ITUB20153877A1 - Method of purification of glycerides of fatty acids, compositions derived from them, and their use - Google Patents

Description

DESCRIPTION

The present invention relates to a new purification method of glycerides of fatty acids (mono- and / or di- and / or triglycerides), of any origin, including natural oils, and in particular those characterized by at least partial esterification with fatty acids long-chain polyunsaturates (PUFAs), in particular C 18 - C22, preferably C20 - C22, and comprising 2-6 double bonds, preferably 5-6 double bonds.

The purification method according to the invention allows to remove from said glycerides products structurally unrelated to them. These foreign products, especially in the case of natural glycerides, are essentially made up, certainly not from the weight point of view but due to their extreme toxicity, of the so-called "persistent" organic pollutants (POPs) now ubiquitously present in the environment (soils, agricultural land, marine and river waters, eco).

Among the numerous POPs known from the literature and present in glyceride compositions, it is possible to mention, for example, the polychlorinated dibenzo-dioxins and the polychlorinated dibenzo-furans, the polychlorinated biphenyls, the polybromo-diphenyl Yeri, the polyethylene aromatic hydrocarbons, and others, also usually present and extremely toxic.

BACKGROUND OF THE INVENTION

The glycerides of the fatty acids (esters with glycerol) object of the purification method of the invention can be mono-, and / or di-, and / or tri-glycerides and are so named according to their degree of substitution. They are also reported in the literature as acyl-glycerols, distinguishing monoacylglycerols in the sn position on the basis of their position and steric structure. 1 or sn-2, or the 1-glycerol diaci in position 671-1,2 or sn-1,3, or again the triadi-glycerols.

If the substituents in position 1 and 3 are different, the carbon in position 2 becomes asymmetric and can be in optically active or racemic form.

The chemical-physical and biological properties of glycerides essentially depend on the number, position and type of acid radical present as glycerine ester.

It is known that the fatty acids contained in the glycerides can be short-chain or more often medium-chain or long-chain, meaning herein as such the acids with 18 or more carbon acids. It is also known that said acids can be saturated (such as C18: 0 stearic acid), or monounsaturated (such as C18: 1 n-9 oleic acid), or polyunsaturated, often containing from 2 to 6 double bonds, and generally belonging to the class of omega-6 (or n-6) acids such as arachidonic acid (ARA, C20: 4 n-6) or to that of omega-3 (or n-3) acids such as e.g. eicosapentaenoic acid (EPA, 020: 5 n-3, all cis) and docosahexaenoic acid (DHA, C22: 6 n-3, all cis), or also docosapentaenoic acid (DPA, C22: 5 n- 3).

