BR102018071692A2 - Process for Obtaining a Microparticle Containing High Bioavailability, Microparticle Effect and Uses of the Microparticle - Google Patents

Abstract

The present invention describes a microencapsulated ingredient for use as a source of high bioavailability iron. The active component is prepared from hydrolysed proteins of bovine whey, where only the low molecular weight fraction is chelated and / or complexed to iron metal ions. The ingredient in the form of microparticles has as its application the production of a water soluble iron mineral supplement with high iron bioavailability. The process is efficient by joining a small organic compound (peptide) to a mineral (iron), resulting in a product of greater stability, solubility and consequently greater absorption by the gastrointestinal tract. The present invention is in the field of food, more precisely in food chemistry.

Description

[0001] The present invention describes about a microencapsulated ingredient, containing Fe-peptides, to be used as a source of iron of high bioavailability, for use as a food supplement of iron source, in the form of powder. The present invention is located in the Food field, more precisely in Food Chemistry.

Background of the Invention [0002] Many nutrients, such as iron, are considered essential to life, since they participate in vital biochemical-metabolic functions. Mineral supplementation has been instituted as a strategy to remedy some nutritional deficiencies. since iron deficiency anemia is one of the biggest public health problems that affects all age groups in the population. minerals conveyed in the form of complexes or chelates, in turn, have been proposed because they have a higher bioavailability. [0003] There are iron compounds used for fortification in their complexed form, such as sodium and iron EDTA (NaFeEDTA), ferric pyrophosphate or ferrous bisglycinate. However, studies point to Fepeptide complexes as a promising alternative, with the potential to increase mineral absorption. The formation of the Fe-peptide complex occurs by the sharing of electrons between the metal and the ligand establishing a coordinated covalent bond, in which the metal is the electron receptor. The specificity of formation of these complexes is determined by the spatial arrangement of the functional groups of the amino acids that participate in the peptide sequence. Interactions can be increased or decreased

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2/32 by modifying the amino acid residues present in the peptide sequence. Chelated minerals provide the animal and human organism with a mineral supplement to be absorbed more quickly and more efficiently, increasing the nutritional benefit. Both the di and the trivalent forms of iron have a high binding affinity for proteins and other complexing agents, at pH around neutral. Several compounds can form complexes with iron, such as carbohydrates, fibers, phytates, amino acids, peptides and proteins.

[0004] Although it is not yet clear the mechanism by which iron in its complexed form is absorbed, it is known that iron is extremely insoluble in physiological pH and strongly influenced by the presence of dietary inhibitory compounds, which can restrict its absorption . Thus, iron needs to be protected during digestion, and the formation of complexes with peptides (Fe-peptides) has been shown to improve iron resistance in gastrointestinal conditions.

[0005] So, the protection of the metal along the gastrointestinal tract, favors its solubility and inhibits its reactivity with other components of the diet. Thus, iron-bound peptides (Fepeptides) can offer advantages over oral ingestion of formulations containing non-heme iron compounds, such as iron sulfate, alleviating low availability problems and side effects in anemic subjects.

[0006] Mineral supplement is defined by the Ministry of Health (MS) as a mineral supply that complements the daily diet of a healthy person, in cases where the intake of these nutrients from the diet is insufficient. In supplements, each nutrient must contain a minimum of 25% and a maximum of up to 100% of the recommended daily intake (IDR), in the daily portion indicated by the manufacturer. It should not replace food, nor should it be considered an exclusive component of the diet. The use of nutritional supplements has become increasingly common and is

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3/32 growing at a very fast pace due to some application advantages, such as low cost, effectiveness, potential for regeneration and even changes in behavior due to the media. In addition, the supplements are subject to a less demanding and lesser amount of regulation, which in turn is less rigid than the laws dealing with prescription drugs and foods with functional claims.

[0007] To be incorporated into a supplement it is crucial that the added iron does not cause unwanted oxidative and sensory changes in the food. Of equal importance, but possibly more challenging, is the addition of a form of bioavailable iron. Exception to the rule, the more bioavailable the more reactive are the complexes and / or chelates of iron and microencapsulated iron, in which iron is protected by physical or chemical means. [0008] In the search for the state of the art in scientific and patent literature, the following documents dealing with the topic were found:

[0009] The document US 20150079268A1 entitled "Formulation for iron supplements" refers to the complexation of iron with a low molecular weight polysaccharide, obtained through extensive hydrolysis of starch. Therefore, the nature of the organic compound and the synthesis reaction are completely different from that used in this work.

