This invention is related to a process for obtaining a concentrate of esters of eicosapentaenoic and docosahexaenoic acids from marine oils.
The importance of long-chain polyunsaturated fatty acids of the ω-3 type, the acids (all cis)- 5, 8, 11, 14, 17 eicosapentaenoic, hereinafter EPA, and (all cis)-4, 7, 10, 13, 16, 19 docosahexaenoic, hereinafter DHA, as a base for pharmaceutical or food ingredients is now well known and documented because of their use, among others, to prevent arteriosclerosis and cardiovascular diseases, alleviate inflammatory conditions and delay the growth of tumors. As a consequence, experts recommend a daily intake of said fatty acids in doses ranging between 0.5 and 10 g.
One of the richest sources of EPA and DHA are oils of marine origin such as fish oils of different origin such as sardines, jack mackerel, anchovy, salmon, codfish and others. Typically, the combined content of EPA and DHA in said oils is between 10 to 35% by weight. Consequently, the first attempts to provide food and pharameutical supplements rich in EPA and DHA were based on fish oils that were refined in order to remove their characteristic unpleasant odor and flavor, for the purpose of developing a type of food or pharmaceutical ingredient fit for human consumption. These refining processes resorted mainly to the classic processes for refining vegetable oils and specific adaptations of said processes to the raw material concerned (Lindsay, USP
Nevertheless, current attempts to provide EPA- and DHA-based food and pharmaceutical products from refined oils of marine origin have not been sucessful to provide a product whose organoleptic properties were acceptable and which was free of the typical side effects such as gastric reflux, stomach and skin irritation and meteor tympanites, among others. These characteristics are accentuated when EPA and DHA are consumed in quantities over 1 g, that is, doses equivalent to about 5 g of fish oil, producing the mentioned side effects in the consumer.
Consequently, the efforts to provide EPA and DHA have been directed towards the production of concentrates of these acids from marine oils. These concentrates may contain between 40 to 95% of EPA and DHA by weight, either in the form of free acids, in the form of esters, typically ethyl esters or mono-, di- or triglycerides. The main aim of these processes is to provide concentrates of EPA and DHA that have better organoleptic properites of flavor, odor and color that can be used directly in products for therapeutic use in humans, as an active pharmaceutical ingredient or as food ingredients in general. Nevertheless, the processes of the state of the art are not capable of providing products able to keep their desirable organoleptic properties over time, that is, products in which undesirable reversion in time of the organoleptic properties, mainly odor and flavor, do not occur and which lack the typical side effects of the marine oils and their derivatives, such as gastric reflux, flattulence, allergy among others. This is obvious from the fact that none of the concentrates of EPA and DHA currently available commercially is intensively used as a food ingredient, but instead their use is restricted to the form of syrups in which the flavor has been disguised or in the form of dragees or in micro-encapsulated form, all this with the purpose of hiding or minimizing the undesirable flavor and odor that develop in said products over time. Additionally, these concentrates are not suitable either for therapeutic uses that normally require relatively high doses of EPA or DHA, such as several grams per day, because at these doses the undesirable side effects of:the concentrates are even more accentuated.
Another approach for providing EPA or DHA for human consumption derived from marine oils has been the development of processes to obtain pure EPA or DHA, as is disclosed in
Numerous processes disclosed in the prior art, addressed to obtaining concentrates of ω-3 fatty acids from oil are shown in Table 1.
European patent No.
Many of the processes disclosed can provide products with acceptable organoleptic properties, but in all of them the above-described side effects are produced and the reversion to fishy smell and flavor occurs over time, unlike in the product obtained by means of the process of the present invention that maintains its neutral organoleptic properties stored at room ambient conditions during a period of at least three months and without significant side effects. Neutral organoleptic properties means a product having acceptable organoleptic properties in the absence of additives to disguise the flavor or odor, while acceptable organoleptic properties are understood to refer to a product organolepticaly and sensoryly evaluated by a sensory panel trained in evaluating edible oils, composed of at least 9 members evaluating product properties such as appearance, aroma and flavor with a qualification of each parameter equal to or greater than 60% of the maximum value of said parameter, and the staleness property, with a qualification equal to or greater than 80% of the maximum value of said parameter.
Stable organoleptic characteristics means a product evaluated both organolepticaly and sensoryly after 24 weeks of storage of the product at room ambient conditions by a trained sensory panel composed of at least 9 members evaluating product properties such as appearance, aroma and flavor with a qualification of each parameter equal to or greater than 60% of the maximum value of said parameter, and the staleness property, with a qualification equal to or greater than 80% of the maximum value of said parameter.
In addition to the requirement of acceptable organoleptic properties and stability during longterm storage, the concentrates of EPA and DHA must also comply with a series of regulatory norms with regard to their content of contaminating organic compounds known as Persistent Organic Pollutants (POP), which are chemical substances which persist in the environment, bioaccumulate in the food chain and imply a risk of causing adverse effects to human health arid to the environment. Among these contaminants, that currently include 17 substances recognized during the third conference of the Parties to the Stockholm Convention of May 2007, are derivatives of dioxins, furans, polychlorinated products, biphenyls, polycyclic aromatic hydrocarbons, etc, whose concentration in fish oils has been increasing over time, requiring effort to be directed toward developing processes specially designed to remove these contaminants from fish oils. Among the Parties to the Stockholm Convention there are currently strict norms with regard to the maximum permissible limits of the POPs in products for human consumption, among which fish oil and products derived from fish oils are included. Processes specifically addressed to the removal of the POPs are found, among others, in the processes disclosed in the patent applications
The processes for the production of concentrates of EPA and DHA described in the European patent No.