In particular, these omega-3 acids and their ethyl esters have assumed increasing importance in recent decades for their biological properties and for their use as nutraceuticals, food and dietary supplements, foods for special medical use, and as drugs. The same is currently happening for their glycerides, considered more "natural * and better absorbed in the human body, which have the additional advantage of being their main source of production. In fact, in relation to their origin, all these acids are found in nature in widely varying ratios with each other and in the form of various chemical structures, such as e.g. in the phospholipids, eoe., but more frequently they occur in the form of natural triglycerides, constituents of vegetable and animal oils and fats. Omega-6 acids are particularly abundant in vegetable oils and seeds, while oinega-3 acids and in particular EPA and DHA are mainly of marine origin and derive essentially from fish oils, also from aquacultures, or from "krill oils" or again from algae and other oil-bearing microorganisms, or from "single celi fermentation" starting from selected strains of algae or other microorganisms. Mono-glycerides (and also di-glycerides) are mainly industrial products, used for their excellent emulsifying properties, and represent 70-80% of the emulsifiers used in the world in food, cosmetic and pharmaceutical products. They are usually obtained, although with low yields and low purity, by chemical glycerolysis of triglycerides (natural oils and fats) at high temperatures and by alkaline catalysis. ; In recent times, the glycerides substituted with polyunsaturated acyls are preferably subjected to hydrolysis or alcoholysis by enzymatic way, by means of lipases - even immobilized - selective for the structure of the acyl and for its position on the glycerol: it is thus possible to act in very reaction conditions. and furthermore - by virtue of the greater resistance of polyunsalide acyls to enzymatic cleavage, compared to saturated and monoin saturated acyls - it is possible to isolate compositions of glycerols still partially acylated and in particular enriched in polyunsaturated acids. It is also known that said enzymatic hydrolysis or alcoholysis, as well as the more traditional chemical splits, can be brought to completion with total separation of PUPA polyunsaturated acids (or their esters) from glycerol, polyunsaturated acids that can be enriched in concentrated compositions through various technologies, also combined, but these phases concern the production technology of polyunsaturated acids and their ethyl esters (see monograph 07/2012: 1250 of the E.P, 8.0 and monograph pages 4059-61 of USP 37), and not the present glyceride purification process. However, as we shall see, they will also find application on glycerides already purified with the method in question. Many thousands of scientific publications are currently available on the various biological processes and effects involving polyunsaturated oils and the various "concentrates" of PUPA and esters that can be obtained. Whenever possible, however, we will refer to the next volume of Breivik H, "Long-Chain Ortiega-3 Special ty Oils" (H Breivik, ed.), The Oily Press, Bridgwater, (2007), hereinafter referred to as "Breivik 2007" . including natural oils, substituted at least partially with long-chain fatty acid radicals, comprising 18-22 carbon atoms, preferably 20-22 carbon atoms, and of the polyunsaturated type, i.e. comprising 2-6 double bonds, preferably 5-6 double bonds. This refers in particular to acids belonging to the class of omega-6 and / or omega-3 acids, more preferably to the class of omega-3 acids, in particular to EPA and / or DHA.; The fatty acid content polyunsaturated as above de finished, e.g. EPA or DHA, or their sum, is between 15-45%, more often 15-30%, and 90%, or between 30-60%, more often 45-60%, and 90%, of the weight of the composition , being 15-30% the average content in EPA or DHA or their sum in a natural oil (fish or similar) used for the production of PUFA, being 45% and 60% the minimum contents of the sum of EPA and DHA and respectively of the sum of the omega-3 acids - all expressed as triglycerides - provided for by monograph 01/2009: 1352 of the E.P.7.0 relating to the glycerides of omega-3 acids, chemically or enzymatically reconstituted and enriched in PUFA, and being around 90% at the maximum possible content for a glyceride having as substituents only polyunsaturated acids as described above, preferably EPA and / or DHA. In more general terms, we will summarize the polyunsaturated acid content as described above, as between 15 and 90% of the weight of the composition, more frequently between 15 and 60%. ; This origin of the glycerides, directly or indirectly, from "natural" and apparently reassuring sources, instead entails serious health hazards in all the uses mentioned above, due to the known presence of numerous environmental pollutants ("dioxins", polychlorinated biphenyls ,etc.). Many of these are particularly fat-soluble and tend to concentrate together with the various fat components (hence the definition of Persistent Organic Pollutants, POPs). The main characteristics of these substances are persistence over time, bioaccumulation through the food chain, the potential for their long-distance diffusion and environmental transport, and their toxicity. Many of these substances are clearly teratogenic, mutagenic and carcinogenic. The danger posed by these substances to humans and animals has led to growing concern about the content of toxic substances in food and the food chain. Food products that do not contain, or have limited content of pollutants, are gaining greater popularity and market capacity. Therefore, the elimination or reduction of pollutants in food products has a great potential to substantially increase their sales potential and their added value, but it also has a high ethical value with particular reference to their use in the "baby foods" and "Infant formulas" and the risk of transmission to the infant through breast milk. ; The first official act against these substances resulted from the "Stockholm Convention on POPs" held in May 2001 in Sweden, which identified 12 distinct chemical classes as the most aggressive and dangerous for human health and the environment, including agriculture and cattle breeding. Many of these substances were in the past used as pesticides (herbicides, insecticides, fungicides, rodenticides, etc.), for others the production was unconscious and unintentional, being unwanted by-products of a series of chemical and / or combustion processes. The conclusion of the Convention was to ban the production and use of numerous of these substances, and the attempt to control all environmental pollutants was then pursued in the following years by the various POPs Review Committecs, as well as by the various Regional Agencies for the Environmental Protection. ; In detail, the "dioxins" are actually made up of the two chemical families of polychlorinated dibenzo-para-dioxins (PCDDs) and polychlorinated dibenzo-furans (PCDFs): of these only 7 PCDDs and 10 PCDFs cause particular concern from the point from a toxicological point of view. ; Generally PCDD / PCDF are detected as mixtures of the latter toxic congeners, having attributed to each of them an appropriate "toxic equivalent factor" (TEF), more precisely the WHO-TEF (European Commission Regulation (EC) 1881/2006 , Off.J.EU, L364 / 5; 20 December 2006, p.20). The maximum level of WHO PCDD / PCDF TEQ (Equivalent Toxicity) for oils of marine origin intended for human consumption according to the aforementioned Regulation (EC) 1881/2006, p. 18, is equal to 2 pg / g oil. According to USP 37, the acceptability criterion corresponds to “no more than” (NMT) 1 pg / g of WHO toxic equivalents. ; Another group of chemical contaminants consists of industrial agents such as polychlorinated biphenyls (PCBs), made up of variously chlorinated biphenyl molecules: only 12 have toxicological characteristics similar to "dioxins" and "furans", and are therefore defined as dio ssino-like (DL-PCBs). The TEF equivalent toxicity factors have also been determined for DL-PCBs and their TEQ equivalent toxicity is usually given cumulatively with that of dioxins. Their maximum level is equal to 10.0 pg / g oil.; The sum of 6 other congeners defined as "indicators" or "markers" is considered by the EFSA (European Food Security Agency) to be an adequate indicator of the presence of non-dioxin-like PCBs (N DL-PCBs) and human exposure According to USP 37, the acceptability of PCB markers - including PCB 118 (IUPAC denomination) - is limited to NMT 0.5 ppm (0.5 micrograms / g).; PBDEs are polybromo-diphenyl ethers, therefore consisting of diphenylether molecules variamen te brominated, and some of them are highly neuro toxic and also can cerogen i. They are used as flame retardants, and are considered as persistent chemical pollutants of the "emerging" type. Of some terms, industrial production has already been banned, but their presence has nevertheless been demonstrated in polluted waters and landfills, and in many fish oil samples (Zennegg M et al, Organohalogen Compounds, 68, 1967,2006; US 7,732,488). ; Polycyclic aromatic hydrocarbons (PAHs or PAHs) are compounds formed by two or more condensed aromatic rings and are of toxicological interest as they are considered possible carcinogens. PAHs are usually expressed as a marker substance benzo [a] pyrene, with a maximum tolerated limit of 2 ng / g in oils and fats (Regolati on (EC) 1881/2006, Section 6, p.18). ; Among the other POPs not discussed in detail here, we still mention by way of non-exhaustive example the 2,2 bis- (p-chlorophenyl) -ethane (DDE), the 2,2 bis- (p-chlorofentl) -l, 1-dichloroethane (DDD), and 2,2 bis- (p-chlorophenyl) -1,1, 1-trichloroethane (DDT), the latter found everywhere in the global environment, the polybromo-biphenyls (PBB), Tesachlor-benzene, the isomers of hydrochlor-cyclohexane, and others. ; Current methods to mitigate the content of these environmental pollutants in glycerides, e.g. in the triglycerides of fish oils, relatively very high if the catch comes from very polluted seas, it is part of the basic routine procedures on raw materials, such as "bleaching" (treatment with activated carbon) and deodorization (stripping in a current of steam) . These standard procedures are however of modest effectiveness (Breivik 2007, page 133), and in a more advanced phase of the treatment, therefore, molecular distillation or distillation or short path is used, which currently represents the most usual method for the purification of environmental pollutants. . ; In more recent times, the patent application W004 / 007654 (EP1523541 and US 7,732,488) described a process for decreasing the quantity of environmental pollutants in a mixture of oils or fats, whereby a "working" volatile fluid is added to the mixture , and then the mixture is subjected to at least one "stripping" phase during which part of the environmental pollutants are distilled together with the "working" volatile fluid. ; This method seems to be an improvement, but it is not decisive with respect to the previous art, as it involves high temperatures for an efficient stripping phase, long heating times, formation of by-products, complex systems such as the molecular still, and therefore overall high costs. Furthermore, the presence of numerous classes and within them of different molecular species of POPs, each with its own chemical-physical characteristics and a wide range of boiling points in. dependence on their degree of substitution (chlorination, bromination, etc.), make this work very difficult, and generally able to reduce the presence of a pollutant without practically ever eliminating it in its entirety, and in the favorable case for a single product without being able to extend the purification of all POPs. ; We have therefore concluded that none of the current techniques is able to purify the compositions under discussion from all the impurities described above, and only if used in combination, repeatedly and with serious losses in yield can they approach the limits imposed by the various legislations, without never reach a substantial absence of any pollutant, as it would be ethically desirable, or even imperative at least in the use of the compositions in early infancy or during breastfeeding. ; The systematic elimination of each and all POPs, on the other hand, appears to be easily achievable with the method of the Invention. ; SUMMARY OF THE INVENTION; In a first aspect, the present invention relates to a method for the purification of a composition comprising monoglycerides, and / or di-glycerides