[0010] US 5002779 A entitled “Iron-containing nutritional supplement“ deals with an oral nutritional supplement of ferric pyrophosphate chelate as an iron fortifying agent. Intended for animals and humans with iron deficiency anemia. The source salt of iron is the pyrosphosphate is joined to the citrate forming the soluble ferric citrate pyrophosphate compound. Therefore, the final compound obtained is different from that shown in this invention.

[0011] The document US 6358544 B1 entitled "COLOR STABLE IRON AND ZINC FORTIFIED COMPOSITIONS" - this patent refers to the color and flavor stability in a powdered chocolate drink containing iron and zinc, with a fruit and / or botanical flavor. This patent does not mention the same

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4/32 chelation process than the present invention, being thus different from the invention disclosed herein.

[0012] The document US 6521247 B1 entitled "Dual iron containing nutritional supplement" - the present invention relates to a nutritional supplement comprising two different iron compounds (ferrous and ferric). Slow and fast release iron compounds. It may contain other non-ferrous vitamins and minerals. Its presentation is in tablet and the administration is for women as a prenatal supplement, during pregnancy and during lactation. Corresponds to the recommended daily intake for this audience. Patent different from the one presented by its administration.

[0013] The document PI 0305871-9 entitled Product based on ferrous sulfate for fortification of dehydrated foods and method of preparation This patent refers to a product, source of iron for use as a food enricher, being able to maintain its sensory properties stable during storage. The product is obtained from the formation of an alginate film on particles of bioavailable iron salts, especially ferrous sulfate, and the method of their preparation. Since iron salts, especially ferrous sulfate monohydrate (FeSO4.H2O), modify their bioavailability in food. The product was developed to circumvent problems with its use in food, and particularly in wheat flour and other cereals and prepared foods such as breads, pasta and cakes. The innovation was to coat the particles with alginate, a natural polysaccharide extracted from algae that is very easy to form a gel with divalent metal ions such as Ca or Fe ^2+, among others. The preparation method consists first of depositing a film of sodium or potassium alginate and on the surface of ferrous sulphate particles or other bioavailable iron salt, followed by successive drying steps, deposition or not of a film of polycations or synthetic polymers. and drying and depositing a new layer of alginate and drying until reaching the

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5/32 desired coating and granulometry.

[0014] The invention PI 0008690-8 entitled "Iron fortification system" - Refers to a hydrolyzed iron-protein complex that can be used to fortify foods and beverages with iron. The complex formed is ferrous ions chelated from partially hydrolyzed egg white protein. The partially hydrolyzed egg white protein has a molecular weight of about 500 to 10,000. The complexes are stable and suitable for use in sterile products, such as applied. Despite the stability, iron in complexes has substantially the same bioavailability as iron sulphate. Therefore, it differs from this proposal, which uses peptides resulting from a different source.

[0015] Thus, from what can be inferred from the researched literature, no documents were found anticipating or suggesting the teachings of the present invention, so that the solution proposed here has novelty and inventive activity in view of the state of the art. Since, in the current state of the art, we can observe that some patented processes and products propose supplementation, however they differ as to the nature of the raw material, applied methodology and iron source salt. [0016] Additionally, the Unified Health System (SUS) uses and recommends supplementation with ferrous sulfate. This compound has an unpleasant, metallic taste, dark coloration and causes gastric-intestinal discomfort in a large percentage of individuals. Additionally, it has a low bioavailability rate of iron. Other products available on the market do not achieve bioavailability efficiency when compared to the product proposed in this patent.

[0017] Thus, there is a clear need for the development of a supplement capable of optimizing iron absorption, increasing its bioavailability to the organism without major side effects, in addition to presenting acceptable palatability.

