ES2684178B1 - Obtaining phospholipids from cephalopods by sequential extraction with supercritical fluids - Google Patents

Description

DESCRIPTION

Obtaining phosphoKids from cephalopods by sequential extraction with supercritical fluids

Field of the invention

The present invention relates to processes for extracting phosphoKids sequentially from cephalopod by-products by using supercritical fluids, as well as with the product obtainable by said methods.

Background of the invention

Fishing and the transforming sector of seafood produce a large volume of by-products. These by-products are widely used for the production of fishmeal and fish oils and are also a source of other high added value products with applications for the pharmaceutical and cosmetics industry, such as collagen peptides, gelatin, pigments, antimicrobial substances and enzymes. Fish oils have traditionally been the main source of omega-3 fatty acids, of which eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been widely recognized for their numerous health benefits [Narayan, B. , et al., Food Reviews International, 2006, 22 (3), 291-307; Riediger, ND, et al., Journal of the American Dietetic Association, 2009, 109 (4), 668-67]. These fatty acids (AGs) are obtained mainly from marine sources.

On the other hand, the management of the by-products generated in the marine products processing sector supposes a high cost due to the fact that the current processes of valorization are not optimized for this type of material.

Among the components of interest within these marine by-products are phospholipids (PLs), whose consumption has increased in recent years due to their potential in the prevention of obesity, cardiovascular diseases and hypercholesterolemia [Blokland, A., et al., Nutrition, 1999, 15 (10), 778-783; Stamler, CJ, et al., Journal of Lipid Research, 2000, 41 (8), 1214-1221; Pandey, NR and DL Sparks, Current Opinion in Investigational Drugs, 2008, 9 (3), 281-285; Sahebkar, A., Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids , 2013, 1831 (4), 887-893]. Several studies have demonstrated the ability of certain phosphoKids to decrease Kpidos in plasma and liver, and also, the prevention of diseases related to obesity, provided by phosphatidylcholine containing long chain omega 3 fatty acids [Buang, Y., et al., Nutrition, 2005, 21 (7), 867-873; Buckley, JD and PRC, Howe, Obesity Reviews, 2009, 10 (6), p. 648 659]. In addition, the consumption of phospholipids has shown an improvement in cognitive functions, stress and depression [Vakhapova, V., et al., Dementia and Geriatric Cognitive Disorders, 2014, 38 (1-2), 39-45] and observed its potential as mediators of inflammation and immunity [Feige, E., et al., Current Opinion in Lipidology, 2010, 21 (6), 525-529; Schmitz, G. and Ruebsaamen K., Atherosclerosis, 2010, 208 (1), 10-18; Banskota, AH, et al., Phytochemistry, 2014, 101, 101-108].

In addition to its biological activity, phospholipids possess numerous technological applications such as stabilizers, texturizers, dispersants, emulsifiers or antioxidants.

For the production of mixtures of phospholipids of marine origin, usually both dry and wet biomass, such as krill, are used, using different extraction processes based mainly on the use of toxic organic solvents and contaminants. In addition, massive krill fishing also has a high ecological impact, as it is a fundamental source of fish and other marine organisms.

The cephalopods are a traditional food in the Spanish diet, being the second market consumer of these species worldwide. In 2013 the consumption "per capita" of squid and frozen pots was 0.44 Kg / person and frozen octopus 0.13 Kg / person.This makes that, within the marine products transformation sector, the processing of cephalopods have relevance

The skin of cephalopods and more particularly the skin of squid (Illex argentinus), is a marine by-product with a relatively high content of phospholipids rich in omega-3 AGs (in particular, EPA and DHA). However, the use of this marine by-product poses difficulties since it is an inadequate material for processing, both in the form of fish meal (more usual and immediate output for by-products of marine origin) and for obtaining bioactive lipids , due to the low performance of the decantation processes usually used. That is why these byproducts are usually eliminated by incineration with a null recovery and revalorization [Martinez, JJ

Secretary of the National Commission of Subproducts of animal origin not intended for human consumption. BOOK white by-products of animal origin not intended for human consumption. Madrid: Ministry of Agriculture, Fisheries and Food. Ministry of Agriculture, Fisheries and Food: Technical Secretary General: Publications Center, 2007. 392 p .; il. col., graf .; 27 cm .: ISBN 978-84-491-0774-0; Nam, KA, et al., Journal of Food Science, 2008, 73 (4), 249-255; Kader, A., et al., Aquaculture Research, 2012, 43 (10), 1427-1438]. This fact supposes an additional cost of management for the companies that generate said by-products, as well as the waste of an important marine resource since, in the transformation of the species of cefalopodos like for example cuttlefish and sepia, around 10% of the weight The whole is made up of the skin.

Furthermore, in the field of lipid extraction, a large part of the extraction processes usually used lack sufficient selectivity and jointly extract neutral lipids (glycerides, fatty acids and cholesterol) and phospholipids, which results in products of low purity and lower added value [Sahena, F., et al., Journal of Food Engineering, 2010, 99 (1), 63-69; Rubio-Rodriguez, N., et al., Journal of Food Engineering, 2012, 109 (2), 238-248].

