ES2684178A1 - Obtaining phospholipids from cephalopods by sequential extraction with supercritical fluids (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Obtaining phospholipids from cephalopods by sequential extraction with supercritical fluids. The present invention relates to a process for obtaining a composition comprising phospholipids from cephalopod products by sequential extraction in two steps with supercritical fluids and with the composition comprising phospholipids obtainable by said process. (Machine-translation by Google Translate, not legally binding)

Description

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DESCRIPTION

Obtaining phospholipids from cephalopods by sequential extraction with supercritical fluids

Field of the Invention

The present invention relates to methods of extracting phospholipids sequentially from by-products of cephalopods through the use of supercritical fluids, as well as with the product obtainable by said procedures.

Background of the invention

Fishing and the processing 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 products of high added value with applications for the pharmaceutical and cosmetic 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, N. D., 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 seafood processing sector implies a high cost because the current recovery processes 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, C.J., et al., Journal of Lipid Research, 2000, 41 (8), 1214-1221; Pandey, N.R. and D.L. Sparks, Current Opinion in Investigational Drugs, 2008, 9 (3), 281-285; Sahebkar, A., Biochimica et Biophysica Acta (BBA) - Molecular and Cell

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Biology of Lipids, 2013, 1831 (4), 887-893]. Several studies have demonstrated the ability of certain phospholipids to decrease lipids in plasma and liver, and in addition, the prevention of obesity-related diseases, provided by phosphatidylcholine containing long-chain omega 3 fatty acids [Buang, Y., et al., Nutrition, 2005, 21 (7), 867-873; Buckley, J.D. and P.R.C., Howe, Obesity Reviews, 2009, 10 (6), p. 648659]. In addition, phospholipid consumption 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 it has been 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, A. H., et al., Phytochemistry, 2014, 101, 101-108].

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

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

Cephalopods are a traditional food in the Spanish diet, being the second consumer market of these species worldwide. In 2013, the "per capita" consumption of squid and frozen potas was 0.44 kg / person and of frozen octopus 0.13 kg / person. This means that, within the marine products processing sector, cephalopod processing be relevant

Cephalopod skin and more particularly the pota skin (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 inappropriate material for processing, both in the form of fishmeal (most common and immediate exit for marine by-products) and for obtaining bioactive lipids , due to the low performance of the decantation processes commonly used. That is why these by-products are usually eliminated by incineration with zero recovery and revaluation [Martínez, J.J.

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Secretariat of the National Commission of Animal By-Products 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: General Technical Secretariat: Publications Center, 2007. 392 p .; il. col., graf .; 27 cm .: ISBN 978-84-491-0774-0; Nam, K. A., et al., Journal of Food Science, 2008, 73 (4), 249-255; Kader, A., et al., Aquaculture Research, 2012, 43 (10), 1427-1438]. This fact implies an additional management cost for companies that generate these by-products, as well as the waste of an important marine resource since, in the transformation of cephalopod species such as pota and sepia, about 10% of the weight The whole is made up of the skin.

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

In this sense, the use of supercritical CO2 to extract lipids has proven to have very interesting properties, especially considering that it is an environmentally friendly technology, which 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 hydrolytic processes. In addition, supercritical CO2 extraction is a very suitable technique for the fractionation of starting materials of a 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]. In addition, it has been described that neutral lipid extraction is hampered as the polar lipid fraction increases [Catchpole, OJ, et al., Journal of Supercritical Fluids, 2009, 47, 591-597] and that lipid solubility 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, in particular cephalopod skin, since in these marine by-products the neutral lipids are minor against polar lipids (phospholipids).

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Therefore, there is a need for selective, efficient and environmentally friendly extraction procedures that allow phospholipids to be obtained from marine by-products comprising both phospholipids and neutral lipids, and thus achieve a better use and recovery of said by-products. sailors

Summary of the invention

The authors of the present invention have found a method of sequential lipid extraction from marine by-products, in particular cephalopod skin, selective, efficient and environmentally friendly by using supercritical fluids. In particular, the authors of the present invention have found that the extraction of lipids from marine by-products, in particular cephalopod skin, using unmodified supercritical fluids (eg 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 a supercritical fluid (for example supercritical CO2) modified with small amounts of alcohol (for example ethanol) as extraction solvent, phospholipids can be extracted quantitatively and with high purity, in addition, said phospholipids they may come to comprise 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 becomes a source of products with high added value, oriented towards emerging markets, proposing strategies that involve the use of clean and environmentally friendly technologies. This increases the potential for recovery of these by-products from the seafood processing sector for possible use in human nutrition.

