Particles for embedding active substances, capsules and process for obtaining them

Abstract

The present invention relates to particles suitable for embedding ingredients such as biologically active substances, vegetal extracts and / or microorganisms and comprising a solid matrix comprising lentil protein. The invention also relates to a process for their preparation and to compositions comprising them.

Description

[0001] PARTICLES FOR EMBEDDING ACTIVE SUBSTANCES, CAPSULES AND PROCESS FOR OBTAINING THEM

[0002] FIELD OF THE INVENTION

[0003]

[0001] The present invention relates to particles suitable for embedding ingredients such as biologically active substances, vegetal extracts and / or microorganisms and comprising a solid matrix comprising lentil protein. The invention also relates to a process for their preparation and to compositions comprising them.

[0004] BACKGROUND

[0005]

[0002] The encapsulation of active substances and materials is of interest in a broad range of industries, including food, nutraceutical, pharmaceutical and cosmeceutical industries as it allows (i) protecting the ingredient from the environment, thus preventing its degradation or volatilization and increasing the shelf life of the product; (ii) controlling the rate and conditions of the release of the encapsulated substance (e.g. enteric coatings); (iii) mask unpleasant odors or flavors of active substances and / or (iv) increase the bioavailability of the encapsulated substance, e.g. by preventing its enzymatic degradation or delivering it in a controlled manner. Encapsulated materials often benefit from an enhanced processing efficiency, as they can be formulated as solid formulations, e.g. powders.

[0006]

[0003] Several methods of encapsulation of ingredients are known in the art, such as spray drying, fluid bed coating, emulsion-based methods, coacervation, extrusion and nanoprecipitation. In emulsion-based methods, the active ingredient is dispersed in a continuous phase (such as oil or water) and then droplets or particles are formed by emulsification. To this end, emulsifying agents may be used. The solvent is then evaporated or extracted, leaving behind encapsulated particles. Emulsion-based methods are versatile and can be used to encapsulate both hydrophobic or hydrophilic compounds. In coacervation-based methods, a liquid-liquid biphasic system is formed by the desolvation of a polymer or the complexation of oppositely charged polymers. The formation of the coacervate is thus often triggered by the addition of an additional substance.

[0007]

[0004] The use of proteins deriving from plants in the formation of coacervates is attractive for several reasons. They are readily available from biomass, thus providing a sustainable raw material in contrast with synthetic polymers. Also, certain plant proteins are hypoallergenic and represent a safer alternative to allergen proteins such as soybean protein or gluten. Certain plant proteins offer functional properties, such as emulsification, gelation, and foaming, which can enhance the performance of coacervates in food, pharmaceutical, and cosmetic applications. For example, while soybean protein may form stable emulsions, pea protein can take part in gel formation. Common plant-derived proteins useful in coacervation processes are for instance, gelatin, albumin, whey proteins, soybean proteins (e.g. glycinin and p-conglycinin), pectin. These proteins also offer a clear nutritional benefit and allow avoiding the use of animal-based protein alternatives.

[0008]

[0005] For instance, Wang et al. disclose in Food Chemistry 2018, 188, 24-29 the encapsulation of quercetin in soybean protein according to the cold-gelation method. In said method a solution of soybean protein isolate is prepared and the pH of the solution is adjusted to 12. After thermal treatment aiming at breaking eventual agglomerates, pH is decreased to a value of 7.4. The formation of particles of soybean protein isolate is triggered by the addition of calcium chloride to the resulting solution. The authors are however silent about the use of lentil protein in the formation of a coacervate.

[0009]

[0006] Wang et al. further disclose in Molecules 2022, 27, 3195 a composition comprising flaxseed oil encapsulated in a matrix comprising lentil protein and maltodextrin prepared by complex coacervation. The solid particles are prepared by spray or freeze drying of an emulsion obtained by mixing flaxseed oil with a solution of lentil protein placed at pH 6, followed by mixing this emulsion with a solution of maltodextrin. The walls of the capsule contain both lentil protein and maltodextrin as wallmaterials, maltodextrin being used as secondary wall material. The authors are however silent about the encapsulation of ingredients other than flaxseed oil.

[0010]

[0007] Karaca et al. disclose in Food Chemistry 2013, 139, 448-457 an alternative composition comprising flaxseed oil encapsulated in a matrix comprising lentil protein and maltodextrin. Said composition is prepared according to a process which comprises the steps of (i) preparing a suspension of lentil protein isolate having a pH of 9.5; (ii) isolating the liquid fraction by centrifugation and acidifying said liquid fraction at pH 4.5 to allow the precipitation of a solid, which is isolated; (iii) addition of maltodextrin to a solution of the solid produced in (ii); and (iv) preparation of oil-in water emulsions using flaxseed oil and the solution obtained in (iii). The thus prepared microcapsules were disclosed as efficient encapsulants of flaxseed oil while protecting it from oxidation and release the oil under simulated gastrointestinal conditions, making them suitable for use in food and bioproduct formulations. The authors are however silent about the encapsulation of ingredients other than flaxseed oil.

[0011]

[0008] A further system for the encapsulation of flaxseed oil is disclosed by Avramenko et al. in Food Research International 2016, 81 , 17-24. This system is based on a capsule composition comprising maltodextrin and lentil protein isolate which forms emulsions with the oil. The composition is prepared by addition of flaxseed oil to a solution comprising a mixture of maltodextrin and lentil protein isolate. The thus prepared microcapsules were disclosed as efficient encapsulants of flaxseed oil while protecting it from oxidation. The authors further conclude that chemical modification of lentil protein isolate (e.g. glycation or phosphorylation) may improve the systems. The authors are however silent about the encapsulation of ingredients other than flaxseed oil.

[0012]

[0009] Encapsulation of canola oil using a system based on lentil protein and maltodextrin has also been reported by Chang et al. in Food Chemistry 2016, 212, 264- 273. Maltodextrin is disclosed as the main component of the microcapsule composition and the formation of the microcapsule is carried out using a solution of lentil protein isolate at acidic pH which forms an emulsion when in contact with maltodextrin and canola oil. The emulsion may further comprise lecithin and / or sodium alginate as emulsifying agents. The authors are however silent about the encapsulation of ingredients other than canola oil.

[0013]

[0010] International patent application WO 2017 / 103072 A1 discloses soybean proteinbased microcapsules suitable for encapsulating microorganisms such as probiotic bacteria prepared by simple coacervation of soybean protein. The formation of the capsule by coacervation is triggered by the use of a divalent or trivalent metal cation, such as Ca2+, by addition of a salt of said metal to a solution comprising soybean protein and the substance to be encapsulated placed in an alkaline medium (pH of about 10). This system is disclosed for the encapsulation of probiotic bacteria. The presence of the capsule around the microorganism is taught to enhance the protection of the microorganism from external agents, such as the acidic conditions of gastric medium. The authors are however silent about the encapsulation of ingredients other than probiotic bacteria.

[0014]

[0011] International patent application WO 2014 / 064591 A1 discloses a microcapsule having a core of a material to be encapsulated (e.g. oil) and a shell comprising a legume protein and a low molecular weight carbohydrate (e.g. maltodextrin). This document also discloses a method for producing microcapsules including combining a material to be encapsulated, a legume protein and a low molecular weight carbohydrate, mixing the resultant solution to form an emulsion, and forming microcapsules. In said method, the pH of the lentil protein isolate solution is adjusted to 3.0 or 7.0 prior to its addition to the low molecular weight carbohydrate and the material to be encapsulated.

[0015]

[0012] Quintero Quiroz, J. et al. disclose in Antioxidants 2020, vol. 9(4), p. 310 a method of encapsulation of annatto extract whereby lentil protein is suspended in water and the pH of the suspension is adjusted to 9.0. After addition of the ingredient to be encapsulated and homogenization, the resulting solution is extruded on a 10% solution of calcium chloride. The resulting capsules are then cross-linked with glutaraldehyde. According to the authors, glutaraldehyde is the most commonly used crosslinking agent to obtain stable microcapsules, and microcapsules with better mechanical properties.

[0016]

[0013] International Patent application WO 2015 / 0193017 A1 discloses microcapsules for the encapsulation of probiotics comprising a cross-linked biopolymer, a plant protein (e.g. lentil protein) and a probiotic. The crosslinker includes calcium or magnesium. The disclosed encapsulation method comprises a step of providing a lentil protein solution of pH 7.0.

[0017]

[0014] Spanish patent ES 2 269 715 T3 discloses a process for manufacturing microcapsules containing a material to be encapsulated, characterized in that a mixture of at least one solubilized plant protein (lentil protein is disclosed as a prophetic example) and a polyelectrolyte of opposite charge to said protein in an aqueous medium is subjected to a complex coacervation optionally followed by a hardening in the presence of such material to be encapsulated. In particular, the method comprises: a) solubilization of at least one plant protein in an aqueous medium at a pH comprised between 2 and 7 and below the isoelectric pH of said protein, b) centrifuging the solution obtained in a), c) mixing the supernatant obtained in b) with an aqueous solution of a polyelectrolyte of opposite charge to that of the plant protein, d) coacervation of the polyelectrolytes in the form of complex polymer, and optionally hardening of the capsules, in the presence of the material to be encapsulated.

[0018]

[0015] From what is disclosed in the art, it derives that there is still a need for improved plant-based compositions for embedding active ingredients, in particular because they offer improved protection of the encapsulated ingredient through the gastrointestinal tract and can be obtained from abundant materials by the means of a simple process which can be implemented at industrial scale.

[0019] SUMMARY OF THE INVENTION

[0020]

[0016] After exhaustive research, the inventors have developed particles having the capacity to encapsulate a broad range of ingredients, including flavouring ingredients, plant extracts and microorganisms such as probiotic bacteria and biologically active ingredients, which can be used in the formulation of food, nutraceutical, pharmaceutical and cosmeceutical products. These particles protect the encapsulated ingredient from being decomposed or inactivated by external agents during (i) the processing of said products, (ii) the storage of said products before their consumption, thus providing a longer shelf-life and (iii) the post-consumption digestion process through the gastrointestinal tract, which typically presents highly acidic conditions. The particles are also suitable for releasing the encapsulated ingredient during the digestion process, particularly in the large intestine and the second portion of the small intestine. The inventors have surprisingly found that the particles according to the invention, which comprise lentil protein, particularly provide higher protection to the encapsulated ingredient from the acidic conditions of the gastrointestinal tract, when compared with the non-encapsulated ingredient or with the ingredient encapsulated with simple coacervate particles of soybean protein known in the art. These particles are stable and inert in the food or in the nutraceutical, cosmeceutical or pharmaceutical formulation in which they are incorporated, preventing the food, nutraceutical, cosmeceutical or pharmaceutical matrix from compromising the viability of the ingredient.

[0021]

[0017] Furthermore, the inventors have developed a method for obtaining these particles in a simple manner, particularly by a method of coacervation, which is applicable at industrial scale. This method does not include the use of surfactants or emulsifiers, synthetic polymers, or any reagent which is not approved as food additive. Furthermore, this process allows particles to be spontaneously formed in the medium in which they are obtained by means of local interactions of the different components thereof, thus leading to a uniform dispersion of self-assembling particles having a matrix-type structure wherein the ingredient is distributed. The particles can be resuspended, but not dissolved, easily in an aqueous medium, protecting the ingredient they contain from the medium. The particles of the invention remain stable in the product in which they are incorporated.