It has been found that, surprisingly, the process of the present invention, unlike the processes of the prior art for producing concentrates of EPA and DHA, is capable of providing a product with acceptable organoleptic properties, without producing a reversion to unpleasant odor and flavor during a time of storage at room ambient conditions of at least three months and also, unlike the processes of the prior art for producing concentrates of EPA and DHA, it is also capable of efficiently reducing or eliminating the POP and heavy metals. Additionally, the disclosed process does not only not promote cis-trans isomerization of EPA and DHA, isomers of unknown metabolic properties, but on the contrary and quite surprisingly, it reduces the trans isomers content when these are found in the raw materials.
Pronova BioPharma (www.pronova.com) discloses another prior art process for the production of concentrates of ethyl esters of EPA and DHA for their use as an active pharmaceutical ingredient.
In said process, crude fish oil is first deacidified to obtain refined oil and this refined oil is subjected to a stripping process directed specifically to the removal of contaminants by means of the process disclosed in the
The process developed by Napro Pharma (www.napro-pharma.no/production) for the production of a concentrate of ethyl esters of EPA and DHA is similar to the process of Pronova BioPharma, but without the stripping step and the urea treatment step, but the product has poor organoleptic properties, it reverts to fishy odor and flavor and the previously mentioned side effects can be observed.
In comparison with the concentrates of EPA and DHA obtained by the process of the state of art, the concentrate obtained by the invented process has surprising and unexpected advantages over the prior art as will be evident from the detailed description of the invention. Among these advantages firstly, is the characteristic of an organoleptically neutral and stable product, since it does not revert to odors and flavors typical of marine oils and is free of side effects and has a level of persistent organic pollutants that meets international regulatory standards. Furthermore, the process does not promote the formation of cis-trans isomers, but on the contrary, in a surprising and unexpected mariner, it reduces the concentration of trans isomers when these are present in the raw material. As a result of all these combined characteristics, the product obtained by the process of this invention is especially adequate for use in therapies that require high doses of EPA and DHA and as a food ingredient.
It is an object of the present invention to provide a novel process for the preparation of a concentrate of esters of EPA and DHA from oils of marine origin, which is organoleptically neutral and stable and has a content of presistent organic contaminants below the standards in force and is consequently suitable for massive and regular human consumption either as a pharmaceutical or as a food ingredient.
Said object is achieved by a process for obtaining a concentrate of esters of EPA and DHA from crude or refined mineral oils, characterized in that it comprises the following steps
The steps of process are synergistically converging toward the objective of the invention in a coordinated manner. None of the steps alone or in combination with less than the total of the steps is able to achieve the objective of the invention as is shown in the examples.
Suitable raw materials for the invention comprise oils from marine animals, such as sardine, anchovy, jack mackerel, Pacific mackerel, tuna fish, cod, salmon, krill and mollusks oils and mixtures of said oils, oils of the sub-products of the processing of marine animals such as the viscera of marine animals, and also oils of microalgae such as, for example, Nannochloropsis sp and plankton. In the current invention the word oil also includes fats or waxes that contain EPA or DHA and their by products, such as glycerides and fatty acids.
Although the use of a raw material with a Totox number lower than 30 is preferred, the process of the current invention can also be carried out with raw materials having a higher Totox number as shown in the examples.
To carry out the process of the current invention, crude or refined marine oil is saponified using an alkali to hydrolyze the glycerides or other esters of fatty acids present in crude or refined marine oil to obtain a saponified mixture comprising the alkaline salts of the saponifiable compounds of the crude or refined marine oil and non saponifiable matter. To this end, crude or refined marine oil is contacted with water and one or more appropriate alkalis, and optionally, one or more solvents such as alcohols and hydrocarbons or with one or more appropriate antioxidants. Appropriate alkalis for the saponification process include sodium, potassium, lithium, magnesium hydroxides and mixtures of said hydroxides. The amount of alkali may range between 5 to 40 grams of alkali for each 100 g of crude or refined marine oil, although the preferred ratio of alkali to crude or refined marine oil is approximately 15 grams of alkali per 100 g of crude or refined marine oil. The amount of water used range between 10 to 500 g of water per 100 g of crude or refined marine oil, although the preferred ratio of water to crude or refined marine oil ranges between 50 to 200 g water per 100 g of crude or refined marine oil. When alcohols such as ethanol are used, the amount of alcohol may range between 10 to 500 g of alcohol per 100 g of crude or refined marine oil, although the preferred ratio of alcohol to crude or refined marine oil ranges between 50 to 200 g of alcohol per 100 g of crude or refined marine oil. When hydrocarbons such as hexane or other solvents are used, the amount of solvent ranges between 10 to 500g of solvent per 100g of crude or refined marine oil, preferably ranges between 50 to 200 g of solvent per 100 g of crude or refined marine oil. When the appropriate antioxidants are used, such as, for example, BHT, tocopheroles or ascorbic acid and their derivatives, the amount of antioxidant used is preferably not greater than 1 g per 100 g of crude or refined marine oil. The contact between crude or refined marine oil, water, one or more alkalis and optionally one or more solvents, can be carried out either continuously or batchwise in an stirred vessel at temperatures between 10 and 100 °C, preferably at temperatures between 40 and 85 °C and at pressures between 0.1 and 5 bar, preferably at atmospheric pressure. This step of the process is a saponification step since it leads to the formation of alkaline salts of the fatty acids from the esters and hydrolyzed fatty acids of the crude or refined marine oil.
