst and 1:10 molar ratio of ethanol as the reacting solvent to produce an FAEE product. The reaction was performed for 3 hrs at 80? C. After 3 hrs, the reaction mixture was cooled to ?40? C. and neutralized with 1:1 v/v of water. After neutralization the reaction mixture was allowed to separate into two layers comprising an ethyl ester layer on top and an aqueous layer on bottom. The ethyl ester layer was separated and was concentrated using a rotary evaporator resulting in ?850 grams of the FAEE product (?85% Yield).
(33) Distillation Step:
(34) For macadamia nut oil FAEE with a composition of at least 20% w/w C16:1n7 (palmitoleic acid) and using a vacuum distillation apparatus in either a one-pass or two-pass system, it is possible to produce an oil product with a concentration of C16:1n7 ethyl esters above 50% w/w. In such a system, the reboiler temperature varied between 170? C. and 220? C., and the vacuum pressure was maintained at 0.3 to 5 mm Hg. Using a single pass vacuum distillation system with the macadamia nut oil FAEE product from the transesterification step resulted in a concentrated oil product mixture of C16:0 and C16:1n7 ethyl esters (second fraction) as shown in Table 4. The resulting second fraction concentrated oil product may be successfully used in the cosmetics field, as the saturated fat along with Omega-7 also has antimicrobial activity. A significant portion of Omega-9 ethyl esters (C18:1n9) were separated from the Omega-7 ethyl esters in the molecular distillation step to form a first fraction, and the first fraction comprising Omega-9 ethyl esters may be used in nutrition products for heart health, blood sugar control, managing cholesterol levels, and promoting healthy inflammation responses.
(35) TABLE-US-00004 TABLE 4 Single pass distillate Analyte % C12:0 1.1 C14:0 5.9 C16:0 19.0 C16:1n7 73.4 (Omega-7) C18:0 0.0 C18:1n9 0.8 (Omega-9) Total Fatty 100.1 Acid Esters
Separation Step:
(36) The second fraction concentrated oil product (20 grams) resulting from the molecular distillation step was treated with urea (2 times w/w) and ethanol (5 times w/v). The resulting reaction mixture was refluxed at 80? C. for 2 hrs and then allowed to crystallize overnight (approximately 10-12 hrs). After overnight crystallization, the resulting crystals comprising saturated fats (C16:0) were separated from the liquid phase using a simple filtration system to form a third fraction of C16:0 ethyl esters. The liquid phase filtrate (fourth fraction) was treated with water to remove the dissolved urea and the Omega-7 ethyl ester product was extracted using hexane. The hexane layer was then concentrated using a rotary evaporator under vacuum to give the product composition as shown in Table 5.
(37) TABLE-US-00005 TABLE 5 Urea crystallization Analyte % C12:0 0.61 C14:0 3.17 C16:0 7.44 C16:1n7 86.37 (Omega-7) C18:1n9 1.43 (Omega-9) Total Fatty 99.02 Acid Esters
(38) Additionally, a nutraceutical fraction comprising C16:1n7 and C18:1n9 ethyl esters may be obtained from macadamia nut oil FAEE by using either a molecular distillation method to remove fatty acids?C16:0 or by using a urea complexation method to remove saturated fat after transesterification, as shown in
Example 2
(39) An experiment was conducted to determine the purity of Omega-7 ethyl esters that could be isolated from natural microalgae oil. Microalgae from genera such as Nannochloropsis, Nitzschia, Thalasossira, and Phaeodactylum are known to be sources of Omega-7 as well as sources of Omega-3 (EPA, DHA) fatty acids. Similar to the macadamia nut oil in Example 1, the oil derived from Nannochloropsis may also be processed to produce a high purity Omega-7 product. Natural Nannochloropsis oil was analyzed and found to contain the fatty acid profile shown in Table 6.