In this sense, the use of supercritical CO2 to extract lipids has shown to have very interesting properties, especially considering that it is an environmentally friendly technology, that leaves no residue in the final product after separation, is not flammable, It is not toxic, it is inert to most materials, it is cheap, and it can be used in relatively mild operating conditions, avoiding oxidations and hydrolic processes. In addition, the extraction with supercritical CO2 is a very suitable technique for the fractionation of starting materials of lipid nature, due to the low polarity of CO2. However, it has been described that supercritical CO2 is only capable of extracting neutral lipids and it is necessary to add a cosolvent, such as ethanol in amounts greater than 5% by weight, for the extraction of phospholipids [Catchpole, OJ, et al. , Journal of Supercritical Fluids, 2009, 47, 591-597]. Furthermore, it has been reported that the extraction of neutral lipids is hindered as the fraction of polar lipids increases [Catchpole, OJ, et al., Journal of Supercritical Fluids, 2009, 47, 591-597] and that the solubility of lipids polar is reduced when neutral lipids have been extracted [Cocero, MJ, Calvo, L., Journal of the American Oil Chemists' Society, 1996, 73 (11), 1573-1578]. These facts make it difficult to take advantage of marine by-products, particularly cephalopod skin, since in these marine by-products neutral lipids are minor compared to polar lipids (phospholipids).

Therefore, there is a need to have selective extraction procedures, efficient and environmentally friendly to obtain phosphoKids from marine by-products that include both phosphoKids and neutral Kpids, and thus get a better use and value of said by-products marine

SUMMARY OF THE INVENTION

The authors of the present invention have found a method of sequential extraction of lipids from marine by-products, in particular cephalopod skin, selective, efficient and environmentally friendly by the use of supercritical fluids. In particular, the authors of the present invention have found that the extraction of lipids from marine by-products, in particular from cephalopod skin, using unmodified supercritical fluids (for example supercritical CO2) and an extraction cell with agitation, surprisingly achieves a practically quantitative extraction of neutral lipids at reduced times in the presence of high concentrations of phospholipids. In a second extraction stage using as extractive solvent a supercritical fluid (for example supercritical CO2) modified with small amounts of alcohol (for example ethanol), it is possible to extract the phospholipids quantitatively and with high purity, where also said phospholipids they can understand a significant percentage of omega-3 fatty acids, in particular, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA).

The process of the present invention is, therefore, selective and effective in the extraction of neutral lipids and phospholipids from marine by-products, in particular cephalopod skin. In this way, a by-product of marine origin with reduced industrial value is converted into a source of high added value products, oriented towards emerging markets, proposing strategies that involve the use of clean technologies and respectful with the environment. This increases the potential for the valorization of these by-products of the marine products processing sector for possible use in human food.

Therefore, the first aspect of the invention is related to a process for obtaining a composition comprising phospholipids from a product of cephalopods comprising phosphoKapidos and neutral Kpidos, wherein said method comprises:

(a) providing a dry and particulate cephalopod product comprising neutral lipids and phospholipids;

(b) subjecting the product of step (a) to extraction with a supercritical fluid by applying agitation to obtain a residue comprising less than 30% by weight of neutral lipids relative to the total weight of neutral lipids and phospholipids in said residue; and (c) subjecting the residue of step (b) to solvent extraction, wherein said solvent comprises a supercritical fluid and from 1% to 10% by volume with respect to the total volume of the supercritical fluid of a C1-C4 alcohol, for obtaining an extract comprising at least 80% by weight of phospholipids relative to the weight of phospholipids present in the product of step (a).

In a particular embodiment, the process for obtaining a composition comprising phospholipids from a cephalopod product comprising neutral phospholipids and lipids, comprises:

(a) providing a dry and particulate cephalopod product comprising neutral lipids and phospholipids;

(b) contacting the cephalopod product of step (a) with a supercritical fluid by agitation;

(c) separating the supercritical fluid containing the soluble components from the insoluble components;

(d) contacting the insoluble components of step (c) with a solvent comprising a supercritical fluid and from 1% to 10% by volume with respect to the total volume of the supercritical fluid of a C 1 -C 4 alkanol by agitation;

(e) separating the solvent containing the soluble components from the insoluble components; Y

(f) isolating the soluble components from the mixture of soluble components and solvent obtained in step (e).

Another aspect of the invention is related to a composition comprising phospholipids obtainable by the process defined in the first aspect.

Brief description of the figures

Figure 1 shows the amount of Kpidos ex ^ two with respect to the extraction time for the skin of freeze-dried and milled Illex argentinus , by means of supercritical CO2 at 350 bars of pressure, CO2 flow rate of 16 g / min and temperature of 40 ° C.

Figure 2 shows the amount of lipids ex dos 2 with respect to the extraction time for the skin of partially defatted Illex argentinus , using supercritical CO2 using 5% ethanol as a modifier, and 350 bar pressure, CO2 flow rate of 25 g / min. and temperature of 40Â ° C.

Detailed description of the invention

Definitions

By "phospholipid" is meant an amphipathic lipid composed of a glycerol or sphingosine molecule to which two fatty acids are attached and a phosphate group linked by a phosphodiester bond to another group such as, for example, choline, ethanolamine. Examples of phospholipids are phosphatidylcholine (PC), lysophosphatidylcholine (PLC), phosphatidylethanolamine (PE) and sphingomyelin (SM).

By "neutral lipid" is meant an apolar, uncharged, mainly hydrophobic lipid. Examples of these lipids include fatty acids, acylglycerides such as mono, di and triglycerides, cerics, cholesterol and cholesterol esters.