and / or tri-glycerides of any origin, in which the glycerol is at least partially esterified with long-chain saturated or unsaturated fatty acids having 16-22 carbon atoms, this composition containing persistent organic environmental pollutants (POPs), in which the method comprises the steps of:; a) treating a part by weight of the above composition with at least 3 parts by weight of urea in a polar solvent, preferably a protic solvent such as a lower alcohol, such as methanol or ethanol, optionally containing up to 20% water, at a temperature close to boiling, to form an ureic complex of essentially total inclusion of the composition ; b) cooling until precipitation of the aforesaid urea complex, and isolating it, by filtration or centrifugation, from the mother liquors containing the aforesaid persistent organic environmental pollutants (POPs); ; c) obtaining a purified composition with a reduced POPs content by dissolving in water the ureic complex of inclusion and separation of the oily phase separated following dissolution or by extraction of the oil phase with an organic solvent immiscible with water, typically hexane or the like , and subsequent evaporation of the solvent until dryness, or by direct extraction from the inclusion urea complex by means of supercritical fluids, in particular carbon dioxide. Preferably, in the cooling step b) the temperature is brought to about 4 ~ 5 <C> C and the isolated urea complex is washed with the aforementioned polar solvent, previously saturated with urea and preferably cooled to a temperature of about 4-5 ° C, before being dissolved in water or extracted with supercritical fluids. Furthermore, the mother liquors and the washing waters of the aforementioned isolated urea complex can be combined and treated, as a second purification step, with an additional 2-3 parts by weight of urea (with respect to the starting composition), repeating the steps from a) a c) formation of the ureic complex of inclusion, cooling, isolation and obtaining the purified composition. ; If necessary, the mother liquors obtained from the second phase of precipitation and isolation can also be collected and combined, as well as the washing waters of the second isolated inclusion urea complex and repeat steps a) to c) again (third purification step). In one aspect of the method according to the present invention, the aforementioned long-chain fatty acids have 18-22 carbon atoms, preferably 20-22 carbon atoms, and are of the polyunsaturated type (PUFA), i.e. containing 2-6 double bonds , preferably 5-6 double bonds, belonging to the class of omega-6 and / or omega-3 acids, preferably to the class of omega-3 acids, more preferably represented by eicosapentaenoic acid (EPA, C20: 5 n-3, all cis ) and / or docosahexaenoic acid (DHA, C22: 6 n-3, all cis). ; In a further aspect, the triglycerides of the above composition have natural origin and come from vegetable seed oils mainly comprising omega-6 acids, or from fish oils, also from aquaculture, or from "krill oils" or from algae and other oil-bearing microorganisms, or from "single celi fermentation" starting from selected strains of algae or other microorganisms and mainly include omega-3 acids, in particular EPA and / or DHA, and in which the content of polyunsaturated fatty acids as defined above, in particular EPA and / or DHA, is greater than or equal to 15%, or is between 15 and 90%, or in particular it is between 15 and 65%. ; in a further aspect of the method according to the present invention, the composition comprising the aforesaid mono-, and / or di-, and / or tri-glycerides is obtained chemically and / or enzymatically, by means of selective lipases for fatty acid and its position on glycerol, starting from natural triglycerides, or from glycerides or glycerol and polyunsaturated fatty acids, through known procedures, and in which the above composition has a content of polyunsaturated fatty acids as defined above greater than or equal to 30%, or including between 30 and 90%, or in particular the content - expressed as triglycerides - of the sum of EPA and DHA is greater than or equal to 45% and the sum of the total omega-3 acids is greater than or equal to 60%, in accordance with monograph 01/2009: 1352 of the E.P.7.0. In another aspect of the method according to the present invention, the above composition comprises diglycerides, wherein the glycerol is esterified with phosphoric acid condensed with amino alcohols to form the corresponding phospholipid, in particular phosphatidylcholine and / or phosphatidylethanolamine. Preferably, step a) of the method according to the present invention, that is the treatment of the above composition with urea, is carried out using methanol as the polar solvent in a quantity equal to 4.5-7 parts by weight, or ethanol in a quantity equal to 45-65 parts by weight. ; The persistent organic environmental pollutants (POPs) mentioned above mainly include polychlorinated dibenzo-paradixins (PCDDs) and polychlorinated dibenzo-furans (PCDFs), and / or dioxin-like polychlorinated biphenyls (PCBs) (DL-PCBs) and markers, and / or polybromodiphenyl ethers (PBDEs), and / or polycyclic aromatic hydrocarbons (PAHs), and their presence in the purified composition obtained in step c) is at least twice reduced, preferably at least 5 times reduced, conveniently at least 10 times reduced, and in any case lower - for each component - to the respective limit of quantification (LOQ) with the standard GC-MS method. In other aspects, the present invention relates to a composition comprising mono-glycerides, and / or di-glycerides and / or triglycerides, as defined in claims 10, 11 and 23, as well as the melt of this composition, for the preparation, from mono-glycerides, and / or diglycerides and / or tri-glycerides, of the corresponding polyunsaturated fatty acids, or their salts, by chemical or enzymatic hydrolysis, or for the preparation of the relative C1-C3 alkyl esters, as specified in claims 12 and 13 Furthermore, the present invention relates to a composition comprising such polyunsaturated fatty acids or their C1-C3 alkyl salts or esters obtained by chemical or enzymatic hydrolysis, as specified in claims 14-16. invention relates to the use of a composition comprising the aforementioned mono-, di- and triglycerides or the corresponding polyunsaturated fatty acids or their salts or C1-C3 alkyl esters for the preparation of formulations useful as food ingredients, food and dietary supplements, foods for special medical purposes (functional foods), foods for animal use and for aquaculture, food formulations for children, cosmetic and pharmaceutical preparations, as specified in claim 17.; Finally, the present invention relates to a composition comprising the aforementioned mono-, di- and triglycerides or the corresponding polyunsaturated fatty acids or their C1-C3 alkyl salts or esters for the therapeutic uses defined in claims 18-21, as well as a pharmaceutical formulation which comprises them according to claim 22.; DETAILED DESCRIPTION; We specify that the use of urea in lipid chemistry is absolutely known. ; A standard procedure provides that an oil or fat is hydrolyzed to give the acid components, or transesterified in an alcoholic medium to give the corresponding esters, by chemical or enzymatic way, and then the polyunsaturated components are concentrated: this generally takes place by molecular distillation, extraction with supercritical fluids, or other methods, but even more often by complication with urea ("inclusion" in urea}. The use of urea, however, is presented only as a suitable means for isolation and removal of saturated and monounsaturated fatty acids, and therefore to the concentration of the polyunsaturated acids in the reaction solvent: this is therefore accompanied - among other things - by an increased concentration of foreign impurities together with the then unsaturated components. urea appears from the literature of increasing difficulty as their concentration and theirs increase relative degree of saturation: in the treatment of a mixture of fatty acids, the PUFA thus appears to be the “passive” component of the complexation. ; The profound difference with the method of the present invention therefore results, for which, for example:; —the "complexation" is carried out directly on glycerides (including natural oils and fats, or other particularly selected glycerides), instead of on fatty acids or their foreigners); - the glycerides are the “active” part of the complexation, while the PUFAs of the literature in their essence are not suitable for complexation, if not marginally, and remain unchanged in the solvent; ; —The literature procedure is aimed at increasing the concentration of PUFAs, enriching them in the mother liquors of the complexation, while it remains essentially constant during our purification process; ; - the purpose of the completion according to the new procedure is to strongly decrease, essentially cancel, the presence of environmental pollutants all officially declared teratogenic, mutagenic and carcinogenic or generally toxic, instead of simply leading to the concentration of PUFAs and their esters, according to the current standard purposes. ; On the use of urea as a complexing agent and related processes, there are many publications, among which we cite the extensive reviews of Schlenk H, "Urea inclusion compounds of fatty acids", in: Progress in the Chernistry of Fats and Other Lipids Voi Il (RT Holmar, ed.), Pergamum n Press, New York, pp. 243-267 (1954), and by Swem D, “Techniques of separation. Urea complexes ", in: Fatty Acids, Pari 3 (KS Markley, ed.), Interscience, New York, pp. 2309-2358 (1963). In particular, mention should be made of the aforementioned “Breivik 2007”, in the chapter “Concentrates”, pages 111-130. ; In relation to the glycerides, subject of this Application, Aylward F and Wood PDS, Chem & fnd. (London), 53 ((1956); Nature, 177, 146 (1956) (ref. 127 and ref. 129 by Swern, cited above, page 2337-8), and others, report the ureic complex of the mono-glyceride in position 1 of stearic acid (CI 8: 0), as well as a series of lower homologs, from caprylic acid C8: 0 to palmitic acid C16: 0. Apparently the rnonoglycerides in position 2 do not form complexes. in all cases of glycerides of saturated acids and attempts at fractionation and enrichment of various components, and not purification from environmental pollutants.; The same authors and others (Martinez Moreno JM et al, Grasas y aceites (Seville, Spain), 7, 285 (1956), ref. 131 by Swern, cited above) report that the di-glycerides in position 1,3 are capable of giving complexes with urea, while the tri-glycerides do not form complexes. tri-glycerides, already anticipated by Schlenk, cited above, page 251, is then also confirmed in the review of "Breivik 2007", page 1 18 , as an expert in the harte. ; Other authors cited by Swern, page 2338 (ref. 128, 132, 133), have studied the formation of monoglycerides by glycerolysis of vegetable oils, usually containing aryls up to C18 in length and up to 3 double bonds, and have attempted with their separation from the other glycerides by complexation with urea under certain conditions is poor. In particular Heckles J.S. and Dunlap L.H., J Am Oil Chernists <7> Soc, 32, 224, 1955 (ref. 133) report that monoglycerides of saturated and monounsaturated acids (oleic acid) form urci complexes, while those with 2 or 3 unsaturation points ( resp. linoleic and linolenic acid) do not complex. When mixed with saturated and unsaturated diglycerides, a separation by complexation is obtainable for the glycerides of saturated acids, while only a slight enrichment is obtainable with the glycerides of unsaturated acids. However, this conclusion is not shared by Mehta T.N. and Shah S.N., J Am Oil Chernists' Soc, 34, 587, 1957 (ref. 128). Also in these cases, and within the limits of the specified products, the discussion focuses only on the possibility of fractionation and enrichment through complexation. ; In recent times, Hayes D.G. et al., JAOCS, 77,207, 2000 report that mono-, di-, and tri-acylglycerols are poor templates for the formation of urea complexes (UC) compared to free fatty acids (FFA) and that their inclusion, in mixtures of acylglycerols containing FFA, even reduces the formation of UC (see Abstract), a conclusion then taken up also by experts in the art, as exemplified by "Breivik 2007", page 118. data reported on acylglycerols appear to us to be absolutely scarce, often in conflict with each other, limited (as only occasionally, if indeed it happens, do they include the PUFAs of greatest interest, with a longer chain and more unsaturated), and almost always linked to specific cases and therefore not