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Summary of the Invention [0018] Thus, the present invention solves the problems of the state of the art from the elaboration of a microencapsulated ingredient to be used as a source of high bioavailability iron. The active component is prepared from hydrolyzed proteins of bovine whey, where only the low molecular weight fraction is chelated and / or complexed to metal iron ions. The ingredient in the form of microparticles has the application of producing a mineral supplement of water-soluble iron with high bioavailability of iron. The process is efficient, because it combines a small organic compound (peptide) with a mineral (iron), resulting in a product of greater stability, solubility and consequently greater absorption by the gastrointestinal tract.

[0019] The present invention has high potential for application as a dietary iron supplement for individuals with iron deficiency anemia, without the sensory characteristics and side effects of ferrous sulfate.

[0020] In a first object the invention describes a process of obtaining a microparticle containing Fe-Peptide of high bioavailability, comprising the steps of:

a) obtaining a liquid protein hydrolyzate, from the hydrolysis of whey proteins;

b) filtering the liquid protein hydrolyzate obtained in (a), to select peptides smaller than 5 kDa;

c) adjustment of the pH of the product obtained in (b) to a pH value between 5.0 to

5.5;

d) addition of iron salt in the product obtained at the end of step (c), in which the proportion of peptide and iron salt varies between 10: 1 to 40: 1 (w / w), where the iron salt is Iron Sulfate;

e) maintaining the product obtained in (d) at a temperature of 3035 ° C in a period of at least 20 minutes for the reaction of

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7/32 synthesis of the Fe-peptide complex;

f) addition of ascorbic acid, in a proportion of 1.6 times the amount of iron used;

g) addition of polymers as wall agents;

h) spray drying to obtain microparticles containing Fe-peptides and ascorbic acid as active;

where the wall agent is selected from maltodextrin, hicap, wallocel, pectin, polydextrose, gum arabic, chitosan, methocel and combination thereof, where the final product of the process is a particle with a diameter between 5.96 to 6.33 microns.

[0021] In a second object, the present invention describes a microparticle, produced according to the first object, comprising:

I. a Fe-peptide composition, comprising at least the following components:

- peptides smaller than 5kDa, in which said peptide is obtained through a hydrolysis reaction of whey protein and an ultrafiltration process,

- iron salt, where the iron salt is ferrous iron sulphate,

- Ascorbic acid;

where peptides smaller than 5kDa and the iron salt are complexed or chelated to form an active Fe-peptide component with a ratio between 20:80 to 25:75 (w / w), preferably 20:80 (w / w), where the proportion of peptides smaller than 5kDa and the iron salt varies between 10: 1 to 40: 1 (w / w);

where the amount of ascorbic acid is 1.6 times the amount of iron used.

II. a capsule comprising composition (I) comprising at least one wall agent, wherein said wall agent is selected from maltodextrin, hicap, wallocel, pectin, gum arabic, polydextrose,

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8/32 chitosan, methocel and combination thereof.

[0022] In a third aspect, the present invention relates to the use of a microparticle, obtained according to the process of the first object, for the production of drinks with high bioavailability of iron.

[0023] In a fourth aspect, the invention refers to the use of microparticles, for the preparation of supplements with high bioavailability of iron for the treatment of iron deficiency anemia.

[0024] Still, the inventive concept common to all the protection contexts claimed is the microparticle containing an active component, Fepeptide, presenting a high bioavailability.

[0025] These and other objects of the invention will be immediately valued by those skilled in the art and will be described in detail below.

Brief Description of the Figures [0026] The following figures are presented:

[0027] Figure 1 shows a flowchart of the steps for obtaining the microparticles, containing the active Fe-peptide. The ingredient refers to a dry ingredient to be incorporated into drinks of high stability and high bioavailability of iron.

[0028] Figure 2 presents a flowchart describing the process of the IMAC technique and isolation of peptides capable of binding to Iron, for amino acid sequencing.

[0029] Figure 3 shows the size distribution by volume (%) at 25 ⁰ C of the peptides present in the hydrolysates obtained from the whey proteins (HPSL) with different enzymes: (red) HPS with Flavourzyme®, ( green) HPS with Alcalase®, (blue) HPS with pancreatin through dynamic light scattering experiments (Dynamic Light Scattering, DLS) on the Dynamic Laser Light Scattering (DLS) equipment provided with a He-Ne laser (633 nm) and a digital correlator, Model ZEN3600.