By "cefalopodo" is meant a class of marine invertebrates comprising, among others cuttlefish (for example Sepia pharaonis and Sepia officinalis), squid (for example Illex argentinus), octopus and nautilus.

By "cephalopod product" is meant one or more parts of one or more cephalopods, such as, for example, skin, viscera, skin and viscera mixtures, or the cephalopod as a whole.

By "dry" it is meant that it comprises less than 15% by weight of water relative to the total weight of the cephalopod product, preferably less than 10%, more preferably less than 5%, even more preferably less than 1%.

By "particulate" is meant a solid product in the form of particles wherein 90% by weight of the particles have a particle size of less than 5 mm, preferably less than 3 mm. Preferably, 95% by weight of the particles have a particle size of less than 5 mm, preferably less than 3 mm. Even more preferably, 99% by weight of the particles have a particle size of less than 5 mm, preferably less than 3 mm. To determine the percentage of particles with a particle size below a specific value, for example 5 mm or 3 mm the material can be subjected to granulometric characterization by using a series of mechanical sieves of specific particle sizes, such as for example 5 mm or 3 mm and determine the weight of particles passing said screen.

By "supercntic fluid" is meant any substance that is in conditions of pressure and temperature above its critical point. Above, but close to this point, the substance is in a fluid state but shares the properties of a liquid and a gas. Thus, the fluid has a density similar to that of a liquid, while its viscosity and diffusivity are similar to those of a gas. An example of supercntic fluid is supercitic CO2.

By "C1-C4 alkanol" is meant a linear or branched alkyl radical of between 1 to 4 carbon atoms attached to a hydroxyl group. Examples of C1-C4 alkanols are methanol, ethanol, n-propanol, iso-propanol, n-butanol, iso-butanol, sec-butanol and tert-butanol.

Procedure of the invention

The first aspect of the invention is related to a process for obtaining a composition comprising phospholipids from a cephalopod product comprising neutral phospholipids and lipids, wherein said process comprises:

(a) providing a dry and particulate cephalopod product comprising neutral lipids and phospholipids;

(b) subjecting the product of step (a) to extraction with a supercitic fluid by applying agitation to obtain a residue comprising less than 30% by weight of neutral lipids relative to the total weight of neutral lipids and phospholipids present in said residue; Y

(c) subjecting the residue of step (b) to solvent extraction, wherein said solvent comprises a supercitic fluid and from 1% to 10% by volume with respect to the volume total of the supercritical fluid of a C1-C4 alkanol to obtain an extract comprising at least 80% by weight of phosphoKids relative to the weight of phosphoKids present in the product of step (a).

The cephalopod product is preferably a cuttlefish product (for example Sepia pharaonis and Sepia officinalis), squid product (for example Illex argentinus), or mixture thereof; more preferably of squid, even more preferably of Illex argentinus. The cephalopod product is preferably selected from the group consisting of viscera, skins and mixtures of viscera and skins. Preferably, the cephalopod product is selected from the group consisting of viscera, skins and mixture of viscera and skins of Illex argentinus; more preferably it is Illex argentinus skin .

The cephalopod product of step (a) is dry and particulate.

The water content of the dry and particulate cephalopod product of step (a) is less than 15% by weight, more preferably less than 10% by weight, even more preferably less than 10% by weight, even more preferably less than 10% by weight. % by weight, most preferred with a water content of less than 1% by weight.

90% by weight of the particulate particles of the dry and particulate cephalopod product of step (a) have a particle size of less than 5 mm, preferably less than 3 mm. Preferably 95% by weight of the particles have a particle size of less than 5 mm, preferably less than 3 mm. Even more preferably, 99% by weight of the particles have a particle size of less than 5 mm, preferably less than 3 mm.

If the cephalopod product has a water content or a particle size higher than the values required in step (a), said cephalopod product is subjected to a preconditioning stage (s) wherein the content in water and / or the particle size is decreased. Advantageously, the water content can be reduced by lyophilization since said technique prevents deterioration of the product. To reduce the particle size the cephalopod product can be chopped and / or milled to the desired size.

In a particular embodiment, the dry and particulate cephalopod product of step (a) is homogenized before being subjected to the extraction of step (b), for example by mixing or agitation of the product.

The cephalopod product comprises neutral phosphoKids and Kpidos. Preferably, the neutral lipids are selected from the group consisting of triglycerides, fatty acids, cholesterol, cholesterol esters and mixtures thereof. Preferably, the phosphoKids are selected from the group consisting of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, sphingomyelin and mixture thereof. A part of the phospholipids present in the cephalopods contain omega-3 fatty acids as fatty acids. Said omega-3 fatty acids are preferably eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). The highest content of these fatty acids is found in squid skins, in particular from Illex argentinus, and cuttlefish skins. Therefore, said cephalopod products are especially advantageous as starting material of the process of the present invention.

In step (b) of the method of the invention, the cephalopod product of step (a) is subjected to extraction with a supercritical fluid by applying agitation. For this, the cephalopod product is put in contact with said supercritical fluid while agitation is applied. In this way, a soluble fraction (extract) is obtained, which essentially comprises neutral lipids and an insoluble fraction (residue), wherein said insoluble fraction or residue comprises less than 30% by weight of neutral lipids with respect to the total weight of neutral lipids and phospholipids. present in said residue.