[0022]

[0018] A first aspect of the invention thus relates to a process for the preparation of a particle for embedding an ingredient selected from the group consisting of a biologically active ingredient, a flavouring ingredient, a vegetal extract, a microorganism and a mixture of one or more thereof comprising the steps of:

[0023] (i) providing a dispersion of lentil protein in an alkaline aqueous medium having a pH above 7 and wherein the amount of dispersed lentil protein in said alkaline aqueous solution is comprised between 5 and 150 grams of lentil protein per each liter of alkaline aqueous solution;

[0024] (ii) isolating the liquid phase from the dispersion provided in (i);

[0025] (iii) optionally, adding said ingredient to the liquid phase isolated in step (ii) while maintaining the pH of the resulting mixture alkaline; preferably as a solution or suspension;

[0026] (iv) adding to the resulting mixture obtained in step (ii) or (iii) an aqueous solution of an agent for coacervating lentil protein selected from the group consisting of (i) polysaccharides such as gum arabic, alginate, carrageenan, and pectin, (ii) surfactants, (iii) synthetic polymers such as polyacrylic acid, poly(ethylene glycol) and poly(ethyleneimine) and multivalent ions such as divalent or trivalent ions, whereby the coacervation agent is in a concentration of between 0.1 and 10 mg / mL; preferably the process does not comprise:

[0027] - the step of adding a carbohydrate to the solution of step (ii);

[0028] - the step of adding a biopolymer to the solution of step (ii); and / or

[0029] - the step of crosslinking the product of step (iv). .

[0030]

[0019] The second aspect of the invention relates to a process for the preparation of a particle for embedding an inorganic salt, which comprises the steps of:

[0031] (i) providing an aqueous solution of an inorganic salt; wherein the weight amount of salt is at least twice the amount of water;

[0032] (ii) adding lentil protein to the solution of step (i) and,

[0033] (iii) adjusting the pH of the solution resulting from (ii) to a value of between 8 and 10; preferably of about 8.8.

[0034]

[0020] In preferred embodiments, the process of the first and second aspect allows the preparation of simple coacervates.

[0035]

[0021] A third aspect of the invention relates to the product obtainable by the process of the first or second aspect of the invention. Thus, preferred embodiments of the second aspect of the invention relate to a product that is a simple coacervate.

[0036]

[0022] The product of the third aspect of the invention may also be formulated as a particle suitable for embedding an ingredient selected from the group consisting of a biologically active ingredient, a flavouring ingredient, a vegetal extract, a microorganism and a mixture of one or more thereof, said particle comprising a solid matrix comprising lentil protein and an agent for coacervating lentil protein.

[0037]

[0023] A fourth aspect of the invention thus relates to a particle comprising a solid matrix consisting essentially of lentil protein and a coacervation agent wherein the coacervation agent is a metal cation selected from the group consisting of Ca2+, Mg2+, Zn2+, Fe2+and combinations thereof; and provided that:

[0038] (i) the particle does not comprise a carbohydrate such as maltodextrin;

[0039] (ii) the particle does not comprise both a biopolymer and a crosslinker including calcium or magnesium ; and

[0040] (iii) the particle is not cross-linked with glutaraldehyde .

[0041]

[0024] In preferred embodiments of the invention, the particles of the third and fourth aspect of the invention comprise an ingredient selected from the group consisting of a biologically active ingredient, a flavouring ingredient, a vegetal extract, a microorganism and a mixture of one or more thereof. In said embodiments, the ingredient is preferably distributed throughout the particle. In other preferred embodiments, the particle of the fourth aspect of the invention does not comprise an oil.

[0025] The fifth asect of the invention relates to a particle comprising sodium chloride and a solid matrix consisting essentially of lentil protein.

[0042]

[0026] The sixth aspect of the invention relates to a composition comprising a particle as defined in the third, fourth or fifth aspect of the invention and further comprising a food, pharmaceutical, cosmeceutical or nutraceutical acceptable carrier and / or wherein the particle is in the form of a dry powder.

[0043]

[0027] Since the particles of the invention are useful in the formulation of consumption products such as food, pharmaceutical, cosmeceutical or nutraceutical products, the seventh aspect of the invention relates to a food, pharmaceutical, cosmeceutical or nutraceutical product comprising at least one particle as defined in the the third, fourth or fifth aspectof the invention, or a composition as defined in the sixth aspect of the invention.

[0044] BRIEF DESCRIPTION OF THE DRAWINGS

[0045]

[0028] Fig. 1 describes the procedure for the preparation of a particle embedding a bacteria population according to the first aspect of the invention.

[0046]

[0029] Fig. 2 shows microscope pictures of a particle encapsulating DHA according to Example 9 after several thermal treatments.

[0047]

[0030] Fig. 3 shows microscope pictures of the particles of Example 14: (a) comparative experiment: no addition of coacervation agent; (b) CaCh as coacervating agent; (c) comparative experiment: no addition of coacervation agent and lentil protein solution at pH 7M; (d) ZnSC>4 as coacervating agent.

[0048]

[0031] Fig. 4 shows the evolution over time of the concentration of a L. casei (CECT 475T) population (lyophilized bacteria - square symbols; or encapsulated bacteria according to Example 15 - circle symbol) exposed to simulated gastric (from 0 to 120 min) and intestinal media (from 120 to 240 min).

[0049]

[0032] Fig. 5 shows the evolution of the fraction of surviving bacteria over time ofL. casei (CECT 475T) exposed to simulated gastric (from 0 to 120 min) and intestinal media (from 120 to 240 min) when (a) non-encapsulated; (b) encapsulated as defined herein but replacing lentil protein for soybean protein; (c) encapsulated as defined in Example 15.

[0050] DETAILED DESCRIPTION

[0051]

[0033] All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly throughout the specification and claims unless an otherwise expressly set out definition provides a broader definition.

[0034] In the context of the present invention, the term “agent for coacervating lentil protein” refers to a chemical substance suitable for interacting with fragments of lentil protein such as ionic parts, polar segments, hydrophobic moieties, by the means of covalent and non-covalent interactions such as ionic bonding, covalent bonding, hydrogen bonding, hydrophobic / hydrophilic interactions, dipolar interactions and dative bonding, thus inducing the desolvation of lentil protein and the formation of selfassembled aggregates of lentil protein and the coacervation agent. In the context of the process of the invention, the term “agent for coacervating lentil protein” thus encompasses substances suitable for forming complex coacervates with lentil protein, for instance polysaccharides such as maltodextrin or chitosan. Also, in the context of the process of the invention, the term “agent for coacervating lentil protein” encompasses substances suitable for forming simple coacervates of lentil protein, which embodiment is preferred. In more preferred embodiments, this term refers to a chemical substance suitable for interacting with ionic and polar groups of lentil protein by the means of ionic bonding, covalent bonding or metal-anion interactions. Such substances are for instance divalent or trivalent metal cations, including metals of the iron group and alkaline earth metals. As is known in the art, the use of metal cations as coacervation agents triggers the formation of simple coacervate systems. The formation of a simple coacervate may also be triggered by desolvation of a macromolecule, for instance by increasing the ionic strength of the solution of said macromolecule.

[0052]

[0035] In the context of the present invention, the term “coacervate” refers to the product of the phase separation of two or more components placed in a solution induced by the association or aggregation of molecules, one of which is a macromolecule, such as a polymer, a protein, a surfactant, a nucleic acid or a polysaccharide. In the context of the invention, one of the components must be lentil protein.

[0053]

[0036] In the context of the present invention, the term “simple coacervate” refers to a coacervate formed from the phase separation of lentil protein in solution.

[0054]

[0037] In the context of the present invention, the term “complex coacervate” refers to a coacervate formed from the phase separation in solution of two different types of macromolecules, one of which is lentil protein.

[0055]

[0038] In the context of the present invention, the term “biologically active ingredient” refers to a chemical substance, such as a small molecule or a peptide sequence, having therapeutic or prophylactic activity against certain health conditions or providing beneficial effects to the organism. Preferred embodiments of such biologically active ingredient include organic molecules having a molecular weight below 1000 g / mol, preferably below 500 g / mol and being biologically active. Biologically active ingredients useful in the formulation of food, nutraceutical, cosmeceutical or pharmaceutical compositions are well known in the art and will become apparent to the skilled person upon reduction to practice of the invention. Those include active pharmaceutical ingredients (APIs), vitamins, antibiotics, minerals, polyphenols, omega-3 fatty acids, antioxidants, sunscreen components (UV absorbers). A particular example of such biologically active ingredient is quercetin, gentamicin, or docosahexaneoic acid (DHA).

[0056]

[0039] In the context of the present invention, the term “flavouring ingredient” refers to a chemical substance useful for providing flavours to food ingredients. Flavouring ingredients useful in the formulation of food, nutraceutical, cosmeceutical or pharmaceutical compositions are well known in the art and will become apparent to the skilled person upon reduction to practice of the invention. Those include for instance essential oils from aromatic plants, synthetic flavours (esters, aldehydes, ketones and lactones), spices, sweeteners (e.g. aspartame), flavour enhancers (e.g. glutamate) and lipid-based flavors. Particular examples include, for instance, sodium chloride and vanillic acid.

[0057]

[0040] In the context of the present invention, the term “vegetal extract” refers to a composition obtainable by performing an extraction of vegetal-based raw materials, such as plant parts, including roots, leaves, seeds, stems, flowers and fruits. Various extraction processes are known in the art and include extractions employing aqueous or organic extraction media. Vegetal extracts useful in the formulation of food, nutraceutical, cosmeceutical or pharmaceutical compositions will become apparent to the skilled person upon reduction to practice of the invention. Examples of particularly useful plant extracts in the formulation of food, nutraceutical, cosmeceutical or pharmaceutical compositions include pomegranate extract, seaweed extract such as C. cogersae extract or seaweed infusion, green tea extract, turmeric extract, grapeseed extract, gingko biloba extract and aloe vera extract.

[0058]

[0041] In the context of the present invention, the term “microorganism” refers to microscopic organisms selected from the taxonomic groups consisting of bacteria, archaea, fungi, protists, and viruses. Examples of particularly useful microorganisms in the formulation of food, nutraceutical, cosmeceutical or pharmaceutical compositions include prebiotic bacteria, probiotic bacteria and yeast. Particular examples of probiotic bacteria include B. longum (DSM 20219), L. salivarius (DSM 24078), L. casei (CECT 475T), L. plantarum (WCFS-1), L. rhamnosus (GG), and L. pentosus (LPG1).

[0059]

[0042] In the context of the present invention, the term “alkaline medium” refers to an aqueous solution having a pH above 7; preferably above 8 and more preferably lower than 11 .

[0060]

[0043] In the context of the present invention, the term “mean size”, when referring to a particle is understood as the average diameter of the particle population, moving together in an aqueous medium. The mean size of these systems can be measured by standard methods known by the person skilled in the art and are described, for example, in the experimental part below.

[0061]

[0044] In the context of the present invention, the term “solid matrix” refers to a solid tridimensional object, such as a sphere, having a matrix-type structure in which the lentil protein and the coacervation agent form a continuous structure and wherein the ingredient is distributed, preferably homogeneously distributed, throughout the entire matrix. The external and internal structures of the particles of the invention are not differentiated, such that the embedded ingredient is distributed throughout the matrix, preferably in a homogeneous manner.

[0062]

[0045] As used herein the term “lentil protein” refers to lentil protein isolate, lentil protein concentrate, extracted lentil protein, fractions of lentil protein and other forms of lentil protein known in the art. Preferably, the term “lentil protein” refers to lentil protein concentrate.