The reaction time for obtaining a mixture comprising saponified crude or refined marine oil in the case of batchwise operation or, the time of residence in the case of a continuous operation may range from 10 to 400 minutes, preferably from 30 and 120 minutes.
The mixture that comprises saponified marine oil is contacted with one or more organic solvents until it forms an extract phase that comprises organic solvent and material dissolved in said phase and a refined phase that is immiscible with the extract phase that comprises the alkaline salts of the fatty acids. Said phases are separated either by settling or centrifugation. The contacting between the mixture comprising saponified crude or refined marine oil and the organic solvents can be carried out either batchwise or in continuous manner at temperatures between 10 and 100 °C, preferably between 20 and 80 °C, and at pressures between 0.1 and 5 bar, preferably at atmospheric pressure. Solvents or mixtures of organic solvents appropriate for the extraction can be elected from the group consisting of petroleum ether, pentane, hexane, heptanes, octane, cyclohexane, methyl cyclohexane, acetone, toluene, xylene, methyl xylene, ethyl benzene, dichloromethane, chloroform, carbon tetrachloride, dichloroethane, trichloroethane, perchloroethylene, dimethyl sulfoxide and tetrahydrofuran. Nevertheless, the preferred solvents comprise aliphatic hydrocarbons such as petroleum ether, pentane, hexane, heptane, octane or mixture of these solvents. The ratio of the solvent or solvents in relation to the mixture comprising saponified marine oil may range between 50 to 1000g per 100g of mixture, preferably from 100 to 500 g per 100 g of mixture. Once the refined phase is separated from the extract phase, if wished, it can be again contacted with one or more solvents in the conditions disclosed to form a second extract phase and a second refined phase. The production of additional phases and refined phases through the described process may be repeated if so desired.
In the acidification step, the refined phase is contacted with a solution of an acid such as sulfuric acid, hydrochloric acid, phosphoric acid, acetic acid, trichloroacetic or carbonic acid, until an aqueous phase and a non-aqueous phase comprising fatty acids are formed. The amount of acid used in the acidification step may be up to 1.5 times the stoichiometric amount of alkali used in the saponification step, preferably 1.05 times the stoichiometric amount of alkali required for the total neutralization of the refined phase. The amount of acid required to acidify the refined phase can be determined measuring the total alkalinity of the refined phase The mixing of the refined phase and the acid solution can be carried out either batchwise or in a continuous manner in an stirred vessel at temperatures between 10 and 100 °C, preferably between 20 and 60 °C, and at pressures between 0.1 and 5 bar, preferably at atmospheric pressure and with a. reaction or residence time, in the case of continuous operation, ranging from 1 to 120 minutes, preferably from 5 to 60 minutes. Optionally, the mixture may also include an antioxidant or a mixture of antioxidants such as, BHT, tocopheroles or ascorbic acid and its derivatives. Next the non-aqueous phase is separated from the aqueous phase by settling or centrifugation. The separated non-aqueous phase is washed with a washing mixture comprising water, a monohydric alcohol, acetone or mixtures thereof, or an aqueous solution of sodium sulfate or sodium chloride, at temperatures between 10 and 100 °C, preferably between 20 and 60 °C and at pressures between 0.1 and 5 bar, preferably at atmospheric pressure. The washed non-aqueous phase can optionally be filtered to eliminate insoluble solids. The term non-aqueous phase used below designates both the washed non-aqueous phase as well as the washed and filtered non-aqueous phase, obtained after the acidification step as described previously.
Optionally, any of the non-aqueous phases is partially or completely desolventized preferably at a reduced pressure and at temperatures below 150°C obtaining what is referred to as a partiallly or totally desolventized non-aqueous phase.
Optionally, any of the non-aqueous or partially or totally desolventized non-aqueous phases can be subjected to a crystallization step. To this end, the phase is mixed with a solvent or mixture of solvents selected from the group consisting of petroleum ether, pentane, hexane, heptane, octane, cyclohexane, methyl cyclohexane, toluene, xylene, methyl xylene, ethyl benzene, dichloromethane, chloroform, carbon tetrachlorid , dichloroethane, trichloroethane, perchloroethylene, dimethyl sulfoxide, dimethyl formamide and tetrahydrofuran, methanol, ethanol, acetone, methy ethyl ketone, diacetone alcohol or mixtures thereof. Preferred solvents for this step are hexane, ethanol, acetone or mixtures of these. The amount of solvent to be used in this step may range between 50 and 1000 g per 100 g of the phase that is crystallized, preferably between 100 and 500 g per 100 g of the phase that is crystallized. The mixture formed is subsequently cooled to a temperature ranging from 0 to -50 °C, preferably from -20 to -40 °C until the formation of a solid crystallized phase and a liquid phase. The.operation of crystallization may be carried out in a batchwise or in a continuous manner and preferably at atmospheric pressure. The crystallized solid phase and the liquid phase are subsequently separated by filtration or centrifugation, preferably at the same final temperature of the crystallization. Then, the solvents of the liquid phase are removed, partially or totally, by evaporation of the solvent to obtain what is then referred to as a first partially or totally desolventized phase produced in the crystallization step comprising EPA and DHA in higher concentration than that of the crude or refined marine oil utilized.