In a preferred embodiment, said residue comprises less than 15% by weight of neutral lipids with respect to the total weight of neutral lipids and phospholipids present in said residue.

For its part, the soluble fraction or extract contains less than 5% by weight of phospholipids relative to the weight of neutral lipids, preferably less than 1%, more preferably less than 0.5%, even more preferably less than 0.1%, even more preferably less than 0.05%, most preferably 0%, ie, the phospholipids are absent from the extract obtained after this first extraction or step (b) of the process of the invention.

The person skilled in the art can determine the amount of neutral phospholipids and lipids by usual techniques, such as, for example, by high resolution liquid chromatography. (HPLC), neutralization of free acidity or gas chromatography using pure patterns of each of the Kpidos classes and quantification by means of calibration lines of each of the detected lipids. The amount of neutral lipids can be determined by HPLC coupled to an evaporative light scattering detector according to the method previously described by Torres et al. [Journal of Chromatography A, 2005, 1078 (1), 28-34]. The amount of polar lipids can be determined by HPLC coupled to an evaporative light scattering detector according to the methodology described by Casado et al. [Journal of Molecular Catalysis B: Enzymatic, 2014, 99, 14-19].

This step therefore allows the selective extraction of neutral lipids with respect to phosphoKids. This fact is surprising since it is known that the extraction of neutral lipids is hindered as the fraction of polar lipids increases. In the cephalopod product generally neutral lipids are minor compared to polar lipids, and yet, by the method of the present invention, the neutral lipids are extracted almost quantitatively in stage (b) of the process.

Step (b) of the process of the invention is preferably carried out in an extraction cell provided with an agitation system, preferably a mechanical agitator. The extraction with agitation is advantageous since it allows to reduce the extraction times and avoids the formation of preferential paths of the supercritical fluid during the extraction. In this way, it is also avoided having to disperse the product of cephalopods in an inert material (for example, sea sand) that would hinder its later recovery and utilization in subsequent processes. In addition, said agitation makes it possible to homogenize the parricule size of the obtained residue that will subsequently be subjected to the second extraction (step (c) of the process of the invention). Thus, the residue from step (b) is recovered and used directly in the next extraction step without further screening.

In a particular embodiment, the cephalopod product of step (a) is introduced into the extraction cell equipped with the agitation system and brought into contact with the supercritical fluid. Preferably the supercritical fluid passes continuously through the extraction cell. In a particular embodiment, the flow rate of said supercritical fluid is from 10 to 30 g / minute, preferably from 10 to 20 g / min, more preferably from 13 to 19 g / min, even more preferably from 15 to 17 g / min, most preferred about 16 g / min. Preferably, the extraction of step (b) is carried out at a pressure of between 150 and 400 bar, more preferably between 300 and 400 bar, even more preferably between 325 and 375 bars, even more preferably between 340 and 360 bars, most preferred about 350 bars. Preferably, the extraction of step (b) is carried out at a temperature between 30 and 50 ° C, more preferably between 35 and 45 ° C, most preferably at about 40 ° C.

Preferably, the supercritical fluid used in step (b) is supercritical CO2.

As an additional advantage, and derived mainly from the application of agitation during the extraction process, this selective extraction can be carried out in reduced times, for example between 20 and 120 minutes, between 30 and 120 minutes, between 40 and 120 minutes, between 50 and 120 minutes. 120 minutes, between 60 and 120 minutes, between 70 and 120 minutes, between 80 and 120 minutes, between 20 and 90 minutes, between 30 and 90 minutes, between 40 and 90 minutes, between 50 and 90 minutes, between 60 and 90 minutes , between 70 and 90 minutes, between 80 and 90 minutes, preferably between 60 and 120 minutes, more preferably between 70 and 90 minutes, even more preferably in about 80 minutes.

Once the extraction of stage (b) is finished, an extract with neutral lipids and a residue is obtained. Neutral lipids can be separated from the supercritical fluid used in the extraction by depressurization, which causes its precipitation and facilitates its recovery. Said neutral lipids can be used in the pharmaceutical, food and cosmetics industry, in particular as steroid hormone precursors in the pharmaceutical industry and also in the preparation of liposomes for their stabilization. These neutral lipids are rich in cholesterol.

Advantageously, the residue of step (b) can be used directly in the next extraction step, step (c) of the process of the invention, since it is not necessary to use any inert material to disperse the starting material of the invention. step (b) (i.e., the product of cephalopods provided in step (a)) and, therefore, it is not necessary to separate said inert material from the residue of step (b) before performing the extraction of the step (c) ). Furthermore, since the agitation applied during the first extraction (step (b)) allows to homogenize the size of parricula, it is also not necessary to grind or crush said residue before performing the extraction of step (c).

The residue obtained after step (b) of the process of the invention contains a weight percentage of fat that ranges between 5 and 20% by weight with respect to the total weight of the product. residue, more preferably between 10 and 15% by weight of fat. Fatty matter is the sum of neutral Kpidos and phosphoKids.

