Nanoparticle (versions), composition containing said nanoparticle, food product including said composition and method of producing said nanoparticle (versions)

FIELD: chemistry.

SUBSTANCE: group of inventions relates to nanoparticles for encapsulating biologically active compounds. The nanoparticle contains a casein matrix, a basic amino acid and a metal selected from a group including a divalent metal, a trivalent metal and combinations thereof. Disclosed is a method of producing nanoparticles, which includes preparing an aqueous solution of a casein source and a basic amino acid and adding to the prepared solution an aqueous solution of a metal selected from a group including a divalent metal, a trivalent metal and combinations thereof to obtain a suspension containing formed nanoparticles. Another version of the method of producing nanoparticles includes mixing an aqueous solution of a casein source and a basic amino acid with a solution of a biologically active compound and adding to the obtained mixture an aqueous solution of a metal selected from the group to obtain a suspension containing formed nanoparticles. Nanoparticles obtained using said methods are used in combination with a carrier to prepare food products, as well as in pharmaceutics and cosmetics.

EFFECT: invention enables to obtain nanoparticles with high stability and low polydispersity.

40 cl, 17 dwg, 13 tbl, 11 ex

 

The technical field to which the invention relates

The present invention is used in food, pharmaceutical and cosmetic industries and in the field of nanotechnology, and it consists of the encapsulation of biologically active compounds using casein as a covering substance.

The level of technology

The food industry needs in technology development to meet the new demands of consumers. Nanotechnology presents great potential for a radical restructuring of the food industry, because through this technology it is possible to encapsulate biologically active substances (BAS), for example, flavorings, vitamins, minerals, essential oils, antioxidants, prebiotics, etc., for the purpose of obtaining numerous advantages, for example, to increase the storage period of the product, reducing the number of used BASS, control of release, improved bioavailability, masking undesirable tastes, etc.

In the development of media suitable for encapsulation BASS that is very important to choose the material used as a covering substance, or matrix, in this respect should be taken into account form of the drug, its toxicity, the product (food, cosmetic, pharmaceutical product, etc.), in which� will be injected, etc. among other factors. In the field of nanotechnology food products is not recommended to use synthetic polymers, since they can carry the problems of toxicity. Natural polymers do not have these drawbacks, but their use involves the development of more sophisticated methods for producing particles, moreover, in most cases, the size of the resulting particles (greater than 100 microns in most cases) is difficult to control, as a result, these nanoparticles can be seen by the consumer and change the organoleptic properties of the target food product.

Proteins are present in materials traditionally used as substances to cover the BASS. Describes the use of casein as a carrier to encapsulate hydrophobic BASS intended for use in food products (see SA 2649788 and EP 2011472).

Folic acid (pteroylmonoglutamic acid or vitamin B9) is a water-soluble b-vitamin that belongs to the group of folate, is the main in important biochemical processes such as DNA synthesis. Its drawback associated with the presence of megaloblastic anemia, Alzheimer's disease, down syndrome, humoral disorders, certain types of cancer (colon cancer, cervical cancer, leukemia, pancreatic cancer), defects of a nervous tube, appearing in the process of p�Suite fruit complications during pregnancy and male infertility. However, it cannot be synthesized by the body, so must be entered with the help of various additives or diet.

Although folates by nature are present in foods (e.g. fruits and vegetables), mainly in the form polyglutamates, their bioavailability, usually 50% or less, is incomplete. As a result, consumption of food products enriched with folic acid, can create additional possibility of increasing the consumption of the specified vitamin in cases where the consumption of folate are lower than recommended. However, the bioavailability of folic acid added to food, is incomplete because (among other reasons) the effect of the matrix (folic acid may be associated with a component of the food product, thereby preventing its absorption) or the presence of any component in the food product, which reduces its bioavailability. In addition, folic acid is not well absorbed if it is not solubilisation, in the gut. Lack of supplements or fortified products with folic acid, which is administered via capsules, tablets, etc., is that they disintegrate in the stomach under the action of gastric acids, folic acid precipitates, becoming its less RA�create the form, however, only part of the entered folic acid reaches the colon.

In addition, fortification of food with folate or folic acid is a complex process, since both the folate and its derivatives, as well as folic acid is sensitive, among other factors, to changes in temperature, light or pH, as a consequence, there is a risk of violation of their stability under processing conditions of the food product, and the bioactive vitamin, needed by the consumer, could decrease significantly. Thus with the fortification of foods with this vitamin is necessary to take into account these aspects, because the biggest losses can occur during storage and cooking of food.

Described fortification of food with folic acid, mainly dairy and grain products. Also described food additives (EP 2002839) or foods fortified with folic acid or folate, such as sausage meat (ES 2302571), dairy products (EP 1941804), baby food (US 4753926) or even canned food based on chicken meat, pork or beef (EN 2223672 and EN 2213493). However, in the described cases do not consider any possible interaction of vitamin a with the matrix of the food product or its bioavailability.

There is also described a method of producing alginate Il� pectin microcapsules, containing folic acid, to protect it from environmental factors that lead to its degradation, such as the conditions in the stomach, with the achievement of its release in the gut. However, the microcapsules obtained is too large, which affects the organoleptic properties of the target food product. In addition, a method is developed for the encapsulation of folic acid in the nanospheres from poly(lactic-coglycolide acid) (PLGA) and achieve sustained release, although the results are positive, their introduction into the food is dangerous due to the use of this polymer, so it is limited to the fields of medicine and pharmacy.

Consequently, a need exists to create systems encapsulate BASS, preferably water-soluble, preferably water-soluble acidic BASS, for example, folic acid, which are wholly or partly overcome the above disadvantages.

Disclosure of the invention

Now unexpectedly discovered that the nanoparticles formed from casein, additionally containing a basic amino acid (e.g. arginine or lysine) and metal, suitable for food (e.g., calcium) to form the new encapsulating and stabilizing system for biologically active compounds (BASS), which are both water and fat soluble, preferred�relatively water-soluble, more preferably, water-soluble acidic BASS intended for use in food, cosmetic and pharmaceutical products.

Consequently, in one aspect, the invention relates to nanoparticles containing casein matrix, a basic amino acid and metal, suitable for use in foods selected from a divalent metal, a trivalent metal and combinations thereof. These nanoparticles can be used as technological additives, in addition, they have the ability to encapsulate a BASS, preferably water-soluble BASS, more preferably water-soluble acidic BASS, such as, for example, vitamin b or C, such as folic acid, Pantothenic acid and ascorbic acid or other hydrophilic compounds, although they may also include liposoluble BASS.

These nanoparticles are stable and able to protect the BASS from decomposition by external factors, e.g. light, pH changes, oxidation, etc., during processing of the product (e.g., food product, pharmaceutical or cosmetic product), and during storage, and, in addition, when used in food products, they protect the BASS from the acidic conditions of the stomach, preventing its release into the gastric tract, making about�way, to avoid his deposition and, consequently, to avoid low bioavailability. Moreover, it was found that these nanoparticles are able to dissolve in (artificial) intestinal environment, providing a full release of BASS in the intestine for proper absorption and, moreover, allowing you to avoid the problems of toxicity of any type. These mostly inert nanoparticles in the food product in which they are entered, allowing, thus, the BASS to avoid reactions with the various components of the matrix and reduce its bioavailability.

In addition, one of the important properties of the nanoparticles provided by the present invention is the use of casein as a natural carrier to protect the BASS from both the ambient conditions and conditions in the stomach, contributing to his release in the gut and increasing, thus, the bioavailability, because the casein in itself demonstrates the nutrient properties, so that it complements the positive effects of the BASS.

In another aspect, the invention relates to a method for producing these nanoparticles. This method is simple and applicable on an industrial scale. Basically, this method does not provide synthetic or reactive polymers that are not approved as food additives, minimizing the inclusion of surfactants or emolga�Directors, and it gives the possibility of obtaining nanoparticles in the nanometer scale with controlled particle size.

In a particular embodiment of the said method, furthermore, provides for the drying of the suspension containing the nanoparticles, with the aim of obtaining the drug in powder form, preserving the BASS stable over time, this type of powder preparation is particularly suitable for use in solid foods. Mainly specified drying treatment is carried out in the presence of substances that protect the nanoparticles. Nanoparticles containing BASS, thus obtained, can easily be suspended in aqueous medium, protecting the BASS from decomposition in solution. Received the final product is stable and protects the BASS for long periods of storage and, in addition, is suitable for various types of foods, liquids (e.g., beverages, etc.), and solid products.

In another aspect, the invention relates to compositions comprising said nanoparticles intended for use in food, pharmaceutical or cosmetic industries. In fact, these nanoparticles can be administered in the form of creams, gels and hydrogels with the aim of obtaining a stable cosmetic preparations suitable for use in the field. These nanoparticles can also get�ü with fillers, suitable for applications of these nanoparticles topically.

In another aspect, the invention relates to a food product containing the specified composition based on casein nanoparticles provided by the present invention. In a specific embodiment of the specified food product is in a liquid, semi-solid or solid form.

Brief description of the drawings

Figure 1 present a schematic representation of a specific embodiment of the method corresponding to the invention, used to obtain casein nanoparticles containing folic acid.

In the Figures 2 and 3 present the image of unloaded casein nanoparticles obtained using transmission electron microscopy (TEM). The black strip located on the lower left border of the image corresponds to the standard 100 nm.

In the Figures 4 and 5 represent the image of casein nanoparticles containing folic acid, obtained using transmission electron microscopy (TEM). The black strip located on the lower left border of the image corresponds to the standard 100 nm.

In Figure 6 represent the ratio of the encapsulated folic acid and the amount of casein for each mg of folic acid added to the preparation. In all preparations, the weight ratio of lysine and �tree before adding a solution of folic acid is 1:12.

In the Figures 7 to 11 are obtained using scanning electron microscopy (SEM) micrograph of casein nanoparticles containing folic acid and consisting of lysine, without high pressure processing (Fig.7 and 8), with treatment at 100 MPa, 5 min (Fig.9), with treatment at 400 MPa, 5 min (Fig.10) and the processing at 800 MPa, 5 min (Fig.11).

In Figure 12 are obtained using scanning electron microscopy (SEM) micrograph of casein nanoparticles containing folic acid and consisting of arginine with treatment at 400 MPa, 5 min.

In Figure 13 represent the release of folic acid from casein nanoparticles without high pressure processing after incubation in artificial gastric fluid (SGF) (for the first 2 hours: 0-2 h) and artificial intestinal fluid (SIF) (2 - 24 h) at 37±1°C. Data are mean mean - standard deviation (n=6).

In the Figures 14 and 15 present the release of folic acid from casein nanoparticles with high pressure processing (Fig.14) 150 MPa, 5 minutes and (Fig.15) 400 MPa, 5 minutes) after their incubation in artificial gastric fluid (SGF) (for the first 2 hours: 0-2 h) and artificial intestinal fluid (SIF) (2-8 hours) at 37±1°C. Data are mean mean - standard deviation (n=4).

In the Figures 16 and 17 represent the Wi�otocol concentration of folic acid (ng/ml) as a function of time after administration of various drugs vitamins laboratory animals. The results show how the mean - standard deviation (n=5).

Intravenous route, dose 1 mg/kg (Fig.16).

Oral route, the dose of 1 mg/kg (Fig.17): unencapsulated folic acid, dissolved in water (•); folic acid encapsulated in casein nanoparticles dispersed in water (■); folic acid encapsulated in casein nanoparticles processed by high pressure, dispersed in water (▲). The implementation of the invention

The present invention provides a casein nanoparticle and methods of encapsulating biologically active compounds (BASS) in order to protect them from decomposition under the action of external factors such as light, pH changes, oxidation, etc.

Definition

To facilitate understanding of the present invention, the meaning of some terms and expressions as used in the context of the invention, below.

As described in this context, the term "basic amino acid include lysine, arginine and histidine.

As described in this context, the term "casein" refers to anywherefrom protein of approximately 80% of all milk proteins. It is a protein phosphoprotein type, which is included in the determination of globulins, it is soluble, has a high water retention ability and precipitates at about pH 4.6 at 0°C. He formed the four main fractions (αs1-casein, (αs2-casein, β-casein and κ-casein), which differ from each other in the compositions of amino acids, the charge distribution and their tendency to form aggregates in the presence of calcium. In milk casein form large colloidal particles with a diameter of from 50 to 600 nm (average of approximately 150 nm), called "casein micelles". These particles form through hydrophobic interactions and aggregation of calcium phosphate with phosphoserine radicals present in the structure of casein. These micelles form a very stable colloidal system in milk, being one of the main reason of his color, thermal stability and coagulation by rennin..