generalizable. The ability to complex is often attributed to a single or a few factors, while in our opinion it depends on all the reaction parameters as a whole, such as e.g. the degree of glyceride substitution, the length and degree of unsaturation of the acyl, the relationship with urea. the type and volume of solvent, the precipitation temperature of the solvent, and so on, each factor interfering with all the others. ; The problem can thus be solved only experimentally, having in mind the goal to be achieved and the products to be investigated. We have thus found, through a series of experiments, that all glycerides in the broadest sense and in an essentially total way and under certain conditions in a way that is little correlated to their structure, can be complexed with urea. This then allows the precipitation of the complex and the total removal, dissolved in the solvent in an uncomplexed form, of all persistent organic pollutants (such as the dreaded dioxins and related substances, pesticides used in agriculture, etc.) that are increasingly found today widely in the animal and vegetable environment and in many foods, in particular in natural oils and fats and in the substances deriving from them, representing a continuous source of danger for human health even if present within the limits imposed by these laws and by the various Pharmacopoeias . A first aspect of the invention provides a purification method which consists in treating the glyceride of a fatty acid in a suitable solvent with an excess quantity of urea, sufficient for an essentially total complexation of the glyceride, usually greater than or equal to 3 parts. by weight and up to 6 parts by weight or more, possibly dividing the treatment into 2-3 consecutive steps, if necessary, preferably into 2 consecutive steps, and in any case until essentially total complexation is reached. The subdivision into several steps is not particularly harmful to the process because it involves only an additional filtration, as it is possible to add a further aliquot of urea directly to the filtered solution and to the washing waters of the inclusion urea complex without further manipulation. ; The treatment results in a precipitation of the complex, possibly divided into the respective 1-3 fractions, then the complex is collected by filtration or centrifugation, and carefully washed with the same solvent saturated with urea and pre-cooled around 4-5 ° C, or less, while the solvent containing all the environmental pollutants (POPs) as described above and others reported in the specialist literature, and any other impurities not covalently linked to the glyceride and not complexable, is eliminated. From the urea complex, or from the combined fractions, the purified glyceride is therefore easily recovered and essentially devoid of the aforementioned POPs. In the context of the present method, the glyceride may be a mono-, and / or di- and / or preferably a tri-glyceride (also called monoacyl-, diacyl-, and triacyl-glycerols), and these will be at least partially esterified - in the far more preferred case - with long-chain fatty acids, comprising 18-22 carbon atoms, preferably 20-22 carbon atoms, and of the polyunsaturated type, i.e. comprising 2-6 double bonds, preferably 5-6 double bonds. It refers in particular to acids belonging to the class of omega-6 and / or omega-3 acids, more preferably to the class of omega-3 acids, in particular to EPA and / or DIIA. These glycerides can have any origin and in the case of triglycerides usually represent natural oils (or fats), in particular vegetable oils, preferably fish oils and the like. ; Omega-6 acids are particularly abundant in vegetable oils and seeds, while omega-3 acids and in particular EPA and ΌΗΑ have mainly marine origin and derive essentially from fish oils, also from aquacultures, or from "krill oils" or even from algae and other oil-bearing microorganisms, or from "single cell fermentation" starting from selected strains of algae or other microorganisms. ; The content of polyunsaturated fatty acids as defined above, in particular EPA and / or DHA, is greater than or equal to 15%, or is between 15 and 90%, or in particular is between 15 and 65%. ; By way of example, we remind you that a typical content of a natural fish oil used for the production of PUFAs is represented by EPA 18% and DHA 12%. while 90% is around the maximum possible content for a glyceride having as substituents only polyunsaturated acids as described, e.g. EPA and / or DHA. Mono-glycerides and di-glycerides, on the other hand, are mainly industrial products, and all the glycerides enriched in polyunsaturated acids are also obtained by chemical and / or enzymatic processes. For this purpose, selective lipases are often used for fatty acid and its position on glycerol, starting from natural triglycerides to hydrolyze saturated and monounsaturated acids, or from various glycerides or glycerol for the action of concentrated polyunsaturated acids and using known procedures. ; In these cases the content of polyunsaturated acids will usually be greater than or equal to 30%, or between 30 and 90%, or in particular the minimum contents of the sum of EPA and DHA and of the sum of the total omega-3 acids - all expressed as triglycerides - will be respectively 45% and 60%, in accordance with monograph 01/2009: 1352 of the E.P.7.0 relating to the glycerides of omega-3 acids chemically or enzymatically reconstituted and enriched in PUFA. These raw material contents do not usually increase substantially in the course of the purification in question, except in special cases. ; From an operational point of view, the present purification process shows some similarities with the enrichment process of PUFAs and their esters by complexation with urea, being able to distinguish:; -a) a reaction phase for which a part by weight of glyceride is treated with 3-6 parts or more by weight of urea (divided into 1-3 steps) in a polar solvent, preferably a practical solvent such as a lower alcohol, such as methanol or ethanol, possibly containing up to 20% water (or others, as indicated by Swern D, already mentioned), at a temperature close to boiling, to form a ureic complex of inclusion of the glyceride; ; - b) a cooling phase at about 4 <5> -5C until precipitation of this ureic complex (or complexes), isolation by filtration or centrifugation (with elimination of the filtered mother liquors) and thorough washing of the complex or complexes combined with the same solvent saturated with urea and pre-cooled to about 4-5 ° C; ; - c) a step of obtaining a purified composition by dissolving in water of said ureic complex of inclusion and direct isolation of the oily phase separated as a result of said dissolution or by extraction of said oily phase with an organic solvent immiscible with water, typically hexane or the like and subsequent evaporation of said solvent to dryness, or again by direct extraction from said inclusion urea complex by means of supercritical fluids, in particular carbon dioxide, operations followed by optional molecular distillation. ; It will also be evident to the expert that the purpose of the entire process is totally different, as well as in phase a) the use of urea in strong excess and possibly in several steps, in phase b) the careful washing of the complex to eliminate the pollutants contained in the mother liquors incorporated, in phase c) the recovery of the purified product from the solid complex, instead of the recovery of the composition enriched in PUFA carried out on the mother liquors as taught by the "prior art". ; The purified composition according to the invention will have essentially the same composition in glycerides and therefore in fatty acids as in the raw material, but will be substantially free from all the toxic environmental pollutants described above, such as dioxins and furans, polychlorinated biphenyls, polybromodiphenyl ethers, cyclic and polycyclic hydrocarbons, simple and condensed, and other POPs known in the specialized literature. ; In particular, it is understood that each of the aforementioned substances and their sum will be at least twice, or at least 5 times reduced, or preferably at least 10 times reduced (reduction of at least 90%) with respect to the compositions from which they come, if initially present in concentrations correspondingly higher than their limit of quantification (LOQ), or still each substance will be lower than its limit of quantification (LOQ) with the standard GC-MS method for which, equating its value to "zero" (lower bound), we will define the They add "essentially zero". A value "zero" corresponds instead to a sum of data below the limit of detection (LOD). congener will be lower than its LOQ limit of quantification (essentially zero).; ~ polychlorinated dibenzo-pàra-dioxins (PCDDs, n = 7) and polychlorinated <1> dibenzo-furans (PCDFs, η <~> 10) in overall concentration less than or equal to 1.0 pg / g, value determined in accordance with the WHO toxic equivalent factors (TEFs) and expressed as toxic equivalents (TEQs}; - similar (DL-PCBs, n = 12) in total concentration lower than or equal to 5.0 pg / g, value determined as defined above (TEQs);; - PCBs marker (n = 6) in total concentration lower than or equal to 5.0 ng / g;; - polybromo-diphenyl ethers (PBDEs, tested n = 9) in complete concentration ssiva less than equal to 5.0 ng / g; ; - a sum of polycyclic aromatic hydrocarbons (PAHs, tested n-4), expressed as benzolapyrene marker substance, less than or equal to 1.0 ng / g; ; - other persistent organic pollutants (POPs) including 2,2 bis- (p-dichlorophenyl) -ethane (DDE), and / or 2,2 bis ~ {p ~ dichlorolenyl) -1,1 -dichloroeta. no (DDD), and / or 2,2 bis- (p-dichlorophenyl) - 1,1,1-trichloroethane (DDT) in a total concentration lower than or equal to 2.0 ng / g, polybromo-biphenyls (PBB) in total concentration less than or equal to 5.0 ng / g; hexachlorobenzene in a concentration less than or equal to 0.1 ng / g, and isomers of and sacloro-hexane in a total concentration less than or equal to 0.1 ng / g. ; Also all the other POPs listed by the Stockholm Convention of 2001 and by the subsequent Review Committees, could be cited here as they are in all cases of spherically "bulky" molecules, not suitable for complexation with urea, and easily eliminated quantitatively during the present purification process. ; It will then be evident that other impurities can also be eliminated in the process, if not covalently linked to the glyceride, and if not complexable with urea, such as some aliphatic hydrocarbons with a long chain and branched structure (typical squalene), some oligomers and polymers of acids fats, some sterol substances, and the like. A second aspect of the invention relates to the glyceride compositions themselves obtained according to the purification method described above. ; Among these compositions we explicitly mention a purified composition of mono-, di-, and tri-esters of omega-3 acids with glycerol, containing mainly tri-esters and a sum of EPA and DHA expressed as rriglycerides of at least 45%, and of Total omega-3 acids expressed as triglycerides of at least 60%, in accordance with the monograph 01/2009: 1352 of the E.P. 7.0, and having a highly reduced content of POPs pollutants, well beyond the limits described above (essentially absent). A third aspect of the invention is the same easily deducible from the availability of glycerides itself, preferably triglycerides, in particular natural triglycerides rich in polyunsaturated PUPA acids and more particularly in omega-3 acids such as fish oils and the others described above, all free from environmental pollutants and highly purified: this aspect therefore concerns the use of said glycerides for the chemical or enzymatic production of other compositions based on PUPA and omega-3 acids and derivatives in more or less concentrated form, according to standard literature procedures described in the chemistry of these substances. ; In fact, the current technology already provides that, starting from natural oils of various origins and therefore from complex mixtures, the most interesting acid components for pharmaceutical use or as food or dietary supplements, such as eg e.g. EPA and / or DHA, and therefore their concentration. by means of alcoholic potash, thus obtaining the corresponding potassium salts and therefore the free acids and - if desired - the alkyl esters; or more often a transesterification reaction is used, e.g. in the presence of excess alitatic alcohols, preferably C1-C3, and of an alkaline or acid catalyst, thus directly obtaining the corresponding alkyl esters of the fatty acids, and from these - if desired * - the relative acids or salts.