Therefore, the first aspect of the invention is related to a process for obtaining a composition comprising phospholipids from a product of

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cephalopods comprising phospholipids and neutral lipids, 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 with respect to the total weight of neutral lipids and phospholipids in said residue; Y

(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, to obtain an extract comprising at least 80% by weight of phospholipids with respect 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 phospholipids and neutral 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 stirring;

(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 C1-C4 alkanol by stirring;

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

(f) isolate 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 procedure defined in the first aspect.

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Brief description of the figures

Figure 1 shows the amount of lipids extracted with respect to the extraction time for the skin of lyophilized and ground Illex argentinus, by means of supercritical CO2 at 350 bar pressure, CO2 flow rate of 16 g / min and temperature of 40 ° C.

Figure 2 shows the amount of lipids extracted with respect to the extraction time for the skin of partially defatted Illex argentinus, by means of supercritical CO2 using 5% ethanol as a modifier, and 350 bars of pressure, CO2 flow of 25 g / min and temperature of 40 ° 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 and a phosphate group linked by a phosphodiester bond to another group such as choline, ethanolamine are attached. Examples of phospholipids are phosphatidylcholine (PC), lysophosphatidylcholine (PLC), phosphatidylethanolamine (PE) and sphingomyelin (SM).

By "neutral lipid" is meant an apolar lipid, without charge, primarily hydrophobic. Examples of these lipids include fatty acids, acylglycerides such as mono, di and triglycerides, cerids, cholesterol and cholesterol esters.

By "cephalopod" is meant a class of marine invertebrates that includes, among other sepias (for example Sepia pharaonis and Sepia officinalis), squid (for example Illex argentinus), octopus and nautilus.

"Cephalopod product" means one or more parts of one or more cephalopods, such as skin, guts, mixtures of skin and viscera, or the cephalopod as a whole.

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

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By "particulate" is meant a solid product in the form of particles in which 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. In order to determine the percentage of particles with a particle size of less than 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 concrete particle sizes, such as 5 mm or 3 mm and determine the weight of particles that pass said sieve.

By "supercritical fluid" is meant any substance that is in conditions of pressure and temperature above its critical point. Above, but close to that 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 supercritical fluid is supercritical 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.

Invention Procedure

The first aspect of the invention relates to a process for obtaining a composition comprising phospholipids from a cephalopod product comprising phospholipids and neutral 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 supercritical fluid by applying agitation to obtain a residue comprising less than 30% by weight of neutral lipids with respect 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 supercritical fluid and from 1% to 10% by volume with respect to volume

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Total supercritical fluid of a C1-C4 alkanol to obtain an extract comprising at least 80% by weight of phospholipids relative to the weight of phospholipids present in the product of step (a).

The cephalopod product is preferably a sepia product (for example Sepia pharaonis and Sepia officinalis), squid (for example Illex argentinus), or mixture thereof; more preferably squid, even more preferably from 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 skin of Illex argentinus.

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 5 % by weight, most preferred with a water content of less than 1% by weight.

90% by weight of the particles of the 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 greater than the values required in step (a), said cephalopod product is subjected to a previous conditioning stage (s) where the water content and / or particle size is decreased. Advantageously, the water content can be reduced by lyophilization since said technique prevents product deterioration. To reduce the particle size, the cephalopod product can be chopped and / or ground until the desired size is obtained.

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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 stirring the product.

The cephalopod product comprises phospholipids and neutral lipids. Preferably, neutral lipids are selected from the group consisting of triglycerides, fatty acids, cholesterol, cholesterol esters and mixtures thereof. Preferably, the phospholipids are selected from the group consisting of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, sphingomyelin and mixture thereof. A part of the phospholipids present in 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 in these fatty acids is found in squid skins, in particular Illex argentinus, and sepia. Therefore, said cephalopod products are especially advantageous as the starting material of the process of the present invention.

In step (b) of the process of the invention, the cephalopod product of step (a) is subjected to extraction with a supercritical fluid using agitation. For this, the cephalopod product is contacted with said supercritical fluid while stirring is applied. In this way a soluble fraction (extract) is obtained, which essentially comprises neutral lipids and an insoluble fraction (residue), where 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.

On the other hand, the soluble fraction or extract contains less than 5% by weight of phospholipids with respect 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%, that is, 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 phospholipids and neutral lipids by usual techniques, such as by means of high performance liquid chromatography

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(HPLC), neutralization of free acidity or gas chromatography using pure standards of each of the lipid 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 phospholipids. 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 product of cephalopods, neutral lipids are generally minor compared to polar lipids, and yet, by the process of the present invention, neutral lipids are extracted almost quantitatively in step (b) of the process.