[0063]

[0046] Lentil protein can be obtained by extraction from milled lentils using aqueous solutions under controlled conditions of temperature pH and time. Said extraction may also be assisted by enzymes. Lentil protein is further obtained by centrifugation and / or precipitation of the soluble protein fraction. While lentil protein may be in the form of protein curds, particle-like separated lentil proteins, particle-like concentrated lentil proteins, spun fiber-like lentil proteins, structural fiber-like lentil proteins, particle-like lentil proteins, hunk-like lentil proteins, flake-like lentil proteins, bar-like lentil proteins, dice-like lentil protein, it is preferred that lentil protein is in the form of a powder. Alternatively, lentil protein may be provided as a protein concentrate.

[0064]

[0047] As used herein, the term “divalent metal cation” refers to a cation originating from any metal element the valence of which is 2, for example, an alkaline earth-metal or a metal from the iron group, e.g., calcium, magnesium, zinc, etc., or if it has several valences, one of them is 2, for example, iron, etc., provided that said cation is suitable for use in pharmaceutical, food, cosmeceutical or nutraceutical compositions. In a preferred embodiment, the divalent metal is selected from Ca2+, Mg2+, Zn2+and Fe2+, more preferably is Ca2+.

[0065]

[0048] As used herein, the term “trivalent metal cation” refers to a cation originating from any metal element the valence of which is 3, or if it has several valences, one of them is 3, for example, iron, aluminium, etc., provided that said cation is suitable for use in pharmaceutical, food, cosmeceutical or nutraceutical compositions.

[0066]

[0049] As will be understood by the person skilled in the art, the di-valent or tri-valent metal can be provided by any suitable source of said metal cation, such as a compound which gives rise to said di- or tri-valent metal cation in an aqueous solution. For example, in the case of using Ca2+, this divalent metal cation can be obtained from calcium chloride, calcium acetate, calcium gluconate, calcium lactate, calcium sorbate, calcium ascorbate, calcium citrate, calcium propionate, calcium sulfate, etc., or mixtures of said compounds.

[0067]

[0050] As defined above, a first aspect of the invention relates to a process for the preparation of a particle for embedding an ingredient selected from the group consisting of a biologically active ingredient, a flavouring ingredient, a vegetal extract, a microorganism and a mixture of one or more thereof comprising the steps of:

[0068] (i) providing a dispersion of lentil protein in an alkaline aqueous medium having a pH above 7 and wherein the amount of dispersed lentil protein in said alkaline aqueous solution is comprised between 5 and 150 grams of lentil protein per each liter of alkaline aqueous solution;

[0069] (ii) isolating the liquid phase from the dispersion provided in (i);

[0070] (iii) optionally, adding said ingredient to the liquid phase isolated in step (ii) while maintaining the pH of the resulting mixture alkaline; preferably as a solution or suspension;

[0071] (iv) adding to the resulting mixture obtained in step (ii) or (iii) an aqueous solution of an agent for coacervating lentil protein selected from the group consisting of (i) polysaccharides such as gum arabic, alginate, carrageenan, and pectin, (ii) surfactants, (iii) synthetic polymers such as polyacrylic acid, poly(ethylene glycol) and poly(ethyleneimine) and multivalent ions such as divalent or trivalent ions, whereby the coacervation agent is in a concentration of between 0.1 and 10 mg / mL; preferably the process does not comprise:

[0072] - the step of adding a carbohydrate to the solution of step (ii);

[0073] - the step of adding a biopolymer to the solution of step (ii); and / or

[0074] - the step of crosslinking the product of step (iv).

[0075]

[0051] In a preferred embodiment of the first aspect of the invention, the dispersion of the lentil protein in the alkaline aqueous solution of step (i) can be obtained by conventional methods known by those skilled in the art, for example by adding the lentil protein to the alkaline aqueous solution.

[0076]

[0052] In a particular embodiment, the alkaline aqueous solution of step (i) is a solution having a pH ranging from 8 to 12, preferably a pH ranging from 8 to 11 ; more preferably about 10. Preferably, said alkaline solution is an aqueous hydroxide solution of an alkaline metal such as sodium.

[0077]

[0053] The amount of lentil protein that can be added to the alkaline aqueous solution in step (i) can vary within a wide range, nevertheless, in a particular embodiment, the amount dispersed in said alkaline aqueous solution is comprised between 5 and 150 grams of lentil protein per each liter of alkaline aqueous solution, preferably between 2 and 100 grams of lentil protein per each liter of alkaline aqueous solution. Said dispersion of lentil protein preferably does not contain any organic solvent.

[0054] In some embodiments, the amount of lentil protein in the alkaline aqueous solution is between 5 and 50 grams of lentil protein per each liter of alkaline aqueous solution; preferably of between 10 and 40 grams of lentil protein per each liter of alkaline aqueous solution; more preferably of about 30 grams of lentil protein per each liter of alkaline aqueous solution; this is particularly the case when the ingredient is a flavouring ingredient that is an inorganic salt, such as sodium chloride.

[0078]

[0055] In other embodiments, the amount of lentil protein in the alkaline aqueous solution is between 90 grams and 120 grams of lentil protein per each liter of alkaline aqueous solution; more preferably of about 100 grams of lentil protein per each liter of alkaline aqueous solution; this is particularly the case when the ingredient is a biologically active ingredient, a vegetable extract, a flavoring ingredient or a microorganism.

[0079]

[0056] In a preferred embodiment of the first aspect of the invention, the dispersion of the lentil protein in the alkaline aqueous solution of step (i) is one wherein the pH of the dispersion is of between 9 and 12, preferably of about 10 and the amount of lentil protein in the alkaline aqueous solution is between 90 grams and 120 grams of lentil protein per each liter of alkaline aqueous solution; more preferably of about 100 grams of lentil protein per each liter of alkaline aqueous solution. This is particularly the case when the particle is used to incorporate an ingredient that is a biologically active ingredient, a vegetable extract, a flavoring ingredient or a microorganism.

[0080]

[0057] In a preferred embodiment of the first aspect of the invention, step (ii) of isolating the liquid phase is carried out employing solid-liquid separation techniques known in the art, and including filtration, microfiltration, ultrafiltration and centrifugation. Preferably, step (ii) is carried out by centrifugation. Said centrifugation is preferably carried out at a temperature of the dispersion of step (i) of between 1 °C and 6 °C; preferably of 4 °C.

[0081]

[0058] In some embodiments, step (i) provides a liquid phase, in which case step (ii) is superfluous.

[0082]

[0059] Step (iii) of the process of the first aspect of the invention comprises adding an ingredient to the liquid phase isolated in step (ii) while maintaining the pH of the resulting mixture alkaline. The ingredient may be added in any form, including a solid, a liquid, a solution or a suspension. Preferably, the ingredient is added as a solution or a suspension.

[0083]

[0060] Any ingredient known in the art as suitable active ingredients in food, nutraceutical, cosmeceutical or pharmaceutical products are suitable for step (iii). Such ingredients include a biologically active ingredient, a flavouring ingredient a vegetal extract, a microorganism and a mixture of one or more thereof.

[0084]

[0061] In some embodiments, the ingredient is a biologically active material selected from the group consisting of active pharmaceutical ingredients (APIs), vitamins, minerals, polyphenols, omega-3 fatty acids, antibiotics, antioxidants, and sunscreen components such as UV absorbers or filters. Preferably, said ingredient is selected from the group consisting of quercetin, gentamicin sulfate, peptides and docosaheaneoic acid (DHA).

[0085]

[0062] In other embodiments, the ingredient is a flavouring ingredient selected from the group consisting of essential oils from aromatic plants, synthetic flavours selected from esters, aldehydes, ketones and lactones, spices, sweeteners such as aspartame, flavour enhancers such as glutamate or sodium chloride and lipid-based flavors. Preferably, said ingredient is selected from the group consisting of sodium chloride and vanillic acid.

[0086]

[0063] In other embodiments, the ingredient is a vegetal extract selected from the group consisting of pomegranate extract, seaweed extract such as C. cogersae extract or seaweed infusion, green tea extract, turmeric extract, grapeseed extract, gingko biloba extract and aloe vera extract. Preferably, said ingredient is selected from the group consisting of pomegranate extract, seaweed extract such as C. cogersae extract and seaweed infusion.

[0087]

[0064] In other embodiments, the ingredient is a microorganism. Said microorganism is preferably selected from the group consisting of prebiotic bacteria, probiotic bacteria and yeast. Preferably, said ingredient is selected from the group consisting of B. longum (DSM 20219), L. salivarius (DSM 24078), L. casei (CECT 475T), L. plantarum (WCFS- 1), L. rhamnosus (GG), and L. pentosus (LPG1).

[0088]

[0065] In some embodiments, it is preferred that the ingredient is not an oil.

[0089]

[0066] The amount of the active ingredient in the solution of step (iii) can vary widely depending on the nature of the ingredient and its efficiency in providing an effect in the product incorporating the composition to be manufactured. Preferably, the amount of the solution of step (iii) added to the liquid solution of step (ii) is such that the final product is suitable for delivering an effective amount of active ingredient by administration of routine amounts of said composition in the context of a food, pharmaceutical, nutraceutical or cosmeceutical product. Such amounts will become apparent to the skilled person upon reduction to practice of the invention.

[0090]

[0067] For instance, biologically active ingredients may be present in the solution of step (iii) in the range of from 0.01 mg / mL to 100 mg / mL of said solution. In such cases, step (iii) produces a solution whereby the concentration of said ingredient varies from 0.1 mg / mL to 500 mg / mL. More specifically, when the ingredient is an antibiotic, such as gentimicin sulfate, the solution of step (iii) comprises the antibiotic in a concentration of between 1 and 100 mg / mL and steps (iii) produces a solution wherein the antibiotic is in a concentration of between 0.5 and 2.5 mg / mL. Similarly, when the ingredient is an omega-3 fatty acid, such as DHA, the solution of step (iii) comprises the omega-3 fatty acid in a concentration of between 1 and 25 mg / mL and steps (iii) produces a solution wherein the omega-3 fatty acid is in a concentration of between 1 and 5 mg / mL. Also, when the ingredient is an omega-3 fatty acid, such as DHA, the solution of step (iii) comprises the omega-3 fatty acid in a concentration of between 1 and 25 mg / mL and steps (iii) produces a solution wherein the omega-3 fatty acid is in a concentration of between 1 and 5 mg / mL. Also, when the ingredient is a flavonoid such as quercetin, the solution of step (iii) comprises the flavonoid in a concentration of between 0.1 and 2 mg / mL and steps (iii) produces a solution wherein the flavonoid is in a concentration of between 0.05 and 0.5 mg / mL. Also, when the ingredient comprises peptides such as peptides extracted from plants, the solution of step (iii) produces a solution wherein the peptides are in a concentration of between 10 and 50 mg / mL.

[0091]

[0068] For instance, flavouring ingredients, such as organic compounds or inorganic salts, may be present in the solution of step (iii). Organic compounds, such as vanilic acid, are preferably present in an amount of between 0.5 mg / mL to 10 mg / mL of said solution. In such cases, step (iii) produces a solution whereby the concentration of said ingredient varies from 0.1 mg / mL to 1.5 mg / mL. Inorganic salts, such as sodium chloride, are preferably present in an amount of between 50 mg / mL to 500 mg / mL of said solution. In such cases, step (iii) produces a solution whereby the concentration of said ingredient varies from 10 mg / mL to 100 mg / mL

[0092]

[0069] Also, vegetal extracts, such as pomegranate extract, seaweed extract such as C. cogersae extract and seaweed infusion, may be present in the solution of step (iii). Vegetal extracts are preferably present in an amount of between 1 mg / mL to 50 mg / mL of said solution. In such cases, step (iii) produces a solution whereby the concentration of said ingredient varies from 0.5 mg / mL to 20 mg / mL.

