HK1237216B - Process for increasing the stability of a composition comprising polyunsaturated omega-3 fatty acids - Google Patents

Description

Method for increasing the stability of compositions containing polyunsaturated omega-3 fatty acids

Extensive evidence has been gathered over the past few decades to link the supplemental intake of polyunsaturated fatty acids (PUFAs) with numerous health benefits. Among the most prominent examples are the prevention of cardiovascular disease and the alleviation of the symptoms of inflammatory disorders. However, positive effects have also been reported, inter alia, in preventing the promotion and progression of certain types of cancer, lowering blood pressure and blood cholesterol, and in the treatment of depression and schizophrenia, Alzheimer's disease, dyslexia, and attention deficit hyperactivity disorder. In addition, since certain PUFAs are considered essential for the growth of the brain, nervous system, and eyes, specific PUFAs are now routinely supplemented in infant nutrition.

However, their high susceptibility to oxidative deterioration hinders the production of food, nutritional, and pharmaceutical products containing PUFAs. Oxidation has negative consequences for both nutritional and organoleptic properties: changes in nutritional value such as the destruction of essential fatty acids, the development of off-flavors and noticeable odors due to rancidity, color changes such as darkening of fats and oils, and loss of flavor. Oxidation of PUFAs produces a complex mixture of volatile secondary oxidation products, and these contribute to particularly objectionable off-flavors.

To stabilize PUFAs against oxidative deterioration, three different strategies have been described in the art and applied in industry (Arab-Tehrany E. et al., Trends in Food Science & Technology 25 (2012) 24-33):

- Added antioxidants,

- microencapsulation, and

- Modified atmosphere packaging.

While these strategies do offer solutions to many oxidation-related problems, new R&D advances are still needed to address the challenges that remain both in the current situation and in potential future settings reflecting specific technological and economic limitations.

It has now been found that a composition comprising polyunsaturated omega-3 fatty acids can be stabilized against oxidation by a process comprising the following steps:

(i) providing a starting composition comprising at least one polyunsaturated omega-3 fatty acid component;

(ii) providing a lysine composition;

(iii) mixing the starting composition with an aqueous, aqueous-alcoholic or alcoholic solution of a lysine composition and subsequently subjecting the resulting compound to spray drying conditions, thereby forming a solid product composition comprising at least one salt of a cation derived from lysine and an anion derived from a polyunsaturated omega-3 fatty acid; said product composition having a solvent content (SC) selected from the group consisting of: SC < 5 wt %, SC < 3 wt %, SC < 1 wt %, SC < 0.5 wt %.

In the context of the present invention, the term PUFA is used interchangeably with the term polyunsaturated fatty acid and is defined as follows: Fatty acids are classified based on the length and saturation characteristics of the carbon chain. Short-chain fatty acids have 2 to about 6 carbons and are typically saturated. Medium-chain fatty acids have about 6 to about 14 carbons and are also typically saturated. Long-chain fatty acids have 16 to 24 or more carbons and can be saturated or unsaturated. In longer chain fatty acids, there can be one or more sites of unsaturation, resulting in the terms "monosaturated" and "polysaturated," respectively. Long-chain polyunsaturated fatty acids having 20 or more carbon atoms are designated as polyunsaturated fatty acids or PUFAs in the context of the present invention.

PUFAs are classified according to the number and position of double bonds in the fatty acid using a well-established nomenclature. There are two main series or families of LC-PUFAs, depending on the position of the double bond closest to the methyl group of the fatty acid: the omega-3 series, which contains a double bond at the third carbon, and the omega-6 series, which has no double bonds before the sixth carbon. Thus, docosahexaenoic acid ("DHA") has a 22-carbon chain length, starting with six double bonds at the third carbon from the methyl group and is designated "22:6n-3" (all-cis-4,7,10,13,16,19-docosaenoic acid). Another important omega-3 PUFA is eicosapentaenoic acid ("EPA"), which is designated "20:5n-3" (all-cis-5,8,11,14,17-eicosapentaenoic acid). An important omega-6 PUFA is arachidonic acid ("ARA"), which is designated "20:4n-6" (all-cis-5,8,11,14-docosatatetraenoic acid).

Other omega-3 PUFAs include:

Eicosatrienoic acid (ETE) 20:3(n-3)(all-cis-11,14,17-eicosatrienoic acid), Eicosatetraenoic acid (ETA) 20:4(n-3)(all-cis-8,11,14,17-eicosatetraenoic acid), Henicosapentaenoic acid (HPA) 21:5(n-3)(all-cis-6,9,12,15,18-eicosapentaenoic acid), Docosapentaenoic acid (DPA) 22:5(n-3)(all-cis-7,10,13,16,19-docosapentaenoic acid), tetracosapentaenoic acid 24:5(n-3)(all-cis-9,12,15,18,21-tetracosapentaenoic acid), tetracosahexenoic acid (nisinic acid) 24:6(n-3)(all-cis-6,9,12,15,18,21-tetracosahexenoic acid).

Other omega-6 PUFAs include:

Eicosadienoic acid 20:2(n-6)(all-cis-11,14-eicosadienoic acid), dihomo-γ-linolenic acid (DGLA) 20:3(n-6)(all-cis-8,11,14-eicosatrienoic acid), docosadienoic acid 22:2(n-6)(all-cis-13,16-docosadienoic acid), adrenoic acid 22:4(n-6)(all-cis-7,10,13,16-docosatetraenoic acid), docosapentaenoic acid (Osbond acid) 22:5(n-6)(all-cis-4,7,10,13,16-docosapentaenoic acid), tetracosatetraenoic acid 24:4(n-6)(all-cis-9,12,15,18-tetracosatetraenoic acid), tetracosatetraenoic acid 24:5(n-6)(all-cis-6,9,12,15,18-tetracosatetraenoic acid).