As described in this context, the term "biologically active compound" or "BASS" refers to any lipid - and water-soluble compound having a nutritional, therapeutic and/or cosmetic activity. Non-limiting illustrative examples of BASS, relevant to the present invention include amino acids, antimicrobial agents, flavors, preservatives, sweeteners, steroids, drugs, hormones, lipids, peptides, polynucleotides, polysaccharides, proteins, proteoglycans, flavorings, vitamins, etc.

As described in this context, the term "water-soluble biological�and active compound" or "BASS water-soluble" refers to the connection, with nutritional, therapeutic and/or cosmetic activity, which is soluble (chorosanctorum, soluble, soluble, tradersforum or malorastvorima) in aqueous solution according to the criteria defined by the Royal Spanish Pharmacopoeia:

Descriptive termsThe approximate volume of solvent in milliliters (ml) per gram of solute, corresponding to a temperature lying in the range from 15°C to 25°C
SolubleLess than 1
Solublefrom 1 to 10

Descriptive termsThe approximate volume of solvent in milliliters (ml) per gram of solute, corresponding to a temperature lying in the range from 15°C to 25°C
Instantfrom 10 to 30
Insolublefrom 30 to 100
Lowfrom 100 to 1000
Very low from 1000 to 10000
Practically insolubleMore than 10000

Non-limiting illustrative examples of water-soluble BASS include vitamins such as vitamins of the groups b or C and their derivatives, salts or esters, hyaluronic acid, chondroitin sulfate, thioctic (lipoic) acid, their salts or esters, etc. In a specific embodiment of the specified water-soluble BASS selected from the group consisting of folic acid, 4-aminobenzoic acid, Niacin, Pantothenic acid, monophosphate, thiamin pyrophosphate, cumintheface, ascorbic acid, pteroylmonoglutamic acids (derivatives of folic acid: volatilisation, polyglutamation), folinic acid, nicotinic acid, hyaluronic acid, thioctic acid (alpha lipoic acid), p-coumaric acid, caffeic acid, and their pharmaceutically or cosmetically acceptable or food grade derivatives, esters or salts, or their mixtures.

As described in this context, the term "fat-soluble biologically active compound" or "BASS fat-soluble" refers to a compound having a nutritional, therapeutic and/or cosmetic activity and which are soluble (chorosanctorum, soluble, soluble, it is difficult�astorino or malorastvorima) in fats and oils according to the criteria defined in the Royal Spanish Pharmacopoeia. Non-limiting illustrative examples of liposoluble BASS include vitamins such as vitamins of the groups A, D, E, K and their derivatives, phospholipids, carotenoids (carotenes, lycopene, lutein, capsanthin, zeaxanthin, etc.), omega-3 fatty acids (docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), etc.), phytostanols and phytosterols (sitosterol, campesterol, stigmasterol, etc.), polyphenols (quercetin, rutin, resveratrol, kaempferol, myricetin, isorhamnetin, etc.) and their derivatives.

They say that the product is "food quality", when its use in food or animal safely in accordance with the food standard of the country or organization, for example, a subsidiary of food and agriculture (FAL) of the United Nations (UN) or the world health Organization (who); therefore, the product is "food quality" is a non-toxic product suitable for use in food products", and therefore both expressions are synonymous and used interchangeably in this description.

As described in this context, the term "ferrous metal" includes any metal element, the valence of which 2, for example, alkaline earth metal, e.g. calcium, magnesium, Cinci etc. or, if it has multiple valences, one of them 2, for example, iron, etc., provided that it is pharmaceutically or cosmetically acceptable or suitable for use in food products.

As described in this context, "trivalent metal" includes any metal element, the valence of which 3, or, if it has multiple valences, one of them 3, for example, iron, etc., provided that it is pharmaceutically or cosmetically acceptable or suitable for use in food products.

As described in this context, the term "nanoparticle" refers to a colloidal system of spherical type or similar form with a size less than 1 micrometer (μm), preferably of the order of magnitude 10-900 nanometers (nm).

As described in this context, the term "average size" refers to the average diameter of a population of nanoparticles, which move together in the aquatic environment. The average size of these systems can be measured by standard methods known to the competent specialist in the field of technology and is described, for example, in the experimental part (see below). Nanoparticles, corresponding to the invention, characterized by the fact that have an average particle size less than 1 micron, typically in the range of 1 to 999 nm, preferably from 10 to 900 nm, more preferably from 50 to 500 nm, even more �predpochtitelno from 100 to 200 nm. In a particular embodiment of the nanoparticles corresponding to the invention have an average particle size in the range from 50 to 200 nm, preferably about 140 nm.

Nanoparticles

In one aspect, the invention relates to the nanoparticles, hereinafter referred to as nanoparticles, corresponding to the invention, containing a casein matrix, a basic amino acid and a metal selected from a divalent metal, a trivalent metal and combinations thereof.

In a specific embodiment of the specified basic amino acid selected from the group consisting of arginine, lysine, histidine and mixtures thereof.

In another specific embodiment of the preferably, when the metal is a divalent metal food quality, selected from the group consisting of calcium, magnesium, zinc, iron (ferrous form) and their combinations.

In another specific embodiment of the specified metal is a trivalent metal food quality, such as, for example, iron in the trivalent form.

Nanoparticles, corresponding to the invention can be used as technological additives, for example, as substitutes for fats, etc. Nanoparticles, corresponding to the invention, in addition, have the ability to encapsulate biologically active .. �moving (BASS).

Thus, in another specific embodiment, the implementation of the nanoparticles corresponding to the invention further contains a biologically active compound (BASS). Specified the BASS can be a BASS water-soluble or fat-soluble BASS; in this case, the nanoparticles according to the invention are sometimes described herein as "loaded nanoparticles according to the invention".

In a specific embodiment of the specified BASS is a BASS water-soluble, preferably water-soluble acidic BASS. In a more specific embodiment of the specified water-soluble BASS selected from the group consisting of:

(a) a vitamin of the group b or C;

(b) a derivative of vitamin corresponding to a);

c) compounds selected from hyaluronic acid, chondroitin sulfate and thioctic acid;

(a) a salt or ester of any of the aforementioned compounds a) to C) and

(e) combinations thereof.

In a special embodiment of the specified water-soluble BASS selected from the group consisting of folic acid, 4-aminobenzoic acid, Niacin or vitamin EOI, Pantothenic acid or vitamin B5, monophosphate, thiamin pyrophosphate, cumintheface, ascorbic acid, pteroylmonoglutamic acids (derivatives of folic acid:

volatilisation, polyglot�of maltolato), folinic acid, nicotinic acid, hyaluronic acid, thioctic acid or alpha-lipoic acid, p-coumaric acid, caffeic acid, and their pharmaceutically or cosmetically acceptable or food grade derivatives, esters or salts, or their mixtures.

In a special embodiment of the specified BASS is a water-soluble acidic BASS, such as folic acid, Pantothenic acid, ascorbic acid, etc.

Without wishing to be bound by any theory, believe that in the presence of metal, such as a divalent metal (e.g. calcium), α - and β-casein are aggregated due to their hydrophilicity, and there is a loss of surface charge, when the radicals of phosphoserine on their surface bind to cationic part. Water-soluble BASS, preferably an acidic (e.g., folic acid), also interacts electrostatically with the specified metal, so it will be captured by the hydrophobic matrix generated by these types of casein. κ-Casein, in turn, react with metal (e.g. calcium), so it is associated with its hydrophobic part with a particle, and its water-soluble fraction of water in contact with the outer environment. The specified water-soluble fraction has in addition to the high proportion of carbonyl groups (�slot groups of amino acids such as glutamic or aspartic acid), polar groups corresponding serialnum and TravelSIM balances associated with three - and tetrasaccharide. Thus, consider that after the formation of the nanoparticles of the basic amino acid (e.g. lysine) in the solution will be linked to the surface of these nanoparticles due to electrostatic interactions [for example, it can be a covalent bond after processing by heating while passing through the spray-drying technique (when required)] to the carboxylic groups of the specified faction. Figure 1 present a schematic representation of the loaded nanoparticles, corresponding to the present invention containing casein matrix, lysine (essential amino acid) and calcium (divalent metal).

In another special embodiment of the specified BASS is a fat-soluble BASS, although in this case it will be necessary to form a preferably homogeneous suspension BASS in an aqueous medium or, more preferably, to dissolve the BASS in an organic solvent, slowly adding the specified aqueous suspension or specified organic solvent in the solution containing a source of casein (e.g. Caseinate), and incubate the mixture.

The mechanism of capture will be different from that described for water-soluble BA�, because fat-soluble BASS will be captured in the inner hydrophobic part of the nanoparticles due to the affinity between both factions, regardless of whether they possess or do not possess the ability to interact with (divalent or trivalent) metal.

In a specific embodiment of the specified BASS is a BASS fat-soluble, selected from vitamins, such as vitamins of the groups A, D, E, K and their derivatives, phospholipids, carotenoids (carotenes, lycopene, lutein, capsanthin, zeaxanthin, etc.), omega-3 fatty acids (e.g., DHA, EPA, etc.), amino acids (e.g., isoleucine, leucine, methionine, phenylalanine, tryptophan, and valine), phytostanols and phytosterols (e.g., sitosterol, campestrini, stigmasterol, etc.) polyphenols (such as quercetin, rutin, resveratrol, kaempferol, myricetin, isorhamnetin, etc.) and their derivatives.

The weight ratio BASS casein in the loaded nanoparticles according to the invention, can vary in a wide interval, in non-limiting illustrative example, the weight ratio BASS casein in the loaded nanoparticles according to the invention, may range from 1:1 to 1:200, preferably from 1:10 to 1:80, more preferably from about 1:15 to 1:35. In a particular embodiment of the BASS is a �vodorastvorimoe BASS and the mass ratio (water-soluble) BASS:casein in the loaded nanoparticles according to the invention, is from 1:1 to 1:50, preferably from 1:10 to 1:30, more preferably from about 1:15 to 1:20. In another specific embodiment of the BASS is a fat-soluble BASS, and the weight ratio of (fat-soluble) BASS:casein loaded in the nanoparticles according to the invention, is from 1:1 to 1:200, preferably from 1:10 to 1:80, more preferably from about 1:20 to 1:35.

In addition, if necessary, the composition of the nanoparticles according to the invention, as those that are loaded with BASS, and those that are not loaded, may be included an antioxidant, such as ascorbic acid (vitamin C), etc., with the purpose to improve their stability in terms of temperature and oxidation. In a particular embodiment of the BASS is a folic acid and the antioxidant is an ascorbic acid, the effect of which, apparently, is to protect the folic acid from decomposition when exposed to ultraviolet radiation, changes in pH, heat, oxygen, etc., and, in addition, provides nutritional support for ascorbic acid. This antioxidant can be coencapsulation with BASS or introduced into the coating of the nanoparticles according to the invention.

A method of producing nanoparticles

In another aspect, the invention relates to FPIC�BU nanoparticles, containing casein matrix, a basic amino acid and a metal selected from a divalent metal, a trivalent metal and combinations thereof (nanoparticles, corresponding to the invention), hereinafter "method [1], corresponding to the invention, which provides for:

(a) obtaining an aqueous solution containing a source of casein and a basic amino acid, and

(b) adding an aqueous solution of metal selected from a divalent metal, a trivalent metal and combinations thereof, to the solution obtained in stage a).

In another aspect, the invention also relates to a method for producing the nanoparticle containing a casein matrix, a basic amino acid, a metal selected from a divalent metal, a trivalent metal and combinations thereof, and the biologically active compound (loaded nanoparticles, corresponding to the invention), hereinafter "method [2], corresponding to the invention, which provides for:

(a) mixing (i) an aqueous solution containing a source of casein and a first basic amino acid, with (ii) a solution containing biologically active compound, and

(b) adding an aqueous solution of metal selected from a divalent metal, a trivalent metal and combinations thereof, to the mixture obtained in stage a).

At the stage a) of the method [1], corresponding to the invention, an aqueous solution containing a source� casein and a basic amino acid, get accepted methods known to the competent specialists in the field of technology, for example, by adding a specified source of casein and a basic amino acid to the aquatic environment.