The subsequent phase of concentration of the most valuable components, and / or adjustment of their ratio according to request, and / or isolation of a single component, is carried out with different methods, also combined, among which mainly:

-the distillation under high vacuum, usually the molecular distillation or short path more suitable for limiting the thermal degradative processes, obviously providing for the appropriate fractionations;

- for chromatographic processes, even under high pressure (HPLC), which are however more suitable for the laboratory scale or for analytical purposes;

- extraction with supercritical fluids (SFE), usually with C02, also in combination with chromatographic processes (SFC) on a preparative scale and with suitable stationary phase, more specific for the separation of single components;

-in the most general and industrially accepted case, a complexation reaction is however used with urea in various solvents, such as alcohols. It is known that urea under particular conditions can crystallize in hexagonal crystals, with the formation of channels capable of including the linear chains of fatty acids, essentially those of saturated and / or monounsaturated acids and esters or with a lower degree of unsaturation. . The formed inclusion complex precipitates by cooling from the alcoholic solution and is isolated and eliminated, thus allowing to recover from the solution a composition enriched in polyunsaturated components, such as e.g. EPA and DHA ("passive" complexation),

This concentration phase is usually followed by a final molecular distillation to remove small low- and high-boiling fractions.

All these procedures, which can be easily adopted also with the purified glycerides of the invention, are extensively described e.g. from “Breivik 2007”, Chapter “Concentrate s”, pag. 111-130 and References cited, and require no further description.

Starting from recent years, said hydrolysis or alcoholysis processes, as well as chemically are also carried out by enzymatic way, by means of selective lipases, even immobilized: this allows to operate in even milder reaction conditions, moreover - by virtue of the greater resistance of PUFAs to enzymatic cleavage, with respect to saturated and monounsaturated acids as well as subsequent optional processes of fractionation of the components and / or enrichment to give compositions of acids or their salts, of esters and even of “structured” glycerides, all enriched and concentrated in certain polyunsaturated components.

These processes for enzymatic catalysis are also well described in “Breivik 2007”, Chapter “Enzvmatic processing of omega-3 specialty oils”, p. 141-164, and are clearly adoptable also with the purified compositions of the invention.

It therefore follows that, starting from the glycerides purified according to the process, it will be possible to obtain all the compositions comprising the corresponding acids in free form, or the relative salts, or the relative esters. In addition, it will still be possible to obtain all the respective compositions enriched in the polyunsaturated components, exactly as feasible e.g. starting from natural "fish oils" or other oils, with the only difference that in this case compositions are obtained that are essentially free from the various pollutants defined as POPs and from the consequent health risks of the humankind.

While the acids are obtained by chemical or enzymatic hydrolysis of the glycerides, as reported above, the salts of the polyunsaturated acids are obtainable from these e.g. by direct salification and are represented by salts with alkali metals, e.g. sodium and potassium, alkaline earth metals, e.g. calcium, with basic amino acids such as lysine and arginyl a, with meglumine, with choline and mono-, di-, and tri-ethanolamine, and the like, if pharmacologically acceptable.

Alkyl esters are obtainable e.g. by chemical or enzymatic alcoholysis and are represented by esters with aliphatic alcohols, even with a very long chain as found in natural "waxes", but they are preferably represented by esters with lower C1-C3 alcohols, in particular ethyl weight.

The reconstituted and concentrated PUFA glycerides can be prepared by esterification e.g. omega-3 acids concentrated and purified with glycerol, or by transesterification of esters with glycerol.

Moving on to greater detail, it is thus possible to obtain, by way of example, a generic composition of lipids and some compositions of the acids or esters that can derive from them as described below.

- A composition comprising mono-glycerides, and / or diglycerides and / or tri-glycerides of any origin, in which the glycerol is at least partially esterified with long chain fatty acids having 18-22 carbon atoms, preferably 20-22 carbon atoms carbon, and polyunsaturated type (PUFA), i.e. containing 2-6 double bonds, preferably 5-6 double bonds, belonging to the class of omega-6 and / or omega-3 acids, preferably to the class of omega-3 acids, plus preferably represented by eicosapentaenoic acid (EPA, C20: 5 n-3, all cis) and / or docosahexaenoic acid (DHA, C22: 6 n-3, all cis), in which the composition further contains the following persistent organic environmental pollutants ( POPs):

polychlorinated dibenzo-para-dioxins (PCDDs) and polychlorinated dibenzo-furans (PGDFs) in a total concentration lower than or equal to 1.0 pg / g, value determined in accordance with the WHO toxicity equivalent factors (TEFs) and expressed as equivalents toxic (TEQs);

PCDDs, PCDFs and polychlorinated biphenyls (PCBs) of non-similar oxydes (DL PCBs) in a total concentration lower than or equal to 5.0 pg / g, a value determined in accordance with the TEF values of the WHO and expressed as TEQs;

- PCBs marker in overall concentration less than or equal to 5.0 ng / g;

- poly bromo-diphenyl yesterday (PBDEs) in a total concentration lower than or equal to 5.0 ng / g; And

- a sum of polycyclic aromatic hydrocarbons (PAIfs), expressed as a marker substance benzo [a] pyrene, less than or equal to 1.0 ng / g;

each of said POPs being preferably contained in said composition in a concentration lower than its quantification limit LOQ with the standard GC-MS method.

- A composition comprising EPA and / or DHA in the form of free acids or in the form of ethyl esters, in which the total concentration is between 15 and 100%, preferably between 50 and 100%, of the weight of the composition, and a content of pollutants as described above and in accordance with the purified compositions of glycerides from which they come.

- A composition in which the concentration by weight of EPA ethyl ester, or DI-IA ethyl ester, is greater than or equal to 80%, preferably greater than or equal to 90%, or their sum is greater than or equal to 80%, preferably greater than or equal to 84%, being the concentration of EPA ethyl ester greater than or equal to 40%, the concentration of DHA ethyl ester greater than or equal to 34% and the sum of the concentrations of all the omega-3 ethyl esters greater than or equal 90% in accordance with the specifications of the European Pharmacopoeia (EP), and a pollutant content as described above.