Step (b) of the process of the invention is preferably carried out in an extraction cell equipped with a stirring system, preferably a mechanical stirrer. Extraction with stirring is advantageous since it allows to reduce extraction times and prevents the formation of preferential paths of the supercritical fluid during extraction. This also avoids having to disperse the product of cephalopods in an inert material (for example sea sand) that would hinder its subsequent recovery and use in subsequent processes. Furthermore, said stirring allows the particle size of the obtained residue to be homogenized and subsequently subjected to the second extraction (step (c) of the process of the invention). Thus, the residue of step (b) is recovered and used directly in the next extraction stage without subjecting it to further screening.

In a particular embodiment, the cephalopod product of step (a) is introduced into the extraction cell provided with the stirring system and is 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 10 to 30 g / min, preferably 10 to 20 g / min, more preferably 13 to 19 g / min, even more preferably 15 to 17 g / min, most preferred about 16 g / min. Preferably, the extraction of step (b) is performed at a pressure of between 150 and 400 bars, more preferably between 300 and 400 bars, even more preferably between 325 and 375

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bars, even more preferably between 340 and 360 bars, most preferably 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 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, 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 step (b) is finished, an extract is obtained with neutral lipids and a residue. Neutral lipids can be separated from the supercritical fluid used in the extraction by depressurization, which causes their precipitation and facilitates their recovery. Such neutral lipids can be used in the pharmaceutical, food and cosmetic industry, in particular as steroid hormone precursors in the pharmaceutical industry and also in the preparation of liposomes for stabilization. Such neutral lipids are high in cholesterol.

Advantageously, the residue of step (b) can be used directly in the next extraction stage, 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 step (b) (i.e., the cephalopod product provided in step (a)) and, therefore, it is not necessary to separate said inert material from the residue of stage (b) before performing the extraction of stage (c ). In addition, since the agitation applied during the first extraction (step (b)) allows the particle size to be homogenized, it is not necessary to grind or crush said residue before extracting the stage (c).

The residue obtained after step (b) of the process of the invention contains a percentage by weight of fat that ranges between 5 and 20% by weight with respect to the total weight of the

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residue, more preferably between 10 and 15% by weight of fat. Fatty means the sum of neutral lipids and phospholipids.

The next stage of the process 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 contacted with a solvent, 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 alkanol. By this step an extract is obtained comprising 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 usual techniques, such as by means of high performance liquid chromatography (HPLC), as described above.

It is surprising that with the process of the present invention polar lipids (phospholipids) can be extracted almost quantitatively using low amounts of C1-C4 alkanol since it is known that the solubility of polar lipids (eg 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 equipped with a stirring system, preferably a mechanical stirrer. Extraction with stirring is advantageous since it allows reducing extraction times and prevents the formation of preferential solvent paths during extraction. This also avoids having to disperse the residue from stage (b) in an inert material (for example, sea sand) that would hinder its subsequent recovery and use in subsequent processes.

For the extraction of stage (c), the residue of stage (b) is introduced into the extraction cell provided with the stirring system, preferably mechanical stirring, and is 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 that in the present invention have not had to be added to achieve selective extraction and efficient of neutral lipids. Preferably the solvent passes continuously through the extraction cell. In a particular embodiment, the flow rate of said supercritical fluid is 10 to 40 g / min, preferably 15 to 35 g / min, plus

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preferably from 20 to 30 g / min, even more preferably from 23 to 27 g / min, most preferably about 25 g / min. Preferably, the extraction of step (c) is carried out at a pressure of between 150 and 400 bars, more preferably between 200 and 300 bars, even more preferably between 225 and 275 bars, even more preferably between 240 and 260 bars, more preferred about 250 bars. Preferably, the extraction of step (c) is carried out at a temperature between 30 and 50 ° C, more preferably between 35 and 45 ° C, most preferably about 40 ° C.

In one embodiment, the solvent of step (c) comprises from 1% to 8% by volume with respect to the total supercritical fluid volume 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 C1-C4 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 supercritical CO2 from ethanol, preferably supercritical CO2 and from 1% to 8% by volume with respect to total volume of supercritical ethanol CO2, more preferably supercritical CO2 and from 1% to 5% by volume with respect to the total volume of supercritical CO2 from ethanol, more preferably supercritical CO2 and from 2% to 5% by volume with respect to total volume of supercritical ethanol CO2, even more preferably supercritical CO2 and from 2.5% to 5% by volume with respect to the total volume of supercritical CO2 from ethanol, most preferred supercritical CO2 and approximately 5% by volume with respect to the total volume of supercritical ethanol CO2.