At the stage a) of the method [2], corresponding to the invention, an aqueous solution (i) containing a source of casein and a basic amino acid, is mixed with solution (ii) containing BASS. The nature and composition of the specified solution (ii) containing BASS, can vary depending on the type and nature of the BASS. Thus, in a particular variant of implementation, when a BASS is a BASS water-soluble, said solution (ii) containing BASS, is an aqueous solution; in another specific embodiment of the when the BASS is a water-soluble acidic BASS, said solution (ii) containing BASS, is an aqueous solution containing, in addition, a second basic amino acid, and in another particular variant of implementation, when a BASS is a BASS fat-soluble, said solution (ii) containing BASS, it is a suspension in an aqueous medium or, preferably, the organic solution, more preferably

organic solution of the water-miscible solvent, such as, for example, ethanol.

Casein, which can be used for practical implementation of both methods [methods [1] and[2], according to the invention], can occur from virtually any source of casein, such as milk, beans, etc. Casein can be found in the specified solution in the form of acid casein or Caseinate. In a specific embodiment of the specified source of casein contains casein in the form of Caseinate, preferably sodium Caseinate. Although you can also use calcium Caseinate and fastcall, they are less suitable in practice, because calcium is used for the formation of nanoparticles after mixing Caseinate with the active ingredient, as a consequence, if the solution already contains Caseinate calcium in the environment, the practical implementation of the method can be severely disturbed.

The amount of casein that can be contained in the aqueous solution formed in step a) of method [1], corresponding to the invention, as well as in aqueous solution (i) [containing a source of casein and a first basic amino acid], used in stage a) of the method [2], corresponding to the invention, can vary within wide limits;

however, in a particular embodiment of the amount of casein contained in the said aqueous solution is from 0.1% to 10% (wt./vol.), preferably from 0.5% to 5%, more preferably from 1% to 3%.

Basic amino acid contributes to the dissolution of casein and, if necessary, BASS, royalty�NGOs water-soluble acidic BASS, therefore, it plays a very important role in getting the BASS that are loaded nanoparticles, corresponding to the invention, and BASS that are not loaded. In fact, apparently, with the increase of pH of the solution basic amino acid gives you the ability to dissolve the Caseinate without having to use inorganic salts, and, in addition, it acts as the basis for preservation of the hydrophilic ends of the Kappa (κ)-casein fractions in anionic form, so that particles with negative surface charge are maintained in suspension and not aggregated due to electrostatic repulsion.

Essential amino acid that can be used for practical implementation of both methods [methods [1] and [2], corresponding to the invention] is selected from the group consisting of arginine, lysine, histidine, and mixtures thereof, preferably from arginine, lysine, and mixtures thereof. Basic amino acid may be inside or outside of the nanoparticles according to the invention, plays an important technological role because it helps dissolve the components before the formation of the nanoparticles, and it maintains a suitable pH after receiving them on both sides of nanoparticles (inside and outside). As an illustration, folic acid slightly soluble in water, but easily soluble in weakly alkaline aqueous solution, so�in the presence of basic amino acid promotes the dissolution of folic acid.

In a particular embodiment of the method [2], corresponding to the invention, when the BASS is a water-soluble acidic BASS, said solution (ii) containing BASS, is an aqueous solution containing, in addition, a second basic amino acid (to prevent deposition of BASS). Although in this case involve the application of two different basic amino acids, in a specific embodiment of the basic amino acid used to obtain an aqueous solution containing a source of casein (the first basic amino acid), and used to obtain the aqueous solution containing the BASS (second basic amino acid), are the same and selected from the group consisting of arginine, lysine, histidine, and mixtures thereof, preferably from arginine, lysine, and mixtures thereof.

The number of basic amino acids that can be contained in the solution formed in step a) of method [1], corresponding to the invention, and in solution (i), corresponding to stage (a) of the method [2], corresponding to the invention, can vary within wide limits and depends largely on the basic amino acids. Consequently, although the mass ratio of basic amino acid:casein can vary, in a particular embodiment of the mass ratio of basic amino acid:�ASEAN in solution, formed in stage a) of the method [1], corresponding to the invention, or in solution (i) of the method [2], corresponding to the invention, is from 1:1 to 1:50, preferably from 1:10 to 1:40, more preferably approximately 1:12, when used the basic amino acid is a lysine, or about 1:25, when used, the basic amino acid is an arginine.

When the BASS is a water-soluble acidic BASS, solution (ii), corresponding to stage (a) of the method [2], corresponding to the invention containing a specified BASS, contains, in addition, a second basic amino acid, which, as mentioned above, may be the same or different from the first basic amino acid, in this case the ratio of the basic amino acid:casein in the method [2], corresponding to the invention, i.e. after mixing the solutions (i) and (ii), corresponding to stage (a) this method is from 1:1 to 1:50, preferably from 1:5 to 1:20, more preferably about 1:6, when used the basic amino acid is a lysine, or about 1:9, when used the basic amino acid is an arginine.

As the method [1], corresponding to the invention and the method of [2] corresponding to the invention include the step of adding (a) an aqueous solution of a metal selected �W divalent metal, trivalent metal and combinations thereof [step b)] to a solution of corresponding stage. Without wishing to be bound by any theory, believe that the specified metal, such as a divalent metal (e.g. calcium), gives the possibility of creating a bridge inside loaded nanoparticles according to the invention, which contributes to the stabilization of the BASS, in particular, when a BASS is a BASS water-soluble, preferably water-soluble acidic BASS, or BASS water-soluble, capable of reacting with said metal (e.g. calcium), for example, folic acid, Pantothenic acid or vitamin b or C or its derivatives, in this case, consider that the specified metal, for example, specified divalent metal (e.g. calcium), acts as a bridge between casein (Caseinate) and BASS, preferably water-soluble BASS, more preferably water-soluble acidic BASS, or BASS water-soluble, capable of reacting with said metal, leaving the specified BASS associated with hydrophobic fraction loaded nanoparticles according to the invention.

In a specific embodiment of the specified metal is a divalent metal selected from calcium, magnesium, zinc, iron in bivalent form, and combinations thereof, preferably ka�of ICI. In another specific embodiment of the specified metal is a trivalent metal such as iron in the trivalent form.

Although in reality for the practical implementation of these methods [1] and [2] according to the invention can be any aqueous solution of calcium, mainly the solution of food quality [see "Codex General Standard for Food Additives" (a Set of basic standards for food additives) GSFA online for the ratio of calcium salts used in microencapsulation of food] in a particular embodiment of the specified solution of a salt of calcium selected from the group consisting of calcium chloride, calcium acetate, calcium gluconate, calcium lactate, calcium sorbate, calcium ascorbate, calcium citrate, of calcium propionate, calcium sulfate and mixtures thereof, preferably calcium chloride. In practice, the calcium carbonate or calcium alginate are not recommended because they are salts that are insoluble or very malorastvorima in the water. Similarly, any aqueous solution of magnesium, zinc or iron in divalent or trivalent form can be used for the practical implementation of these methods [1] and [2] according to the invention.

The weight ratio of metal:casein, where "metal" refers to the specified metal selected from divalent IU�Alla, trivalent metal and combinations thereof, can vary within wide limits, however, in a particular embodiment of the mass ratio of metal:casein is from 1:5 to 1:15, preferably from 1:7 to 1:10, more preferably about 1:8,5. In a specific embodiment of the specified metal is a divalent metal.

Method [2], corresponding to the invention leads to obtaining loaded nanoparticles according to the invention, and in this case, step a) involves mixing (i) an aqueous solution containing a source of casein and a first basic amino acid, with (ii) a solution containing BASS. The properties of the specific BASS mentioned above. In a specific embodiment of the specified BASS is a BASS water-soluble, preferably water-soluble acidic BASS, for example, folic acid, 4-aminobenzoic acid, Niacin or vitamin B3, Pantothenic acid or vitamin B5, monophosphate, thiamin pyrophosphate, cumintheface, ascorbic acid, pteroylmonoglutamic acid (derivatives of folic acid: volatilisation, polyglutamate), folinic acid, nicotinic acid, hyaluronic acid, thioctic acid, p-kumarovuû acid, caffeic acid, and their pharmaceutically or cosmetically acceptable or food grade origin�water, esters or salts, and mixtures thereof. In another specific embodiment of the specified BASS is a BASS fat-soluble, such as vitamin A, D, E, K and derivatives thereof, a phospholipid, a carotenoid (e.g., carotenes, lycopene, lutein, capsanthin, zeaxanthin, etc.), omega-3 fatty acid (e.g., DHA, EPA, etc.), amino acid (e.g., isoleucine, leucine, methionine, phenylalanine, tryptophan, and valine), phytosterol or phytosterols (such as sitosterol, campesterol, stigmasterol, etc.), polyphenols (e.g., quercetin, rutin, resveratrol, kaempferol, myricetin, isorhamnetin, etc.) or their derivatives.

The weight ratio BASS casein in the loaded nanoparticles according to the invention, can vary within wide limits, in non-limiting illustrative case, the weight ratio BASS casein in the loaded nanoparticles according to the invention, may range from 1:1 to 1:200, preferably from 1:10 to 1:80, more preferably from about 1:15 to 1:35. In a particular embodiment of the BASS is a water-soluble BASS, and the mass ratio (water-soluble) BASS:casein loaded in the nanoparticles according to the invention, is from 1:1 to 1:50, preferably from 1:10 to 1:30, more preferably from about 1:15 to 1:20. In another specific embodiment of Bespectacled a fat-soluble BASS, and the weight ratio of (fat-soluble) BASS:casein loaded in the nanoparticles according to the invention, is from 1:1 to 1:200, preferably from 1:10 to 1:80, more preferably from about 1:20 to 1:35.

Similarly, the mass ratio of basic amino acid:BASS (corresponding aqueous solution (ii) containing water-soluble acidic BASS and the second basic amino acid used in stage (a) of the method [2], corresponding to the invention), can vary within wide limits, however, in a particular embodiment of the mass ratio of basic amino acid:(acid soluble) BASS in this solution (ii) ranges from 1:0.1 to 1:3, preferably from 1:0.5 to 1:1, more preferably about 1:0.75, respectively.

As mentioned above, the composition of the nanoparticles according to the invention, as those that are loaded with BASS, and those that are not loaded, you can enter an antioxidant, e.g., ascorbic acid (vitamin C), etc., with the purpose to improve their stability in relation to temperature and oxidation. In this case, the antioxidant may be coencapsulation with the BASS (if necessary) or be in the coating of the nanoparticles according to the invention, in this case these methods [1] and [2], corresponding to the invention, will be accordingly adapted to be introduced�I antioxidant in the composition of the nanoparticles for example, by adding an antioxidant to the aqueous solution containing the specified BASS and a basic amino acid.

In a particular embodiment of the BASS is a folic acid and the antioxidant is an ascorbic acid, the effect of which, apparently, is to protect the folic acid from decomposition by the action of ultraviolet radiation, changes in pH, heat, oxygen, etc., and, in addition, provides nutritional support for ascorbic acid. The mentioned antioxidant may be coencapsulation with BASS or introduced into the coating of the nanoparticles according to the invention.

In addition, if necessary, as a way [1], corresponding to the invention and the method of [2] corresponding to the invention, can include one or more additional stages aimed at stabilizing the nanoparticles obtained by using different treatments.

In a specific embodiment of the specified stabilizing treatment involves exposure to the slurry containing the formed nanoparticles, corresponding to the invention, as those that are loaded with BASS, and those that are not loaded, high pressure, e.g. a pressure lying in the range from 100 to 800 MPa, typically from 350 to 600 MPa. In a specific embodiment, the implementation of such processing includes�Ivan doesn impact on the nanoparticle suspension cycles from 3 to 5 minutes of pressure from 100 MPa to 800 MPa, typically from 350 to 600 MPa; in fact, the pressure of 400 MPa provides positive results.

In another specific embodiment of the specified stabilizing treatment involves exposure to the slurry containing the formed nanoparticles, corresponding to the invention, as those that are loaded with BASS, and those that are not loaded, UHT (ultra high temperature), for example, temperatures from 130°C to 140°C for 2-5 seconds followed by rapid cooling.