[0093]

[0070] In further embodiments, the solution of step (iii) comprises microorganisms such as probiotic bacteria, including B. longum (DSM 20219), L. salivarius (DSM 24078), L. casei (CECT 475T), L. plantarum (WCFS-1), L. rhamnosus (GG), and L. pentosus (LPG1). In such cases, the solution of step (iii) comprises the microorganisms in an amount of at least 106CFU / mL, generally between 106and 5x1012CFU / mL, preferably between 109and 1012CFU / mL. In such cases, step (iii) produces a solution whereby the concentration of said ingredient varies from 105and 1012CFU / mL, preferably between 108and 1011CFU / mL.

[0094]

[0071] In some embodiments, the process of the invention thus further comprises the previous preparation of a suspension of probiotic bacteria. Although virtually any probiotic bacteria can be used, in a particular embodiment, said probiotic bacteria are bacteria of the genus Bifidobacterium or Lactobacillus. In a more particular embodiment, said probiotic bacteria are B. longum (DSM 20219), L. salivarius (DSM 24078), L. casei (CECT 475T), L. plantarum (WCFS-1), L. rhamnosus (GG), and L. pentosus (LPG1). The bacterial suspension of step (iii) may comprise, in addition to the probiotic bacteria, a medium suitable for the corresponding probiotic bacteria. Said media are known by the persons skilled in the art, including MRS medium. The amount of probiotic bacteria which may present in the bacterial suspension can vary within a wide range; nevertheless, in a particular embodiment, the amount of probiotic bacteria present in the bacterial suspension is at least 106CFU / mL, generally between 106and 5x1012CFU / mL, preferably between 107and 1012CFU / mL.

[0095]

[0072] In a particular embodiment, once the bacteria have been cultured in the culture medium until early stationary phase growth, the bacterial suspension is subjected to centrifugation in order to eliminate the culture medium. Then, the bacteria are washed to remove any metabolite that would have been formed as well as residues of the culture medium. The washed bacteria are resuspended in an aqueous solution containing a saccharide, such as sucrose, sucralose or other suitable disaccharide, such as for example, maltose or trehalose. If said bacterial suspension contains a disaccharide, for example, a sucrose, the amount of disaccharide (e.g., sucrose) present in said bacterial suspension will be comprised between 0.1% and 10% (w / v) of disaccharide (e.g., sucrose), preferably between 0.1 % and 0.5% (w / v).

[0096]

[0073] In further embodiments, the weight ratio of lentil protein to the ingredient in the solution produced in step (iii) is comprised from 1 :100 to 100:1.

[0097]

[0074] In some embodiments, the weight ratio of lentil protein to the ingredient in the solution produced in step (iii) may be comprised between 1 :100 and 1 :10; preferably of about 3:100; this is particularly the case when the ingredient is an inorganic salt such as sodium chloride.

[0098]

[0075] In other embodiments, the weight ratio of lentil protein to the ingredient in the solution produced in step (iii) is comprised between 100:1 and 20:1 ; this is particularly the case when the ingredient is (i) a biologically active ingredient that is a flavonoid such as quercetin or an antibiotic such as gentimicin sulfate or an omega-3 fatty acid such as DHA, (ii) a flavouring ingredient such as vanilic acid, (iii) a plant extract such as C. cogersae extract.

[0099]

[0076] In other embodiments, the weight ratio of lentil protein to the ingredient in the solution produced in step (iii) is comprised between 100:1 and 20:1 ; this is particularly the case when the ingredient is (i) a biologically active ingredient that is a flavonoid such as quercetin or an antibiotic such as gentimicin sulfate or an omega-3 fatty acid such as DHA, (ii) a flavouring ingredient such as vanilic acid, (iii) a plant extract such as C. cogersae extract.

[0100]

[0077] In other embodiments, the weight ratio of lentil protein to the ingredient in the solution produced in step (iii) is comprised between 2:1 and 1 :2; this is particularly the case when the ingredient is (i) a biologically active ingredient that is a composition consisting essentially of peptides, (ii) a plant extract such as pomegranate extract or organic seaweed infusion.

[0078] In further embodiments of the first aspect of the invention, step (iii) of the process of the invention is carried out at room temperature i.e., at a temperature comprised between 18 °C and 25 °C, preferably between 20 °C and 22 °C.

[0101]

[0079] In further embodiments of the first aspect of the invention, step (iii) of the process of the invention is carried out by dropwise addition of the solution or suspension comprising the ingredient to the solution of step (ii).

[0102]

[0080] In further embodiments of the first aspect of the invention, the solution or suspension of the ingredient employed in step (iii) is in a polar protic solvent, such as water and ethanol or mixtures thereof.

[0103]

[0081] In further embodiments of the first aspect of the invention, the pH of the solution produced in step (iii) is maintained alkaline, preferably at a value comprised between 8 and 12; preferably between 8 and 11 ; more preferably of about 10.

[0104]

[0082] As mentioned above, the process of the first aspect of the invention comprises step (iv) of adding to the resulting mixture obtained in step (ii) or (iii) an agent for coacervating lentil protein in an amount sufficient to allow for the formation of coacervate.

[0083] In preferred embodiments, the agent for coacervating lentil protein is not maltodextrin.

[0105]

[0084] Suitable coacervation agents for vegetal proteins are known in the art and include (i) polysaccharides such as gum arabic, alginate, carrageenan, and pectin, (ii) surfactants, (iii) synthetic polymers such as polyacrylic acid, poly(ethylene glycol) and poly(ethyleneimine) and multivalent ions such as divalent or trivalent ions, including Ca2+or Mg2+. Additional factors such as pH and temperature may also play a role in the coacervation process. Ionic strength of the solution also plays a role in the coacervation process - particularly, in the embodiments whereby the ingredient is sodium chloride, said ingredient may be in such an amount that the ionic strength of the medium is sufficient to allow coacervation of the lentil protein, such that no further coacervation agent may advantageously be required.

[0106]

[0085] In preferred embodiments of the first aspect of the invention, the agent for coacervating lentil protein is chitosan or a di-valent or tri-valent metal cation; preferably it is a di-valent or tri-valent metal cation selected from the group consisting of Ca2+, Mg2+, Zn2+, Fe2+and combinations thereof; preferably Ca2+. The calcium cation can be provided by an aqueous solution of calcium chloride, calcium acetate, calcium gluconate, calcium lactate, calcium sorbate, calcium ascorbate, calcium citrate, calcium propionate, calcium sulfate, calcium carbonate etc., or mixtures of said compounds.

[0107]

[0086] The amount of the coacervation agent must be sufficient to allow for the formation of a coacervate. Such amount will become apparent to the skilled person upon reduction to practice of the process of the invention.

[0087] In preferred embodiments, the amount of coacervation agent is such that the weight ratio of lentil protein to coacervation agent is comprised between 200:1 and 100:40; preferably between 100:1 and 100:10; more particularly from 1 :1 to 100:1. This is particularly the case when the coacervation agent is a calcium(ll) salt or chitosan.

[0108]

[0088] The coacervation agent is preferably added as an aqueous solution of the coacervation agent, whereby the coacervation agent in in a concentration of between 0.1 and 10 mg / mL; preferably between 0.1 and 1 mg / mL. This is particularly the case when the coacervation agent is a calcium (II) salt.

[0109]

[0089] In further embodiments, the process of the first aspect of the invention further comprises (v) drying the mixture resulting from step (iv); preferably by spray-drying.

[0110]

[0090] In a particular embodiment, step (v) further comprises adding a protecting agent to the particles of the invention once they are formed. This protecting agent protects the matrix and the ingredient during the drying process and may be, for example, a saccharide or a suitable acceptable additive or excipient for food, nutraceutical, cosmeceutical or pharmaceutical compositions. Non-limiting, illustrative examples of saccharides which can be used as protecting agents within the context of the present invention include lactose, mannitol, sucrose, maltodextrin, glucose, sorbitol, etc., as well as polysaccharides with prebiotic characteristics, such as for example, oligofructose, pectin, inulin, oligosaccharides (e.g. galacto-oligosaccharides, human milk oligosaccharides), lactulose, dietary fiber, etc. and mixtures thereof. In a particular embodiment, the protecting agent is selected from maltodextrin, inulin, fructooligosaccharides (FOS) and combinations thereof. If the particles of the invention include a protecting agent, the latter is added in a suitable amount. Although the weight ratio of the matrix of particles to the protecting agent can vary within a wide range, in a particular embodiment, the matrix lentil protein protecting agent weight ratio is comprised between 1 :0.1 and 1 :5, typically between 1 :0.5 and 1 :4, preferably about 1 :1. During the spray drying process, the suspension containing the particles of the invention and, eventually the protecting agent, is introduced in a spray-dryer and the processing conditions [air inlet temperature, air outlet temperature, air pressure, sample pumping rate, suction, and airflow] are controlled. The person skilled in the art can set the processing conditions that are most suitable for each case without undue burden.

[0111]

[0091] The method of the invention allows obtaining the particles of the invention in the form of a dry powder, which contributes to the stability of the particles of the invention during long storage periods under controlled or environmental conditions and it can also be easily incorporated in different intended solid and liquid products (e.g., foods, etc.).

[0112]

[0092] Since the particles are formed previously to the addition of the protecting agent, this does not form any conjugate or complex with the lentil protein.

[0093] As mentioned above the process of the second aspect of the invention comprises the steps of:

[0113] (i) providing an aqueous solution of an inorganic salt; wherein the weight amount of salt is at least twice the amount of water;

[0114] (ii) adding lentil protein to the solution of step (i) and,

[0115] (iii) adjusting the pH of the solution resulting from (ii) to a value of between 8 and 10; preferably of about 8.8.

[0116]

[0094] In a preferred embodiment of the second aspect of the invention, the dispersion of the lentil protein in the alkaline aqueous solution of step (i) is one wherein the amount of lentil protein added in step (ii) in the solution of step (i) is between 10 and 40 grams of lentil protein per each liter of the solution of step (i); more preferably of about 30 grams of lentil protein per each liter of the solution of step (i). This is particularly the case when the particle is used to incorporate an ingredient that is a flavouring ingredient that is an inorganic salt, such as sodium chloride.

[0117]

[0095] As mentioned above, the third aspect of the invention relates to a product obtainable according to the process of the first or second aspect of the invention.

[0118]

[0096] In preferred embodiments, the product of the third aspect of the invention is obtainable according to the process as defined in any of the embodiments and alternatives described above for the first and second aspects of the invention.

[0119]

[0097] This product is preferably in the form of a dry powder.

[0120]

[0098] As mentioned above, the fourth aspect of the invention relates to a particle comprising a solid matrix consisting essentially of lentil protein and a coacervation agent wherein the coacervation agent is a metal cation selected from the group consisting of Ca2+, Mg2+, Zn2+, Fe2+and combinations thereof; and provided that:

[0121] (i) the particle does not comprise a carbohydrate such as maltodextrin;

[0122] (ii) the particle does not comprise both a biopolymer and a crosslinker including calcium or magnesium ; and

[0123]

[0099] (iii) the particle is not cross-linked with glutaraldehyde .

[0124]

[0100] In preferred embodiments of the fourth aspect of the invention, each of the ingredient, the coacervation agent, the amount of ingredient and the amount of coacervation agent are independently as defined in any of the embodiments described above for the first aspect of the invention.