Preferred omega-3 PUFAs in embodiments of the present invention are docosahexaenoic acid ("DHA") and eicosapentaenoic acid ("EPA").

Without being bound by theory, the increased stability against oxidation achieved by the method of the present invention is a result of salt formation between lysine and the PUFA. No corresponding increase in stability was observed for PUFAs that retain the free acid, or form ester moieties or inorganic salts such as Na+, K+, Ca2+ or Mg2+.

The composition comprising polyunsaturated omega-3 fatty acids that can be stabilized for oxidation by the method of the present invention can be any composition comprising free polyunsaturated omega-3 fatty acids in substantial amounts. This composition can further comprise other naturally occurring fatty acids in free form. In addition, this composition can further comprise a composition that is solid, liquid or gaseous at room temperature and under standard atmospheric pressure. The corresponding liquid component comprises a composition that can be easily removed by evaporation and can therefore be considered as a volatile component, and a composition that is difficult to remove by evaporation and can therefore be considered as a non-volatile component. In the context of the present invention, the gaseous component can be considered as a volatile component. Typical volatile components are water, alcohol and supercritical carbon dioxide.

The composition comprising polyunsaturated omega-3 fatty acids that can be stabilized against oxidation by the methods of the present invention can be obtained from any suitable raw material, which can further be processed by any suitable method for processing such raw materials. Typical raw materials include any part of fish, vegetables and other plants, and materials derived from microbial and/or algal fermentation. Typically, such materials also contain substantial amounts of other naturally occurring fatty acids. Typical methods for processing such raw materials may include steps for obtaining crude oil, such as extraction and separation of the raw material, as well as steps for refining the crude oil, such as precipitation and degumming, deacidification, bleaching, and deodorization, and steps for producing omega-3 PUFA concentrates from the refined oil, such as further steps of deoxygenation, transesterification, concentration, and deodorization (see, for example, the EFSA Scientific Opinion on Fish Oil for Human Consumption). Any processing of the raw materials may further include a step for at least partially converting the omega-3 PUFA esters into the corresponding free omega-3 PUFAs or their inorganic salts.

Preferred compositions comprising polyunsaturated omega-3 fatty acids that can be stabilized against oxidation by the methods of the present invention can be obtained from compositions consisting primarily of esters of omega-3 PUFAs and other naturally occurring fatty acids by cleaving the ester bonds and subsequently removing the alcohol previously bound to the esters. Preferably, ester cleavage is performed under alkaline conditions. Methods for ester cleavage are well known in the art.

In the context of the present invention, stabilizing a composition against oxidation means that the composition has increased stability against oxidation. One measure of the stability of a composition against oxidation is the induction time in a rancidity test. Protocols for conducting rancidity tests are well known in the art and/or are provided by the manufacturers of the instruments used to conduct rancidity tests. An alternative measure of the stability of a composition against oxidation can be obtained as follows: the stability against oxidation of two or more samples of any composition or compound can be obtained by (1) initially measuring the degree of oxidation of the samples, then comparing the degree of oxidation of the samples by (2) subjecting the samples to comparable (oxidizing) conditions and (3) measuring the degree of oxidation of the samples thereafter. The sample with the smallest increase in degree of oxidation exhibits the highest stability against oxidation under the given conditions, while the sample with the largest increase in degree of oxidation exhibits the lowest stability against oxidation under the given conditions. The increase in the degree of oxidation of the sample can be expressed in absolute terms, i.e., as the difference between the values obtained before and after subjecting the sample to the oxidizing conditions, or, alternatively, the increase can be expressed in relative terms, i.e., as the ratio of the values obtained before and after subjecting the sample to the oxidizing conditions. Clearly, a reduction in the degree of oxidation resulting from exposure to oxidizing conditions indicates a very high level of stability against oxidation, which should be interpreted as an even higher level of stability than if the sample were exposed to comparable oxidizing conditions resulting in an unchanged degree of oxidation.

Several measurements are known in the art for quantifying the degree of oxidation of a sample. In the broadest sense of the present invention, any of these measurements may be used. In preferred embodiments of the present invention, one or more of the following measurements are used to quantify the degree of oxidation: peroxide value (PV), anisidine value (AV), and Totox value. PV is a measure of primary oxidation products (hydroperoxides formed at double bonds), and AV is a measure of secondary degradation products (carbonyl compounds). The Totox value is calculated as Totox = 2*PV + AV (where PV is expressed as milliequivalents of _O₂ per kg of sample). Methods for determining the peroxide value (PV) and the anisidine value (AV) have been described in the literature (see, for example, Official Methods and Recommended Practices of the AOCS, ^6th Edition 2013, Edited by David Firestone, ISBN 978-1-893997-74-5; or, for example, PV can be determined according to Ph. Eur. 2.5.5 (01/2008: 20505) and AV can be determined according to Ph. Eur. 2.5.36 (01/2008: 20536)).

An exemplary method for determining the peroxide value (PV) of a sample is performed as follows:

Reagents and solutions:

1. Acetic acid-chloroform solution (7.2 ml of acetic acid and 4.8 ml of chloroform).

2. Saturated potassium iodide solution. Store in the dark.

3. Sodium thiosulfate solution, 0.1 N. Commercially available.

4. 1% starch solution. Commercially available.