Similarly, if necessary, as a way [1], corresponding to the invention and the method of [2] corresponding to the invention may include the step of drying the slurry containing the formed nanoparticles, with the aim of producing nanoparticles according to the invention, as those that are loaded with BASS, and those that are not loaded, in powder form. This form of representation of these nanoparticles contributes to their stability and, in addition, are particularly useful for their possible use in solid foods, such as flour, bread, cakes, cereal products, dry milk, etc., as well as in cosmetic and/or pharmaceutical products.

Actually adopted any technology or method that are suitable for drying of suspensions containing nanoparticles can be used to implement this studiously, however, in a particular embodiment of the drying of the suspension containing the nanoparticles is carried out using spray drying or by lyophilization. This treatment is usually carried out by adding a suitable protective component for the nanoparticles, such as saccharide, for example, lactose, trehalose, mannitol, sucrose, maltodextrin, glucose, sorbitol, maltose, etc., and mixtures thereof to the suspension of nanoparticles. The security component protects the nanoparticles, corresponding to the invention, from the decomposition under the action of heat and oxidation during the drying process.

The weight ratio casein:saccharide can vary widely, however, in a particular embodiment of the mass ratio of casein:the saccharide is from 1:1 to 1:4, preferably about 1:2.

Similarly, in a particular embodiment of the solution containing the saccharide may also contain an antioxidant, such as ascorbic acid (vitamin C), etc., in this case, the weight ratio casein:saccharide:an antioxidant, such as vitamin C, may be 1:0,75-2,5:0,01-1,5, preferably 1:2,0:0,10.

Nanoparticles, corresponding to the invention, obtained according to the method [1], corresponding to the invention, i.e., the nanoparticles containing a casein matrix, a basic AMI�akisato and metal, selected from a divalent metal, a trivalent metal and combinations thereof, formed by a method which involves: a) obtaining an aqueous solution containing a source of casein and a basic amino acid, and (b) adding an aqueous solution of metal selected from a divalent metal, a trivalent metal and combinations thereof, to the solution corresponding to stage (a), form an additional aspect of the present invention.

Similarly loaded nanoparticles, corresponding to the invention, obtained according to the method [2], corresponding to the invention, i.e., the nanoparticles containing a casein matrix, a basic amino acid, a metal selected from a divalent metal, a trivalent metal and combinations thereof, and BASS, obtained by using method, which involves (a) mixing (i) an aqueous solution containing a source of casein and a first basic amino acid with (ii) a solution containing BASS, and b) adding an aqueous solution of metal selected from a divalent metal, a trivalent metal and combinations thereof, to the mixture, resulting from stage a), form an additional aspect of the present invention.

Application options

Nanoparticles, corresponding to the invention can be used as technological additives, for example, alternate fat, etc. They also possess the ability to encapsulate BASS, for example, BASS water-soluble or fat-soluble BASS.

In a particular embodiment of the nanoparticles corresponding to the invention allow encapsulation of BASS, preferably water-soluble BASS, more preferably water-soluble acidic BASS, and its inclusion in the pharmaceutical, cosmetic composition and the composition of food, as other ingredients that are not natural polymers (to prevent the toxicity associated with synthetic polymers) and don't have food quality, do not use when they are received and in the final product (the nanoparticles). These nanoparticles protect the BASS from the decomposition of external factors (light, pH changes, oxidation, etc.).

Predominantly nanoparticles, corresponding to the invention, have an average size less than 1 μm, preferably of 50 to 200 nm, more preferably about 140 nm, to prevent the change of the organoleptic properties (structure, feel the sky).

Similarly, nanoparticles, corresponding to the invention, increase bioavailability BASS in the intestine, protecting the specified BASS from peptic acidic conditions of the stomach and promoting their dissolution and release in the intestine.

Nanoparticles, corresponding to the invention, can be resuspendable in�Noah environment while protecting the BASS from decomposition when dissolved. They, moreover, can be represented in the form of a dry powder that retains the BASS in a stable condition and enabling it to be stored for a long period of time (especially for their inclusion in solid foods).

In addition, the nanoparticles corresponding to the invention, suitable for obtaining cosmetic and pharmaceutical compositions for local application.

Consequently, in another aspect, the invention relates to a composition, hereinafter composition corresponding to the invention, containing at least one nanoparticle according to the invention in a particular embodiment of the nanoparticle, corresponding to the invention is a nanoparticle containing a casein matrix, a basic amino acid and a metal selected from a divalent metal, a trivalent metal and combinations thereof, in another specific embodiment of the nanoparticle, corresponding to the invention, is a loaded nanoparticle according to the invention, i.e., the nanoparticles containing a casein matrix, a basic amino acid, a metal selected from a divalent metal, trivalent metal and combinations thereof, and BASS with nutritional, therapeutic and/or cosmetic activity, as well as pharmaceutically or cosmetically acceptable carrier or wear�spruce, suitable for food products.

In a specific embodiment of the specified BASS selected from the group consisting of amino acids, antimicrobial agents, flavors, preservatives, sweeteners, steroids, drugs, hormones, lipids, peptides, polynucleotides, polysaccharides, proteins, proteoglycans, flavorings, vitamins and mixtures thereof.

In a specific embodiment of the specified BASS is a BASS water-soluble, preferably water-soluble acidic BASS. Non-limiting illustrative examples of water-soluble BASS include vitamins, e.g., vitamin b or C and their derivatives, salts or esters, hyaluronic acid, chondroitin sulfate, thioctic acid, their salts or esters, etc. In a specific embodiment of the specified water-soluble BASS selected from the group consisting of folic acid, 4-aminobenzoic acid, Niacin, Pantothenic acid, monophosphate, thiamin pyrophosphate, cumintheface, ascorbic acid, pteroylmonoglutamic acids (derivatives of folic acid: volatilisation, polyglutamation), folinic acid, nicotinic acid, hyaluronic acid, thioctic acid, p-coumaric acid, caffeic acid, and their pharmaceutically or cosmetically acceptable or food to�the number of derivatives, esters or salts, or their mixtures.

In another specific embodiment of the specified BASS is a fat-soluble BASS. Non-limiting illustrative examples of liposoluble BASS include vitamins, for example, groups A, D, E, K and their derivatives, phospholipids, carotenoids (carotenes, lycopene, lutein, capsanthin, zeaxanthin, etc.), omega-3 fatty acids (e.g., DHA, EPA, etc.), amino acids (e.g., isoleucine, leucine, methionine, phenylalanine, tryptophan, and valine), phytostanols and phytosterols (e.g., sitosterol, campesterol, stigmasterol, etc.), polyphenols (such as quercetin, rutin, resveratrol, kaempferol, myricetin, isorhamnetin, etc.) or their derivatives.

In a specific embodiment of the composition corresponding to the invention is a pharmaceutical composition suitable for administration by local, in this case, the specified composition comprises a pharmaceutically acceptable carrier that contains one or more excipients suitable for topical administration, for example, in the form of a gel, ointment, cream, etc. Information about excipients suitable for pharmaceutical compositions for local administration, as well as on obtaining these pharmaceutical compositions can be found in the monograph by C. Fauli i Trillo "Tratado de Farmacia Galenica" (Handbook of Galen�new drugs), 10 ed., 1993, Luzán 5, S. A. de Ediciones. The dose of nanoparticles according to the invention, intended for the introduction, can vary within wide limits, e.g. from about 0.5 (g/cm2the area exposed to treatment) to about 2 (g/cm2the area exposed to treatment) of the composition according to the invention, containing from 0.1% to 30% of the nanoparticles according to the invention, preferably from 0.5% to 5%.

In another specific embodiment of the composition corresponding to the invention is a cosmetic composition suitable for topical administration, in this case, the specified composition comprises a cosmetically acceptable carrier containing one or more excipients suitable for topical administration, for example, in the form of a gel, cream, shampoo, lotion, etc. Information about excipients suitable for receiving the cosmetic compositions intended for topical administration, as well as on obtaining these pharmaceutical compositions can be found in the monograph by Octavio Diez Sales Manual de Cosmetologia" (Guide for cosmetologists and), 1 ed., 1998, Editorial Videocinco, S. A.

In another specific embodiment of the composition corresponding to the invention, is a composition of food product, such as solid, liquid or semi-solid food product.

In a particular embodiment, be implemented thr�of the composition, corresponding to the invention, contains:

casein, 10% to 50% based on weight;

folic acid, from 0.9% to 2.5% based on weight;

calcium, from 1% to 6% based on weight; and

the basic amino acid, from 1% to 7% based on weight; and

saccharide, from 30% to 80% in terms of mass,

all ratios lead in terms of mass relative to the total weight of the composition.

In another specific embodiment of the composition corresponding to the invention, contains:

casein, 10% to 50% based on weight;

folic acid, from 0.9% to 2.5% based on weight;

calcium, from 1% to 6% based on weight; and

the basic amino acid, from 20% to 55% based on weight; and

saccharide, from 20% to 55% based on weight; and

ascorbic acid, from 1% to 25%,

all ratios lead in terms of mass relative to the total weight of the composition.

Alternatively, the composition according to the invention can be introduced into the food product, consequently, in another aspect, the invention relates to a food product containing the composition according to the invention. The specified food product can be found in liquid, semi-solid or solid form. Primarily to prevent or minimize the total or partial dissolution of the nanoparticles according to the invention, and, still� way contribute to their stability, the specified food product has an acidic pH, i.e. less than 7, preferably less than or equal to 6, more preferably less than or equal to 5. Illustrative examples of foods that can enrich or vitaminsyou composition according to the invention, include milk and its derivatives (yogurt, cheese, cottage cheese products, etc.), juices, jams, bakery products, fermented meat, sauces, etc. Similarly, the composition according to the invention can be inserted into the food for animals, for example, in the feed.

Examples

The following examples describe how to get the casein particles, which can comprise a biologically active compound, in particular folic acid. They are able to protect the compound from the destruction to which it may be subjected in product supply due to the previously mentioned a number of factors. These examples demonstrate the ability of these nanoparticles to protect the folic acid from the conditions existing in the stomach, after their admission and release it into the intestines.

General method of obtaining the unloaded casein nanoparticles

A method of producing casein nanoparticles provides for the dissolution of sodium Caseinate (ANVISA, Madrid, Spain) in an aqueous medium together with a certain amount of basic amino�of islote followed by the addition with stirring using a magnetic stirrer and a constant flow of a certain volume of a solution of calcium which leads to the early formation of the nanoparticles with the appearance of the breast suspension.

Physico-chemical characterization of nanoparticles

Various studies necessary to obtain a complete physico-chemical characterization of nanoparticles, described below.

The size and surface charge of the nanoparticles is determined on the basis of physico-chemical tests, the latter set by measuring the Zeta potential. The first parameter is obtained by photon correlation spectroscopy using Zetasizer nano Z-S (Malvern Instruments/Optilas, Spain), whereas the Zeta potential is measured using the analyzer Zeta potential (Zeta Potential Analyzer (Brookhaven Instruments Corporation, New York, USA).

The output produced by the method of formation of the nanoparticles is calculated by quantifying the remaining free casein after receipt of the nanoparticles collected in the supernatants obtained by centrifugation of the drug (17000×g, 20 minutes). Thus, the amount of casein, which forms particles in the product, estimated as the difference between the initial added amount and the amount determined in supernatants collected in the cleaning stage. This quantification is carried out using ultraviolet (UV) spectrometry at a wavelength of 282 nm (Agilent 8453, system spectroscopy in the UV-visible region). Wychodzenia as:

Insxod(%a)=[(mgobuegoKazeandnatamgKazeandnatainCIpepnatante)/mgobuegoKazeandnata]×100[Ipainnenande1]

For the purpose of various calculations using the calibration curve from 150 to 1500 µg/ml (R2=0,9992; and LD=36 µg/ml; LQ=119 μg/ml).

In addition, conducting a study aimed at the quantification of precipitation, obtained after centrifugation, in order to confirm the results obtained on the basis of the difference between the total Caseinate and Caseinate contained in the supernatant�. In this case, for the destruction of the particles using 0.05 M NaOH, which is the same environment that is used to produce the calibration curve. Consequently, in this case, the output is estimated as:

Insxod(%a)=[(mgKazeandnainoCadKe)/mgobuegoKazeandnata]×100[Ipainnenande2]

Maximum absorption, is indicated for the Caseinate obtained in a given environment, is 300 nm. The concentration used to construct the calibration curve, also lie in the range from 150 to 1500 µg/ml (R2=0,9996; LD=26 µg/ml; LQ=85 µg/ml).