Optionally, any of the non-aqueous phases or any of the non-aqueous partially or totally desolventized phases or the first partial or totally desolventized phase produced in the crystallization step can be treated with urea or another compound forming complexes or adducts with fatty acids or their derivatives through methods for forming complexes or adducts with fatty acids with urea for the fractioning of fatty acids from vegetable and animal oils. To this end, a solution is formed at a temperature between 50 and 100 °C where the solution consists of between 5 and 40 g of the phase being subjected to the urea treatment for every 100 g of a solution of the compound that forms complexes or adducs in an organic solvent, preferably urea in ethanol in ethanol, that contains, at the dissolution temperature, approximately 30 g of urea per 100 g of ethanol. Then the solution is cooled down to room temperature or less, forming a solid phase comprising complexes or adducts and a solid free liquid phase. The complexes or adducts and the solid free liquid phase are separated via known techniques such as filtration or centrifugation, and the solid free liquid phase is washed with water or an acidic solution until the extraction of the remaining compound that forms complexes or adducts dissolved in that phase. Then, the solvents of the solid free liquid phase are removed, either totally or partially, to obtain what is referred to as a second partiallly or totally desolventized phase produced in the complex forming step, and comprising EPA and DHA in a concentration higher to that in the raw material.
Then, any of the non-aqueous phases or any of the non-aqueous partially or totally desolventized phases or the first partial or totally desolventized phase produced in the crystallization step or the second partial or totally desolventized phase produced in the complex formation are subjected to an esterification step. To this end, the phase is mixed with a monohydric alcohol such as methanol or ethanol or with a polyhydric alcohol such as glycerol in a proportion that may be up to 500g of alcohol for every 100g of the phase, preferably from 20 to 200g of alcohol for 100 g of the phase, and with a catalyst such as sulfuric acid, p-toluene sulfonic, methane sulfonic, ethane sulfonic or with a resin such as amberlite, in a ratio of 0.05 to 10 g of catalyst for every 100 g of phase. The esterification step can be carried out either batchwise or continuously in a stirred reactor, at a temperature between 10 and 150 °C, preferably between 30 and 80 °C and at a pressure between 0,1 and 5 bar, preferably at atmospheric pressure. The time of esterification in the case of batchwise operation or residence time in the case of continuous operation may range between 30 and 600 minutes, preferably between 60 and 240 minutes. At the end of the esterification step an esterified mixture is obtained comprising esters of fatty acids.
The catalyst used is removed from the esterified mixture by processes known in the state of the art, such as filtration or by neutralizing and washings with aqueous solutions to form a catalyst-free esterified mixture. The catalyst-free esterified mixture is desolventized evaporation, preferably at a reduced pressure and temperatures lower than 150 °C, to obtain a desolventized mixture comprising esters of fatty acids.
Subsequently, the mixture of esters of desolventized fatty acids is distilled in a short-path distillation column to obtain a distillate and a residue. Thereafter the distillate or the residue may be submitted to a new phase of distillation to obtain a second distillate and a second residue. The process can be repeated until a distillate or a residue is obtained as final product containing the desired concentration of esters of EPA and DHA or a concentrate of EPA and DHA. The distillations can be carried out at a temperature lower than 180 °C, preferably lower than 150 °C and a pressure lower than 1 mbar, preferably lower than 0.1 mbar. In this way, ester concentrates of EPA and DHA with a content among both esters of between 40 and 95% by weight of the concentrate may be obtained.
The concentrate of EPA and DHA or any ester stream can be subjected to one or more additional steps of purification such as, fractionation by means of cooling at a temperature lower than -5 °C and separating the solids by filtration or centrifugation; deodorization in packed columns or tray columns at reduced pressure, and temperature below 200 °C, preferably lower than 150 °C using either nitrogen or steam for deodorizing; adsorption by means of the use of infusorial earth, active carbon, zeolites and molecular sieve among others.
Likewise, optionally, if so desired, the concentrates of EPA and DHA can be transesterified with glycerin to form concentrated glycerides of EPA and DHA through processes known in the prior art to obtain a new final product of EPA and DHA glycerol esters.
One or more appropriate antioxidants may be added to the concentrate of EPA and DHA, such as tocopherol, esters of tocopherol, ascorbic acid and their derivatives, extract of rosemary, extract of boldo, boldine, among others. Preferably, the amount of antioxidant is less than by 1% by weight, preferably less than 0.5%.
The concentrates of EPA and DHA obtained through the process disclosed have superior properties, since they eliminate or significantly reduce the undesirable side effects associated with the consumption of derivatives of fish oil, such as gastric reflux, stomach and skin irritation and meteorism, among others. Additionally, and surprisingly, the concentrates of EPA and DHA obtained by the process of the present invention do not present organoleptic reversion to marine oils, allowing their utilization as ingredients of food or pharmaceutical products, without the need of resorting to flavor and odor maskers, encapsulations, and micro-encapsutation, among others. Moreover, the process disclosed significantly reduces the content of Persistent Organic Pollutants and heavy metals below the maximum levels permitted internationally.