- A composition in which the sum of the concentrations by weight of ethyl ester EPA and ethyl ester DHA is between 80.0 and 88.0%, the concentration of ethyl ester EPA being between 43.0 and 49.5%, ia concentration of DHA ethyl ester between 34.7 and 40.3%, and the sum of the concentrations of all omega-3 ethyl esters greater than or equal to 90% in accordance with the specifications of the United States Pharmacopoeia (USP) , and a pollutant content as described above.

Having defined some of the compositions obtainable chemically or enzymatically starting from the purified glycerides, a fourth aspect of the invention comprises the addition to the purified glycerides and their derivatives of suitable diluents, excipients, suspensions, eoe and / or preservatives, antioxidants, etc., according to all the technologies known in the part to give all the formulations known in the part to allow their use in all the proposed uses. These formulations include, in addition to their direct inclusion, p. And. in the various foods, or in the form of micro-encapsulated or nano-encapsulated or cochleate obtained according to the known art, also the formulations for oral fusion - such as drops, soft gelatin capsules, hard gelatin self-sealing capsules, tablets, if necessary after adsorption on support solid or as an inclusion complex, if necessary also in a gastro-resistant formulation, etc. - as known in the part, or for topical fuso <.> such as creams, ointments, etc. ~ as also known in the part, or again for injection fuso - as vials for fuso intramuscular, slow intravenous infusion drip, etc., after sterilization and / or if necessary after chemical and / or physical modification, e.g. such as glycerides in emulsion, as well known in the art. With reference to their use, said purified compositions and their formulations will be addressed to the preparation and use as food or food ingredient, of every type and for every purpose, as a food and dietary supplement, food for special medical purposes (functional food) , both new and deductible from pharmaceutical use, food for animal use and aquaculture, baby food formula, cosmetic and pharmaceutical preparation, all containing or enriched in long-chain fatty acids or their derivatives, with a polyunsaturated character or specifically of omega-3 series.

For all uses, but in particular the preparation for pharmaceutical use, it will preferably be enriched and concentrated in the polyunsaturated components, in particular in EPA and / or DHA, or their salt, or their ethyl ester, or their glyceride.

Of great and growing importance are foods for special medical purposes, or functional foods - including drinks and supplements - which include specific ingredients, such as omega-3 acids in all their forms, capable of imparting certain specific benefits for the health (fortified foods). These functional foods are currently known under the English term FOSHU (Foods for Specified Health Use) or the Japanese term Tokuho.

Going into detail, the composition of the compositions will be aimed at the prevention and treatment of risk factors for heart, cardiovascular and cardiovascular diseases, such as piperten sione, severe and moderate hypertriglyceridemia (resp.> 500mg / dl and> 200mg / dl) and 1 hypercholesterolemia , in particular the familial and genetic forms, also in association with other drugs and in particular with statins, and as the defects of coagulation and platelet aggregation.

A very important use is that for the prevention and treatment of heart, cardiovascular and cardiovascular diseases, such as coronary-atherosclerotic and ischemic cardiac and cerebral states, including myocardial and cerebral linfarction and the reduction of the risk of sudden cardiac mortality resulting from myocardial infarction; those of electrical origin and involving the onset and propagation of the heart rhythm, including arrhythmia and atrial and / or ventricular fibrillation; and those due to mechanical defects of the heart pump, such as heart failure and heart failure and / or congestive "heart failure".

In other pharmaceutical uses the compositions are used for the prevention and treatment of disorders of the central nervous system (CNS), including epilepsy, various forms of depression, bipolar disorders, pediatric attention deficit disorders and hyperactivity disorders. (ADHD), learning and memory defects, various forms of schizophrenia, Alzheimer's disease and various forms of dementia. Memory improvements, particularly in children, are of great interest.

Still other pharmaceutical uses include the prevention and treatment of retinopathy and symptoms of dryness, ocular, metabolic syndrome, metabolic and obesity-related defects, type 2 diabetes, liver dysfunction, connective tissue disease and joints, inflammatory states, autoimmune diseases, ulcerative colitis, psoriasis and cancer.

A further aspect of the invention still relates to the purification method as described above, but applied to a composition comprising diglycerides, in which the glycerol is esterified with phosphoric acid condensed with amino alcohols to form the corresponding phospholipid, in particular phosphatidylcholine and / or phosphatid. happy n oi am i na.

This aspect of the invention obviously also relates to the purified phospholipid composition resulting therefrom.

The final aspect of the invention finally concerns the purification of a composition comprising a mono-glyceride, and / or di-glyceride and / or tri-glyceride of any origin, esterified with long-chain fatty acids consisting of 16-20, preferably 18-20 carbon atoms, of the saturated and / or monounsaturated type, and the purified composition obtained.

EXPERIMENTAL EXAMPLES

The invention will now be illustrated by means of some examples that will not have any limiting character.

EXAMPLE 1

A commercial sample of "fish oil" (fatty acid triglycerides) presented in the form of 0.5 g soft gelatin capsules and indicated as a food supplement was used.

The declared content for a daily dose of 4 "pearls" is reported here per gram of product (2 capsules) and corresponds to total omega-3 equal to 350 mg, of which EPA 180 mg and DHA 120 mg, a composition we have confirmed. by transesterification under standard conditions to medium fatty acid esters (FAME) and GC / MS analysis. The other components were found to be predominantly C16-C18 saturated fatty acids and mono unsaturated fatty acids.

In the absence of other excipients, the oil was taken directly from the capsules by syringe or by puncture and direct squeezing and from the control of the main quality parameters (value of peroxides, anisidine, color, etc.) it was judged as fish oil of good quality and duly refined for nutraceutical use.

Some informative data on the content of POPs will be given in comparison with the purified product obtained at the end of Step 1 of the Procedure.

Procedure - Step 1

200 mL of methanol and 100 g of urea are loaded into a flask, then heated under reflux under stirring to obtain a substantially clear solution.

Then 25 g of the triglycerides of fish oil are added (ratio by weight of oil: urea 1: 4), and it is then left under stirring and boiling for a further 5 min, obtaining a slightly yellowish mixture with a lot of oil still in suspension.

It is then left to cool under stirring and at about 45 ° C the precipitation of the desired complex rapidly begins, which is cooled for a few minutes with running water and then at rest for 1 hour in a refrigerator at about 5 <n> C to complete the precipitation.

At the end the precipitate is collected by filtration on buckner and squeezed well to separate as much as possible the mother liquors, which are kept apart for a second treatment. No separation of oily substances is noted in methanol.

The collected precipitate is washed thoroughly on a buckner with 2 portions of 50 mL of a solution of urea in methanol, obtained by dissolving 16 g of urea in 100 mL of hot ethanol and then cooling to 5 "C, thus obtaining at the end water of colorless washing.

98 g still wet of the precipitate are obtained, from which the purified glycerides are recovered directly by dissolution in 200 mL of 5% solution of NaCl in water and subsequent extraction with 2 portions each of 50 mL of iv hexane.

The 2 organic phases are separated from the aqueous phase and reunited, then the n-hexane is evaporated to dryness at about 35 ° C and at reduced pressure to constant weight, obtaining 21.2 g of residue consisting of purified fish oil which is stored around at 5 ° C in a nitrogen atmosphere.

The product is usually used as it is, any traces of solvents can be eliminated, if desired, with known methods and with quantitative yields.

The GCMS analysis of the corresponding FAME methyl esters shows that the composition is similar to that of the starting product.

The analysis of POPs is reported here only as the number of undetectable congeners, as their content is lower than their LOQ, compared to the number of checks performed. For comparison, the corresponding values of the raw material in the original capsules are reported, as a precaution without reporting the absolute values since it is a product duly authorized for marketing.

Product Purified raw material

Dioxins 7/7 7/7

Furani 10/10 8/10

Dioxin-like PCBs 12/12 1/12

PCB markers 6/6 3/6

PBDE 9/9 2/9

PAH 4/4 0/4

In the purified product, all the congeners of toxic and carcinogenic environmental pollutants (POPs: persisi ent organic pollutants) were found to be below the measurable limit and essentially absent, in 48 out of 48 cases. This leads us to believe that the complexation with urea acts as a sort size exclusion chromatography and that this purification can be extended to all classes of environmental pollutants of similar structure and serum size, without risk of exceptions.