The morphology of the nanoparticles observed using scanning electron microscopy (Zeiss DSM 940A, Germany). In this case, the lyophilized nanoparticles coated with a molecular layer of gold 9 �m (Emitech K550 Team, Sputter-Coater, United Kingdom) and the photos make using a microscope Zeiss DMS 940 A (United States).

A common method of producing casein nanoparticles containing folic acid

A method of producing casein nanoparticles containing folic acid, provides for the dissolution of sodium Caseinate in the aqueous medium together with a certain amount of basic amino acids followed by the addition with stirring magnetic stirrer certain volume of folic acid solution, previously prepared in an aqueous medium with a certain amount of basic amino acids. After incubation of the mixture for a few minutes last stage consists of adding calcium salts, leading to the formation of nanoparticles, having the appearance of a milky yellowish suspension.

The formed nanoparticles can optionally be subjected to the treatment of high hydrostatic pressure (Stansted Fluid Power, model NO. FPG11500B110; series No.: 7844) cycles from 1 to 5 minutes at 100-800 MPa for the purpose of stabilization.

Then after 3 minutes of homogenization by mixing add a certain volume of a solution of the saccharide (lactose, trehalose, mannitol, glucose, sorbitol, maltodextrin or maltose), without stopping the stirring. Finally, the suspension lyophilizer or sprayed into the spray-drying (minireplicon dryer Buchi B-191, Buhi Labortechnik AG, Switzerland) under the following conditions:

- Inlet air temperature: 60-100°C

Is the outlet air temperature: 30-90°C

- Air pressure: 2-10 bar [2-10×105PA]

- The speed of pumping of the sample: 2 to 9 ml/min

Aspiration: 30-100%

- Air flow: 200-900 l/h.

Optional preparations can be dried after the addition of the saccharide through drying is spray drying.

Determination of the amount of folic acid that is associated with the casein particles

The amount of folic acid associated with nanoparticles, determined using high performance liquid chromatography (HPLC) according to the method described Faye [see Faye Russell, L, Quantitative Determination of Water-Soluble Vitamins (Quantitative determination of water-soluble vitamins) in the monograph Food Analysis by HPLC (Analysis of food products using HPLC), edited by Nollet, L. M. L, Marcel Dekker, Inc., New York, 2 ed., Chapter 10 (2000) pp. 444-445]. The analysis is carried out on the chromatograph model 1100 series LC (Agilent, Waldbornn, Germany) associated with the system for UV detection diode array. Data were analyzed using a computer to Hewlett-Packard software Chem-Station G2171. For the isolation of folic acid is heated to 40°C column (Alltech Inc 18 Alltima™ (5 μm, 150 mm × 2.1 mm) compatible with Gemini column®C18AJO-7596. Mobile phase prepared from a mixture of H3PO4(33 mm, pH 2,3)/acetonitrile in a gradient (see Table ) and pumped at a flow rate of 0.25 ml/min. Detection is carried out at a wavelength of 290 nm. The amount of injection of sample is 10 ál. The retention time of folic acid is 22.6±0.5 minutes.

Table 1
The conditions of the gradient for the mobile phase (A: H3RO433 mm, b: Acetonitrile)
Time (min)A (%)(%)
095,05,0
895,05,0
3382,517,5
4595,05,0

Before quantification of the sample are the calibration curves of concentrations from 2 to 400 μg/ml, and getting some accurate results, confirmed more than 95% of what the presence of casein and/or amino acids in solution will not violate any accurate quantitative determination of folic acid.

For the analysis of their sample (before drying), the supernatants obtained after filtering a certain amount of prepar�the one quantify using tubes for dialysis Vivaspin® 300,000 MWCO (VIVASPIN 2, Sartorius stedim Biotech, Germany). The precipitate, in turn, was dissolved in 0.05 M NaOH to destroy the particles and save the casein and folic acid, and amino acid in solution and, thus, to exercise their quantification. The amount of folic acid, found in both fractions (supernatant and sediment), coincides in all cases with the total of the original amount added. Moreover, it is also possible to determine the total amount of folic acid by dissolving 1 ml of the drug in 1 ml of 0.05 M NaOH. This study helps confirm that the difference between the amount of added folic acid and folic acid, obtained by quantitative determination using the described chromatographic method, exceed 10% in all cases.

In addition, 10 mg of nanoparticles take for the quantitative determination of powder samples, their was resuspended in 2 ml of water and centrifuged, then treated in the same way as their samples.

Study of release kinetics for the release of folic acid from the nanoparticles in artificial gastrointestinal environment

The release kinetics for the release of folic acid from the nanoparticles determine dispersive approximately 10 mg of the compound in 2 ml �artificial gastric environment (0-2 hour) (USP (USP XXIII) at 37±1°C. At the specified time of suspension of nanoparticles was centrifuged (17000×g, 20 minutes) and the amount of folic acid in the supernatants was quantitatively determined by the above HPLC method. After removal of supernatants from gastric environment add artificial intestinal medium (2-24 h) (USP XXIII) at 37±1°C, then treated in the same manner as in the above case.

The proportion of folic acid, liberated in all cases, expect, taking into account the total content of the vitamin present in the drug is taken for each study.

Pharmacokinetic study. The bioavailability of folic acid encapsulated in casein nanoparticles

Pharmacokinetic studies were performed according to the rules of the ethics Committee of the Institute (Institution Ethics Committee), as well as European legislation on experimental animals (European legislation on experimental animals (86/609/EU). In this case, 25 male Wistar rats with an average weight of 200 g is placed in the normal conditions of alternating light and darkness (12 hours - 12 hours), and during the week before the study they are fed without restriction of food with folic acid deficiency (diet with folic acid deficiency. TD. 95247. Harlan, USA) and water. Twelve hours prior to drug administration rats are isolated in cells for metabolic studies without excessive feed, but with free access to drinking�water howl.

Animals are divided into 5 treatment groups (5 rats/group). The first group was orally administered only 1 ml of PBS (phosphate buffer pH 7.4). The following three groups treated orally with doses of only 1 mg/kg (200 μg/rat) of folic acid included in any of the following drugs: (i) free folic acid (unencapsulated) (Aditio, Panreac Quimica, Barcelona, Spain); (ii) the casein nanoparticles with encapsulated folic acid; (iii) casein nanoparticles processed by high pressure, with encapsulated folic acid. 1 ml of each of various drugs dispersed in water, is injected through the gastroesophageal cannula (catheter). Finally, the same dose of free folic acid (1 mg/kg) dissolved in saline serum (0.5 ml), administered intravenously in the fifth group of the saphenous vein of the thigh.

Before the introduction of drugs take blood from caudal saphenous vein for the purpose of verification baseline vitamin each rat. After the introduction, take the blood in a volume of approximately 500 μl in different time periods, while using tubes for serum separator (SARSTEDT Microtube 1.1 ml Z-Gel). In all cases, blood sampling was carried out after immersion of the animal in a dream when you are using inhalation anesthesia (isoflurane:oxygen) to prevent pain in rats, checking them constant at all points of time.

Then volun� blood fill by intraperitoneal injection of 500 μl of serum-based physiological solution, pre-heated to body temperature of the animal. In the study in this period the condition of the animals (motility, aggressiveness, allergic reactions, and temperature) do not detect any significant changes.

Pre-processing and quantification of folic acid in serum samples

Quantitative determination of folic acid in serum samples obtained after centrifugation of the tubes with blood (6000 rpm, 20 min, 20°C), carried out by the method of enzyme immunoassay. In this case, use the set Elisa Kit (Diagnostic automation, INC. Calabasas, California, USA), approved FDA (Management on control over products and medicines of the USA) for the quantitative determination of folic acid in foods. Conduct quantitative determination of serum samples without pre-treatment and following the manufacturer's instructions.

Since the set is designed for use in food products, will hold a series of preliminary studies to prove its ability to quantify vitamin in the serum samples. These studies consist of an exhaustive comparison of the results obtained with the set, and the results obtained by the method of high performance liquid chromatography described in the preceding sections, with the following� way advance preparation: different quantities (0-300 μl) of folic acid, dissolved in a 50 mm solution of sodium tetraborate obtained in 1% (wt./about.) the sodium ascorbate are added to 50 μl of serum. The resulting solution is adjusted to a final volume of 350 µl (serum dilution 1:7) 50 mm solution of sodium tetraborate. Each mixture is boiled for 30 minutes and then cooled to 2°C and kept overnight at this temperature.

After centrifugation the resulting samples at 20,000 rpm for 20 minutes and filtering them through a filter of 20 μm quantitatively determine the content of folic acid by using the previously described method of high performance liquid chromatography. In this case, and due to the low concentration of vitamin a in serum using a standard way to add to minimize errors in quantifying and to exclude any influence of the matrix.

In all studied cases, the difference in concentrations of folate in serum in both methods was less than 10%. Consequently, choose the method of enzyme immunoassay for the quantitative assessment of the set of the samples, because it requires a smaller amount of serum for analysis and it is an easier and faster way, the limit of detection (2 ng/ml) which is considerably less than the chromatographic method.

a Common method of producing casein nanoparticles containing fat-soluble active substance quercetin

A method of producing casein nanoparticles containing quercetin, provides for the dissolution of sodium Caseinate in the aqueous medium together with a certain amount of basic amino acids followed by the addition with stirring using a magnetic stirrer certain volume of ascorbic acid solution and then quercetin, previously dissolved in ethanol. After incubation of the mixture for a few minutes last stage consists of adding calcium salts, causing the formation of nanoparticles with the appearance of milky-yellowish suspension.

The formed nanoparticles can optionally be podvegnut the treatment of high hydrostatic pressure (Stansted Fluid Power, model NO. FPG11500B110; series No.: 7844) cycles from 1 to 5 minutes at 100-800 MPa for the purpose of stabilization.

Then after 3 minutes of homogenization by mixing add a certain volume of a solution of the saccharide (lactose, trehalose, mannitol, glucose, sorbitol, maltodextrin or maltose), without stopping the stirring. Finally, the suspension lyophilizer or sprayed into the spray-drying (minireplicon dryer Buchi B-191, Buchi Labortechnik AG, Switzerland) under the following conditions:

- Inlet air temperature: 60-100°C

Is the outlet air temperature: 30-90°C

- Yes�ing: air: 2-10 bar [2-10×10 5PA]

- The speed of pumping of the sample: 2 to 9 ml/min

Aspiration: 30-100%

- Air flow: 200-900 l/h.

Optional preparations can be dried after the addition of the saccharide through drying is spray drying.

Determination of the amount of quercetin that is associated with the casein particles

The amount of quercetin that is associated with the nanoparticles, determined using high performance liquid chromatography (HPLC) according to the method described Lacopini (Lacopini et al., J Food Comp Anal 2008;21:589-598), although with some changes. The analysis is carried out on the chromatograph model 1100 series LC (Agilent, Waldbornn, Germany) associated with the system for UV detection diode array. Data were analyzed using a computer to Hewlett-Packard software Chem-Station G2171. For the isolation of folic acid is heated to 40°C column (Alltech Inc C18Alltima™ (5 μm, 150 mm × 2.1 mm) with a compatible column Gemini® C18 AJO-7596 and a mixture of water/methanol/glacial acetic acid in a gradient (see Table 2) as the mobile phase, pumped at a flow rate of 0.25 ml/min. Detection was carried out at a wavelength of 260 nm, injection volume of the sample is 10 µl and the retention time of quercetin is 24.2±0.2 minutes.

Table 2
Conditions hail�cient for the mobile phase (A: water, In: methanol: glacial acetic acid)
Time (min)A (%)(%)C (%)
080155
1570255
2010855
3010855
3580155
4080155

Before quantification of the samples are calibration curves of concentrations from 1 to 100 µg/ml in aqueous-alcoholic medium (75% ethanol), and getting some accurate results higher than 95%.

For the analysis of freshly prepared samples (before drying) of the supernatants obtained after the treatment process of the nanoparticles by filtration (17000 rpm, 20 min), diluted to obtain a water-alcohol solution with the content of ethanol 50% (wt./vol.).