5. Distilled or deionized water.

method:

Perform a reagent blank determination.

1. Weigh 2.00 (± 0.02) g of sample into a 100 ml glass-stoppered conical flask. Record the weight to the nearest 0.01 g.

2. Add 12 ml of acetic acid-chloroform solution through a measuring cylinder.

3. Swirl the flask until the sample is completely dissolved (careful heating on a hot plate may be necessary).

4. Using a 1 ml Mohr pipette, add 0.2 ml of saturated potassium iodide solution.

5. Stopper the flask and swirl the contents for 1 minute.

6. Immediately add 12 ml of distilled or deionized water via graduated cylinder, stopper and shake vigorously to release iodine from the chloroform layer.

7. Fill the burette with 0.1N sodium thiosulfate.

8. If the solution starts out dark orange, titrate slowly, mixing until the color lightens. If the solution starts out light amber, proceed to step 9.

9. Using a dispersion apparatus, add 1 ml of starch solution as an indicator.

10. Titrate until the bluish-grey color disappears in the aqueous (upper layer).

11. Accurately record the milliliters of titrant used to 2 decimal places.

calculate:

S = Sample titration

B = Blank titration

Peroxide value = (S-B) * N thiosulfate * 1000 / sample weight

An exemplary method for determining the Anisidine Value (AV) of a sample is performed as follows:

The anisidine value is defined as 100 times the optical density of a solution containing 1 g of the substance to be tested in 100 ml of a mixture of solvent and reagents measured in a 1 cm cuvette according to the following procedure: Perform the procedure as quickly as possible, avoiding exposure to actinic light.

Test solution (a): Dissolve 0.500 g of the substance to be tested in trimethylpentane and dilute to 25.0 ml with the same solvent.

Test solution (b): To 5.0 ml of test solution (a), add 1.0 ml of a 2.5 g/l solution of p-anisidine in glacial acetic acid, shake and store protected from light.

Reference solution: Add 1.0 ml of a 2.5 g/l solution of p-anisidine in glacial acetic acid to 5.0 ml of trimethylpentane, shake, and store protected from light. Measure the absorbance of the test solution (a) at a maximum of 350 nm using trimethylpentane as a calibration solution. Measure the absorbance of the test solution (b) at 350 nm for 10 minutes using the reference solution as a calibration solution. Calculate the anisidine value (AV) using the following expression:

AV＝(25*(1.2*A1-A2))/m

A1＝absorbance of test solution (b) at 350nm,

A2 = absorbance of test solution (a) at 350 nm,

m = amount of substance to be measured in the test solution (a) in grams.

When comparing the stability of samples against oxidation by (1) measuring the degree of oxidation, (2) subjecting them to oxidizing conditions and (3) measuring the degree of oxidation again, in the context of the present invention, preferably, the degree of oxidation in steps (1) and (3) is assessed by determining the peroxide value (PV) and/or the anisidine value (AV); furthermore, preferably, the oxidizing conditions in step (2) are selected from one of the following: storage in an open container exposed to air at room temperature for a defined period of at least ten days; storage in an open container exposed to air at 50° C. for a defined period of at least three days.

Increasing the stability of a composition towards oxidation by a method in the context of the present invention means that at least one measure describing the stability of the composition towards oxidation, eg at least one measure as described above, is increased after said composition has been subjected to the method.

In the context of the present invention, the starting composition comprising at least one polyunsaturated omega-3 fatty acid component can be any composition comprising a substantial amount of at least one polyunsaturated omega-3 fatty acid component, wherein each type (i.e., molecular species) of free omega-3 PUFA ("free" being used to refer to the presence of free carboxylic acid functional groups) constitutes a different polyunsaturated omega-3 fatty acid component. Such a composition may further comprise other naturally occurring fatty acids in free form. In addition, such a composition may further comprise components that are solid, liquid, or gaseous at room temperature and standard atmospheric pressure. The corresponding liquid components include components that can be easily removed by evaporation and can therefore be considered volatile components, as well as components that are difficult to remove by evaporation and can therefore be considered non-volatile components. In the context of the present invention, gaseous components can be considered volatile components. Typical volatile components are water, alcohol, and supercritical carbon dioxide.

Accordingly, a typical starting composition, not taking into account the volatile components, has a PUFA content PC (i.e., the total content of one or more free polyunsaturated omega-3 fatty acids) of at least 25% by weight, up to 75% by weight of other naturally occurring fatty acids in free form, and up to 5% by weight of other components that are solid or liquid at room temperature and standard atmospheric pressure. However, higher levels of polyunsaturated omega-3 fatty acids can be obtained by purifying the respective starting materials. In a preferred embodiment of the present invention, the starting composition, not taking into account the volatile components, has a PUFA content PC (i.e., the total content of one or more free polyunsaturated omega-3 fatty acids) of at least 50% by weight, up to 50% by weight of other naturally occurring fatty acids in free form, and up to 5% by weight of other components that are solid or liquid at room temperature and standard atmospheric pressure. In another preferred embodiment of the present invention, the starting composition, not taking into account the volatile components, has a PUFA content PC (i.e., the total content of one or more free polyunsaturated omega-3 fatty acids) of at least 75% by weight, up to 25% by weight of other naturally occurring fatty acids in free form, and up to 5% by weight of other components that are solid or liquid at room temperature and standard atmospheric pressure. In a further preferred embodiment of the invention, the starting composition, not taking into account the volatile components, has a PUFA content PC (i.e. the total content of one or more free polyunsaturated omega-3 fatty acids) of at least 90% by weight, up to 10% by weight of other naturally occurring fatty acids in free form, and up to 5% by weight of other components that are themselves solid or liquid at room temperature and standard atmospheric pressure. In a further preferred embodiment of the invention, the starting composition, not taking into account the volatile components, has a PUFA content PC (i.e. the total content of one or more free polyunsaturated omega-3 fatty acids) of at least 90% by weight, up to 10% by weight of other naturally occurring fatty acids in free form, and up to 1% by weight of other components that are themselves solid or liquid at room temperature and standard atmospheric pressure.