(40) TABLE-US-00006 TABLE 6 Natural Nannochloropsis Oil Analyte % C14:0 Myristic 4.0 C16:0 Palmitic 32.1 C16:1n7 Palmitoleic (Omega-7) 23.8 C18:1n9 Oleic (Omega-9) 12.7 C18:2 Linoleic 1.0 C20:4n6 Arachidonic (Omega-6) 1.6 C20:5n3 Eicosapentaenoic (Omega-3) 5.1 Others 1.4 Total Fatty Acids 84.4
Transesterification Step:
(41) A traditional two stage transesterification method may be used with microalgae oil. During the first stage, the Nannochloropsis oil (1 kg) was transesterified in a 5 L, 3-neck round bottom flask using 8% w/w concentration. In the first transesterification stage, sulphuric acid was used as the acid catalyst and a 1:7 molar ratio of ethanol was used as the reacting solvent. The first stage reaction was performed for 3 hrs at 80? C. After 3 hrs, the second stage of transesterification was performed. In the second transesterification stage, the reaction mixture used 2.5% sodium ethoxide as the base catalyst and a 1:7 molar ratio of ethanol as the reacting solvent. The second stage reaction was performed for 3 hrs at 80? C. After completion of both reaction stages (6 hrs), the reaction mixture was cooled to ?40? C. and neutralized with 1:1 v/v of water. The resulting mixture was extracted using hexane, forming a hexane layer on top comprising the target Omega-7 ethyl ester product which was separated and was concentrated using rotary evaporator resulting in ?700 grams of the FAEE product (?70% Yield).
(42) Distillation Step:
(43) With a microalgae oil mixture that contains less than 85% total fatty acids, as with the Nannochloropsis oil shown in Table 6, it is recommended that the first distillation step comprise a simple distillation under high vacuum in order to create a distillate with a total fatty acid content greater than 85%. The first step distillation may be performed with a short path distillation unit or simple distillation unit operated at a temperature at which all the fatty acids present will vaporize but below a temperature that causes thermal decomposition. With the Nannochloropsis FAEE product, the first step distillation was performed at a reboiler temperature of 275? C., a pressure of 2 mmHg, and a distillate temperature of 168? C.
(44) After the simple or short path distillation step, a fractional distillation step may be performed on the purified FAEEs to fractionate C16:0 and C16:1 ethyl esters using conditions similar to conditions used for macadamia nut oil in Example 1. Variations from the parameters in Example 1 may be required due to the different molar fraction of C16:0 and C16:1 in microalgae oil as compared to the nut oil. With the Nannochloropsis FAEE product, the fractional distillation was performed at a reboiler temperature of 200? C., a pressure of 1 mmHg, and a distillate temperature of 144? C. The method of processing Nannochloropsis oil is shown in
(45) TABLE-US-00007 TABLE 7 Distillate Residue (Omega 7) % (Omega 3) % C14:0 7.19 C14:1 0.41 C15:0 0.56 C16:0 41.31 8.71 C16:1n7 (Omega-7) 35.13 4.79 C18:1n9 (Omega-9) 5.93 22.78 C18:2 1.02 3.49 C18:3n6 0.19 0.59 C20:1 0.33 C20:3n6 0.92 C20:4n6 (Omega-6) 0.23 10.39 C20:5n3 (Omega-3) 0.66 33.37 Total Fatty Acid 98.53 93.9 Esters
(46) The process shown in
(47) The method shown in
(48) Additionally, the microalgae FAEE produced with the method shown in
Example 3
(49) An experimental plan was formed to determine for isolating an Omega-7 ethyl esters product from natural fish oil. Natural fish oil from sources such as menhaden and anchovy are also known to be sources of nutritional fatty acids, including Omega-7 fatty acids. Natural menhaden oil was analyzed and found to contain the fatty acid profile shown in Table 8, and may be processed in a method similar to the methods described for macadamia nut oil in Example 1 to produce high purity fractions of Omega-3, 6, 7 & 9 ethyl esters.