[0030] Figure 4 shows the size distribution (pm) of the

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9/32 microparticles of the samples with different combinations of wall material, by laser diffraction in a particle size analyzer LV950V2 (Horiba, Kyoto, Japan): maltodextrin (MD), MD + HC (malt dextrin and hicap) and MD + PD (maltodextrin and polydextrose).

[0031] Figure 5 shows Photomicrographs of microparticles with different wall materials obtained at different times: A) newly produced (time 0) and B) after 180 days of storage, C) 360 days of storage, through scanning electron microscopy . Increase: SEM: 1000X. The figure shows the monitoring of the microparticles during the shelf life for 360 days. The microparticles obtained with 100% maltodextrin and Maltodextrin: polydextrose (80:20), compared to microparticles without active (Fe-peptide). The samples were compared by SEM (scanning electron microscopy), being A) right after processing at zero time (right after drying), B) at 180 days of storage and C) 360 days after storage. During the storage period there were no changes in shape and size.

[0032] Figure 6 shows the solubility of Fe-peptide samples at pH 7.0 after centrifugation of samples with different proportions of Fe: peptides (<5kDa) after their gastro-intestinal digestion in vitro and compared to ferrous sulfate, in same conditions.

[0033] Figure 7 shows the recovery of iron through the in vitro dialysability technique, after gastric-intestinal digestion, using 6-8 kDa dialysis membrane.

Detailed Description of the Invention

Definitions [0034] In the present invention, the term "Fe-peptide" can also be understood as an "active component", it is better defined as a chelated or complexed mineral. Where the mineral introduces a mineral supplement to the body to be absorbed more quickly and with greater

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10/32 efficiency, increasing the nutritional benefit. Furthermore, the process involves combining minerals with chelating agents of an organic nature, which can be small food peptides and free amino acids.

[0035] In the present invention, the term "substrate" can also be understood as "whey proteins", which in turn were selected in this study as a source of iron chelating peptides. These proteins have high biological value, high digestibility and rapid absorption by the body. From the amino acid point of view, whey proteins have almost all essential amino acids in excess of the recommendations, except for the aromatic amino acids (phenylalanine, tyrosine), but they meet the recommendations for all ages. They have high concentrations of the amino acids tryptophan, cysteine, leucine, valine, isoleucine and lysine. They have high levels of branched chain amino acids (valine, isoleucine and leucine). Whey proteins are highly digestible and rapidly absorbed by the body, stimulating the synthesis of blood and tissue proteins, to the point that, some researchers have classified these proteins as rapidly metabolizing proteins, suitable for situations of metabolic stresses, in which the protein replacement becomes emergency. They are widely used by practitioners of physical activity to rebuild injured tissue and increase muscle mass.

[0036] In the present invention the "protein hydrolysis process" consists of the chemical or enzymatic cleavage of protein molecules into small peptides of different sizes and, eventually, free amino acids. The control of the conditions of enzymatic hydrolysis and the appropriate choice of enzyme condition the obtaining of products with characteristics appropriate to a given application in food formulation.

[0037] In the present invention "proteolytic enzymes" or "proteases" are called those that catalyze the breaking of peptide bonds in proteins. They are class 3 enzymes, hydrolases, and subclass 3.4, the

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11/32 peptide-hydrolases or peptidases. These enzymes form a large family, divided into endopeptidases or proteinases and exopeptidases, according to the position of the peptide bond capable of cleaving the polypeptide chain. The choice of the proteolytic enzyme is very important, since its specific action will influence the final composition of the hydrolysis products, mainly in relation to the average size of the peptides and exposure of the hydrophobic side groups. Small molecular size peptides, produced in vivo by the action of proteases, can have different physiological activities such as: antimicrobial effect, hypotensive, antioxidant, anti-inflammatory, mineral transporters, in addition, affect gastric hormones controlling intestinal and gastric motility . [0038] In the present invention the term "liquid protein hydrolyzate" refers to the product generated when the "whey proteins" or the "substrate" are subjected to the protein hydrolysis process ".