As an additional advantage, and derived mainly from the application of agitation during the extraction process, this practically quantitative 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, 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 step (c) is finished, an extract is obtained with the phospholipids and a residue. Phospholipids can be separated or isolated from the supercritical fluid used in the

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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 exceeding 40 ° C, thus obtaining a composition comprising phospholipids.

Said composition preferably comprises at least 70% by weight of phospholipids with respect 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 cosmetic industry, in particular for obtaining 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) can be the same or different, preferably 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 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 procedure 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. of 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.

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This composition preferably comprises at least 70% by weight of phospholipids with respect 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 with respect 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 that 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 provided for illustrative purposes, and do not imply a limitation of the present invention.

Materials and methods

Cephalopod products were ceded by the company CEFRICO S.L consisted of pota skins of the species Illex argentinus from Argentina.

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The CO2 was acquired and supplied by the company Metallic Carbides in liquid form and bottle format with siphon or sprat and 99.98% purity.

Extractions with supercritical fluids were performed on the TharSFC team (Thar SFC, a Waters company). The extraction cell has a volume capacity of 104 mL, and in its upper part houses a stirring shaft that can be optionally activated or not. The CO2 is vehiculized into the cell through several holes. The CO2 working pressure ranges from 73 bar to 400 Bar and is controlled by a pressure regulator (Automated Back Pressure Regulator TharSFC) and the defined work flow is maintained by a hydraulic pump (High pressure P-series pump TharSFC ) being established in a range between 10 and 50 g / min. The process temperature is controlled by an electrical resistor. The CO2 is pre-cooled to liquefy it before pumping it through 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 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 phenolphthalein.

Neutral lipid analysis 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 by Agilent Technologies 1200 Series HPLC (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].

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Conditioning of the cephalopod product

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

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

The ground material was homogenized and kept protected from light, vacuum packed and refrigerated at 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 conditioned Illex argentinus (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 required for complete extraction of neutral lipids. The results are shown in Figure 1.

That first neutral lipid extract was quantified and chemically characterized 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 standards of each of the lipid classes and quantification by means of calibration lines of each 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

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 Neutral lipids

 Half of

 Free fatty acids

 16.2 0.79

 Cholesterol

 78.4 4.96

 Triglycerides and cholesterol esters

 14.8 0.57

SD = standard deviation

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

The partially defatted Illex argentinus product 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 performed using commercial standards of different phospholipids, such as phosphatidylcholine (PC), lysophosphatidylcholine (PLC), phosphatidylethanolamine (PE) and sphingomyelin (SM). The quantification of these was carried out by means of calibration lines of each of the lipids detected, resulting in the composition shown in Table 4.

Table 4: Percentage by weight of phospholipids

 Phospholipids

 Half of

 PC

 63.8 3.18

 PLC

 5.83 0.63

 PE

 6.2 0.6

 YE

 7.7 0.39

SD = standard deviation

The material balance was closed with a small fraction (10% approx.) 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 (omorochrome), a pigment present in the skin of some cephalopods, such as Illex argentinus. In addition, this substance could protect the extract itself from oxidation after the supercritical CO2 extraction process.

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

EXAMPLE 3. Skin degreasing of Illex argentinus ground by supercritical CO2 in cell with agitation

It started from 10 g of the skin of Illex argentinus preconditioned. The granulometry obtained for this material was 7.9% of particles smaller 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 TharSFC equipment extraction cell and extracted with mechanical agitation. The applied CO2 pressure was 350 bar, circulated CO2 flow rate 16 g / min and process temperature 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. Skin degreasing of Illex argentinus ground by supercritical CO2 in cell without agitation (comparative example)

It started from 10 g of the previously prepared ground pota skin. The granulometry obtained for this material was 7.9% of particles smaller 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 mechanical agitation. It was extracted under conditions of CO2 pressure of 350 bar, circulated CO2 flow rate 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 dehydrated starting material.