The next step of the method of the invention is step (c). In said process step, the residue of step (b) is subjected to solvent extraction. For this, said residue is put in contact with a solvent, where said solvent comprises a supercritical fluid and from 1% to 10% by volume with respect to the total volume of the supercritical fluid of a C 1 -C 4 alkanol. By means of this step an extract is obtained which comprises at least 80% by weight of phospholipids with respect to the total weight of phospholipids present in the product of step (a), preferably at least 90% by weight of the phospholipids with respect to the total weight of phospholipids present in the product of step (a). The person skilled in the art can determine the amount of phospholipids by customary techniques, such as, for example, by high performance liquid chromatography (HPLC), as described above.

It is surprising that with the method of the present invention it is possible to extract the polar lipids (phospholipids) almost quantitatively using low amounts of C 1 -C 4 alkanol since it is known that the solubility of polar lipids (for example phospholipids) is reduced when the Neutral lipids have been extracted.

Step (c) of the process of the invention is preferably carried out in an extraction cell provided with an agitation system, preferably a mechanical agitator. The extraction with agitation is advantageous since it allows to reduce the extraction times and avoids the formation of preferential paths of the solvent during the extraction. In this way, it is also avoided having to disperse the residue of stage (b) in an inert material (for example, sea sand) that would make it difficult to later recover and utilize it in subsequent processes.

For the extraction of step (c), the residue of step (b) is introduced into the extraction cell equipped with the agitation system, preferably mechanical agitation, and brought into contact with the solvent. Advantageously, in the present invention it is not necessary to perform any additional screening after the extraction of step (b) to separate, for example, inert dispersion materials which in the present invention have not had to be added to achieve a selective extraction and efficient neutral lipids. Preferably the solvent passes continuously through the extraction cell. In a particular embodiment, the flow rate of said supercritical fluid is from 10 to 40 g / minute, preferably from 15 to 35 g / min, more preferably from 20 to 30 g / min, even more preferably from 23 to 27 g / min, most preferably approximately 25 g / min. Preferably, the extraction of step (c) is carried out at a pressure of between 150 and 400 bar, more preferably between 200 and 300 bar, even more preferably between 225 and 275 bar, even more preferably between 240 and 260 bar, most preferred about 250 bars. Preferably, the extraction of step (c) is carried out at a temperature of between 30 and 50 ° C, more preferably between 35 and 45 ° C, most preferred of about 40 ° C.

In one embodiment, the solvent of step (c) comprises from 1% to 8% by volume with respect to the total volume of the supercritical fluid of an alkanol, preferably from 1% to 5%, more preferably from 2% to 5%, even more preferably from 2.5% to 5%, most preferably about 5%. Preferably, the supercritical fluid used in step (c) is supercritical CO2. Preferably, the C 1 -C 4 alkanol used in step (c) is ethanol. Particularly preferably, the solvent of step (c) is supercritical CO2 and from 1% to 10% by volume with respect to the total volume of the supercritical CO2 of ethanol, preferably supercritical CO2 and from 1% to 8% by volume with respect to the total volume of the supercritical ethanol CO2, more preferably supercritical CO2 and from 1% to 5% by volume with respect to the total volume of the supercritical CO2 of ethanol, more preferably supercritical CO2 and from 2% to 5% by volume with respect to the total volume of the supercritical ethanol CO2, even more preferably supercritical CO2 and 2.5% to 5% by volume with respect to the total volume of the supercritical CO2 of ethanol, most preferred supercritical CO2 and approximately 5% by volume with respect to the total volume of the supercritical CO2 of ethanol.

As an additional advantage, and derived mainly from the application of agitation during the extraction process, this practically quantitative extraction can be done in reduced times, for example between 20 and 120 minutes, between 30 and 120 minutes, between 40 and 120 minutes, between 50 and 120 minutes, between 60 and 120 minutes, between 70 and 120 minutes, between 80 and 120 minutes, between 20 and 90 minutes, between 30 and 90 minutes, between 40 and 90 minutes, between 50 and 90 minutes, between 60 and 90 minutes minutes, between 70 and 90 minutes, between 80 and 90 minutes, preferably between 60 and 120 minutes, more preferably between 70 and 90 minutes, even more preferably in about 80 minutes.

Once the extraction of stage (c) is finished, an extract with the phospholipids and a residue is obtained. The phospholipids can be separated or isolated from the supercritical fluid used in the extraction by depressurization and evaporation of the C1-C4 alkanol, preferably by evaporation under reduced pressure (for example less than 10 mbar) and temperature not higher than 40 ° C, thus obtaining a composition comprising phosphoKids.

Said composition preferably comprises at least 70% by weight of phosphoKids relative to the total weight of the composition, more preferably at least 75% by weight, even more preferably at least 80% by weight. The phospholipids of the composition of the invention are preferably selected from the group consisting of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, sphingomyelin and mixture thereof. Preferably, the composition comprises from 55% to 75% by weight of phosphatidylcholine, from 3% to 10% by weight of lysophosphatidylcholine, from 3% to 10% by weight of phosphatidylethanolamine and from 5% to 12% by weight of sphingomyelin, in where the percentages by weight are expressed with respect to the total weight of the composition. Said composition comprising phospholipids can be used in the pharmaceutical, food and cosmetic industry. The residue of step (c) can be used in the pharmaceutical, food and cosmetics industry, in particular for the production of collagen and protein hydrolysates.