In step (ii) of the method of the present invention, the lysine composition provided is a composition comprising a substantial amount of free lysine (Lys). The lysine composition may further comprise a component that is solid, liquid or gaseous at room temperature and standard atmospheric pressure. The corresponding liquid component includes a component that can be easily removed by evaporation and can therefore be considered as a volatile component, as well as a component that is difficult to remove by evaporation and can therefore be considered as a non-volatile component. In this context, the gaseous component is considered to be a volatile component. Typical volatile components are water, alcohol and supercritical carbon dioxide. Typical lysine compositions not considering volatile components contain at least 95wt%, 97wt%, 98wt% or 99wt% free lysine. Preferred lysine compositions not considering volatile components contain at least 98wt% free lysine.

In a preferred embodiment of the present invention where volatile components are not considered, the starting composition comprises primarily free PUFAs and other naturally occurring fatty acids in free form, and the lysine composition comprises primarily free lysine, so that the resulting product composition consists primarily of salts of lysine with PUFAs and other naturally occurring fatty acids.

Accordingly, in a preferred embodiment of the present invention, the starting composition in step (i) and the lysine composition in step (ii) are provided in such a way that at least sp wt% of the product composition consists of one or more salts of cations derived from lysine and anions derived from one or more polyunsaturated omega-3 fatty acids and other naturally occurring fatty acids, wherein sp is selected from 90, 95, 97, 98, 99, 100.

In step (iii) of the method of the present invention, the starting composition is combined with the lysine composition. This combination can be achieved by any means that allows the formation of a product composition comprising a cation derived from lysine and at least one salt of an anion derived from a polyunsaturated omega-3 fatty acid. Accordingly, a typical approach for combining the starting composition and the lysine composition is to mix each aqueous, hydroalcoholic, or alcoholic solution and subsequently remove the solvent. Alternatively, depending on the remaining components of the starting composition and the lysine composition, it may be sufficient to directly combine the starting composition and the lysine composition without adding a solvent. In the context of the present invention, a preferred approach for combining the starting composition and the lysine composition is to mix each aqueous, hydroalcoholic, or alcoholic solution and subsequently remove the solvent.

In the context of the present invention, a cation derived from lysine is a cation obtained from the protonation of lysine.

In the context of the present invention, anions derived from polyunsaturated omega-3 fatty acids are anions obtained from the deprotonation of polyunsaturated omega-3 fatty acids.

It should be noted that salts of lysine with polyunsaturated fatty acids are known per se in the art (cf. EP 0 734 373 B1), however, it was not known that such salts exhibit a higher stability against oxidative degradation than free PUFAs or PUFA-esters.

Given the inherent stability of salts of lysine and omega-3 PUFAs, it is not necessary to add substantial amounts of antioxidants to these salts. Accordingly, in a preferred embodiment of the present invention, the product composition obtained in step (iii) comprises an insubstantial amount of antioxidant, wherein insubstantial amount means that the composition comprises less than 5 wt%, 3 wt%, 1 wt% or 0.1 wt% of antioxidant. In a further preferred embodiment, the product composition comprises no antioxidant at all. In a preferred embodiment of the present invention, the product composition comprises an insubstantial amount of antioxidant, wherein insubstantial amount means that the product composition comprises less than 5 wt%, 3 wt%, 1 wt% or 0.1 wt% of antioxidant and wherein the antioxidant is selected from the group consisting of vitamin C and its esters, isoascorbic acid and its esters, vitamin E and its esters, polyphenols and its esters, carotenoids, gallic acid and its esters, butylated hydroxyanisole and its esters, butylated hydroxytoluene and its esters, rosemary oil, and hexylresorcinol and its esters. In a further preferred embodiment, the product composition does not contain any antioxidant at all, wherein the antioxidant is selected from vitamin C and its esters, isoascorbic acid and its esters, vitamin E and its esters, polyphenols and its esters, carotenoids, gallic acid and its esters, butylated hydroxyanisole and its esters, butylated hydroxytoluene and its esters, rosemary oil, hexylresorcinol and its esters.

According to the present invention, the product composition exhibits a higher stability towards oxidation than the starting composition. This means that at least one measure describing the stability of a composition towards oxidation, such as at least one measure as described above, indicates that the product composition is more stable towards oxidation than the starting composition.

In a preferred embodiment of the present invention, in order to promote the formation of quantitative salts, free carboxylic acid functional groups and lysine are provided in approximately equimolar amounts. Accordingly, in a preferred embodiment of the method of the present invention, the lysine composition in step (ii) is provided in the following manner, i.e., the ratio R=n(ca)/n(lys) of the amount of carboxylic acid functional groups n(ca) in the starting composition provided in step (i) and the total amount of free lysine n(lys) in the lysine composition provided in step (ii) is selected from the range of 0.9<R<1.1, 0.95<R<1.05, 0.98<R<1.02. In a particularly preferred embodiment, R is in the range of 0.98<R<1.02. The amount n(ca) of the carboxylic acid functional groups in the starting composition provided in step (i) can be determined by standard analytical methods well known in the art, such as acid-base titration.