Finally, the amount of quercetin that is associated with nanoparticles [encapsulation efficiency (E. E.)], calculated as the difference between the initial amount added quercetin (Q) and the quantity specified in the supernatants according to the following equation:

E.E(%a)=mgobuegoQmgQinCIpepnatantemgobuegoQ*100

Example 1. Obtaining and characterization of unloaded casein nanoparticles. The output of the method of their preparation. The influence of the type of amino acids on the stability and physico-chemical properties of nanoparticles

1 g of sodium Caseinate was dissolved together with 90 mg of lysine in 75 ml of water. Then to this solution was added 40 ml of 0.8% CaCl2with stirring using a magnetic stirrer and constant flow. This process is carried out in three replicates.

In the Figures 2 and 3 show obtained by electron transmission microscopy image KAZ�inovah particles prepared in this way.

In addition, the same study was conducted in the absence of amino acids or by using 50 mg of arginine instead of lysine to understand the influence of the type of amino acids on the physico-chemical properties of the particles.

In Table 3 summarize the main physico-chemical parameters of the resulting nanoparticles.

Table 3
Physico-chemical properties of casein nanoparticles (mean ±SD, n=10). The weight ratio in terms of the mass of the amino acid, lysine or arginine and protein is 1:11 and 1:20, respectively
DrugSize (nm)PDI8Zeta potential (mV)O/o)
Casein bass (without amino acids)154±300,24±0,04-17,6±0,3
Casein LF (lysine)138±130,19±0,02-14,0±0,595±3
Casein LF (arginine) 157±190,21±0,03-17,5±0,697±1
aPDI: polydispersity;
bOutput: Percentage of protein, transformed into nanoparticles.

Conducted statistical research (non-parametric test of independent samples Kruskal-Wallis) show that there is no statistically significant evidence that there is a difference between the physicochemical parameters of drugs. Consequently, we can conclude that the type of amino acids does not violate the properties of unloaded nanoparticles.

The same study was conducted with variation of the ratio of amino acids added to the drug, coming to similar conclusions, i.e. the ratio and type of amino acids does not violate the end-properties of the unloaded particles.

For understanding the stability of the preparations was measured physico-chemical parameters of three types of nanoparticles over time. The results obtained are included in Table 4.

Table 4
Physico-chemical properties of casein nanoparticles (mean ±SD, n=6) over time. The weight ratio of amino�slots lysine or arginine and protein is 1:11 and 1:20, respectively
Time (hours)Casein bass (without amino acids)Casein LF (lysine)Casein LF (arginine)
SizePDISizePDISizePDI
0165±400,25±0,05138±130,19±0.02157±210,21±0,04
2323±640,45±0,15155±11Of 0.14±0.02176±120,1 b±0,04
16317±60,40±0,03157±5Of 0.18±0.03175±20,14±0,02
24231±50,36±0,03155±5A 0.13±0.02 183±40,25±0,02
30295±600,73±0,06157±3A 0.13±0.02195±40,32±0,03
48255±200,79±0,02157±4Of 0.16±0.01205±30,33±0,04
PDI: polydispersity.

During their receipt of three types of nanoparticles have dimensions of the same order of magnitude and relatively low polydispersity (considering that the PDI values less than 0.3, the distribution of particle size is homogeneous). These size values and variance did not show significant changes throughout the study in the case of nanoparticles obtained with amino acid. However, two hours after their receipt nanoparticles that get without amino acids, have a significant increase in both the average size and polydispersity (for the value of polydispersity more than 0.3, the value of particle size is not representative, it is only indicative, since there is a high heterogeneity of the diameters), reaching very high values poly�isperia after the end of the study. These elevations suggest the existence of the phenomenon of aggregation of the particles. This phenomenon is even confirmed at the microscopic level, as when three of the drug is followed over time, confirm that the nanoparticles without amino acids are precipitated, leading to the formation of a milky layer, whereas the nanoparticles obtained with an amino acid to form a homogeneous suspension. In light of these results, consider that the presence of an amino acid important for the production of particles that are stable over time.

In addition, all three types of drugs again receive and study their physico-chemical properties after they are dried by a spray drying method. The process conditions are the following:

- Inlet air temperature: 90°C

Is the outlet air temperature: 49°C

- Air pressure: 6 bar [6×105PA]

- The speed of pumping of the sample: 4.5 ml/min

Aspiration: 100%

- Air flow: 600 l/h.

This study is carried out with the aim of understanding the impact of an amino acid, when dried nanoparticles during their production, although in this case none of the drugs does not demonstrate the phenomenon of aggregation. The results obtained are included in Table 5.

Table 5
Physico-chemical�technical properties of casein nanoparticles (mean ±SD, n=3), dried by spray drying. The weight ratio of the amino acid, lysine or arginine and protein is 1:11 and 1:20, respectively
DrugSize (nm)PDI8Zeta potential (mV)
Casein bass (without amino acids)305±560,45±0,02-9,8±0,2
Casein LF (lysine)170±70,25±0,02-11,9+0,9
Casein LF (arginine)184±20,25±0,01-9,4±0,2

When resuspending nanoparticles with amino acid, dried into powder, in an aqueous medium note that the size distribution remains monodisperse and their sizes are slightly larger than their homologues before they are dried spray drying. However, the nanoparticles obtained without amino acids, size and value of polydispersity more, indicating that they may be subjected to a phenomenon of aggregation during drying. Thus, the presence of amino acids is also necessary when the particles are dried by �especialley drying.

In light of the specified conclude that the physico-chemical properties of nanoparticles differ from the amino acid properties not containing particles, they have a lower tendency to aggregate and, consequently, represent drugs selected for the encapsulation of biologically active compounds.

Example 2. Obtaining and characterization of casein nanoparticles containing folic acid. The influence of the content of lysine and Folic acid on the efficiency of encapsulation

Various solutions, all of which contain 100 mg of sodium Caseinate and different levels of lysine (0-8,5 mg) are given in the final volume of 7.5 ml of water.

In addition, 300 mg of folic acid are dissolved together with 400 mg of lysine in 50 ml of water.

Then 1 ml of a solution of folic acid is added to a solution of Caseinate. After 5 minutes of incubation to the mixture add 4 ml of 0.8% CaCl2with stirring using a magnetic stirrer and constant flow. This process is carried out in three replicates for each type of drug.

In the Figures 4 and 5 show the images obtained by electron transmission microscopy casein particles with encapsulated folic acid obtained by this method.

Physico-chemical properties obtained in each case, included in Table 6:

Table 6
Physico-chemical properties of the casein nanoparticles with folic acid and variable amounts of lysine (mean ±SD, n=6). The weight ratio of folic acid and protein is 1:17
The mass ratio of lysine: caseinaSize (nm)PDIZeta potential (mb)The content of folic acid mcg FC/mg LFEncapsulation efficiency
0:100159±60,16±0,04-7,9±2,4......
1:26139±10,11±0,05-17,5±0,522,1±0,932,2±0,8
1:22140±10,10±0,05Is 16.8±0,722,3±0,432,3±0,4
1:12136±40,08±0,02Is 16.8±0,725,7±3,2 37,6±4,8
andBefore adding a solution of folic acid
FC: Folic acid; LF: Nanoparticle

Conducted statistical research (non-parametric test of independent samples Kruskal-Wallis) show that there is no statistically significant evidence that there is a difference in the physicochemical properties of the latter three drugs included in the table (with the lysine content of 3.9, 4.5 and 8.5 mg). In the first case, this confirms the fact that, although the solution of folic acid contains lysine, the lack of amino acid in the initial solution of Caseinate contributing to the partial deposition of folic acid calcium that errors in the quantitative determination of vitamin, because not all folic acid encapsulated in the sediment after centrifugation.

Additional studies have shown that, when the solution contains vitamin amino acid, but the solution of Caseinate does not contain, maximum quantity of folic acid that can be entered in the drug without her deposition, is 4 mg, and then get similar results as shown in Table 6 (25,5±1 μg FC/mg LF and encapsulation efficiency: 68,7±0,5). Thus confirm that the presence and�of inability not affect the amount of encapsulated vitamin. However, since the nanoparticles obtained without amino acids, less stable and have a higher tendency to aggregation (see Example 1), the drugs get in the presence of this amino acid.

In order to understand the influence of the amount of folic acid added to the preparation, physico-chemical properties of the particles, carried out a similar study by varying only the amount of added folic acid solution, wherein the amount of amino acid in the initial solution of casein remains constant, averaging 8.5 mg.

Figure 6 shows the ratio of the encapsulated folic acid as a function of the amount of vitamin added to the drug.

The dimensions found with the studied drugs, lie in the range from 132 to 140 nm at polydispersity less than 0.2 in all cases. In this example, the values of encapsulation efficiency are incomparable, because the amount of added folic acid is different. The maximum value is 73,1±7,5 for the weight ratio casein:folic acid is 13.5:1.

As a result of this study it can be concluded that, since the ratio of mg casein/mg FC in the product is reduced (i.e., because the original amount of folic acid added to the drug increases), receive an increased amount of folic acid encapsulated HV�three nanoparticles. However, when the amount of casein in the drug (in mg) for each mg of folic acid less than the experimental values, see the formation of precipitation and unstable drugs, such as occur in the absence of lysine.

Example 3. Obtaining and characterization of casein nanoparticles containing folic acid, dried by spray drying. The effect of method of drying on the final drug

Two solutions, both containing 1000 mg of sodium Caseinate and 90 mg of lysine, prepared in a final volume of 75 ml of water.

In addition, 600 mg of folic acid are dissolved together with 800 mg of lysine in 100 ml of water.

Then to each solution of Caseinate is added 7.5 ml of a solution of folic acid. After 5 minutes of incubation with 40 ml of 0.8% CaCl2add to the mixture with stirring using a magnetic stirrer and constant flow.

Finally, one of the drugs centrifuged for quantitative estimation of folic acid in the supernatant and the sediment, whereas in the other add 1900 mg of lactose before dried using spray drying. The process conditions are the following:

- Inlet air temperature: 90°C

Is the outlet air temperature: 45°C

- Air pressure: 6 bar [6×105PA]

- The speed of pumping of the sample: 4.5 ml/min

Aspiration: 95%

- Air flow: 600 l/h.

Physico-chemical properties observed � both cases, included in Table 7.

Table 7
Physico-chemical properties of the casein nanoparticles with folic acid, quantitatively defined in the recently obtained nanoparticles or after drying in the spray-drying (mean ±SD, n=6). The weight ratio of lysine and protein in the final preparation is 1:7 and the ratio of folic acid and casein is 1:22
Type of drug isSize (nm)PDIZeta potential (mV)The content of folic acid mcg FC/mg LFEncapsulation efficiency
Spray drying157±50,17±0,01-15,7±0,318,6±3,441,4±7,6
Recently obtained137±30,08±0,02Of-16,7±0,727,6±0,758,7±1,4
FC: Folic acid;

Conducted statistical research (non-parametric test of independent samples Kruskal-Wallis) show that there is a statistically significant difference (p<0.05) between the encapsulation efficiencies obtained for both drugs. This difference may be due to the drying method of the preparation of spray drying at these temperatures, causing partial decomposition of the casein nanoparticles, resulting in the release of part of the previously encapsulated folic acid.

These results demonstrate the necessity of application of the method the cross-linkage of the particles, which is in the process of development, in order to increase their stability and to prevent the above-mentioned decrease in the efficiency of encapsulation in the way of centrifugation or drying of the drug.

Example 4. Obtaining and characterization of the casein nanoparticles with lysine containing folic acid, stable high pressure and dried by a spray drying method. The effect of treatment on the physico-chemical properties of nanoparticles

Various solutions, all of which contain 1000 mg of sodium Caseinate and 90 mg of lysine, get in the final volume of 75 ml of water.

In addition, dissolved 600 mg of folic acid together with 800 mg of lysine in 100 ml of water.

Then 7.5 ml of a solution of folic acid �dobavlaut to a solution of Caseinate. After 5 minutes of incubation with 40 ml of 0.8% l2add to the mixture with stirring using a magnetic stirrer and constant flow.

After the formation of particles of drugs are transferred into sealed plastic containers and subjected to the treatment of high hydrostatic pressure (0 MPa 100 MPa, 5 min; 200 MPa, 5 min; 400 MPa, 5 min; 600 MPa, 5 minutes or 800 MPa, 5 minutes).