The weight (in grams) of cephalopod product that is introduced into the extraction cell in step (b) as in step (c) may be the same or different, preferably is between 0.05 and 0.5 times the volume (in milliliters) of the extraction cell, more preferably between 0.05 and 0.2 times, more preferably between 0.05 and 0.1 times, even more preferably between 0.05 and 0.2 times, most preferably preferred between 0.05 and 0.1 times.

Extract and composition of the invention

In another aspect, the present invention relates to an extract obtainable according to the process defined in the first aspect. Said extract contains at least 80% by weight of phospholipids with respect to the total weight of phospholipids present in the product of step (a), preferably at least 90% by weight of the phospholipids with respect to the total weight of phospholipids present in the product from stage (a)

In a further aspect, the invention relates to a composition comprising phospholipids, said composition being obtainable by the process of the invention which further comprises the step of isolating or separating the soluble components of the solvent extract used in the second extraction.

This composition preferably comprises at least 70% by weight of phosphoKids relative to the total weight of the composition, more preferably at least 75% by weight, even more preferably at least 80% by weight. The composition may also comprise a minor portion of neutral lipids, preferably less than 15% by weight relative to the total weight of the composition, more preferably less than 10% by weight, even more preferably less than 5% by weight. Said composition may also contain other components, such as water and optionally xanthomatin [Williams, T. L. et al., 2016, Langmuir, 2016, 32 (15), 3754-3759].

The phospholipids of the composition of the invention are preferably selected from the group consisting of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, sphingomyelin and mixture thereof. Preferably, the composition comprises from 55% to 75% by weight of phosphatidylcholine, from 3% to 10% by weight of lysophosphatidylcholine, from 3% to 10% by weight of phosphatidylethanolamine and from 5% to 12% by weight of sphingomyelin, in where the percentages by weight are expressed with respect to the total weight of the composition.

The phospholipids of the composition of the invention preferably contain omega-3 fatty acids as fatty acids which are attached to the glycerol or sphingosine of said phospholipids. Preferably, the omega-3 fatty acids are eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA) or mixture thereof.

Said composition can be used in the pharmaceutical, food and cosmetic industry.

Examples

The following examples are given for illustrative purposes, and do not imply a limitation of the present invention.

Materials and methods

The cephalopod products were ceded by the company CEFRICO SL consisted of squid skins of the species Illex argentinus from Argentina.

The CO2 was acquired and supplied by the company Carburos Metalicos in Kquida form and bottle format with siphon or espadm and purity of 99.98%.

Extractions with supercritical fluids were carried out in the TharSFC team (Thar SFC, to Waters company). The extraction cell has a volume capacity of 104 mL, and in its upper part it houses an agitation axis that can be optionally activated or not. The CO2 is transported to the interior of the cell through several holes. The working pressure of the CO2 ranges between 73 bar and 400 Bar and is controlled by a pressure regulator (Automated Back Pressure Regulator TharSFC) and the defined working flow is maintained by the action of a hydraulic pump (High pressure P-series pump TharSFC ) being established in a range between 10 and 50 g / min. The temperature of the process is controlled by an electrical resistance. The CO2 is pre-cooled to liquefy it prior to its pumping by means of a cryostat (Huber CC508) and subsequently heated to the process temperature by means of a heat exchanger (Heat exchanger TharSFC).

The absolute ethanol used as a co-solvent or modifier was obtained from AppliChem Panreac (Barcelona, Spain), with a purity of 99.5%.

The concentration of free fatty acids was determined by neutralization with potassium hydroxide in the presence of phenolphthalema.

The analysis of neutral lipids by gas chromatography was performed using an Agilent gas chromatograph (6890N Network GC System) coupled to a triple axis detector (5975C) and an automatic sampler (Agilent 7683B). For this, the method previously described by Torres et al [Chromatographia, 2009, 69 (7-8), 729-734] was used.

Neutral lipid analysis was also performed by HPLC using an Agilent Technologies 1200 Series chromatograph (Santa Clara, CA, USA) coupled to an evaporative light scattering detector (ELSD) (Agilent 1260 Infinity) according to the previously described method by Torres et al. [Journal of Chromatography A, 2005, 1078 (1), 28-34].

The analysis of polar lipids using HPLC Agilent Technologies 1200 Series (Santa Clara, CA, USA) coupled to an evaporative light scattering detector (ELSD) (Agilent 1260 Infinity) according to the methodology described by Casado et al. [Journal of Molecular Catalysis B: Enzymatic, 2014, 99, 14-19].

Conditioning of the cephalopod product

For the preparation of the product, the Illex argentinus skins were initially frozen and stored in a freezer chamber at -20 ° C until their subsequent lyophilization. The frozen product was placed in stainless steel trays and placed in a lyophilizer for 4 days. 6 kg of Illex argentinus skins were started and a freeze-dried weight of 720 g was obtained.

Next, the lyophilized material was chopped and ground using a hammer mill with 3 mm sieve. The ground material was subjected to physical characterization by granulometry using a series of 4 mechanical sieves of particle sizes of 3 mm, 1 mm, 500 mm and 250 mm.

The ground material was homogenised and kept protected from light, packed at the ford and at a refrigeration temperature of 4 ° C for subsequent extraction with supercritical fluids divided into two stages.

EXAMPLE 1. Study of the extraction times of stage (b)

The product of Illex argentinus conditioned (10 g) was extracted with supercritical CO2 at 350 bar pressure, CO2 flow rate of 16 g / min and temperature of 40 ° C at different times in order to define the time necessary for complete extraction of neutral lipids. The results are shown in Figure 1.