In a preferred embodiment of the present invention, the starting composition provided in step (i) does not contain a substantial amount of fatty acid esters, thereby producing a product composition that also avoids substantial amounts of fatty acid esters. Accordingly, in a preferred embodiment of the present invention, the starting composition provided in step (i) does not contain more than x(fe) wt% of fatty acid esters, thereby producing a product composition that contains a maximum of x(fe) wt% of fatty acid esters, wherein x(fe) is selected from 5, 3, 1, 0.3, 0. In a particularly preferred embodiment, x(fe) is 1.

As noted above, salts of lysine with polyunsaturated fatty acids are known in the art (see EP 0 734 373 B1). What was not known was that such salts exhibited a higher stability against oxidative degradation than free PUFAs or PUFA esters. Importantly, lysine-PUFA salts are described as "very thick, transparent oils that transform at low temperatures into solids with a waxy appearance and consistency" (see EP 0 734 373 B1, page 1, lines 47-48). Consequently, a person skilled in the art would not have foreseen that salts of lysine with omega-3 PUFAs could be obtained by spray drying. Instead, a skilled person would have expected such salts to (a) deteriorate under spray drying conditions due to oxidative damage at elevated temperatures in the absence of substantial amounts of solvent, antioxidant, and protective coating, and (b) mechanically hinder spray drying due to the supposed waxy solid appearance of such salts. It is therefore remarkable that salts of lysine with omega-3 PUFAs can now be obtained in a gentle manner by spray drying. The conditions for spray drying always have to be adapted to the specific spray drying equipment used. However, such an adaptation in the described case is well within the scope of routine laboratory work for a person skilled in the art.

To carry out the spray-drying step of the method according to the present invention, aqueous, hydroalcoholic, or alcoholic solutions are used. Lys-salts of PUFAs have been found to be poorly soluble in anhydrous alcoholic solvents. Such salts have also been found to exhibit a gel-like appearance when dissolved at high concentrations in purified water. Therefore, a hydroalcoholic solvent system can be employed to avoid this problem. Accordingly, in a preferred embodiment of the present invention, the mixed solvent subjected to spray-drying conditions is a hydroalcoholic solvent system comprising 20 to 90 wt% water and 80 to 10 wt% alcoholic solvent.

The implementation of the solid product composition will vary depending on the spray-drying conditions and the substrate used. However, it has now been discovered that even very low solvent contents in the solid product composition do not cause oxidative damage. As noted above, this was unexpected. Preferably, therefore, according to the present invention, a solid product composition having a low solvent content is obtained. Thus, according to the present invention, in step (iii), an aqueous, aqueous-alcoholic, or alcoholic solution of the starting composition and the lysine composition are first mixed and then subjected to spray-drying conditions, thereby producing a solid product composition comprising at least one salt of a cation derived from lysine and an anion derived from a polyunsaturated omega-3 fatty acid, having a solvent content (SC) selected from the group consisting of: SC < 5 wt%, SC < 3 wt%, SC < 1 wt%, and SC < 0.5 wt%. In a particularly preferred embodiment of the present invention, SC is selected from the group consisting of SC < 1 wt%.

The present invention further includes compositions obtainable from any of the methods of the present invention.

The present invention further comprises the use of a composition obtainable from any method of the present invention in the manufacture of a food product comprising polyunsaturated omega-3 fatty acids.

In the context of the present invention, food products include, but are not limited to, baked goods, vitamin supplements, dietary supplements, beverage powders, doughs, batters, baked goods including, for example, cakes, cheesecakes, pies, cupcakes, cookies, bars, breads, rolls, biscuits, muffins, pastries, scones, and croutons; liquid foods such as beverages, energy drinks, infant formula, liquid foods, juices, multivitamin syrups, meal replacements, medical foods, and syrups; semi-solid foods such as infant food, yogurt, cheese, cereals, pancake mixes; prepared food bars including energy bars; processed meats; ice cream; frozen desserts; frozen yogurt; waffle mixes; salad dressings; and egg replacement powders. and other cookies, crackers, sweets, snacks, pies, ready-to-eat cereals/snack bars, and toaster pastries; salty snacks such as potato chips, corn chips, nachos, extruded foods, popcorn, pretzels, potato crisps, and nuts; specialty snacks such as dips, dried fruit snacks, meat snacks, smoked pork rinds, health food bars, and rice/corn cakes; confectionery snacks such as candy; and instant foods such as instant noodles, instant soup cubes, or instant soup granules.

The present invention further comprises the use of a composition obtainable from any of the methods of the present invention for the manufacture of a nutritional product comprising polyunsaturated omega-3 fatty acids.

Nutraceuticals in the context of the present invention include any type of nutraceutical, nutritional or dietary supplement, for example for the supplementation of vitamins, minerals, fiber, fatty acids or amino acids.

The present invention further comprises the use of a composition obtainable by any method of the present invention for the manufacture of a pharmaceutical product comprising polyunsaturated omega-3 fatty acids.

In the context of the present invention, the pharmaceutical product may further comprise pharmaceutically acceptable excipients and other pharmaceutically active agents including, for example, cholesterol-lowering agents such as statins, antihypertensive agents, antidiabetic agents, antidementia agents, antidepressants, antiobesity agents, appetite suppressants and agents that enhance memory and/or cognitive function.