After 1900 mg of lactose dissolved in water, is added to each drug, and dried using spray drying under the following conditions:

- Inlet air temperature: 85°C

Is the outlet air temperature: 45°C

- Air pressure: 6 bar [6×105PA]

- The speed of pumping of the sample: 4.5 ml/min

Aspiration: 95%

- Air flow: 600 l/h.

In Table 8 summarize the main physico-chemical properties of the resulting nanoparticles.

Table 8
Physico-chemical properties of the casein nanoparticles with folic acid and processing of various high pressure (mean ±SD, n=6). The final mass ratio of lysine and casein is 1:7 and the ratio of folic acid and casein is 1:22
Type of drug is Size (nm)PDIZeta potential (mV)Output (% by weight)The content of folic acid mcg FC/mg LFEncapsulation efficiencyμg FC/mg composition
Without high pressure157±50,17±0,01-15,7±0,356,418,6±3,441,4±7,612,1±0,4
100 MPa 5 min144±30,13±0,01-13,6±0,254,125,3±4,555,1±7,611,5±1,4
200 MPa 5 min139±10,22±0,02-13,2±0,567,623,2±0,952,1±2,111,2±1,4

Type of drug isSize (nm)PDIThe Zeta-potential�al (mV) Output (% by weight)The content of folic acid mcg FC/mg LFEncapsulation efficiencyμg FC/mg composition
400 MPa, 5 min121±30,14±0,01-13,3±0,568,225,5±3,258,7±4,811,9±1,4
600 MPa, 5 min111±20,15±0,01-12,8±0,447,730,8±3,067,8±5,511,8±0,9
800 MPa 5 min115±30,12±0,01-14,2±0,831,4±3,565,8±8,112,1±1,5
FC: Folic acid; LF: Nanoparticle

As can be seen from Table 8, regardless of the type of processing drugs, nanoparticles have similar surface charges. However, the results allow us to determine that with increasing pressure�tion, used for processing, reduces the size of the obtained particles, reaching a maximum reduction of 7%. However, the amount of encapsulated vitamin (and as a result, the encapsulation efficiency) reaches higher values with increasing of pressure used, and get a growth of 65% relative to the drugs that is not processed (in the case of the samples treated 800 MPa).

In addition, in the Figures 7 to 11 show photomicrographs of drugs that is not processed by the high pressure, and those that processed 100, 400 and 800 MPa, obtained using scanning electron microscopy. They give evidence of how the nanoparticles obtained without hydrostatic high pressure processing, partially affected by the different ways in which they're influenced after receiving (drying spray drying, centrifugation, obtaining photomicrographs way in which reach high temperatures), and the particles are subjected to high pressure processing, more stable.

These results show that the hydrostatic high pressure processing of cross stitch nanoparticles, making them more stable and, consequently, preventing their decomposition after centrifugation, drying and photographing. All these facts Clare�tween increased encapsulation efficiency, obtained in the treated samples, since the partial decomposition of the nanoparticles in some of these methods of drying or centrifugation will result in the release of folic acid and, consequently, to obtain the reduced efficiency of encapsulation.

Example 5. Obtaining and characterization of the casein nanoparticles with arginine containing folic acid, when using high pressure, dried by spray drying.

The influence of amino acids used in the end result

The solution 3,065 mg of sodium Caseinate with 123 mg of arginine get in the final volume of 210 ml of water.

Furthermore, 605 mg of folic acid are dissolved together with 800 mg of arginine in 100 ml of water.

Then to a solution of Caseinate is added 27 ml of a solution of folic acid. After 5 minutes of incubation to the mixture was added to 120 ml of 0.8% CaCl2with stirring using a magnetic stirrer and constant flow.

After the formation of the drug particles is transferred to a sealed plastic container and subjected to the treatment of high hydrostatic pressure, consisting of a 5 minute cycle at 400 MPa.

After the process is added 5,880 mg of mannitol dissolved in water to 300 ml product processed by high pressure, and dried it using spray drying method under the following conditions:

- Temperature post�ment of air: 85°C

Is the outlet air temperature: 45°C

- Air pressure: 6 bar [6×105PA]

- The speed of pumping of the sample: 4.5 ml/min

Aspiration: 95%

- Air flow: 600 l/h.

In Table 9 summarize the main physico-chemical properties of the resulting drug.

Table 9
Physico-chemical properties of the casein nanoparticles with arginine and folic acid, processed by high pressure and dried by spray drying (mean ±SD, n=6). The final mass ratio of arginine and protein is 1:9 and the ratio of folic acid and casein is 1:19
Type of drug isSize (nm)PDIZeta potential(MB)Output (% by weight)The content of folic acid mcg FC/mg LFEncapsulation efficiencyμg FC/mg product
400 MPa, 5 min137±60,20±0,01-11,9±0,133,5±2,259,8±3,9 13,9±0,6
FC: Folic acid; LF: Nanoparticle

Figure 12 shows a micrograph obtained by scanning electron microscopy (SEM) of casein nanoparticles containing folic acid, and including in the preparation of arginine in the treatment of 400 MPa for 5 minutes.

As you can see, the resulting preparation has properties similar to the properties of the nanoparticles were obtained using a lysine instead of arginine.

Example 6. Study of release kinetics for the release of folic acid from the nanoparticles in artificial gastrointestinal environment. The effect of high pressure processing on the kinetics of the release

For research release take powder preparations described in Example 4 (without high pressure processing, processed 100 MPa and 400 MPa).

Figure 13 shows the release kinetics obtained in the case of samples treated for high blood pressure. It shows that after two hours of incubation in the gastric environment, reach maximum values of the release of folic acid 4%. However, in the conditions of the intestine particles of casein are dissolved, releasing a higher percentage of vitamin (reaching 90% after 24 hours of study). Moreover, in this environment, the samples centrifuged after them and�of kupirovaniya, have virtually no precipitate of casein, which attests to their dissolution and, consequently, the release of vitamin. Thus, it is clear that developed the drug stores folic acid encapsulated in the gastric tract, preventing the decrease in its bioavailability in the environment of the stomach. In addition, the nanoparticles dissolve in the gut, promoting the release of vitamin and eliminating the problem of any toxicity that may occur due to the presence of nanoparticles.

In the case of the samples treated by high pressures in the Figures 14 and 15 show the kinetics of their release. They can see that the profile is very close to that found for samples obtained without high pressure processing, and the maximum percentage release after 6 hours in artificial intestinal environment is slightly lower (70%) than found for the samples not treated at the moment (80%).

Thus, the use of high hydrostatic pressure for casein nanoparticles with the aim of cross-linkage does not significantly modifies the release profile of the ingredient from the sample, although the total amount of vitamin released after 6 hours, is reduced by 10%.

Example 7. Pharmacokinetic study of folic acid encapsulated in casein nanoparticles

In Table 10 summarize the basic physical and chemical properties of the nanoparticles tested in pharmacokinetic study. Both types of nanoparticles (with treatment and without treatment high blood pressure) is prepared according to the method described in Example 5.

Table 10
Physico-chemical properties of the casein nanoparticles with folic acid (mean ±SD, n=6) used with pharmacokinetic studies
Type of drug isSize (nm)PDIZeta potential (mV)The content of folic acid mcg FC/mg LF
KAZ LF FC134±30,17±0,02-11,8±0,224,2±1,1
KAZ LF VD FC134±30,23±0,03-14,4±2,329,5±1,8
FC: Folic acid; LF: Nanoparticle; KAZ LF
FC: Casein nanoparticles with encapsulated folic acid; KAZ
LF VD FC: casein nanoparticles with encapsulated folic acid, treated with high pressure (400 MPa, 5 min).

Pharmacokinetic study is divided into three phases. The first phase consists of intravenous administration of 1 mg/kg folic acid, dissolved in phosphate buffer; the second phase consists of oral administration of 1 ml of phosphate buffer rats groups of 5 male Wistar rats (initial levels of vitamin over time are examined in this group of rats). Finally, the third phase consists of oral administration of 1 mg/kg (i), folic acid, dissolved in water, (ii) folic acid encapsulated in casein nanoparticles, and (iii) of folic acid encapsulated in casein nanoparticles processed by high pressure, groups of rats, made up of 5 animals.

After the introduction of different time periods(0, 1, 2, 3, 8 and 24 hours) take the blood in a volume of approximately 500 ml and collected in tubes for serum separator with subsequent restoration of blood volume of the animal by intraperitoneal injection of an equal volume of saline serum. Pharmacokinetic analysis of data obtained after the introduction of folic acid, is carried out using compartmental settings program settings pharmacokinetic WiNNonlin 1.5 (Pharsight Corporation, Mountain View, United States).

The results obtained (after �of citania baseline) together represented in the Figures 16 and 17. As you can see, the intravenous injection of folic acid (see Fig.16) shows a peak serum concentration of the drug in the absorption of the first sample, followed by a sharp decrease serum levels of the drug. The profiles obtained by oral administration of vitamin (see Fig.17), are different, because the maximum concentration is significantly below;

they appear for a longer time and a more gradual decrease. However, when comparing the levels of vitamin shown after oral administration of folic acid in the free form (without encapsulation) or encapsulated in casein nanoparticles (with or without treatment high pressure processing), find the concentration profile in similar time periods, but the maximum values are higher with the introduction of encapsulated vitamin.

The values of pharmacokinetic parameters obtained after compartmental analysis of the experimental data of the present study are included in Table 11.

Table 11
Pharmacokinetic parameters tested different drugs (mean ±SD, n=5)
Drug Tmax(min)Withmax(ng/ml)AUC (×104) (ng×min/ml)MRT (min)FR(%)
Maincaps.FC58.8±36.0191.3±41.07.8±1.5383.8±47.536.3±7.2
Incaps.LF to FC70.0±24.5240.9±71.711.2±2.8*485.8±267.152.1±13.0*
Incaps.LF VD FC52.8±20.8331.3±45.7"11.3±2.5*560.4±124.7*52.7±11.6*
Intravenous4227,1±1651.5**21.5±2.8**57.8±15.5**100**
* p<0,5 relative to non-encapsulated folic acid. U-Mann-Whitney test.
** p<0.01 vs. non-encapsulated folic acid. U-Mann-Whitney test.
AUC: PLO�ad under the curve of serum concentration
Withmax: the maximum concentration
Tmax: the time at which reaches Withmax
MRT: mean residence time
FR: relative bioavailability in percent.

As you can see, the values of AUC are subject to significant changes depending on the type of drug. When the vitamin is encapsulated in casein nanoparticles, AUC values are significantly higher than the values obtained after the introduction of free folic acid, and, in addition, they are saved in the period up to 24 hours after injection. Observe that the average time of stay (MW) of folic acid in the plasma close to the two preparations of nanoparticles and larger compared with the free form (oral and intravenous drugs).

According to the calculated results that the oral bioavailability of casein nanoparticles with encapsulated folic acid, which is 52% in both drugs, 45% higher than the values obtained after oral administration of free folic acid.

Example 8. Cosmetic product HZ with casein nanoparticles with encapsulated folic acid

A solution containing 200 mg �Caseinate sodium and 18 mg of lysine, get in a final volume of 15 ml of water.

In addition, 600 mg of folic acid are dissolved together with 800 mg of lysine in 100 ml of water.

Then to a solution of Caseinate is added 1.5 ml of a solution of folic acid. After 5 minutes of incubation to the mixture is added 8 ml of 0.8% CaCl2while stirring the mixture with a magnetic stirrer and constant flow.

Finally, the preparation was centrifuged at 17000×g, 20 minutes. The discard supernatant and pellet resuspended in 25 ml of water.

The additional benefit of a solution containing 7 g of glycerol and 0.2 g of dissolved sodium in 42 ml of water. The solution was heated on a water bath to 50°C and then added an aqueous solution of casein nanoparticles containing folic acid, getting the final aqueous solution, which serves a cosmetic product.

In addition, also heat 25 g of Neo PCL O/W at 70°C until it is completely melted. After this the fat phase is melted, add the above aqueous solution under constant stirring to obtain O/W (oil in water) emulsions, which is the true and stable over time. Organoleptic evaluation of the resulting cream is positive, it has a uniform appearance and contains no lumps.

A similar study is carried out, in addition, with the use of the drug nanoparticles processed by high pressure (400 MPa, 5 minutes) and you�Osennij using spray drying, described in Example 4. Take 600 mg of the drug and was resuspended in 25 ml of water, then processing in the same manner as described above. The resulting cream has a homogeneous appearance and contains no lumps.