This first extract of neutral lipids was quantified and characterized chemically by neutralization of free acidity, gas chromatography and high efficiency liquid chromatography (HPLC coupled to an evaporative light scattering detector). The identification was made using pure patterns of each of the lipid classes and the quantification by means of calibrated lines of each one of the detected lipids, resulting in the composition shown in Table 3. It is possible to observe the total absence of polar lipids in these first extracts, which indicates the selectivity of the sequential extraction method of the invention.

Table 3: Weight percentage of neutral lipids

DE = standard deviation

EXAMPLE 2. Study of the extraction times of the stage (c)

The product of partially defatted Illex argentinus from Example 1 (7.5 g) was extracted with supercritical CO2 using 5% ethanol as a modifier at 350 bar pressure, CO2 flow rate of 25 g / min and temperature of 40 ° C at different times in order to define the time necessary for the complete extraction of polar lipids. The results are shown in Figure 2.

The extract obtained in this second stage was quantified and chemically characterized by HPLC coupled to an evaporative light scattering detector. The identification of polar lipids was carried out using commercial patterns of different phospholipids, such as phosphatidylcholine (PC), lysophosphatidylcholine (PLC), phosphatidylethanolamine (PE) and sphingomyelin (SM). The quantification of them was carried out by calibrated lines of each of the detected lipids, resulting in the composition shown in Table 4.

Table 4: Percentage by weight of phospholipids

DE = standard deviation

The material balance was closed with a small fraction (approximately 10%) corresponding mainly to neutral lipids that were not extracted in the first extraction stage.

The extract obtained showed high coloration, which could be due to the presence of residual xanthomatin (omocromo), a pigment present in the skin of some cephalopods, such as Illex argentinus. In addition this substance could protect the own extract of its oxidation after the extraction process with supercritical CO2.

The oxidation state of these extracts was determined by measuring the peroxides index using the "Food-Lab Fat" rapid measurement equipment The results of the determined peroxides index show a low oxidation state in these extracts, being approximately 1.3 ± 0.21 mEq / Kg.

EXAMPLE 3. Degreasing of Illex argentinus skin milled by supercritical CO2 in cell with agitation

We started with 10 g of the skin of Illex argentinus previously conditioned. The granulometry obtained for this material was 7.9% of parricula less than 250 ^ m, 7.8% between 500 and 250 ^ m, 18.2% between 500 ^ m and 1 mm and 66% between 1 and 3 mm. This material was introduced into the extraction cell of the TharSFC equipment and extracted with mechanical agitation. The pressure of the CO2 applied was 350 bar, flow of CO2 circulated 16 g / min and temperature of the process 40 ° C. The extraction time was 80 min. Under these conditions, a residue of 416.9 ± 15.50 mg was obtained, which represents 4.17% of the starting dehydrated material.

EXAMPLE 4. Degreasing of ground Illex argentinus skin by means of supercritical CO2 in cell without agitation (comparative example)

10 g of skin of ground squid conditioned previously was started. The granulometry obtained for this material was 7.9% of parricula less than 250 ^ m, 7.8% between 500 and 250 ^ m, 18.2% between 500 ^ m and 1 mm and 66% between 1 and 3 mm. This material was introduced into the extraction cell of the TharSFC equipment without applying mechanical agitation. It was extracted under CO2 pressure conditions of 350 bar, flow rate of CO2 circulated 16 g / min, temperature of 40 ° C and extraction time of 80 min. A residue of approximately 354 mg was obtained, which represents 3.54% of the starting dehydrated material.

EXAMPLE 5. Extraction of the fraction of phospholipids present in the skin of Illex argentinus ground and defatted by the application of supercritical CO2 and 5% ethanol as a modifier

7.5 g of partially defatted ground Illex argentinus skin obtained in example 3 was started, which was introduced into the extraction cell with agitation of the TharSCF equipment. The granulometry obtained for this material was 8.8% of parricula less than 250 ^ m, 19.1% between 500 and 250 ^ m, 37.9% between 500 ^ m and 1 mm and 33.6% between 1 and 3 mm. The The extraction process was carried out in the same way as that defined in Example 3, with the difference that on this occasion, together with the CO2, an amount of ethanol corresponding to 5% of the volume of CO2 used was pumped using a Dosapro Milton hydraulic pump. Roy, so that by mixing these two solvents in a premix chamber it is possible to extract the phospholipids present in the starting material. The working parameters were: pressure 350 bar, flow rate of CO225 g / min, flow rate of ethanol 1.58 mL / min, and 40 ° C. The extraction time was 80 min. The ethanol present in the obtained extract was evaporated under conditions of ford (less than 10 mbar) and maximum temperature of 40 ° C, to avoid the oxidation of the same. An extract weight of 826.6 ± 8.13 mg, representing 11.02% of the skin of defatted ground Illex argentinus, was obtained under these conditions.

EXAMPLE 6. Extraction of the fraction of phospholipids present in the skin of defatted Illex argentinus by the application of supercritical CO2 and 2.5% of ethanol as modifier

7.5 g of partially defatted ground Illex argentinus skin obtained in Example 3 was started, which was introduced into the extraction cell with agitation of the TharSCF equipment. The extraction process was carried out for 80 min, in the same way as that defined in Example 5, this time using 2.5% ethanol. The working parameters were: pressure of CO2350 bar, flow rate of CO225 g / min, flow rate of ethanol 0.79 mL / min, and 40 ° C. The weight of the extract obtained was approximately 605.56 mg, which represents 8.06% of the skin of defatted ground squid.