[0125]

[0101] In further preferred embodiments of the fourth aspect of the invention, the particle does not comprise an oil.

[0126]

[0102] In other embodiments, the particle of the fourth aspect of the invention is devoid of any coating material or protecting material.

[0103] In more particular embodiments, the particle of the fourth aspect of the invention consists essentially of said solid matrix.

[0127]

[0104] In more particular embodiments, the particle of the fourth aspect of the invention comprises said solid matrix wherein said solid matrix consists essentially of the lentil protein and, optionally, the coacervation agent. Preferably, the ratio of lentil protein to coacervation agent is as defined in the first or second aspect of the invention. In such embodiments, the particle of the fourth aspect of the invention further comprises an ingredient embedded in the solid matrix. Preferably, the ingredient is distributed throughout the matrix; more preferably, said distribution is essentially homogeneous. Said ingredient may be as defined above in the first aspect of the invention.

[0128]

[0105] In further particular embodiments the particle of the fourth aspect of the invention consists essentially of said solid matrix wherein said solid matrix consists essentially of the lentil protein and, optionally, yet preferably, the coacervation agent. Preferably, said particle is devoid of any coating. These embodiments correspond in particular to hollow coacervates.

[0129]

[0106] In further particular embodiments, the particle of the fourth aspect of the invention has a size between 0.1 and 30 .m. Different factors have an impact on the size of the particle. Those include, in a non-limiting manner, the degree of ionization of lentil protein in said particle, the nature and size of ingredient embedded in said particle, the strength of the non-covalent interactions between the ingredient and the solid matrix, the chemical nature of the coacervation agent.

[0130]

[0107] In specific embodiments, the particle of the fourth aspect of the invention is a coacervate, i.e. a self-assembled particle. More particularly, the particle of the third aspect of the invention is preferably a simple coacervate.

[0131]

[0108] In preferred embodiments, the amount of coacervation agent is such that the weight ratio of lentil protein to coacervation agent is comprised between 200:1 and 2:1 ; preferably between 100:1 and 10:1.

[0132]

[0109] In further embodiments, the particle of the fourth aspect of the invention further comprises an ingredient selected from the group consisting of a biologically active ingredient, a flavouring ingredient a vegetal extract, a microorganism and a mixture of one or more thereof. Said ingredient is preferably as defined in any of the embodiments of the first aspect of the invention and / or is present in an amount as defined in any of the embodiments of the first aspect of the invention defined above. In these embodiments, the ingredient is preferably distributed throughout the matrix; more preferably, said distribution is essentially homogeneous.

[0133]

[0110] As defined above, the fifth aspect of the invention relates to a particle comprising sodium chloride and a solid matrix consisting essentially of lentil protein.

[0111] In a preferred embodiment of the fifth aspect of the invention, sodium chloride is embedded in said solid matrix.

[0134]

[0112] As defined above, the sixth aspect of the invention relates to a composition comprising a particle as defined in the third or the fourth aspect of the invention and further comprising a food, pharmaceutical, cosmeceutical or nutraceutical acceptable carrier and / or wherein the particle is in the form of a dry powder.

[0135]

[0113] In particular embodiments of the sixth aspect of the invention, the composition is one wherein the particle, its components and their relative amounts are as defined in any of the embodiments of the third or the fourth aspect of the invention.

[0136]

[0114] In particular embodiments of the sixth aspect of the invention, the particle is in the form of a dry powder.

[0137]

[0115] The composition of the sixth aspect of the invention may be in the form of a liquid, a solid or semi-solid composition.

[0138]

[0116] The composition of the sixth aspect of the invention further comprises a carrier that is acceptable in a food, pharmaceutical, cosmeceutical or nutraceutical composition. Such carriers are known in the art and will become apparent to the skilled person upon reduction to practice of the invention.

[0139]

[0117] As defined above, the seventh aspect of the invention relates to a food, pharmaceutical, cosmeceutical or nutraceutical product comprising at least one particle as defined in the third or the fourth aspect of the invention, or a composition as defined in the sixth aspect of the invention.

[0140]

[0118] In a particular embodiment, the product of the seventh aspect of the invention is a food or feed comprising i) at least one particle according to the third or the fourth aspect of the invention, or ii) a composition comprising at least one particle according to the the third or the fourth aspect of the invention, and a food acceptable vehicle or carrier.

[0141]

[0119] As used herein, the term “food” is any substance or product of any nature, solid or liquid, natural or processed which due to its characteristics, applications, components, preparation and state of preservation, can usually or ideally be used for some of the following purposes: a) as normal nutrition for human beings or animals or as pleasurable foods; or b) as dietetic products, in special cases of human or animal food. The term “feed” includes all the natural materials and finished products of any origin which, separately or conveniently mixed with one another, are suitable as animal food. Examples include cattle feed, chicken feed, horse feed, poultry feed.

[0142]

[0120] A ready-to-eat food is one which does not need to be treated prior to comsumption, for instance by dilution by means of an aqueous solution suitable for consumption. In principle, the ingredients present in a ready-to-eat food are balanced and there is no need to add additional ingredients to the food to make it edible. A concentrated food is one wherein one or more ingredients are present at a higher concentration than in a ready-to-eat food, therefore for use it is necessary to dilute it by means of an aqueous solution suitable for consumption for example. Non-limiting, illustrative examples of foods provided by this invention include both dairy products and derivatives, for example, fermented milks, yoghurt, kephir, curd, cheeses, butters, ice creams, milk-based desserts, etc., and non-dairy products, such as baked products, cakes and pastries, cereals, chocolates, jams, juices, other fruit derivatives, oils and margarines, prepared dishes, etc.

[0143]

[0121] In another particular embodiment, the product of the invention is a nutraceutical product comprising i) at least one particle according to the second or third aspect of the invention, or ii) a composition comprising at least one particle according to the second or third aspect of the invention, and a nutraceutical acceptable carrier.

[0144]

[0122] As used herein, the term “nutraceutical product” refers to a product suitable for use in human beings or animals, comprising one or more natural products with therapeutic activity which provide a health benefit or have been associated with disease prevention or reduction, for example, probiotic bacteria, etc., and it includes dietary supplements presented in a non-food matrix (e.g., capsules, powder, etc.) of a concentrated bioactive product usually present (or not) in the foods and which, when taken in a dose higher than that existing in those foods, exerts a favorable effect on health which is greater than the effect which the normal food may have. Therefore, the term “nutraceutical product” includes isolated or purified food products as well as additives or food supplements which are generally presented in dosage forms normally used orally, for example, capsules, tablets, sachets, drinkable phials, etc.; such products provide a physiological benefit or protection against diseases, generally against chronic diseases. If desired, the nutraceutical product provided by the invention can contain, in addition to the ingredient embedded in the matrix, one or more nutraceuticals (products or substances associated with disease prevention or reduction), for example, flavonoids, omega-3 fatty acids, etc., and / or one or more prebiotics (non-digestible food ingredients which stimulate probiotic activity and / or growth), for example, oligofructose, pectin, inulin, galacto-oligosaccharides, lactulose, human milk oligosaccharides, dietary fiber, etc.

[0145]

[0123] In another particular embodiment, the product of the invention is a pharmaceutical product comprising i) at least one particle according to the second or third aspect of the invention, or ii) a composition comprising at least one particle according to the second or third aspect of the invention, and a vehicle or carrier suitable for oral, topical, rectal or vaginal administration; to that end, said product comprises a pharmaceutically acceptable vehicle or carrier comprising one or more excipients suitable for oral administration, for example, in the form of capsule, powder, granulate, tablet (coated or non-coated), sachet, matrix, suspension, etc., or a pharmaceutically acceptable vehicle or carrier comprising one or more excipients suitable for topical administration, for example, in the form of cream, ointment, salve, etc., or a pharmaceutically acceptable vehicle or carrier comprising one or more excipients suitable for rectal administration, for example, in the form of suppository, etc., or a pharmaceutically acceptable vehicle or carrier comprising one or more excipients suitable for vaginal administration, for example, in the form of bolus, suppository, etc.

[0146]

[0124] In another particular embodiment, the product of the invention is a cosmeceutical product comprising i) at least one particle according to the second or third aspect of the invention, or ii) a composition comprising at least one particle according to the second or third aspect of the invention, and a cosmeceutical acceptable vehicle or carrier. As used herein, the term “cosmeceutical product” refers to a product suitable for use in the body or animal body comprising one or more cosmeceutical products (functional cosmetics, dermaceuticals or active cosmetics), i.e., topical hybrid products with cosmetic-pharmaceutical characteristics containing active ingredients having effect on user’s skin, hair and / or nails, at higher and more effective concentrations, therefore they are located in an intermediate level between cosmetic and drug. Illustrative examples of cosmeceutical products include essential oils, ceramides, enzymes, minerals, peptides, vitamins, etc.

[0147]

[0125] Throughout the description and claims the word “comprises" and variations of the word, are not intended to exclude other technical features, additives, components or steps. Furthermore, the word “comprise” encompasses the cases of “consist of” and “consists essentially of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention.

[0148] EXAMPLES

[0149]

[0126] In the following examples, the following reagents have been employed in the preparation of particles according to the invention:

[0150] - lentil protein concentrate: lentil protein 55 or lentil protein 55 Deflav (Univar), used as received from supplier

[0151] - calcium(ll) chloride or chitosan as coacervating agent

[0152] - sodium hydroxide

[0153] - ethanol 96%

[0154] - eventual protecting agents: mannitol or maltodextrin. General procedure: The process of preparation of a particle according to the invention comprises the steps of:

[0155] (i) providing a dispersion of lentil protein in water whereby the concentration of lentil protein is of 10 mg / mL or 100 mg / mL and wherein the pH is adjusted by addition of NaOH to a value of 8.8 or 10;

[0156] (ii) isolating the liquid phase from the dispersion provided in (i) by centrifugation (at least 3000 g, e.g. 3214 g, during 10 min); the resulting solution may be stored at 4 °C;

[0157] (iii) optionally, adding said ingredient to the solution isolated in step (ii) while maintaining the pH at the value of 8.8 or 10 by addition of NaOH 1 N; preferably as a solution or suspension;

[0158] (iv) adding to the resulting mixture obtained in step (ii) or (iii) an aqueous solution of an agent for coacervating lentil protein in an amount sufficient to allow for the formation of coacervate: the coacervation agent may be calcium(ll) chloride at a concentration of 2 mg / mL or 8 mg / mL; or chitosan. The coacervation agent may also be sodium chloride.

[0159] (v) atomizing the suspension resulting from step (iv) by spray drying.

[0160] Figure 1 summarizes said process for the encapsulation of a bacteria B. longum (DSM 20219).

[0161] Preparative Example 1 : Preparation of solution of lentil protein concentrate

[0162]

[0127] An amount of 10 g or 100 g of lentil protein concentrate was weighed and dispersed in type II distilled water (1 kg) and the resulting dispersion was stirred for 20 minutes. During this period, an aqueous solution of 5 M NaOH was added until reaching a pH value of 10. After this time, the resulting dispersion was centrifugated at 40C at a rate of 3000- 6000 g (e.g. 3214 g or 5000 g) during 8-10 minutes. The recovered solid was removed and the supernatant was recovered. The resulting lentil protein solution was stored at 4 °C. This step allows removing insoluble fibers from lentil protein concentrate. As it will be obvious to the skilled person, this procedure may be adapted by varying the amounts of lentil protein and water to prepare solutions having different concentrations of lentil protein.