With reference to
The washing mixture of reactor (18) is fed into a settler (21) via line (20) to separate the light phase from the heavy phase of the washing mixture. The settler (21) operates at a temperature between 20 and 70 °C, at a pressure between 1 and 2 bar and at a residence time between 5 and 60 minutes. The heavy phase is removed via line (22) for its subsequent treatment, to recover solvents or to recirculate it or part of it to the washing reactor (18). Via line (23), the light phase separated in the settler (21) is fed to an esterification reactor (24) also fed via line (25) with a stream of a solution of p-toluene sulfonic acid dissolved in ethanol. The reactor (24) operates at a temperature between 40 and 85 °C under stirring, at a pressure between 0.5 and 2 bar and with residence time of 180 minutes to produce an esterified mixture. The esterified mixture is fed via line (26) to the washing and neutralization reactor (27) where it is contacted under stirring, at a temperature between 20 and 70 °C, at a pressure of 1 and 2 bar and with residence time of between 1 and 30 minutes, with the stream (28) comprising a solution of sodium carbonate at 5% in water, to generate a neutralized washing mixture. The neutralized washing mixture of the washing reactor (27) is fed to a settler (31) via line (30) to separate a mixture of esters of fatty acids and an aqueous phase. The settler (31) operates at a temperature between 20 and 70 °C, at a pressure between 1 and 2 bar and residence time between 5 and 60 minutes. The aqueous phase is removed via line (32) for its subsequent treatment to recover solvents. Via line (33) the mixture of esters of fatty acids separated in the settler (31) is fed to a falling film evaporator (34) that operates at a temperature between 50 and 180 °C, at a pressure between 1 and 100 mbar and residence time no greater than 30 minutes, to obtain a distillate stream and a desolventized stream comprising esters of fatty acids. Via line (35) the distillate is pumped to a storage tank not shown. The desolventized fatty acid esters are fed via line (36) to a short path evaporator (37) operating at a temperature between 50 and 180 °C, at a pressure between 0.001 and 1 mbar. Via line (38) the distillate is removed from the short path evaporator (37) and via line (39) the residue of the distillation of the short path evaporator (37) is removed and fed to a short path evaporator (40). The short path evaporator (40) operates at a temperature between 50 and 180 °C, at a pressure between 0.001 and 1 mbar. Via line (41) the residue of the distillation of the short path evaporator (40) is removed and via line (42) the distillate of the short path evaporator (40), comprising a concentrated mixture of organoleptictilly neutral and stable esters of EPA and DHA is removed that have no reversion to odors and flavors typical of fish oil, are free of side effects, with a level of persistent organic pollutants and heavy metals that complies with the international regulatory standards.
The following examples illustrate ways in which this invention may be carried out as well as the outstanding characteristics of the product of the process of the invention.
300 g of salmon oil (sample M1) whose characteristics are shown in Table 2, were placed into a 2000 ml Erlenmeyer flask. 150 g ethanol and 150 g of a 28% solution of sodium hydroxide in distilled water were added.
Then, with stirring and under a nitrogen atmosphere, the mixture was refluxed for one hour, resulting in full saponification of the salmon oil. Immediately afterwards 160 g of an aqueous solution of hydrochloric acid at 26% was added and the mixture was shaken vigorously for 5 minutes. Then, 450 ml of petroleum ether were added and it was shaken once again. The mixture was placed in a 2000 ml separating funnel and allowed to separate. The settled upper phase was separated and the aqueous phase was extracted twice with 450 ml of petroleum ether. The extracts of petroleum ether were collected in a 2000 ml funnel and were washed with water until pH neutrality. The washed extract was evaporated in a rotary evaporator operating at 10 mbar and 60 °C. Subsequently, traces of petroleum ether were removed feeding the residue of the evaporation of the rotary evaporator to a short path distillator KDL5 UIC, at a flow rate of 1250 ml/h, jacket temperature of 90 °C, condenser temperature at -4 °C, roller speed of 350 rpm and pressure of 4 mbar. A mixture of fatty acids of salmon oil was obtained with 30.1% of long chain ω-3 fatty acids (sample M2).
The mixture of fatty acids of salmon was fed into a short, path distillator KDL5 UIC, at a flow rate of 100 ml/h, jacket temperature of 65°C, condenser temperature at 4 °C, roller speed of 350 rpm, pressure of 0.005 mbar and a first distillate and a first residue Were obtained. The first residue was subjected to a second step of short path distillation, at a temperature of 85 °C obtaining a second distillate and a second residue. The second distillate contained 52.2% of long chain ω-3 fatty acids (sample M3).
The results of the analysis of the samples of Comparative Example 1 are shown in Table 2.
300 g of the salmon oil used in Comparative Example 1 and 200 g of a solution of sulfuric acid at 5% in absolute ethanol were placed in a 2000 ml Erlenmeyer flask. The mixture was refluxed under stirring and purged with nitrogen for 8 hours. Excess ethanol was removed by distillation at a reduced pressure while the reaction mixture Was cooled to room temperature.
The residue of the distillation was diluted with 400 ml of hexane and was washed with 500 ml of water. The heterogeneous mixture was stirred vigorously. The aqueous phase was separated from the hexane phase in a separating funnel and the hexane phase was repeatedly washed with water until the pH of the aqueous phase was neutral. The washed hexane extract was purified by passing it through a column of silica gel. The purified hexane extract was subsequently evaporated in a rotary evaporator up to 10 mbar and 60 °C. Traces of solvent were removed feeding the residue of the evaporation stage carried out in the rotary evaporator into a short path distillator KDL5 UIC, at a flow rate of 1250 ml/h, jacket temperature of 90 °C, condenser temperature of - 4 °C, roller speed of 350 rpm and pressure of 4 mbar. A mixture of ethyl esters was obtained containing 27.9% of long-chain ω-3 fatty acids (sample M4).
The mixture of ethyl esters was fed into a short path distillator KDL5 UIC, at a flow rate of 100 ml/h, jacket temperature of 65 °C, condenser temperature of 4 °C, roller speed of 350 rpm, pressure of 0.005 mbar by using a diffusion pump and a first distillate and a first residue were obtained. The first residue was subjected to a second stage of short path distillation, at a temperature of 85 °C in order to obtain a second distillate and a second residue. The second distillate contained 51.6% of long chain ω-3 fatty acids (sample M5).