In relation to the reference product, it can be noted that the dioxin and furan content is acceptable, as the control legislation has been more rigorous and has been in force for the longest time. For other pollutants, equally toxic, the situation is decidedly worse and presents some concentration peaks with high risk due to latent and poor adherence to ethical and legislative standards.

Sten 2

The methanolic mother liquors isolated by filtration of the inclusion complex of step 1 in urea, combined with the washing methanol as described above, are added with another 5Q g of urea (total ratio by weight of triglycerides: urea of 1: 6) and flow re-boil for 5 minutes under slow stirring obtaining a solution with a little oily material in suspension.

It is allowed to cool, always under stirring, obtaining rapid precipitation at about 45 ° C, and then at rest for 1 hour in the refrigerator at about 5 "C.

At the end the precipitate is filtered on buckner, the mother liquors are removed thoroughly, and washed well with 2 portions of 50 mL of a solution of urea in methanol obtained as described above and pre-cooled to 5 “C.

An abundant precipitate is thus obtained, from which further purified product is recovered as described in Step 1, by dissolving in 200 mL of a 5% aqueous solution of NaCl and extraction with 2 portions of 50 mL of n-hexane.

The dry evaporation of the n-hexane extracts as described above leads to a further oily residue of 3.3 g, also consisting of purified fish oil, which is stored at 5 ° C under nitrogen.

The GCMS analysis confirms the purity of the product and shows that its composition is essentially the same as that obtained in Step 1, so the products are combined, thus leading to a total yield of 24.5 g.

The analysis of POPs shows that their content is still undetectable and "essentially zero <5>", in 48 cases out of 48.

EXAMPLE 2

A commercial sample of "fish oil" (fatty acid triglycerides) was used, supplied in bulk and defined as if mi-refined, batch 200907. EPA content (evaluated as GC area, FAME) 17%, DHA content 11.5%, normal GC framework. It is not known which physical and chemical treatments it had already undergone, among those usual in this technology (deodorization in a current of steam, distillation, winterization, bleaching and treatment with coal, treatment with silica, alkaline washing, degumming}.

At the analytical control many values were higher or much higher than normal values (peroxides, anisidine, Totox, etc.), the color was intense orange. The analysis of POPs showed that 42 congeners among those controlled (n = 48) were present and above the LOQ, in some cases with individual values so high as to bring the overall TEQs values tens of times above the commonly tolerated limits, and others again .

The purification of this product was carried out following the procedure of Example 1, in 2 steps. The overall yield was 95%, the light yellow color and the first quality analyzes indicated that the chemical and organoleptic properties of the composition following the purification process were greatly improved (deodorization, bleaching, absence of oxidation products and material polymeric and oligomeric, etc.), thus making a part of the standard technological procedures cited above superfluous.

Above all, as a consequence of the specific purpose of the purification, the analyzes of the 6 classes of POPs routinely checked confirmed that the 48 congeners of the various families were all below their LOQ quantification limit and therefore essentially absent.

Also absent were some pesticides of the family of 2,2 bis - (p-dichlorofcnil) - ethane variously chlorinated, such as DDT, as well as polybrominated biphenyls and hexachlor derivatives of benzene and cyclohexane, present instead in concentrations around l-2ng / g in the starting product.

EXAMPLE 3

A commercial sample of "blue fish oil" (based on polyunsaturated fatty acids of the omega-3 series) presented in the form of soft gelatin capsules of 1.0 g (total weight 1313 mg) was used and indicated as a food supplement that contributes to normal heart, brain and visual function Composition (expressed as ethyl esters): EPA 360mg, DHA 180mg, total omega-3 630mg, vitamin E 5.5mg.

It is therefore a normal fish oil refined and enriched in omega-3 presumably by catalysis induced by specific lipases and in the presence of a composition of EPA and DHA, or by another method of the literature.

After purification by total complexation in urea and recovery of glycerides from the complex, according to the procedure of Example 1, the substantial absence of all controlled POPs was once again confirmed, as reported above.

EXAMPLE 4

By direct esterification with glycerol of a composition concentrated at about 80% of omega-3 polyunsaturated acids (PUFA n-3: EPA 40% about, DHA 30%, DPA 5%, C18: 4 8%, and others), plus others saturated and monounsaturated components, a preparation of approximately equal content of monoglycerides, diglycerides and triglycerides rich in good quality omega-3 acids was obtained, but confirmed for the presence of environmental pollutants POPs (28/48). The synthesis was carried out in the presence of an immobilized preparation of antarctic Candida lipase (NV-435), and the component ratios, temperatures and reaction times adopted were those reported for Experiment 14 by Noriega-Rodriguez J.A. et al, J. Food Res. 2 (6), 97, 2013.

The product obtained and analyzed by HPLC, as indicated by the same Author, was used as it is, without further manipulation, and was subjected to purification as described in Example 1.

Once again it was confirmed that the 48 congeners of the 6 classes of POPs checked were all without exception below their limit of quantification.

Claims (23)