[0163] Example 1 : preparation of a lentil protein coacervate embedding sodium chloride

[0164]

[0128] Procedure 1 : A solution of sodium chloride in water was prepared by dissolving 100 g of sodium chloride in 33.3 mL of water. Then, 3 g of lentil protein conjugate were added to the resulting solution. NaOH 50% in water (w / w) was added until the pH of the solution reached the value of 8.8 and the resulting mixture was stirred for 20 minutes. The resulting suspension was then atomized using the following parameters:n: 145 °C, Tout: 78 °C, feed: 3.9 l / h; atomizing frequency: 65 Hz, fan frequency: 60 Hz. More than 80% of the recovered solid particles exhibit a particles size higher than 40 .m.

[0129] Procedure 2: 240 mL of a dispersion of lentil protein conjugate at a concentration of 10 mg / mL as prepared according to Preparative Example 1 were placed in a 500 mL beaker glass. Then, 200 mL of a NaCI solution at 20% (w / v) were added dropwise using gravity to said solution via a 20 mL needleless syringe pointing towards the vortex of the stirring solution of the lentil protein conjugate. After 15 minutes, 20 mL of a solution of calcium chloride at 0.9% (w / v) were added to the solution according to the same procedure. After 15 minutes incubation, the resulting dispersion was atomized by spray drying using the following parameters:n: 200 °C, Tout: 99 °C, drying gas: air, pressure: ca. 50 mbar; pumping: 15%; aspiration: 80%, small cyclone.

[0165]

[0130] Procedure 3: 240 mL of a dispersion of lentil protein conjugate at a concentration of 10 mg / mL as prepared according to Preparative Example 1 were placed in a 500 mL beaker glass. Then, 200 mL of a NaCI solution at 20% (w / v) were added dropwise using gravity to said solution via a 20 mL needleless syringe pointing towards the vortex of the stirring solution of the lentil protein conjugate. After 15 minutes incubation, the resulting dispersion was atomized by spray drying using the following parameters:n: 200 °C, Tout: 99 °C, drying gas: air, pressure: ca. 50 mbar; pumping: 15%; aspiration: 80%, small cyclone.

[0166]

[0131] The inventors have found that Procedures 2 and 3 provide encapsulated salt as a white, fine, odourless powder which exhibit a surprisingly high salted taste while the particles are not perceived by the mouth or the tongue. The observed perceived increased salty flavour surprisingly allows decreasing the amount of salt of up to 25% in certain food matrices. The choice of the procedure mainly depends on the nature of the food matrix hosting the particle embedding sodium chloride.

[0167] Example 2: preparation of a lentil protein coacervate embedding quercetin

[0168]

[0132] 100 mL of a solution of lentil protein conjugate at a concentration of 10 mg / mL as prepared according to Preparative Example 1 were placed in a beaker glass. Then, 47.5 mL of a quercetin solution in absolute ethanol at a concentration of 0.67 mg / mL were added dropwise using gravity to said solution via a 20 mL needleless syringe pointing towards the vortex of the stirring solution of the lentil protein conjugate. After 5 minutes, 10 mL of a solution of calcium chloride at 0.2% (w / v) were added to the solution according to the same procedure. After 20 minutes incubation, the resulting dispersion was atomized by spray drying using the following parameters:n: 120 °C, Tout: 68 °C, drying gas: air.

[0169] Example 3: preparation of a lentil protein coacervate embedding vanilic acid

[0133] 100 mL of a solution of lentil protein conjugate at a concentration of 10 mg / mL as prepared according to Preparative Example 1 were placed in a beaker glass. Then, 80 mL of an aqueous solution of vanilic acid at a concentration of 1.5 mg / mL were added dropwise using gravity to said solution via a syringe pointing towards the vortex of the stirring solution of the lentil protein conjugate. After 5 minutes, 5 mL of a solution of calcium chloride at 0.2% (w / v) were added to the solution according to the same procedure. After 20 minutes incubation, the resulting dispersion was atomized by spray drying using the following parameters:n: 150 °C, Tout: 87 °C, drying gas: air.

[0170] Example 4: preparation of a lentil protein coacervate embedding nile red

[0171]

[0134] 50 mL of a solution of lentil protein conjugate at a concentration of 10 mg / mL as prepared according to Preparative Example 1 were placed in a beaker glass. Then, 3 mL of a solution of nile red in ethanol 70% (w / v) at a concentration of 1 mg / mL were added dropwise using gravity to said solution via a syringe pointing towards the vortex of the stirring solution of the lentil protein conjugate. After 5 minutes, 20 mL of a solution of calcium chloride at 0.2% (w / v) were added to the solution according to the same procedure. After 20 minutes incubation, 10 mL of an aqueous solution of mannitol was added (50 mg / mL concentration). The resulting dispersion was then atomized by spray drying using the following parameters:n: 150 °C, Tout: 89 °C, drying gas: nitrogen.

[0172] Example 5: preparation of a lentil protein coacervate embedding pomegranate extract

[0173]

[0135] 100 mL of a solution of lentil protein conjugate at a concentration of 10 mg / mL as prepared according to Preparative Example 1 were placed in a beaker glass. Then, 64 mL of a solution of pomegranate extract in ethanol 70% (w / v) at a concentration of 17.5 mg / mL were added dropwise using gravity to said solution via a syringe pointing towards the vortex of the stirring solution of the lentil protein conjugate. After 5 minutes, 10 mL of a solution of calcium chloride at 0.2% (w / v) were added to the solution according to the same procedure. The resulting dispersion was then atomized by spray drying using the following parameters: Tjn: 110 °C, Tout: 60 °C, drying gas: nitrogen. of a lentil protein coacervate embedding gentamicin sulfate

[0174]

[0136] 50 mL of a solution of lentil protein conjugate at a concentration of 10 mg / mL as prepared according to Preparative Example 1 were placed in a beaker glass. Then, 2 mL of a solution of gentamicin sulfate in water at a concentration of 50 mg / mL were added dropwise using gravity to said solution via a syringe pointing towards the vortex of the stirring solution of the lentil protein conjugate. After 5 minutes, 64 mL of a solution of calcium chloride at 0.2% (w / v) were added to the solution according to the same procedure. The resulting dispersion was then atomized by spray drying using the following parameters: Tjn: 120 °C, Tout: 66 °C, drying gas: nitrogen.

[0175] Example 7: preparation of a lentil protein coacervate embedding DHA

[0176]

[0137] 100 mL of a solution of lentil protein conjugate at a concentration of 10 mg / mL as prepared according to Preparative Example 1 were placed in a beaker glass. Then, 40 mL of a solution of DHA in ethanol 96% (w / v) at a concentration of 10 mg / mL were added dropwise using gravity to said solution via a syringe pointing towards the vortex of the stirring solution of the lentil protein conjugate. After 5 minutes, 50 mL of a solution of calcium chloride at 0.2% (w / v) were added to the solution according to the same procedure. Optionally, 6.25 mL of an aqueous solution of mannitol can be added (100 mg / mL concentration). The resulting dispersion was then atomized by spray drying using the following parameters:n: 150 °C, Tout: 77 °C, drying gas: air.

[0177]

[0138] CaCh may be replaced by a solution of chitosan in the above procedure, said solution of chitosan having a concentration of 1-5 mg / mL in water comprising 10% v / v acetic acid and having a pH of 3.

[0178]

[0139] General method for particle size measurement: The size of the microparticles was determined by means of confocal microscopy using a Nikon ECLIPSE 55 microscope with Colorview Soft Imaging Systems Camera DS-L2 (Tokyo, Japan). Morphology analysis was additionally performed with a Transmission Electron Microscopy (TEM) Darwin 208 Philips 6080-100 kV coupled to an AMT camera. The particle size distribution and PDI were measured by dynamic light scattering (DLS) using a Zetaplus apparatus (Brookhaven Instrument Corporation, USA). The diameter of the nanoparticles was determined after dispersion in distilled water (1 :10) and measured at 25 °C with a scattering angle of 90°C.

[0179]

[0140] Table 1 below summarizes the obtained results:

[0180] Table 1 Example 8: preparation of a lentil protein coacervate embedding an extract of Coelastrella Cogersae

[0181]

[0141] 100 mL of a solution of lentil protein conjugate at a concentration of 100 mg / mL as prepared according to Preparative Example 1 were placed in a beaker glass. Then, 10 mL of an ethanol solution of an extract of Coelastrella Cogersae at a concentration of 10 mg / mL comprising 43.19% (w / w) omega-3 fatty acids and provided by NeoAlgae were added dropwise using gravity to said solution via a syringe pointing towards the vortex of the stirring solution of the lentil protein conjugate. After 5 minutes, 49.92 mL of a solution of calcium chloride at 0.8% (w / v) were added to the solution according to the same procedure. The resulting dispersion was then atomized by spray drying using the following parameters: Tjn: 110 °C, drying gas: air.

[0182]

[0142] The resulting particles have a particle size of between 1788 ± 253 and 6900 ± 1799 nm and a polydispersity of between 0.477 ± 0,002 y 0.417 ± 0,092. The average omega-3 content of said particles is of 3.52% w / w.

[0183]

[0143] Further tests have shown that the produced particles are not essentially altered when different processing conditions of moisture and temperature have been used, as shown in Table 2 and Figure 2:

[0184] Table 2

[0185] Example 9: preparation of a lentil protein coacervate embedding peptides proceeding from algae

[0186]

[0144] 100 mL of a solution of lentil protein conjugate at a concentration of 20 mg / mL or 100 mg / mL as prepared according to a procedure adapted from Preparative Example 1 were placed in a beaker glass. Then, 2.62 g of powder of protein hydrolysis product provided by Iwi life were added as a suspension towards the vortex of the stirring solution of the lentil protein conjugate. After 5 minutes, 50 mL of a solution of calcium chloride at 0.8% (w / v) were added to the solution using gravity via a syringe pointing towards the vortex of the stirring solution. The resulting dispersion was then atomized by spray drying using the following parameters:n: 120 °C, Tout: 66-75 °C drying gas: air.

[0187]

[0145] Table 3 below summarizes the particle size of the obtained particles:

[0188] Table 3

[0189]

[0146] Process yield, expressed as a percentage, was obtained by gravimetry and was calculated as the ratio of the mass of the obtained powder at the end of the process to the theoretical amount of powder in the final formulation. Theoretical IWI content, expressed as a percentage, was calculated as the ratio of the IWI content added to the formulation (g) to the total amount of powder (theoretical) (g) in the final formulation (g).

[0190] Example 10: preparation of a lentil protein coacervate embedding organic seaweed infusion

[0191]

[0147] 100 mL of a solution of lentil protein conjugate at a concentration of 100 mg / mL as prepared according to Preparative Example 1 were placed in a beaker glass. Then, 18.37 mL iodine rich organic seaweed infusion were added as a suspension towards the vortex of the stirring solution of the lentil protein conjugate. Said organic seaweed infusion was prepared by infusing of powdered dried thallus of Ascophyllum nodosum from outher Hebrides (Scotland) having a content of about 800 mg / kg of iodine in water. After 5 minutes, 19.4 mL of a solution of calcium chloride at 0.2% (w / v) were added to the solution using gravity via a syringe pointing towards the vortex of the stirring solution. The resulting dispersion was then atomized by spray drying using the following parameters: Tjn: 105 °C, Tout: 66-76 °C drying gas: air.