The results of the analysis of the samples of Comparative Example 2 are shown in Table 3.
300 g of salmon oil used in Comparative Example 1, 150 g of ethanol and 150 g of sodium hydroxide solution in distilled Water at 28% were placed in a 2000 ml Erlenmeyer flask. The mixture was then refluxed under stirring and nitrogen atmosphere for one hour, resulting in full saponification of the salmon oil.
The saponified mixture was placed in a 3000 ml separating funnel and 150 g of ethanol, 150 g of distilled water and 900 g of hexane were added to the funnel. The resulting mixture was stirred vigorously and was left to settle. The upper hexane phase was separated out and the aqueous phase was extracted three more times with 700 ml of hexane. The hexane extracts were desolventized in a rotary evaporator at a reduced pressure.
The extracted aqueous phase was acidulated by adding 200 g of an aqueous solution of hydrochloric acid at 20%. The resulting organic was washed with portions of an aqueous solution of ethanol at 50% until pH 4-5 and was then evaporated in a rotary evaporator at 10 mbar and 60 °C. A mixture of salmon fatty acids was obtained containing 29.6% of long-chain ω-3 fatty acids (sample M6).
The salmon fatty acids was mixed with 100 g of a solution of sulfuric acid at 1.0% in anhydrous ethanol and was refluxed for 2 hours. The reaction was considered finalized, as determined by titration, upon the mixture reaching a constant acid number. The reacted mixture was neutralized with 40 g of a solution of sodium carbonate at 10% in distilled water followed by washings with portions of 40 g of distilled water. Subsequently, the mixture was evaporated in a rotary evaporator at 10 mbar and 60°C. Traces of solvent were removed feeding the residue of the evaporation into a short path distillation column KDL5 UIC, at a flow rate of 1250 ml/h, jacket temperature of 90°C, condenser temperature at -4°C, roller speed of 350 rpm and pressure of 4 mbar. A mixture of ethyl esters was obtained containing 30.9% of long chain ω-3 fatty acids (sample M7).
The mixture of ethyl esters was fed into a short path distillator KDL5 UIC, at a flow rate of 100 ml/h, jacket temperature of 65 °C, condenser temperature 4°C, roller speed of 350 rpm, pressure of 0.005 mbar by using a diffusion pump, obtaining a first distillate and a first residue. The first residue was again distilled in the short path distillator at a temperature of 85 °C obtaining a second distillate, and a second : residue. The second distillate contained 52.3% of long-chain ω-3 fatty acids (sample M8).
The results of the analysis of the samples of Example 1 of the present invention are shown in Table 4.
As can be seen in the above examples, only the process of the present invention was able to obtain a concentrate of EPA arid DHA within the specifications according to the internationally proposed regulations.
The test of Example 1 was repeated using sardine oil as raw material with a Totox value of 45. The results of the example are shown in Table 5:
The test of Example 1 was repeated using jack mackerel oil as a raw material having a Totox value of 33. The results of the example are shown in Table 6:
A 200-liter baffled, steam-jacketed, water-cooled, turbine-stirred stainless-steel reactor with a stainless-steel condenser was charged with 15 kg of ethanol, 15 kg of an aqueous solution of sodium hydroxide at 18.7% and 15 kg of sardine oil (sample M10) whose characteristics are shown in Table 7. The mixture was heated up to 55 °C for one hour and was then cooled to 45 °C. Then 45 kg of hexane were added and it was stirred for 10 minutes. The mixture was left to settle for 15 minutes and the organic phase was separated from the aqueous phase. The aqueous phase was
extracted twice more using the same procedure. The hexane extracts were collected and desolventized at a reduced pressure, thus generating a hexane residue. The aqueous or refined phase was acidulated at a temperature of 25 °C with 28 kg of a solution of hydrochloric acid at 10% and stirring the mixture for 5 minutes. The acidulated mixture was left to settle for 15 minutes, in order to separate out the aqueous phase The organic phase was washed with 10 kg of an aqueous solution of ethanol at 50% to pH 5. The washed organic phase was filtered to separate suspended solids. The organic phase, washed and filtered, was diluted with hexane up to 20% by weight and was transferred to a second reactor of 150 liters, provided with a mechanical stirrer, an anchor type agitator and cooling jacket, and was cooled down to -25 °C. The cooled mixture at -25 °C was filtered through a bag filter using a polyester mesh of 10 microns as filtration means. The filtrate from the filtration step was charged into the 200 liters reactor and was heated at 55 °C at a pressure of 200 mbar. The mixture of desolventized fatty acids was contacted with a solution of 20 kg of urea dissolved in 55 kg of ethanol at 80 °C. The mixture was stirred to form the complex with urea and then it was cooled to 15 °C. The precipitated solids were separated by filtration and a solid free filtrate was obtained. The solid free filtrate was cooled to 1 °C and filtered to obtain a second solid free filtrate. The second solid free filtrate was mixed with 3 kg of hydrochloric acid dissolved in . 50 kg of water and 20 kg of hexane, was stirred and left to settle. The acidic aqueous phase was separated and the organic phase was washed with 5 kg of water until the pH was neutral. The organic phase was desolventized at 80 °C and 50 mbar. 2.3 kg of a mixture of fatty acids were obtained containing 77.2% of ω-3 fatty acids (sample M-11).