Example 9. Cosmetic product [2] with the casein nanoparticles with encapsulated folic acid

A solution containing 200 mg of sodium Caseinate and 18 mg of lysine, get in a final volume of 15 ml of water.

In addition, 600 mg of folic acid are dissolved together with 800 mg of lysine in 100 ml of water.

Then to a solution of Caseinate is added 1.5 ml of a solution of folic acid. After 5 minutes of incubation to the mixture is added 8 ml of 0.8% CaCl2while stirring the mixture with a magnetic stirrer and constant flow.

Finally, the preparation was centrifuged at 17000×g, 20 minutes. The discard supernatant and pellet resuspended in 25 ml of water.

An additional 0.5 g of Carbopol Ultrez 10 was dissolved in 75 ml of water. The suspension of nanoparticles is added to the solution. After homogenization of the mixture is added a sufficient amount of trimethylamine to obtain a pH of 10. The mixture was homogenized to obtain a homogeneous and stable slightly yellowish gel Carbopol.

The same test is carried out, in addition, with the use of the drug nanoparticles processed by high pressure (400 MPa, 5 min) and dried using spray drying as described in Example 4. Take 600 phpreports and was resuspended in 25 ml of water, processing then in the same manner as described above. The resulting gel has a slightly yellowish color and a homogeneous and stable appearance.

Example 10. Cosmetic product [3] with the casein nanoparticles with encapsulated folic acid

3 g of glyceryl monostearate mixed with 5 g of isopropylmyristate and 2 g of catelouge alcohol. The mixture was heated in a water bath at 70°C.

In addition, 87 g of Carbopol gel containing casein nanoparticles with folic acid, described in Example 8, was heated to 50°C in water together with 3 g of sorbitol solution. This solution was added to the first, gently stirring to obtain a homogeneous emulsion.

Example 11. Obtaining and characterization of casein nanoparticles. contains quercetin

A solution containing 100 mg of sodium Caseinate and 8.5 mg of lysine (or 5.5 mg of arginine), get in 7.5 ml of water.

In addition, get a solution of sodium ascorbate in water with a concentration of 12 mg/ml, 0.5 ml of which was added to a mixture of Caseinate and lysine. The reason for using the sodium ascorbate is to prevent the oxidation of quercetin in the process of obtaining nanoparticles.

In addition, 50 mg of quercetin was dissolved in 5 ml of ethanol.

Then 0.15 ml of a solution of quercetin is added to a solution of Caseinate. After 5 minutes of incubation with 4 ml of 0.8% l2add to the mixture with stirring using�Yu a magnetic stirrer at a constant flow. This process is carried out in three replicates for each type of drug.

Physico-chemical properties obtained in each case, included in Table 12.

Table 12
Physico-chemical properties of the casein nanoparticles with quercetin, amino acid and ascorbic acid (mean±SD, n=3). The weight ratio of quercetin and protein is 1:67; the weight ratio of quercetin and ascorbic acid is 1:3,4
DrugSize (nm)PDIZeta potential (mV)The content of quercetin µg/mg LFEncapsulation efficiency
Casein LF (lysine)115±50,21±0,03Occupies 15,5±0,311,1±0,386,7±2,6
Casein LF (arginine)112±30,20±0,02-17,1±0,311,7±0,488,1±2,5
K: Quercetin; LF: Nan�particle

The results show that the nanoparticles corresponding to the invention, is also suitable for the encapsulation of biologically active compounds with fat-soluble properties and give the possibility of obtaining high percent encapsulation efficiency.

In addition, the results allow to confirm that the presence of one or the other amino acids do not affect the physicochemical properties of the resulting nanoparticles.

With the aim of increasing the number of encapsulated quercetin repeat the study using lysine as amino acids and various amounts of quercetin (0.05 to 0.50 ml of a solution of quercetin in ethanol). The results obtained are included in Table 13.

Table 13
Physico-chemical properties of the casein nanoparticles with lysine, ascorbic acid and varying amounts of quercetin (mean ±SD, n=3). The weight ratio of ascorbic acid and protein (casein) is 1:17
The mass ratio of quercetin: caseinSize (nm)PDIZeta potential (mV)The content of quercetin µg/mg N� Encapsulation efficiency
1:180147±140,21±0,03-17,6±0,34,3±0,283,2±4,2
1:67115±50,21±0,03Occupies 15,5±0,311,1±0,386,7±2,6
1:20---------38,0±1,389,3±3,1
To: Quercetin; LF: Nanoparticle

The results show that with increasing the amount of quercetin in the product, the amount of encapsulated quercetin increases in the same proportion, whereas the encapsulation efficiency remains constant.

In addition, conduct tests in accordance with the previously described manner, but with the dispersion of quercetin in water (instead of its dissolution in ethanol) before adding it to the solution of Caseinate. The results show that some of quercetin encapsulated in casein nanoparticles, although the encapsulation efficiency is less than in the previous case�e, where quercetin was dissolved in ethanol before adding to a solution of Caseinate.

1. Of nanoparticles comprising a casein matrix, a basic amino acid and a metal selected from the group comprising a divalent metal, a trivalent metal and combinations thereof.

2. Nanoparticle according to claim 1, in which the basic amino acid selected from the group comprising arginine, lysine, histidine, and mixtures thereof.

3. Nanoparticle according to claim 1, in which the divalent metal is selected from the group including calcium, magnesium, zinc, iron in bivalent form, and combinations thereof, wherein said divalent metal is preferably a calcium.

4. Nanoparticle according to any one of claims.1-3, which further comprises a biologically active compound.

5. Nanoparticle according to claim 4, in which the biologically active compound selected from the group comprising water soluble biologically active compound and fat-soluble biologically active compound.

6. Nanoparticle according to claim 5, in which the water-soluble biologically active compound is selected from the group including vitamin b group, vitamin C, a derivative of vitamin b group, a derivative of the b vitamin C, hyaluronic acid, chondroitin sulfate, thioctic acid, and their salts, esters, or combinations thereof.

7. Nanoparticle according to claim 5, in which the water-soluble biological�and active compound selected from the group including folic acid, 4-aminobenzoic acid, Niacin, Pantothenic acid, monophosphate, thiamin pyrophosphate, cumintheface, ascorbic acid, pteroylmonoglutamic acid, folinic acid, nicotinic acid, hyaluronic acid, thioctic acid, p-kumarovuû acid, caffeic acid, and their pharmaceutically, cosmetically acceptable, food quality derivatives, or esters, salts, and their mixtures.

8. A method of producing the nanoparticle containing a casein matrix, a basic amino acid and a metal selected from the group comprising a divalent metal, a trivalent metal and combinations thereof, comprising preparing an aqueous solution of a source of casein and a basic amino acid, and add to cooked aqueous solution of a metal selected from the group comprising a divalent metal, a trivalent metal and combinations thereof, to obtain a suspension containing the formed nanoparticles.

9. A method according to claim 8, wherein said source contains casein sodium Caseinate.

10. A method according to claim 8, in which specified basic amino acid selected from the group comprising arginine, lysine, histidine, and mixtures thereof.

11. A method according to claim 8, wherein said metal is a divalent metal selected from the group including calcium, magnesium, zinc, iron double-shaft�Noi form, and combinations thereof, wherein said divalent metal is preferably a calcium.

12. A method according to claim 8, wherein said aqueous solution of the metal is an aqueous solution of the calcium salt, wherein said calcium salt is selected from the group comprising calcium chloride, calcium acetate, calcium gluconate, calcium lactate, calcium sorbate, calcium ascorbate, calcium citrate, calcium propionate, calcium sulfate and mixtures thereof.

13. A method according to claim 8, which additionally comprises treating a suspension containing nanoparticles formed of at least one cycle of hydrostatic pressure at a pressure of 100÷800 MPa.

14. A method according to claim 8, which further comprises drying the slurry containing the formed nanoparticles.

15. A method according to claim 14, in which drying is performed in the presence of protective substances and optionally in the presence of an antioxidant.

16. A method according to claim 15, wherein said protective substance is a saccharide.

17. A method according to claim 15, wherein said antioxidant contains vitamin C.

18. A method of producing the nanoparticle containing a casein matrix, a basic amino acid, the metal selected from the group comprising a divalent metal, trivalent metal, and combinations thereof, and the biologically active compound comprising mixing an aqueous solution of a source of casein and basically� amino acid with a solution of biologically active compounds and adding to this mixture an aqueous solution of the metal, selected from the group, obtaining a suspension containing the formed nanoparticles.

19. A method according to claim 18, wherein the source of casein contains sodium Caseinate.

20. A method according to claim 18, in which specified basic amino acid selected from the group comprising arginine, lysine, histidine, and mixtures thereof.

21. A method according to claim 18, wherein said metal is a divalent metal selected from the group including calcium, magnesium, zinc, iron in bivalent form, and combinations thereof, wherein said divalent metal is preferably a calcium.

22. A method according to claim 18, wherein said aqueous solution of the metal is an aqueous solution of the calcium salt, wherein said calcium salt is selected from the group comprising calcium chloride, calcium acetate, calcium gluconate, calcium lactate, calcium sorbate, calcium ascorbate, calcium citrate, calcium propionate, calcium sulfate and mixtures thereof.

23. A method according to claim 18, wherein said biologically active compound selected from the group comprising water soluble biologically active compound and fat-soluble biologically active compound.

24. A method according to claim 23, wherein said water-soluble biologically active compound is selected from the group including vitamin b group, vitamin C, a derivative of vitamin of group b, PROIZVODITELYA group, hyaluronic acid, chondroitin sulfate, thioctic acid, and their salts, esters, or combinations thereof.

25. A method according to claim 24, wherein said water-soluble biologically active compound is selected from the group including folic acid, 4-aminobenzoic acid, Niacin, Pantothenic acid, monophosphate, thiamin pyrophosphate, cumintheface, ascorbic acid, pteroylmonoglutamic acid, folinic acid, nicotinic acid, hyaluronic acid, thioctic acid, p-kumarovuû acid, caffeic acid, and their pharmaceutically, cosmetically acceptable, food quality derivatives, or esters, salts, and their mixtures.

26. A method according to claim 18, which further comprises treating a suspension containing nanoparticles formed of at least one cycle of hydrostatic pressure at a pressure of 100÷800 MPa.

27. A method according to claim 18, which further comprises drying the slurry containing the formed nanoparticles.

28. A method according to claim 27, in which drying is performed in the presence of protective substances and optionally in the presence of an antioxidant.

29. A method according to claim 28, wherein said protective substance is a saccharide.

30. A method according to claim 28, wherein said antioxidant contains vitamin C.

31. Of nanoparticles comprising a casein matrix, a basic amino acid�and the metal, selected from the group comprising a divalent metal, a trivalent metal and combinations thereof, characterized by obtaining by the method according to claim 8.

32. Of nanoparticles comprising a casein matrix, a basic amino acid, the metal selected from the group comprising a divalent metal, a trivalent metal and combinations thereof, and the biologically active compound, characterized by obtaining by the method according to claim 18.

33. Composition comprising at least one nanoparticle according to any one of claims.1-7 or the nanoparticles obtained by the method according to any one of claims.8-30, and a carrier acceptable for food products, pharmaceuticals or cosmetics.

34. A composition according to claim 33, in which the nanoparticles are characterized by an average size of 50÷200 nm, preferably about 140 nm.

35. A composition according to claim 33, which is characterized by containing the following components, wt.%:

casein10÷50
folic acid0,9÷2,5
calcium1÷6
essential amino acid1÷7
saccharide30÷80

36. A composition according to claim 33, characterized by a content of a trace�of existing components, wt.%:

casein10÷50
folic acid0,9÷2,5
calcium1÷6
essential amino acid1÷7
saccharide20÷55
ascorbic acid1÷25

37. A composition according to claim 33, in which the media contains a pharmaceutical or cosmetic excipient that is acceptable for local use.

38. A composition according to any one of claims.33-37, in which the nanoparticle is made in the form of dry powder.

39. A food product comprising the composition according to any one of claims.33-36, 38.

40. The food product according to claim 39, which is made in liquid, semi-solid or solid form.



 

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4 ex

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3 ex

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7 tbl, 10 ex, 12 dwg

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3 cl, 4 ex

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7 ex

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EFFECT: preparing the composition for neuronal protection.

14 cl, 1 ex, 4 dwg

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