[0192]

[0148] Table 4 below summarizes the properties of the obtained particles:

[0193] Table 4

[0194] 1zeta potential of the above formulations were measured by electrophoretic laser Doppler anemometry using a Zetaplus apparatus (Brookhaven Instrument Corporation, USA). The zeta potential was measured after dispersion of the dried nanoparticles in 1 mM pH 6 KCI solutions.

[0195] 2Iodine Content was measured by ICP-MS.

[0196] Example 11 : preparation of a lentil protein coacervate embedding sodium propionate

[0197]

[0149] 100 mL of a solution of lentil protein conjugate at a concentration of 10 mg / mL as prepared according to Preparative Example 1 were placed in a beaker glass. Then, 874 mg of sodium propionate were added as a suspension towards the vortex of the stirring solution of the lentil protein conjugate. After 5 minutes, 24 mL of a solution of calcium chloride at 0.2% (w / v) were added to the solution using gravity via a syringe pointing towards the vortex of the stirring solution. The resulting dispersion was then atomized by spray drying using the following parameters: Tjn: 105 °C, Tout: 66-76 °C drying gas: air. The resulting particles have an average size of 509.82±9.64 nm and polydispersity index (PDI) of 0.323±0.011.

[0198] Example 12: preparation of a lentil protein coacervate embedding B. lonqum (DSM 20219)

[0199]

[0150] 100 mL of a solution of lentil protein conjugate at a concentration of 25 mg / mL as prepared according to a procedure adapted from Preparative Example 1 were placed in a beaker glass. Then, 15 mL of a suspension of B. longum (DSM 20219) at a concentration of 1010-1011CFU / mL in a 0.2% (w / v) sucrose solution were added towards the vortex of the stirring solution of the lentil protein conjugate. Alternatively, 3 g of lyophilized bacteria dispersed in 10 mL 0.2% (w / v) sucrose solution were added likewise. After 5 minutes, 30 mL of a solution of calcium chloride at 0.2% (w / v) were added to the solution using gravity via a syringe pointing towards the vortex of the stirring solution. After 20 minutes incubation, an aqueous solution of mannitol (100 mg / mL) was optionally added. The resulting dispersion was then atomized by spray drying using the following parameters: Tjn: 100 °C, Tout: 70 °C drying gas: air.

[0200]

[0151] General method for quantifying viable bacteria present in the formulation, and determining the bacterial death cycle throughout the process: In order to determine the viable bacteria in the particles, 20 ml of a solution of NaOH 15 mM was added to a known weight particles (100 mg), weighted with accurate precision in an analytical balance (Sartorius, SAR ME 235S, Gottingen, Germany). Particles were resuspended using a vortex for 5 minutes at 2500 rpm. Then, 1 mL of this solution was added to 9 mL of Ringer solution and vortexed for 15 minutes at 2500 rpm. After the disruption of particles which was checked by optical microscopy, the corresponding decimal dilutions were performed in BPW and seeded in MRS plates. After incubation at 37 °C under anaerobic conditions (MACS 500 Airlock chamber, AEX Chemunex, Spain) for 24-48 hours, colony counts were performed. Taking into account the amount of bacteria initially included in the formulation before spray-drying per each gram of formulation and the counts obtained at the end of the process, the bacterial death cycles were determined by means of the following equation:

[0201] Bacterial death cycles=log(initial CFL / g)- log(recovered CFL / g)

[0202]

[0152] Table 5 below summarizes the obtained results:

[0203]

[0153] The tolerance to gastric medium was then tested according to the following general procedure: The gastrointestinal resistance of probiotic assays were carried out according to the method described by Vinderola et al. (Food Research International, 2003, 36, 895-904).

[0204]

[0154] For the evaluation of gastrointestinal resistance of bacteria, 10 pl of the bacterial suspension in culture medium or 1-2 mg (weighted with accurate precision) of particles were added to PVC tubes with 990 pl of simulated gastric fluid at pH 2.5. As many tubes were used as treatment times planned to be evaluated and taking into account three replicates, specifically 18 tubes (6 times x 3 replicates). Nine of them with simulated gastric fluid corresponding to the times: 0.1 , 0.5 and 2 hours (resistance to simulated gastric fluid) and 2.1 , 5 and 8 hours (containing for 2 hours simulated gastric fluid and during 0.1 , 2, 3 and 6 hours under simulated intestinal fluid).

[0205]

[0155] The simulated gastric fluid was prepared according to pharmacopeia and had the following composition for 1 liter of solution:

[0206] 2 g NaCI (Sigma, Barcelona, Spain)

[0207] 3.2 g pepsin from porcine gastric mucosa (Sigma, Barcelona, Spain)

[0208] 37% HCI (v / v) to adjust the pH at 1.2

[0209] Sodium chloride and pepsin were dissolved in almost 1 liter of type I water and the pH was adjusted to 1.2. The solution was made up to 1 litre volume of type I water and filtered by 0.22 pm sterilized filter.

[0210]

[0156] The simulated intestinal fluid was also prepared according to pharmacopeia:

[0211] 6.8 g KH2PO4 (Panreac, Madrid, Spain) dissolved in 750 ml of type I water

[0212] 10 g of pancreatin from porcine pancreas (Sigma, Barcelona, Spain)

[0213] 0.2 N NaOH / O.2 HCI solutions to adjust the pH at 6.8

[0214]

[0157] The samples were kept at 37 °C in an orbital shaker at 150 rpm for the corresponding time. Then the samples were extracted each time for survivor evaluation.

[0215]

[0158] After 2 hours under simulated gastric conditions, samples were centrifuged at 10,000 rpm for 10 minutes and the supernatant was removed. The pellet was washed twice with 1 ml 0.1 % BPW (w / v) before adding 990 pl of simulated intestinal fluid. The samples were kept in contact with the simulated intestinal fluid for 0.1 , 3 and 6 hours (2.1 , 5 and 8 hours after from the start of the assay). After those times were lapsed, the samples were centrifuged; the supernatants were discarded and the pellets were treated according to the method for the particle disruption described in Section VIII. The viable bacteria count was performed using the method of counting in a MRS agar plate describe in the same section.

[0216]

[0159] The fraction of surviving bacteria was calculated according to the following equation:

[0217] Log survivor fraction = Log / N ) where Nt represents the total viable bacteria after each time of treatment, and No represents the initial number of inoculated bacteria (Bao et al., Food Control, 2010, 21 (5): 695-701).

[0218]

[0160] After two hours incubation at 37 °C in simulated gastric medium, the composition of entry 3 was shown to have undergone 3.45 bacteria death cycles while the control non-encapsulated lyophilized bacteria has undergone 5.24 cycles of bacteria death. These results suggest that a sample of 2.7 x 108CFU / g whereby the bacteria are embedded in a particle according to the invention is equivalent to a sample of 1.96 x 1012CFU / g of non-encapsulated bacteria.

[0219] Example 13: preparation of a lentil protein coacervate embedding L. salivarus

[0220]

[0161] 100 mL of a solution of lentil protein conjugate at a concentration of 25 mg / mL as prepared according to a procedure adapted from Preparative Example 1 were placed in a beaker glass. Then, 2.5-5 mL of a suspension of L. salivarus at a concentration of 101°- 1011CFU / mL in a 0.2% (w / v) sucrose solution were added towards the vortex of the stirring solution of the lentil protein conjugate. Alternatively, 1 g of lyophilized bacteria dispersed in 10 mL 0.2% (w / v) sucrose solution were added likewise. After 5 minutes, 25 mL of a solution of calcium chloride at 0.2% (w / v) were added to the solution using gravity via a syringe pointing towards the vortex of the stirring solution. Alternatively to CaCh, a solution of ZnSCL (50 mL 0.3% (w / v)) can be added. After 20 minutes incubation, an aqueous solution of mannitol (100 mg / mL) was added. The resulting dispersion was then atomized by spray drying using the following parameters: Tjn: 100 °C, Tout: 70 °C drying gas: air.

[0221]

[0162] Figure 3 shows microscope pictures of the particles prepared according the method described above: (a) comparative experiment: no addition of coacervation agent; (b) CaCh as coacervating agent; (c) comparative experiment: no addition of coacervation agent and lentil protein solution at pH 7M; (d) ZnSCL as coacervating agent.

[0222]

[0163] Table 6 below summarizes the obtained results:

[0223] Table 6

[0224]

[0164] Table 7 below shows the measured particles sizes of systems prepared in entries 2 and 3 of Table 6:

[0225] Table 7

[0226]

[0165] After two hours incubation at 37 °C in simulated gastric medium: - the composition of entry 2 of Table 6 was shown to have undergone 2.42 bacteria death cycles;

[0227] - the composition of entry 3 of Table 6 was shown to have undergone 2.24 bacteria death cycles;

[0228] - a composition comprising non-encapsulated lyophilized bacteria was shown to have undergone 4.06 bacteria death cycles;

[0229] - a composition comprising non-encapsulated atomized bacteria was shown to have undergone 4.38 bacteria death cycles.

[0230] These results suggest that the particle protect the bacteria from aggressive gastric medium. .

[0166] The inventors also found that the prepared encapsulated bacteria were stable upon storage at 4 °C for periods of at least one month. In this regard, encapsulated bacteria prepared from suspensions of bacteria appear to be more stable:

[0231] Table 8

[0232] Example 14: preparation of a lentil protein coacervate embedding L. casei (CECT 475T)

[0233]

[0167] Procedure 1 : 100 mL of a solution of lentil protein conjugate at a concentration of 10 mg / mL as prepared according to a procedure adapted from Preparative Example 1 were placed in a beaker glass. Then, 4 mL of a suspension of L. casei (CECT 475T) at a concentration of 101°-1011CFU / mL in a 0.2% (w / v) sucrose solution were added towards the vortex of the stirring solution of the lentil protein conjugate. After 5 minutes, 40 mL of a solution of calcium chloride at 0.9% (w / v) were added to the solution using gravity via a syringe pointing towards the vortex of the stirring solution. After 20 minutes incubation, an aqueous solution of maltodextrin 21 DE (40 mg / mL) was added. The resulting dispersion was then atomized by spray drying using the following parameters: Tjn: 90 °C, Tout: 48 °C drying gas: air.

[0234]

[0168] Figure 4 shows the evolution over time of the concentration of a L. casei (CECT 475T) population (lyophilized bacteria or encapsulated bacteria according to this example) exposed to simulated gastric (from 0 to 120 min) and intestinal media (from 120 to 240 min) as defined in Example 13. The results of Figure 4 show that a larger number of bacteria survive the gastric and intestinal environments when embedded in a matrix according to the invention.

[0235]

[0169] Figure 5 shows the evolution of the fraction of surviving bacteria over time of L. casei (CECT 475T) exposed to simulated gastric (from 0 to 120 min) and intestinal media (from 120 to 240 min) as defined in Example 13 when (a) non-encapsulated; (b) encapsulated as defined herein but replacing lentil protein for soybean protein (purchased from Univar); (c) encapsulated as defined in the present Example. The results of figure 5 show that the containers according to the invention provide unexpected advantages over similar containers based on soybean proteins, particularly in that the embedded ingredient is more protected from degradation in the gastrointestinal tract, thus allowing to obtain a similar effect of the ingredient by administration of a lesser dose. The inventors also found that the lentil protein based capsules favour the late-delivery of the ingredient through the gastrointestinal tract, in particular in the second portion of the small intestine and large intestine.