[0039] In the present invention, the term "wall agent" can also be understood as a "wall-forming polymer" and refers to a polymer capable of enveloping a "Fe-peptide" nucleus to form a "microencapsulated" .

[0040] In the present invention, the term "microencapsulation" is defined as a process that creates a barrier, from a polymeric matrix or covering that surrounds the active component, protecting it against chemical interactions and effects of environmental factors such as temperature , pH, enzymes and oxygen, allowing, when desirable, the controlled release of the active component. Additionally, the advantages of microencapsulation are associated with the stabilization of bioactive compounds, reaching the asset at the appropriate levels, inhibiting the formation of harmful and undesirable compounds due to chemical changes over time, and with the action of masking undesirable sensory attributes.

[0041] In the present invention, the "Spray drying" technique, also understood as "spray drying" is a method of producing dry powder at

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12/32 from a liquid or suspension, by rapid drying with hot air. It is a preferred method of drying various thermally sensitive materials. [0042] The present invention presents the differential of the use in an inventive way of small fragments of proteins, obtained from a food naturally present in the human diet, which are animal whey proteins. Only the small fragments of the hydrolyzed proteins are added to the metal iron ions, which can be from different sources, to form complexes and chelates under controlled conditions. The conditions were standardized according to the bioavailability of iron.

It is noteworthy that in the present invention an initial enzymatic process is used, followed by fractionation by size, where the enzyme and processing are decisive for obtaining the Fe-peptide complexes or chelates. The degree of hydrolysis achieved and the conditions of the chelation / complexation reaction, as well as the drying of the material with the addition of other compounds to increase the rate of iron absorption by the body, make up a sequence of essential events for obtaining the powdered product. This invention uses chemical and biochemical techniques, and proves the increased absorption in tests carried out in vitro. The final product has applicability to health with the potential to be used as food supplements by the population and animals deficient in iron.

[0043] In the present invention it is understood that the generic name of Flavourzyme® marketed by Sigma-Aldrich is fungal protease from Aspergillus oryzae. It is formed with an enzymatic complex, composed of a mixture of eight enzymes, two aminopeptidases, two dipeptidylpeptidases, three endopeptidases, and an α-amylase from the strain of A. oryzae (yellow koji fungus). While the Alcalase® enzyme must be understood as being subtilisin.

Examples - Embodiments [0044] The examples shown here are intended only to exemplify one of the countless ways of carrying out the invention, however

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13/32 without limiting its scope.

Example I - Process for obtaining a microparticle containing Fe-Peptide with high bioavailability [0045] The present invention relates to the preparation of a microencapsulated ingredient to be used as a source of high bioavailability iron. The active component is prepared from hydrolyzed proteins of bovine whey, where only the low molecular weight fraction is chelated and / or complexed to metal iron ions. The process is efficient, because it combines a small organic compound (peptide) with a mineral (iron), resulting in a product of greater stability, solubility and consequently greater absorption by the gastrointestinal tract.

[0046] Thus, in a first object the invention describes a process of obtaining a microparticle containing Fe-Peptide of high bioavailability, comprising the steps of:

a) obtaining a liquid protein hydrolyzate, from the hydrolysis of whey proteins

b) filtering the liquid protein hydrolyzate obtained in (a), to select peptides smaller than 5 kDa.

c) Adjustment of the pH of the product obtained in (b) to a pH value between 5.0 to 5.5 to perform the synthesis reaction of the chelates and complexes;

d) Addition of iron salt in the product obtained at the end of step (c), in which the proportion of peptide and iron salt varies between 10: 1 to 40: 1 (w / w), where the iron salt is Iron Sulfate;

e) Maintenance of the product obtained in (d) at a temperature of 3035 ° C at pH 5.5 for a period of at least 20 minutes for the synthesis of the Fe-peptides complex to take place;

f) Addition of ascorbic acid, in a proportion of 1.6 times the amount of iron used.

g) Addition of polymers as wall agents;

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h) Spray drying to obtain microparticles containing Fe-peptides and ascorbic acid as active;

wherein the wall agent is selected from maltodextrin, hicap, wallocel, pectin, gum arabic, chitosan, polydextrose, methocel and combination thereof.

where the final product of the process is a particle with a diameter between 5.96 to 6.33 microns.