Preferred methods of the invention are characterized by one of the following options:

·0.90<R<1.10,x(fe)=5; PC=25; SC<1wt%

·0.90<R<1.10,x(fe)=3; PC=25; SC<1wt%

·0.90<R<1.10, x(fe)=2; PC=25: SC<1wt%

·0.90<R<1.10,x(fe)=1; PC=25; SC<1wt%

·0.95<R<1.05, x(fe)=5; PC=25; SC<1wt%

·0.95<R<1.05, x(fe)=3; PC=25; SC<1wt%

·0.95<R<1.05, x(fe)=2; PC=25; SC<1wt%

·0.95<R<1.05, x(fe)=1; PC=25; SC<1wt%

·0.98<R<1.02,x(fe)=5; PC=25; SC<1wt%

·0.98<R<1.02,x(fe)=3; PC=25; SC<1wt%

·0.98<R<1.02,x(fe)=2; PC=25; SC<1wt%

·0.98<R<1.02,x(fe)=1; PC=25; SC<1wt%

·0.90<R<1.10,x(fe)=5; PC=50; SC<1wt%

·0.90<R<1.10,x(fe)=3; PC=50; SC<1wt%

·0.90<R<1.10, x(fe)=2; PC=50: SC<1wt%

·0.90<R<1.10,x(fe)=1; PC=50; SC<1wt%

·0.95<R<1.05,x(fe)=5; PC=50; SC<1wt%

·0.95<R<1.05, x(fe)=3; PC=50; SC<1wt%

·0.95<R<1.05,x(fe)=2; PC=50; SC<1wt%

·0.95<R<1.05, x(fe)=1; PC=50; SC<1wt%

·0.98<R<1.02,x(fe)=5; PC=50; SC<1wt%

·0.98<R<1.02,x(fe)=3; PC=50; SC<1wt%

·0.98<R<1.02,x(fe)=2; PC=50; SC<1wt%

·0.98<R<1.02,x(fe)＝1; PC＝50; SC<1wt%

·0.90<R<1.10,x(fe)=5; PC=75; SC<1wt%

·0.90<R<1.10,x(fe)=3; PC=75; SC<1wt%

·0.90<R<1.10, x(fe)=2; PC=75: SC<1wt%

·0.90<R<1.10,x(fe)=1; PC=75; SC<1wt%

·0.95<R<1.05,x(fe)=5; PC=75; SC<1wt%

·0.95<R<1.05,x(fe)=3; PC=75; SC<1wt%

·0.95<R<1.05, x(fe)=2; PC=75; SC<1wt%

·0.95<R<1.05, x(fe)=1; PC=75; SC<1wt%

·0.98<R<1.02,x(fe)=5; PC=75; SC<1wt%

·0.98<R<1.02,x(fe)=3; PC=75; SC<1wt%

·0.98<R<1.02,x(fe)=2; PC=75; SC<1wt%

·0.98<R<1.02,x(fe)=1; PC=75; SC<1wt%

·0.90<R<1.10,x(fe)=5; PC=90; SC<1wt%

·0.90<R<1.10,x(fe)=3; PC=90; SC<1wt%

·0.90<R<1.10, x(fe)=2; PC=90: SC<1wt%

·0.90<R<1.10,x(fe)=1; PC=90; SC<1wt%

·0.95<R<1.05,x(fe)=5; PC=90; SC<1wt%

·0.95<R<1.05,x(fe)=3; PC=90; SC<1wt%

·0.95<R<1.05,x(fe)=2; PC=90; SC<1wt%

·0.95<R<1.05, x(fe)=1; PC=90; SC<1wt%

·0.98<R<1.02,x(fe)=5; PC=90; SC<1wt%

·0.98<R<1.02,x(fe)=3; PC=90; SC<1wt%

·0.98<R<1.02,x(fe)=2; PC=90; SC<1wt%

·0.98<R<1.02,x(fe)=1; PC=90; SC<1wt%

Preferred compositions obtainable from the process of the invention using spray drying in step (iii) as disclosed in the specification are characterized by one of the following options:

·0.90<R<1.10, x(fe)＝1, SC<3wt%, sp＝90

·0.90<R<1.10, x(fe)＝1, SC<3wt%, sp＝95

·0.90<R<1.10, x(fe)＝3, SC<3wt%, sp＝90

·0.90<R<1.10, x(fe)＝3, SC<3wt%, sp＝95

·0.95<R<1.05, x(fe)＝1, SC<3wt%, sp＝90

·0.95<R<1.05, x(fe)＝1, SC<3wt%, sp＝95

·0.95<R<1.05, x(fe)＝3, SC<3wt%, sp＝90

·0.95<R<1.05, x(fe)＝3, SC<3wt%, sp＝95

·0.98<R<1.02, x(fe)＝1, SC<3wt%, sp＝95

·0.98<R<1.02, x(fe)＝1, SC<3wt%, sp＝97

·0.98<R<1.02, x(fe)＝3, SC<3wt%, sp＝95

·0.98<R<1.02, x(fe)＝3, SC<3wt%, sp＝97

·0.90<R<1.10, x(fe)＝1, SC<1wt%, sp＝90

·0.90<R<1.10, x(fe)＝1, SC<1wt%, sp＝95

·0.90<R<1.10, x(fe)＝3, SC<1wt%, sp＝90

·0.90<R<1.10, x(fe)＝3, SC<1wt%, sp＝95

·0.95<R<1.05, x(fe)＝1, SC<1wt%, sp＝90

·0.95<R<1.05, x(fe)＝1, SC<1wt%, sp＝95

·0.95<R<1.05, x(fe)＝3, SC<1wt%, sp＝90

·0.95<R<1.05, x(fe)＝3, SC<1wt%, sp＝95

·0.98<R<1.02, x(fe)＝1, SC<1wt%, sp＝95

·0.98<R<1.02, x(fe)＝1, SC<1wt%, sp＝97

·0.98<R<1.02, x(fe)＝3, SC<1wt%, sp＝95

·0.98<R<1.02, x(fe)＝3, SC<1wt%, sp＝97

·0.90<R<1.10, x(fe)＝1, SC<0.5wt%, sp＝90

·0.90<R<1.10, x(fe)＝1, SC<0.5wt%, sp＝95

·0.90<R<1.10, x(fe)＝3, SC<0.5wt%, sp＝90

·0.90<R<1.10, x(fe)＝3, SC<0.5wt%, sp＝95

·0.95<R<1.05, x(fe)＝1, SC<0.5wt%, sp＝90

·0.95<R<1.05, x(fe)＝1, SC<0.5wt%, sp＝95

·0.95<R<1.05, x(fe)＝3, SC<0.5wt%, sp＝90

·0.95<R<1.05, x(fe)＝3, SC<0.5wt%, sp＝95

·0.98<R<1.02, x(fe)＝1, SC<0.5wt%, sp＝95

·0.98<R<1.02, x(fe)＝1, SC<0.5wt%, sp＝97

·0.98<R<1.02, x(fe)＝3, SC<0.5wt%, sp＝95

·0.98<R<1.02, x(fe)＝3, SC<0.5wt%, sp＝97

experiment

Analytical methods:

The primary oxidation products (hydroperoxides at double bonds) were quantified by determining the peroxide value (PV) according to Ph. Eur. 2.5.5 (01/2008: 20505). The secondary oxidation products (carbonyl compounds) were quantified by determining the anisidine value (AV) according to Ph. Eur. 2.5.36 (01/2008: 20536).

The oligomeric PUFA components and their derivatives (collectively referred to as oligomer content) were quantified by gel permeation chromatography (GPC, styrene divinylbenzene phase with trifluoroacetic acid containing tetrahydrofuran as eluent). A refractive index (RI) detector was used for detection. Since the specific response factors of the sample components were unknown, the ratios were calculated based on the fraction ratios relative to the total chromatographic area.

The water content was determined by Karl-Fischer titration.

The ethanol content was determined by 1-H-NMR spectroscopy.

The acid value was determined by titration with potassium hydroxide.

Experiment 1: Eicosapentaenoic acid (EPA) from eicosapentaenoic acid ethyl ester (EPA-OEt)

5.00 kg (commercially available, standard mass) of eicosapentaenoic acid ethyl ester (EPA-OEt) with a calculated EPA content of 92.0% (92.0 wt% of the total weight is free EPA), an anisidine value of 5.0 A/g, a peroxide value of 6.5 mmol/kg, and an oligomer content of 0.2 area % (gel chromatography, RI detector) were placed in a 30 L double-jacketed vessel (made inert with nitrogen) and diluted with 5.0 L of ethanol. 1.6 kg of 50% NaOH solution were added, and the resulting solution was stirred at 30° C. to 50° C. for 30 minutes. Subsequently, the reaction mixture was diluted with 15 L of water, and then 1.4 L of 85% phosphoric acid was added. After stirring for 10 minutes, the phases were separated, and the product phase was washed with 5 L of water. 4.639 kg of eicosapentaenoic acid were obtained as an oil with an anisidine value of 3.1 A/g and a peroxide value of 8.6 mmol/kg. The oligomer content was not determined.

Experiment 2: Docosahexaenoic acid (DHA) from docosahexaenoic acid ethyl ester (DHA-OEt)

5.00 kg of ethyl docosahexaenoate (commercially available, standard quality) with a calculated DHA content of 82.8% (82.8 wt% of the total weight as free DHA) and a total omega-3 PUFA content of 92.8% (92.8 wt% of the total weight as free omega-3 PUFAs), an anisidine value of 16.0 A/g, a peroxide value of 26.1 mmol/kg, and an oligomer content of 0.4 area % (gel chromatography, RI detector) were placed in a 30 L double-jacketed vessel (made inert with nitrogen) and diluted with 5.0 L of ethanol. 1.6 kg of a 50% NaOH solution were added, and the resulting solution was stirred at 30° C. to 50° C. for 30 minutes. Subsequently, the reaction mixture was diluted with 15 L of water, and then 1.4 L of 85% phosphoric acid was added. After stirring for 10 minutes, the phases separated, and the product phase was washed with 5 L of water. 4.622 kg of docosahexaenoic acid were obtained in the form of an oil with an anisidine value of 1.7 A/g and a peroxide value of 7.9 mmol/kg. The oligomer content was not determined.

Experiment 3: Eicosapentaenoic acid-L-lysine salt derived from eicosapentaenoic acid (EPA) and L-lysine (L-Lys) (EPA-Lys)

2.00 kg of eicosapentaenoic acid from Experiment 1, exhibiting an acid value of 177.8 mg KOH/g based on titration, was dissolved in 2 kg of ethanol and combined with 1.69 kg of an aqueous L-lysine solution (51.3 wt-%). The resulting homogeneous solution was spray-dried using a custom-made spray dryer equipped with a two-substance nozzle and a 300 mm x 900 mm drying chamber with an inlet temperature of 170° C. and an outlet temperature of 80° C. 1.798 kg of a beige powder was obtained with a water content of 0.24% and an ethanol content of <0.1%. The salt exhibited an anisidine value of 2.1 A/g and a peroxide value of 1.3 mmol/kg. The oligomer content was not determined.