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

It started from 7.5 g of partially defatted ground Illex argentinus skin obtained in Example 3, which was introduced into the extraction cell with agitation of the TharSCF equipment. The particle size obtained for this material was 8.8% of particles smaller 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. He

The extraction process was carried out in the same way as defined in Example 3, with the difference that on this occasion an amount of ethanol corresponding to 5% of the volume of CO2 used was pumped together with CO2, using a Dosapro Milton hydraulic pump Roy, so that by mixing these two solvents in a premix chamber 5 it is possible to extract the phospholipids present in the starting material. The working parameters were: pressure 350 bar, CO2 flow 25 g / min, ethanol flow 1.58 mL / min, and 40 ° C. The extraction time was 80 min. The ethanol present in the extract obtained was evaporated under vacuum conditions (less than 10 mbar) and a maximum temperature of 40 ° C, to avoid oxidation. Under these conditions, an extract weight of 10 826.6 ± 8.13 mg was obtained, which represents 11.02% of the skin of Illex argentinus milled fat.

EXAMPLE 6. Extraction of the fraction of phospholipids present in the skin of Illex argentinus milled degreased by applying supercritical CO2 and 2.5% ethanol as modifier 15

It started from 7.5 g of partially defatted ground Illex argentinus skin obtained in Example 3, 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 defined in Example 5, using 2.5% ethanol this time. The working parameters were: CO2 pressure 350 20 bar, CO2 flow 25 g / min, ethanol flow 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 pota.

Claims (35)

5 10 fifteen twenty 25 30 35 1. A process for obtaining a composition comprising phospholipids from a cephalopod product comprising phospholipids and neutral 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 supercritical fluid by applying agitation to obtain a residue comprising less than 30% by weight of neutral lipids with respect to the total weight of neutral lipids and phospholipids in said residue; Y (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 alkanol to obtain a composition comprising at least 80% by weight of phospholipids with respect to the weight of phospholipids present in the product of step (a). 2. A 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. A method 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 cephalopod viscera. 5. A 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 sepia and squid. 7. A method according to claim 6, wherein the cephalopod is Illex argentinus. 5 10 fifteen twenty 25 30 35 8. The method according to any of the preceding claims, wherein the dry and particulate cephalopate product of step (a) defined in claim 1 comprises 90% of particles with a particle size of less than 3 mm. 9. Method according to any of the preceding claims, wherein the supercritical fluid is supercritical CO2. 10. The method according to any 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 C1-C4 alkanol. 11. A method 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 C1-alkanol- C4 12. The method 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 C1-alkanol- C4 13. Process according to any of the preceding claims, wherein the C1-C4 alkanol is ethanol. 14. A method according to any 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 with respect to the total weight of neutral lipids and phospholipids in said residue. 15. A method according to any of the preceding claims, wherein the step (b) defined in claim 1 is carried out for at least 60 minutes. 16. The method according to any of the preceding claims, wherein the step (b) defined in claim 1 is carried out for a period of time between 60 minutes and 120 minutes. 17. The method of claim 16, wherein the period of time is between 70 minutes and 90 minutes. 5 10 fifteen twenty 25 30 18. The method according to any of the preceding claims, wherein the extraction of the step (b) defined in claim 1 is carried out at a pressure between 150 and 400 bar. 19. The method according to claim 18, wherein the extraction of the step (b) defined in claim 1 is carried out at a pressure of between 300 and 400 bar. 20. Method according to any of the preceding claims, wherein the extraction of the step (b) defined in claim 1 is carried out at a temperature between 30 and 50 ° C. 21. A method according to any of the preceding claims, wherein after step (c) defined in claim 1, a composition comprising at least 90% by weight of the phospholipids is obtained relative to the total weight of phospholipids present in the product of stage (a). 22. Method according to any of the preceding claims, wherein the step (c) defined in claim 1 is carried out for at least 60 minutes. 23. Method according to any of the preceding claims, wherein the step (c) defined in claim 1 is carried out for a period of time between 60 minutes and 120 minutes. 24. Method according to claim 23, wherein the time period is between 70 minutes and 90 minutes. 25. The method according to any of the preceding claims, wherein the extraction of the step (c) defined in claim 1 is carried out at a pressure of between 150 and 400 bar. 26. A method according to claim 25, wherein the extraction of the step (c) defined in claim 1 is carried out at a pressure of between 200 and 300 bar. 27. The method according to any of the preceding claims, wherein the extraction of the step (c) defined in claim 1 is carried out at a temperature between 30 and 50 ° C. 5 10 fifteen twenty 25 28. A method according to any one of the preceding claims, further comprising after step (c), isolating the soluble components of the solvent extract used in step (c). 29. An extract comprising phospholipids obtainable by the method defined in any one of claims 1 to 27. 30. Extract according to claim 29, wherein the phospholipids comprise omega-3 fatty acids. 31. 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 method defined in claim 28. 33. Composition according to claim 32, comprising at least 80% by weight 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.