Claims (35)

A process for obtaining a composition comprising phosphoKids from a cephalopod product comprising neutral phosphoKids and Kpidos, wherein said process comprises: (a) providing a dry and particulate cephalopod product comprising neutral lipids and phospholipids; (b) subjecting the product of step (a) to extraction with a supercritical fluid by applying agitation to obtain a residue comprising less than 30% by weight of neutral lipids relative to the total weight of neutral lipids and phospholipids in said residue; and (c) subjecting the residue of step (b) to solvent extraction, wherein said solvent comprises a supercritical fluid and from 1% to 10% by volume with respect to the total volume of the supercritical fluid of a C 1 -C 4 alkanol to obtain a composition comprising at least 80% by weight of phospholipids relative to the weight of phospholipids present in the product of step (a). 2. Method according to claim 1, wherein the neutral lipids are selected from the group consisting of triglycerides, fatty acids, cholesterol, cholesterol esters and mixtures thereof. 3. Process according to claim 1 or 2, wherein the phospholipids are selected from the group consisting of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, sphingomyelin and mixture thereof. 4. Method according to any of the preceding claims, wherein the cephalopod product is selected from skin and viscera of cephalopods. 5. Method according to claim 4, wherein the cephalopod product is cephalopod skin. 6. Method according to any of the preceding claims, wherein the cephalopod is selected from cuttlefish and squid. 7. Process according to claim 6, wherein the cephalopod is Illex argentinus. The method according to any one of the preceding claims, wherein the dry and particulate cephalopod product of step (a) defined in claim 1 comprises 90% particles with a parricula size of less than 3 mm. 9. Process according to any of the preceding claims, wherein the supercritical fluid is supercritical CO ² . The process according to any one of the preceding claims, wherein the solvent of step (c) comprises from 1% to 8% by volume with respect to the total volume of the supercritical fluid of a C ¹ -C ⁴ alkanol. 11. Process according to claim 10, wherein the solvent of step (c) comprises from 1% to 8% by volume with respect to the total volume of the supercritical fluid of a C ¹ -C ⁴ alkanol. The process according to claim 11, wherein the solvent of step (c) comprises from 2% to 5% by volume with respect to the total volume of the supercritical fluid of a C ¹ -C ⁴ alkanol. The process according to any one of the preceding claims, wherein the C ¹ -C ⁴ alkanol is ethanol. The method according to any one of the preceding claims, wherein after step (b) defined in claim 1 a residue with less than 15% by weight of neutral lipids is obtained relative to the total weight of neutral lipids and phospholipids in said residue. The method according to any one of the preceding claims, wherein step (b) defined in claim 1 is carried out for at least 60 minutes. The method according to any one of the preceding claims, wherein step (b) defined in claim 1 is carried out for a period of time between 60 minutes and 120 minutes. 17. The method according to claim 16, wherein the time period is between 70 minutes and 90 minutes. The method according to any one of the preceding claims, wherein the extraction of step (b) defined in claim 1 is carried out at a pressure of between 150 and 400 bar. 19. The method according to claim 18, wherein the extraction of step (b) defined in claim 1 is carried out at a pressure of between 300 and 400 bar. The method according to any one of the preceding claims, wherein the extraction of step (b) defined in claim 1 is carried out at a temperature between 30 and 50 ° C. The method according to any one of the preceding claims, wherein after the step (c) defined in claim 1 there is obtained a composition comprising at least 90% by weight of the phospholipids with respect to the total weight of phospholipids present in the product of stage (a). The method according to any one of the preceding claims, wherein step (c) defined in claim 1 is carried out for at least 60 minutes. The method according to any one of the preceding claims, wherein step (c) defined in claim 1 is carried out for a period of time between 60 minutes and 120 minutes. 24. The method according to claim 23, wherein the time period is between 70 minutes and 90 minutes. 25. Method according to any of the preceding claims, wherein the extraction of step (c) defined in claim 1 is carried out at a pressure of between 150 and 400 bar. 26. The method according to claim 25, wherein the extraction of step (c) defined in claim 1 is carried out at a pressure of between 200 and 300 bar. Method according to any of the preceding claims, wherein the extraction of step (c) defined in claim 1 is carried out at a temperature between 30 and 50 ° C. Process according to any of the preceding claims, which further comprises, after step (c), isolating the soluble components of the solvent extract used in step (c). 29. An extract comprising phospholipids obtainable by the process defined in any of claims 1 to 27. 30. Extract according to claim 29, wherein the phospholipids comprise omega-3 fatty acids. 31. An extract according to claim 30, wherein the omega-3 fatty acids are selected from eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA) and mixture thereof. 32. A composition comprising phospholipids obtainable by the process defined in claim 28. 33. Composition according to claim 32, comprising at least 80% by weight of phospholipids. 34. Composition according to any of claims 32 or 33, wherein the phospholipids comprise omega-3 fatty acids. 35. Composition according to claim 34, wherein the omega-3 fatty acids are selected from eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA) and mixture thereof.