Experiment 4: Docosahexaenoic acid-L-lysine (DHA) derived from docosahexaenoic acid (DHA) and L-lysine (L-Lys) DHA-Lys

2.00 kg of docosahexaenoic acid from Experiment 2, exhibiting an acid value of 166.3 mg KOH/g based on titration, was dissolved in 2.0 kg of ethanol and combined with 1.81 kg of an aqueous L-lysine solution (51.3 wt-%). The resulting homogeneous solution was spray-dried using a custom-made spray dryer equipped with a two-substance nozzle and a 300 mm x 900 mm drying chamber with an inlet temperature of 170° C. and an outlet temperature of 80° C. 1.892 kg of a beige powder was obtained with a water content of 0.27% and an alcohol content of <0.1%. The salt exhibited an anisidine value of 3.1 A/g and a peroxide value of 1.7 mmol/kg. The oligomer content was not determined.

Experiment 5: Eicosapentaenoic acid-sodium salt (EPA-Na) from eicosapentaenoic acid (EPA) and NaOH

50 g of eicosapentaenoic acid, obtained analogously to Experiment 1 and exhibiting an acid number of 183.3 mg KOH/g based on titration, were dissolved in 50 ml of ethanol and combined with 6.54 g of sodium hydroxide in 30 ml of water under stirring. The resulting homogeneous solution was spray-dried using a Büchi B190 laboratory spray dryer with an inlet temperature of 140°C and an outlet temperature of approximately 80°C. 28.6 g of a light beige powder were obtained. After 3 months of storage at room temperature, the salt acquired a gray appearance and, at this time, exhibited an anisidine value of 41.1 A/g and a peroxide value of 5.0 mmol/kg. The oligomer content was determined to be 2.4 area % (gel chromatography, RI detector).

Experiment 6: Docosahexaenoic acid-sodium salt (DHA-Na) derived from docosahexaenoic acid (DHA) and NaOH

50 g of docosahexaenoic acid, obtained analogously to Experiment 2 and exhibiting an acid number of 169.5 mg KOH/g based on titration, were dissolved in 50 ml of ethanol and combined with 6.04 g of sodium hydroxide in 30 ml of water under stirring. The resulting homogeneous solution was spray-dried using a Büchi B190 laboratory spray dryer with an inlet temperature of 140°C and an outlet temperature of approximately 80°C. 27.5 g of a light beige powder were obtained. After 3 months of storage at room temperature, the salt acquired a gray appearance and, at this time, exhibited an anisidine value of 77.9 A/g and a peroxide value of 6.9 mmol/kg. The oligomer content was determined to be 3.4 area % (gel chromatography, RI detector).

Experiment 7: Stability of PUFAs and their derivatives by storage at elevated temperature (50°C) and exposed to air Qualitative

About 50 g each of the liquid ethyl esters EPA-OEt and DHA-OEt used in Experiments 1 and 2 and the liquid fatty acids EPA and DHA obtained from Experiments 1 and 2 were filled into 250 ml Schott Duran bottles with an inner diameter of about 60 mm (filling height about 20 mm).

About 50 g each of the solid lysine-salts EPA-Lys and DHA-Lys obtained from Experiments 3 and 4 were filled into a 250 ml polyethylene wide-necked bottle (55 mm*55 mm*80 mm) (filling height about 60 mm-70 mm).

All bottles were placed together with the caps opened in a drying oven at 50°C with the ventilation valves open and stored under these conditions for 26 days. The results of the subsequent analyses and the results obtained from the sodium salt stored at room temperature (see experiments 5 and 6) are summarized in the table below (Table 1).

Claims (7)

1. A method for increasing the oxidative stability of a composition containing polyunsaturated omega-3 fatty acids, comprising the following steps: (i) Providing a starting composition comprising at least one polyunsaturated omega-3 fatty acid component; (ii) Provide a lysine composition; (iii) A solution of the starting composition and the lysine composition in an aqueous, water-alcohol or alcoholic solution is mixed, and the resulting mixture is then subjected to spray drying conditions to form a solid product composition comprising a salt of at least one lysine-derived cation and an anion derived from a polyunsaturated Ω-3 fatty acid; the product composition exhibiting a solvent content SC selected from the following: SC < 5 wt%, SC < 3 wt%, SC < 1 wt%, SC < 0.5 wt%; The starting composition in step (i) and the lysine composition in step (ii) are provided in such a way that at least 90 wt% of the product composition consists of one or more salts of lysine-derived cations and anions derived from one or more polyunsaturated omega-3 fatty acids and other naturally occurring fatty acids. 2. The method of claim 1, wherein the starting composition provided in step (i) does not contain more than x(fe)wt% of fatty acid esters, wherein x(fe) is selected from 5, 3, 1, 0.3, 0. 3. The method according to any one of claims 1 to 2, wherein the starting composition in step (i) and the lysine composition in step (ii) are provided in such a way that at least sp wt% of the product composition consists of one or more salts of a lysine-derived cation and anions derived from one or more polyunsaturated omega-3 fatty acids and other naturally occurring fatty acids, wherein sp is selected from 95, 97, 98, 99, 100. 4. A composition available from the method according to any one of claims 1 to 3. 5. Use of the composition according to claim 4, for the manufacture of food containing polyunsaturated omega-3 fatty acids. 6. Use of the composition according to claim 4, for the manufacture of a nutrient containing polyunsaturated omega-3 fatty acids. 7. Use of the composition according to claim 4, for the manufacture of pharmaceutical products comprising polyunsaturated omega-3 fatty acids.