(50) TABLE-US-00008 TABLE 8 Natural Menhaden Oil Composition Analyte % (Average) C14:0 8 C16:0 18 C16:1n7 11 (Omega-7) C18:0 3.1 C18:1n9 8 (Omega-9) C20:5n3 15 (Omega-3) C22:6n3 7 (Omega-3) Total Fatty 70.1 Acids
Transesterification Step:
(51) Fish oil (1 kg) may be transesterified in a 5 L, 3-neck round bottom flask using 2.5% w/w sodium ethoxide as the catalyst and a 1:10 molar ratio of ethanol as the reacting solvent. The reaction may be performed for 3 hrs at 80? C. After 3 hrs, the reaction mixture will be cooled to ?40? C. and neutralized with 1:1 v/v of water. After neutralization the reaction mixture will separate into two layers comprising an ethyl ester layer on top and an aqueous layer on bottom. The ethyl ester layer will be separated and concentrated using a rotary evaporator resulting in ?850 grams of the FAEE product (?85% Yield).
(52) Distillation Step:
(53) A fish oil FAEE product with a composition of at least 20% w/w C16:1n7 ethyl esters may be concentrated to above 50% w/w C16:1n7 ethyl esters using a vacuum distillation apparatus in either a one-pass or two-pass system. The two-pass distillation system may be similar to the distillation system and method described in Example 2. In such a system, the reboiler temperature varies between 170? C. and 220? C., and the vacuum pressure is maintained at 0.3 to 5 mmHg. Using the single pass distillation system in conditions similar to Example 1 for the macadamia nut oil FAEE will result in a second fraction mixture of C16:0 and C16:1n7 ethyl esters. The resulting concentrated oil product may be successfully used in the cosmetics field, as the saturated fat along with Omega-7 also has antimicrobial activity.
(54) Separation Step:
(55) The second fraction distillate mixture (20 grams) resulting from the molecular distillation step may be treated with urea (2 times w/w) and ethanol (5 times w/v). The resulting reaction mixture will be refluxed at 80? C. for 2 hrs and then allowed to crystallize overnight (approximately 10-12 hrs). After overnight crystallization, the resulting crystals comprising saturated fats (C16:0) will be separated using a simple filtration system to form a third fraction of C16:0 ethyl esters. The fourth fraction liquid phase filtrate (fourth fraction) will be treated with water to remove the dissolved urea and the Omega-7 product will be extracted using hexane. The hexane layer will be concentrated using a rotary evaporator under vacuum to give the Omega-7 ethyl ester product as shown in
(56) Additionally, the Omega-3, 6, & 9 first fraction separated during the simple distillation step may be further purified using adsorption chromatography to further separate the Omega-3, Omega-6, and Omega-9 FAEE from each other as shown in
(57) A nutraceutical fraction (Omega-3) may also be produced from fish oil FAEE using either a molecular distillation method to remove fatty acids?C16:0 or a urea complexation method to remove saturated fat. The Omega fraction separated from saturated fat may comprise Omega-3, 6, 7 & 9 FAEE, similar to the fraction separated from microalgae oil in Example 2. The Omega-3, 6, 7, & 9 fraction has potential value in a nutraceutical product as an Omega mixture. As shown in
(58) All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law), regardless of any separately provided incorporation of particular documents made elsewhere herein. The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.
(59) The use of the terms a and an and the and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
(60) Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by about, where appropriate). All provided ranges of values are intended to include the end points of the ranges, as well as values between the end points.
(61) The description herein of any aspect or embodiment of the invention using terms such as comprising, having, including, or containing with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that consists of, consists essentially of, or substantially comprises that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).
(62) All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.
(63) The use of any and all examples, or exemplary language (e.g., such as) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
(64) This invention includes all modifications and equivalents of the subject matter recited in the claims and/or aspects appended hereto as permitted by applicable law.