[0236]

[0170] Procedure 2: 100 mL of a solution of lentil protein conjugate at a concentration of 100 mg / mL as prepared according to a procedure adapted from Preparative Example 1 were placed in a beaker glass. Then, 10.7 mL of a suspension of L. casei (CECT 475T) at a concentration of 101°-1011CFU / mL in a 0.2% (w / v) sucrose solution were added towards the vortex of the stirring solution of the lentil protein conjugate. After 5 minutes, 22.16 mL of a solution of chitosan at 4.77 mg / mL were added to the solution using gravity via a syringe pointing towards the vortex of the stirring solution After 20 minutes incubation, 10.67 mL of an aqueous solution of mannitol (100 mg / mL) was added. The resulting dispersion was then atomized by spray drying using the following parameters: Tjn: 100 °C, Tout: 70 °C drying gas: air.

[0237] Example 15: preparation of a lentil protein coacervate embedding L. plantarum (WCFS- 11

[0238]

[0171] 100 mL of a solution of lentil protein conjugate at a concentration of 10 mg / mL as prepared according to a procedure adapted from Preparative Example 1 were placed in a beaker glass. Then, 5.97 mL of a suspension of L. plantarum (WCFS-1) at a concentration of 1010-1011CFU / mL in a 0.2% (w / v) sucrose solution were added towards the vortex of the stirring solution of the lentil protein conjugate. After 5 minutes, 23.78 mL of a solution of chitosan at a concentration of 5 mg / mL were added to the solution using gravity via a syringe pointing towards the vortex of the stirring solution. After 20 minutes incubation, 0.4 mL of an aqueous solution of mannitol (100 mg / mL) was added. The resulting dispersion was then atomized by spray drying using the following parameters: Tjn: 105 °C, Tout: 70 °C drying gas: air.

[0239]

[0172] The resulting solid product presents a bacteria concentration of 1.37 x 1010CFU / g.

[0240] Example 16: preparation of a lentil protein coacervate embedding L. rhamnosus (GG)

[0241]

[0173] 100 mL of a solution of lentil protein conjugate at a concentration of 100 mg / mL as prepared according to a procedure adapted from Preparative Example 1 were placed in a beaker glass. Then, 4.8-5 mL of a suspension of L. rhamnosus (GG) at a concentration of 1010-1011CFU / mL in a 0.2% (w / v) sucrose solution were added towards the vortex of the stirring solution of the lentil protein conjugate. After 5 minutes, 24.25- 50 mL of a solution of calcium chloride at 0.8% (w / v) were added to the solution using gravity via a syringe pointing towards the vortex of the stirring solution. After 20 minutes incubation the resulting dispersion was atomized by spray drying using the following parameters: Tjn: 100 °C, Tout: 60 °C drying gas: air.

[0242]

[0174] The resulting solid product presents a bacteria concentration of 1.79-2.80 x 1010CFU / g. in coacervate embedding L.

[0243]

[0175] 100 mL of a solution of lentil protein conjugate at a concentration of 100 mg / mL as prepared according to a procedure adapted from Preparative Example 1 were placed in a beaker glass. Then, 6.67-12 mL of a suspension of L. pentosus (LPG1) at a concentration of 1010-1011CFU / mL in a 0.2% (w / v) sucrose solution were added towards the vortex of the stirring solution of the lentil protein conjugate. After 5 minutes, 17.78- 28 mL of a solution of calcium chloride at 0.8% (w / v) were added to the solution using gravity via a syringe pointing towards the vortex of the stirring solution. After 20 minutes incubation, 1.60-3.38 mL of an aqueous solution of mannitol (100 mg / mL) was added. The resulting dispersion was then atomized by spray drying using the following parameters: Tjn: 100 °C, Tout: 70 °C drying gas: air.

[0244]

[0176] The resulting solid product presents a bacteria concentration of 1.20-6.94 x 1010CFU / g.

Claims

CLAIMS1 . Process for the preparation of a particle for embedding an ingredient selected from the group consisting of a biologically active ingredient, a flavouring ingredient, a vegetal extract, a microorganism and a mixture of one or more thereof comprising the steps of:(i) providing a dispersion of lentil protein in an alkaline aqueous medium having a pH above 7 and wherein the amount of dispersed lentil protein in said alkaline aqueous solution is comprised between 5 and 150 grams of lentil protein per each liter of alkaline aqueous solution;(ii) isolating the liquid phase from the dispersion provided in (i);(iii) optionally, adding said ingredient to the liquid phase isolated in step (ii) while maintaining the pH of the resulting mixture alkaline; preferably as a solution or suspension;(iv) adding to the resulting mixture obtained in step (ii) or (iii) an aqueous solution of an agent for coacervating lentil protein selected from the group consisting of (i) polysaccharides such as gum arabic, alginate, carrageenan, and pectin, (ii) surfactants, (iii) synthetic polymers such as polyacrylic acid, poly(ethylene glycol) and poly(ethyleneimine) and multivalent ions such as divalent or trivalent ions, whereby the coacervation agent is in a concentration of between 0.1 and 10 mg / mL; preferably the process does not comprise:- the step of adding a carbohydrate to the solution of step (ii);- the step of adding a biopolymer to the solution of step (ii); and / or- the step of crosslinking the product of step (iv).

2. Process according to claim 1 wherein the alkaline aqueous solution of step (i) is is an aqueous hydroxide solution of an alkaline metal having a pH ranging from 8 to 12.

3. Process according to any one of claims 1 to 2 wherein the amount of lentil protein in the alkaline aqueous solution of step (i) is between 90 grams and 120 grams of lentil protein per each liter of alkaline aqueous solution.

4. Process according to any one of claims 1 to 3 wherein step (ii) of isolating the liquid phase is carried out by a method selected from the group consisting of filtration, microfiltration, ultrafiltration and centrifugation.

5. Process according to any one of claims 1 to 4 wherein step (iii) of the process of the first aspect of the invention comprises adding an ingredient to the liquid phase isolated in step (ii) while maintaining the pH of the resulting mixture alkaline.

6. Process according to any one of claims 1 to 5 wherein the ingredient is selected from the group consisting of:(i) a biologically active material selected from the group consisting of active pharmaceutical ingredients (APIs), vitamins, minerals, polyphenols, omega-3 fatty acids, antibiotics, antioxidants, and sunscreen components such as UV absorbers or filters; said ingredient is preferably selected from the group consisting of quercetin, gentamicin sulfate, peptides and docosaheaneoic acid (DHA);(ii) a flavouring ingredient selected from the group consisting of essential oils from aromatic plants, synthetic flavours selected from esters, aldehydes, ketones and lactones, spices, sweeteners such as aspartame, flavour enhancers such as glutamate, sodium chloride and lipid-based flavors; said ingredient is preferably selected from the group consisting of sodium chloride and vanilic acid;(iii) a vegetal extract selected from the group consisting of pomegranate extract, seaweed extract such as C. cogersae extract or seaweed infusion, green tea extract, turmeric extract, grapeseed extract, gingko biloba extract and aloe vera extract; preferably, said ingredient is selected from the group consisting of pomegranate extract, seaweed extract such as C. cogersae extract and seaweed infusion; and(iv) a microorganism selected from the group consisting of prebiotic bacteria, probiotic bacteria and yeast; preferably, said ingredient is selected from the group consisting of B. longum (DSM 20219), L. salivarius (DSM 24078), L. casei (CECT 475T), L. plantarum (WCFS-1), L. rhamnosus (GG), and L. pentosus (LPG1).

7. Process according to any one of claims 1 to 6 wherein the weight ratio of lentil protein to the ingredient in the solution produced in step (iii) is comprised from 1 :100 to 100:1.

8. Process according to any one of claims 1 to 7 further comprising drying the mixture resulting from step (iv); preferably by spray-drying.

9. Process according to any of claims 1 to 8 wherein the agent for coacervating lentil protein is chitosan or a di-valent or tri-valent metal cation.

10. Process according to any of claims 1 to 9 wherein the coacervation agent is selected from the group consisting of Ca2+, Mg2+, Zn2+, Fe2+and combinations thereof; preferably Ca2+.11 . Process according to any of claims 1 to 10 wherein the pH of the mixture of steps(i) and (iii) is comprised between 8 and 12; preferably between 8 and 11.

12. Process according to any of claims 1 to 11 wherein the pH of the mixture of steps (i) and (iii) is about 10.

13. Process for the preparation of a particle embedding an inorganic salt comprising the steps of:(a) providing an aqueous solution of an inorganic salt; wherein the weight amount of salt is at least twice the amount of water;(b) adding lentil protein to the solution of step (i) and,(c) adjusting the pH of the solution resulting from (ii) to a value of between 8 and 10; preferably of about 8.8.

14. Process according to claim 13 wherein the amount of lentil protein added in step (b) in the solution of step (a) is between 10 and 40 grams of lentil protein per each liter of the solution of step (i); more preferably of about 30 grams of lentil protein per each liter of the solution of step (i).

15. Process according to any of claims 13 to 14 wherein the inorganic salt is sodium chloride.

16. Product obtainable by the process according to any one of the claims 1 to 15.

17. Particle comprising a solid matrix consisting essentially of lentil protein and a coacervation agent wherein the coacervation agent is a metal cation selected from the group consisting of Ca2+, Mg2+, Zn2+, Fe2+and combinations thereof; and provided that:(i) the particle does not comprise a carbohydrate such as maltodextrin;(ii) the particle does not comprise both a biopolymer and a crosslinker including calcium or magnesium; and(iii) the particle is not cross-linked with glutaraldehyde.

18. Particle according to claim 17 wherein the solid matrix is devoid of any coating material.

19. Particle according to any one of claims 17 to 18 wherein the particle has a size of between 0.1 and 30 .m.

20. Particle according to any one of claims 17 to 19 wherein the weight ratio (lentil protein) / (coacervation agent) ranges from 100:1 to 10:1.

21. Particle according to any one of claims 17 to 20 further comprising an ingredient selected from the group consisting of:(i) a biologically active material selected from the group consisting of active pharmaceutical ingredients (APIs), vitamins, minerals, polyphenols, omega-3 fatty acids, antibiotics, antioxidants, and sunscreen components such as UV absorbers or filters; said ingredient is preferably selected from the group consisting of quercetin, gentamicin sulfate, peptides and docosaheaneoic acid (DHA);(ii) a flavouring ingredient selected from the group consisting of essential oils from aromatic plants, synthetic flavours selected from esters, aldehydes, ketones and lactones, spices, sweeteners such as aspartame, flavour enhancers such as glutamate and lipid-based flavors; said ingredient is preferably selected from the group consisting of sodium chloride and vanilic acid;(iii) a vegetal extract selected from the group consisting of pomegranate extract, seaweed extract such as C. cogersae extract or seaweed infusion, green tea extract, turmeric extract, grapeseed extract, gingko biloba extract and aloe vera extract; preferably, said ingredient is selected from the group consisting of pomegranate extract, seaweed extract such as C. cogersae extract and seaweed infusion; and(iv) a microorganism selected from the group consisting of prebiotic bacteria, probiotic bacteria and yeast; preferably, said ingredient is selected from the group consisting of B. longum (DSM 20219), L. salivarius (DSM 24078), L. casei (CECT 475T), L. plantarum (WCFS-1), L. rhamnosus (GG), and L. pentosus (LPG1).

22. Particle comprising sodium chloride and a solid matrix consisting essentially of lentil protein.

23. Composition comprising a particle as defined in anyone of claims 16 to 22 and further comprising a food, pharmaceutical, cosmeceutical or nutraceutical acceptable carrier, preferably wherein the particle is in the form of a dry powder.

24. A food, pharmaceutical, cosmeceutical or nutraceutical product comprising at least one particle as defined in anyone of claims 16 to 22, or a composition as defined in claim 23.

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