Then, 40 g of sulfuric acid dissolved in 10 kg of ethanol were introduced and heated controlling the temperature of the reactor through distillation until reaching 80 °C. Subsequently the reactor was cooled to 40 °C and diluted with 10 kg of hexane. Then 70 g of sodium carbonate dissolved in 5 kg of water were added to the diluted mixture and it was stirred for 10 minutes. The aqueous phase was separated. The organic phase was washed with 5 kg of water and then desolventized at 80 °C and 50 mbar. 2.5 kg of a mixture of ethyl esters of fatty acids were obtained containing 70.9 % of ω-3 fatty acids (sample M-12).
Traces of solvent from the ethyl esters were removed feeding the mixture to a short path distillator KDL5 UIC, at a flow rate of 1250 ml/h, jacket temperature of 80 °C, condenser temperature at -5 °C, roller speed of 350 rpm and pressure of 4 mbar. The mixture of ethyl esters from the preceding distillation was fed to a short path distillator column KDL5 UIC, at a flow rate of 90 ml/h, jacket temperature of 85 °C, condenser temperature at 4 °C, roller speed of 350 rpm, pressure of 0.005 mbar using a diffusion pump, obtaining a first distillate and a first residue.
The first residue was mixed with a ratio of 1% Tonsil at 70°C and at reduced pressure for 30 minutes and was filtered obtaining a purified filtrate which was subjected to a second stage of short path distillation, at a temperature of 98 °C obtaining a second distillate and a second residue. The second distillate contained 86.2% of long chain ω-3 fatty acids. The second distillate was cooled to -25 °C for 12 hours and then filtered. The resulting filtrate was fed to a deodorization column, at a temperature of 100 °C using nitrogen at 130 °C and at the pressure of 15 mbar for the deodorization. A deodorized concentrate of ethyl esters of long chain ω-3 fatty acids (sample M13) was obtained to which a mixture of esters, of tocopherol, ascorbyl palmitate and extract of rosemary at a concentration of 2550 ppm was added.
The results of the analysis of the samples of Example 4 are shown in Table 7.
A 200-liter baffled, steam-jacketed, water-cooled, turbine-stirred stainless-steel reactor with a stainless-steel condenser was charged with 15 kg of ethanol, 15 kg of an aqueous solution of sodium hydroxide at 17.4% and 15 kg of jack mackerel oil of example 3 and whose characteristics are shown in Table 8. The mixture was heated at 75°C for one hour and then was cooled to 45 °C. Next, 45 kg of hexane were added and stirred for ten minutes, The mixture was left to settle for 15 minutes and the organic phase was separated from the aqueous phase. The aqueous phase was extracted twice more by the same procedure. The aqueous phase or refined phase was acidulated at a temperature of 25 °C with 26 kg of a solution of hydrochloric acid at 10% and stirred for 5 minutes. The acidulated mixture was left to settle for 15 minutes, and then the aqueous phase was separated out. The organic phase was washed with 10 kg of 50 % aqueous ethanol solution until pH 5. The washed organic phase was mixed with 100 g of sulfuric acid dissolved in 10 kg of ethanol and was heated while controlling the temperature of the reactor distilling a mixture of solvents until the temperature of 80 °C was reached. Subsequently, the reactor was cooled to 40 °C, 350 g of sodium carbonate dissolved in 5 kg of water was added and was stirred for 10 minutes. The aqueous phase was separated. The organic phase was washed with 5 kg of water and was desolventized at 80 °C and 50 mbar. 14.6 kg of a mixture of ethyl esters of fatty acids were obtained containing 24.7% of ω-3 fatty acids. (sample M-14).
Traces of solvent of the ethyl esters were removed feeding the mixture to a short path distillator KDL5 UIC, at a flow rate of 1250 ml/h, jacket temperature of 80 °C, condenser temperature at -5 °C, roller speed of 350 rpm and pressure of 4 mbar. The mixture of ethyl esters from the preceding distillation was fed to a short path distillator KDL5 UIC, at a flow rate of 90 ml/h, jacket temperature of 85 °C, condenser temperature at 4 °C, roller speed of 350 rpm, pressure of 0.005 mbar by means of a diffusion pump, obtaining a first distillate and a first residue. Subsequently, the first residue was subjected to a second short path distillation, at a temperature of 96 °C and a second distillate and a second residue were obtained. The second distillate contained 51.2% of long chain ω-3 fatty acids (Sample M15). Finally, 2000 ppm of tocopherol acetate (Grindox Toco 70, Danisco) was mixed with the second distillate.
The results of the analysis of samples of Example 5 are shown in Table 8.
For the study of the formation of trans isomers of ω-3 fatty acids of marine oils the Cis/trans isomerism of EPA of Examples 2 and 3 was evaluated.
One gram of the second distillate of Example 2 was saponified with a solution of potassium hydroxide in aqueous methanol at 10 °C for 24 hours. Then, the saponified mixture was acidified with a dilute solution of hydrochloric acid at 1%, at 10 °C. The acidified mixture was extracted with petroleum ether three times. The extracts of petroleum ether were collected, washed with an aqueous solution of methanol at 20% and the washed extract was desolventized in a rotary vaporizer at 20 °C and 5 mbar. The residue was methylated using boron trifluoride 870 mg of methyl esters of long chain ω-3 fatty acids were obtained. A sample of methyl esters of long chain ω-3 fatty acids of the second distillate of example 3 was prepared in a similar manner.