CLAIMS X, Method for the purification of a composition comprising mono-glycerides, and / or di-glycerides and / or tri-glycerides of any origin, in which the glycerol is at least partially esterified with long-chain saturated or unsaturated fatty acids having 16- 22 carbon atoms, said composition containing persistent organic environmental pollutants (POPs), in which said method comprises the steps of: a} treating a part by weight of said composition with at least 3 parts by weight of urea in a polar solvent, preferably a protic solvent such as a lower alcohol, such as methanol or ethanol, possibly containing up to 20% of water, at a temperature close to aH boiling, to form an essentially total inclusion urea complex of said composition; b) cooling until precipitation of said urea complex, and isolating it, by filtration or centrifugation, from the mother liquors containing said persistent organic environmental pollutants (POPs); c) obtaining a purified composition with a reduced POPs content by dissolving in water of said ureic complex of inclusion and separation of the oily phase separated following said dissolution or by extraction of the oil phase with an organic solvent immiscible with water, typically hexane or the like and subsequent evaporation of said solvent until dryness, or by direct extraction from said inclusion urea complex by means of supercritical state fluids, in particular carbon dioxide. 2. Method according to claim 1, in which in said cooling step b) the temperature is brought to about 4-5 ° C and said isolated urea complex is washed with said polar solvent, previously saturated with urea and cooled to a temperature equal to at about 4-5 ° C, before being dissolved in water or extracted by means of fluids in the supercritical state. 3. Method according to claim 2, in which said mother liquors and the washing waters of said isolated urea complex are combined and treated with further 2-3 parts by weight of urea (with respect to the starting composition), repeating steps from to ) a c) forming the ureic complex of inclusion, cooling, isolation and obtaining the purified composition. Method according to any one of claims 1-3, wherein said long-chain fatty acids have 18-22 carbon atoms, preferably 20-22 carbon atoms, and are of the polyunsaturated type (PUFA), i.e. containing 2-6 double bonds, preferably 5-6 double bonds, belonging to the class of omega-ó and / or omega-3 acids, preferably to the class of omega-3 acids, more preferably represented by eicosapentaenoic acid (EPA, C20: 5 n-3, ditto cis) and / or docosahexaenoic acid (DHA, C22: 6 n-3, all cis). 5. Method according to claim 4 in which the triglycerides of said composition have natural origin and come from vegetable seed oils comprising mainly omega-6 acids, or from fish oils, also from aquaculture, or from "krill oils" or from algae and other oil-bearing microorganisms, or from "single cell fermentation" starting from selected strains of algae or other microorganisms and mainly include omega-3 acids, in particular EPA and / or DHA, and in which the polyunsaturated fatty acid content as above defined, in particular EPA and / or DHA, is greater than or equal to 15%, or is between 15 and 90%, or in particular is between 15 and 65%. 6. Method according to claim 4 wherein said composition comprising said mono-, and / or di-, and / or tri-glycerides is obtained chemically and / or enzymatically, by means of lipases selective for the fatty acid and its position on glycerol, starting from natural triglycerides, or from glycerides or glycerol and polyunsaturated fatty acids, through known procedures, and in which said composition has a content of polyunsaturated fatty acids as defined above greater than or equal to 30%, or between 30 and 90%, or in particular the content - expressed as triglycerides - of the sum of EPA and DHA is greater than or equal to 45% and the sum of the total omega-3 acids is greater than or equal to 60%, in accordance with monograph 01 / 2009: 1352 of the E.P.7.0. Method according to any one of claims 1-4, wherein said composition comprises diglycerides, wherein the glycerol is esterified with phosphoric acid condensed with amino alcohols to form the corresponding phospholipid, in particular phosphatidylcholine and / or phosphatidylethanolamine. 8. Method according to any one of claims 1-7, wherein step a) of treating said composition with urea is carried out using methanol in a quantity equal to 4.5-7 parts by weight, or ethanol in a quantity equal to 45- 65 parts by weight. 9. Method according to any one of claims 1-8 wherein said persistent organic environmental pollutants (POPs) mainly comprise polychlorinated dibenzo-para-dioxins (PCDDs) and polychlorinated dibenzo-urans (PCDFs), and / or polychlorinated biphenyls (PCBs) dioxins - similar (DL-PCBs) and markers, and / or polybromo-diphenyl ethers (PBDEs), and / or polycyclic aromatic hydrocarbons (PAHs), and their presence in the purified composition obtained in step c) is at least 2 times reduced, preferably at least 5 times reduced, conveniently at least 10 times reduced, and in any case lower - for each component - to the respective limit of quantification (LOQ) with standard GC-MS method. 10. Composition obtained by the method of any one of claims 4-9, comprising mono-glycerides, and / or di-glycerides and / or tri-glycerides of any origin, wherein the glycerol is at least partially esterified with long-lasting fatty acids chain having 18-22 carbon atoms, preferably 20-22 carbon atoms, and of polyunsaturated type (PIJFA), i.e. containing 2-6 double bonds, preferably 5-6 double bonds, belonging to the class of omega-6 and / o omega-3, preferably to the class of omega-3 acids, more preferably represented by eicosapentaenoic acid (EPA, C20: 5 n-3, all cis) and / or docosahexaenoic acid (DHA, C22: 6 n-3, all cis ), said composition further containing the following persistent organic environmental pollutants (POPs): polychlorinated-benzo-para-dioxins (PCDDs) and polychlorinated dibenzo-furans (PCDFs) in a total concentration lower than or equal to 1.0 pg / g, value determined in accordance with the WHO toxic equivalent factors (TEFs) and expressed as toxic equivalents (TEQs); PCDDs, PCDFs and dioxin-like polychlorinated biphenyls (PCBs) (DL-PCBs) in a total concentration lower than or equal to 5.0 pg / g, value determined in accordance with the WHO TEF values and expressed as TEQs; - PCBs marker in overall concentration less than or equal to 5.0 ng / g; - polybromo-diphenyl ethers (PBDEs) in a total concentration lower than or equal to 5.0 ng / g; And - a sum of polycyclic aromatic hydrocarbons (PAHs), expressed as a marker substance benzo [a] pyrene, less than or equal to 1.0 ng / g; each of said POPs being preferably contained in said composition in a concentration lower than its quantification limit LOQ with the standard GC-MS method. 11. A composition according to claim 10, wherein the total content of said polyunsaturated fatty acids is comprised between 15 and 90% of the weight of the composition. Use of a composition according to any one of claims 10 and 11 for the preparation from said mono-glycerides, and / or di-glycerides and / or tri-glycerides of the corresponding polyunsaturated fatty acids, or their salts, by chemical or enzymatic hydrolysis , or for the preparation of the relative C1-C3 alkyl esters, preferably ethyl esters, by chemical or enzyme-catalysed transesterification, in the presence of C1-C3 alitatic alcohols, preferably ethanol. 13. Use according to claim 12, in which the acids or esters obtained are subjected to a concentration process of the polyunsaturated components by means of fractional distillation, or molecular distillation or "short patir", or by fractionation by progressive complexation with urea of the saturated components and possibly of part of the components with a lower degree of unsaturation, followed by elimination of the complexes and final distillation of the concentrate, or again by extraction and / or fractionation with supercritical fluids, or with combined techniques. 14. Composition comprising said polyunsaturated fatty acids or their salts or C1-C3 alkyl esters obtained by chemical or enzymatic hydrolysis according to any one of claims 12 and 13, comprising EPA and / or DHA in the form of free acids or ethyl esters, in concentration total comprised between 15 and 100%, preferably between 50 and 100% of the weight of the composition, and a content of POPs pollutants as defined in any one of claims 9 and 10. 15. Composition according to claim 14, comprising EPA ethyl ester, or DHA ethyl ester, wherein the concentration of EPA ethyl ester or DHA ethyl ester is greater than or equal to 80%, preferably greater than or equal to 90% of the weight of the composition, or the sum of the concentrations of EPA ethyl ester and DHA ethyl ester is greater than or equal to 80%, preferably greater than or equal to 84%, being the concentration of EPA ethyl ester greater than or equal to 40%, the concentration of DHA ester ethyl greater than or equal to 34% and the sum of the concentrations of all the omega-3 ethyl esters greater than or equal to 90% of the weight of the composition in accordance with the specifications of monograph 1250 of EP 8.0. 16. Composition according to claim 14, comprising EPA ethyl ester and DHA ethyl ester in a total concentration comprised between 80.0 and 88.0%, the concentration of EPA ethyl ester being comprised between 43.0 and 49.5%, the concentration of DHA ethyl ester between 34.7 and 40.3%, and the sum of the concentrations of all the omega-3 ethyl esters greater than or equal to 90% of the weight of the composition in accordance with the specifications of USP 37, pages 4059- 61. 17. Use of a composition according to each of claims 10, 11, 14, 15 and 16 for the preparation of formulations useful as food ingredients, food and dietary supplements, food for special medical purposes (functional food), food for animal use and for aquaculture, food formulations for infancy, cosmetic and pharmaceutical preparations, by virtue of their high content of long-chain fatty acids or their derivatives, with a polyunsaturated nature or specifically of the omega-3 series. 18. A composition according to any one of claims 10, 11, 14, 15 and 16 for use in the prevention and treatment of risk factors for heart, cardiovascular and cardiovascular diseases, such as hypertension, coagulation defects and 'platelet aggregation, severe and moderate hypertriglyceridemia (resp.> 500mg / dl and> 200mg / dl) and hypercholesterolemia, in particular the familial and genetic forms, also in association with other drugs and in particular with statins. 19. Composition according to any one of claims 10, 11, 14, 15 and 16 for use in the prevention and treatment of heart, cardiovascular and cardiovascular diseases, such as coronary-atherosclerotic and ischemic cardiac and cerebral states, including myocardial and cerebral infarction and the reduction of the risk of sudden cardiac mortality following myocardial infarction; those from electrical causes and involving the onset and propagation of the heart rhythm, including arrhythmia and atrial and / or ventricular fibrillation; and those due to mechanical defects of the heart pump, such as heart failure and heart failure and / or congestive "heart fai Iure". 20. Composition according to any one of claims 10, 11, 14, 15 and 16 for use in the prevention and treatment of disorders of the central nervous system (CNS), including epilepsy, various forms of depression, bipolar disorders, the various forms of schizophrenia, Alzheimer's disease and the different forms of dementia, pediatric pathologies of attention deficit and hyperactivity disorder (ADHD), and in particular to improve learning defects and memory capacity. 2 1. Composition according to any of claims 10, 1, 14, 15 and 16 for use in the prevention and treatment of retinopathy and symptoms of dry eye, metabolic syndrome, metabolic and obesity-related defects, diabetes type 2, liver dysfunction, connective tissue and joint diseases, inflammatory states, autoimmune diseases, ulcerative colitis, psoriasis and cancer. Pharmaceutical formulation comprising a composition according to any one of claims 10, 11, 14, 15 and 16 and and a pharmaceutically acceptable carrier. 23. Composition obtained with the method according to any one of claims 1-3 and 7, comprising mono-glycerides, and / or di-glycerides and / or tri-glycerides of any origin, in which the glycerol is at least partially esterified with fatty acids long-chain saturated or unsaturated having 16-22 carbon atoms.