Similarly, methyl esters of fatty acids of sardine oil of Example 2 and jack mackerel oil of Example 3 were prepared using the technique described.
A standard of methyl ester of all cis (5, 8, 11, 14, 17) eicosapentaenoic acid was injected in a gas chromatograph series 7890A provided with selective mass detector of 5975Cinert, of Agilent Technologies, using a 100-meter SP 2560 column, having an internal diameter of 0.25 mm and a film thickness of 0.20 microns. The chromatographic program was: initial temperature 140 °C for 5 minutes; increasing temperature at the rate of 2 °C/min up to 240 °C and maintained at 240. °C for 30 minutes. Injector and detector temperature was 250 °C. Extra pure helium was used as carrier. The mass spectrums of the standard were stored in the data library.
Subsequently, the samples of the methyl esters prepared from the samples of the second distillate of Examples 2 and 3 and of the methyl esters obtained from the original oils of Examples 2 and 3 were injected into the chromatograph. The software Chemstation was used to obtain a "Match Quality" of 99% for each one of the samples methylated. Additionally, the chromatograms and spectrograms of all the methyl esters chromatographied were compared arid no new peaks were detected associated to the isomerization of EPA.
Additionally, the content of trans isomers of fatty acids of 16 to 22 carbon atoms was determined according to the methodology described in the process AOCS Ce 1h-05. The results are shown in Table 9. As can be seen, in the tests of Examples 2 and 3, no trans isomers were generated and unexpectedly there was a decrease of said isomers via the process of the present invention.
Sensory evaluation of the quality and determination of the stability of samples of ethyl esters of fatty acids.
Organoleptic qualities and stability were evaluated by a trained panel of 12 panelists. Samples to be evaluated were given to each panelist in small glasses coded with 3 random digits and containing 15 ml of samples per panelist. The evaluation was carried out at a Sensory Evaluation Laboratory about 11:00 in the morning.
In order to assess the sensory quality of the product the following parameters were considered: appearance, aroma, flavor, rancidity and presence of extraneous flavors and odors. The measuring scale for evaluating said parameters had a 9-point range, in which 9 indicated that the parameter evaluated was optimal for the product and 1 indicated that it was very poor. For example, 9 stands, in the case of flavor for "excellent, typical, exceptionally agreeable" and 1 for "strange, disagreeable, putrid". In the case of rancidity, the measuring scale had a range of 5 point to which 5 meant "without rancidity" and 1 "extremely rancid".
Table 10 presents the average results obtained in the organoleptic assessment of the fresh sample of ethyl esters of fatty acids M15 of Example 5 (A) and the same sample after 29 weeks of storage at room temperature (B). No flavor, odor or appearance masking agents were added to the samples.
As can be seen, the concentrate of EPA and DHA generated by the present invention has acceptable organoleptic Quality and stability.
In order to compare the side effects caused by concentrates of EPA and DHA obtained from fish oil, 10 volunteers were recruited and divided into two groups, A and B, of 5 individuals each. 900 g of yoghurt were mixed with 100 grams of commercial 32/22 EPA/DHA ethyl esters of fatty acids, to generate a sample of yogurt 1. In parallel 100 g of ethyl esters of Example 5, Sample M15, were mixed with a separate 900 g of yogurt, to generate a sample of yogurt 2. Each member of group A ingested 150 g of yogurt mixture 1 and each member of group B ingested 150 g of yoghurt mixture 2. Three hours later, the test volunteers were asked about the presence or absence of gastric reflux. In group A, 4 positive cases were detected, while in group B, 1 positive case was detected.
A week later the test was repeated with the same individuals of groups A and B. this time each member of group A ingested 150 g of a new yoghurt mixture 2 and each member of group B ingested a new yoghurt mixture 1. Three hours later, the test volunteers were asked about the presence or absence of gastric reflux. In group A, no positive cases were detected, while in group B, 5 positive cases were detected. The test group shows that the concentrates of EPA and DHA obtained by the process of the present invention do not exhibit the characteristic side effects of marine derivatives such as gastric reflux, probably: due to the efficient removal of allergens originally present in the raw material.
A Petri dish of 15 cm in diameter with 20 g of sample M15 of Example 5 was kept in a forced convection oven at 45°C for 6 hours. Subsequently, the sample was removed from the oven and allowed to cool.
The sample was evaluated by a panel of 5 people. No fishy smell was detected.
The test was repeated with Sample M3 of Comparative Example 1. A characteristic rancid fishy smell was noticed.
The test was repeated with Sample M5 of Comparative Example 2. Again, a characteristic rancid fishy smell was noticed.
The test was repeated with the sample of ethyl esters of fatty acids 33/22 EPA/DHA used in Example 8. A characteristic rancid fishy smell was noticed.
The oxidation stability of a portion of Sample M15 of Example 5 was measured by means of the Rancimat Test method. The induction time at 80 °C was 28.11 ± 0.97 hours. In parallel, the Rancimat tests were carried out on the sample of ethyl esters 33/22 used in Example 8. The induction time at 80 °C was 1.67 ± 0.10 hours.
A sample of the hexane residue of Example 4 was determined by GC-MS in a HP7890 Chromatograph coupled to a 5975Cinert mass detector. The chromatographic report indicated the presence in the sample of over 50. compounds in a concentration higher than 1000 ppm as can be seen in Table 11.
As can be seen in Table 11, the process of the present invention is capable of removing a large family of compounds present in the raw material, which are not ω -3 fatty acids, and which could be responsible for side effects and undesired reversions of flavor and odor.
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