[0047] In step (f) ascorbic acid is used to promote better absorption of iron, since it is a reducing agent. It is added on drying to act as an additive with the active ingredient and ensure its greater absorption. The effect of ascorbic acid was observed in the iron bioavailability tests by dialysability, as shown in Table 1.

Table 1. Iron bioavailability test by dialysability in hydrolysates with pancreatin added with ascorbic acid and without the acid.

T ratements (%) Fe dialysed ^to Peptides <5 kDa (Pancreatin) Buffer PIPES + Fe 27.85 ± 1.10 Fe-chelated peptides without AA (ascorbic acid) 41.17 ± 2.40 AA-chelated Fe-peptides (ascorbic acid) 47.21 ± 0.40

- treatments performed in duplicate (mean and standard deviation) [0048] In one embodiment of the first object, obtaining a liquid protein hydrolyzate in step (a) consists of the steps:

i) dilution of the substrate in water, in the proportion of 8% to 12% of substrate;

ii) homogenization of the sample at room temperature (22 to 25 ° C), iii) adjustment of pH and temperature according to the enzyme specification for the hydrolysis of the substrate to be used, iv) addition of a protease for hydrolysis of the substrate.

v) maintenance of the substrate and enzyme for a period of 120 to 340 minutes for the hydrolysis reaction to occur, vi) inactivation of the enzymes by cooling in an ice bath and

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15/32 kept cool until the ultrafiltration process.

[0049] In the invention any protease can be used, regardless of the origin, animal, vegetable or microbial, what matters are the peptide fragments formed by extensive hydrolysis of the enzymes. The HPLC / Ms / MS mass analyzes reinforce this understanding, showing that regardless of the enzyme used, the amino acid sequence in the peptide chain (originated from the same region of the protein) is responsible for the iron chelating capacity. Because, the amino acid sequences that make up the peptides that were isolated by iron ion affinity chromatography (metal ion affinity column - IMAC-Fe), regardless of the enzyme cut, were mostly repeated for the different hydrolysates obtained with the different enzymes. Tables 2 and 3 exemplify the results discussed here.

Table 2. Peptides identified from a- Lactoalbumin (a- La), from different enzymatic treatments (Alcalase® (subtilisin), pancreatin and Flavourzyme® (peptidase prepared from Aspergillus oryzae)). Isolation by metal ion affinity chromatography (IMAC) immobilized with iron (III). Identification performed by reverse phase liquid chromatography and tandem mass spectroscopy (HPLC -FR / MS / MS).

Enzyme: Alcalase® Enzyme: Pancreatin Enzyme: Flavourzyme® Fragment of a- La Amino Acid Sequence Fragment of a- La Amino Acid Sequence Fragment of a- La Amino Acid Sequence 10 - 15 RELKDL 81 - 89 LDDDLTDDI 14- 26 DLKGYGGVSLPEW 13 - 18 KDLKGY 81- 90 LDDDLTDDIM 30 -45 TFHTSGYDTQAIVQ NN 43 - 54 QNNDSTEY GLFQ 82 - 90 DDDLTDDIM 62-71 KDDQNPHSSN 94 - 99 KILDKV 82 - 88 DDDLTDD 81 - 88 LDDDLTDD 94- 103 KILDKVGINY 82 - 89 DDDLTDDI 114 - 118 KLDQW

[0050] It can be seen in Table 2 that the region 81 to 88 of αlactoalbumin appears in both the hydrolyzate obtained from pancreatin and from Flavorzyme. Fragments of similar regions of whey protein that appear as iron ligands, obtained by

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16/32 hydrolysis with different enzymes.

Table 3. Peptides identified from β-Lactoglobulin (B-Lg), from different enzymatic treatments (Alcalase® / subtilisin), pancreatin and Flavourzyme® (peptidase prepared from Aspergillus oryzaé). Isolation by metal ion affinity chromatography (IMAC) immobilized with iron (III). Identification performed by reverse phase liquid chromatography and tandem mass spectroscopy (HPLC -FR / MS / MS).

Enzyme: Alcalase® Enzyme: Pancreatin Enzyme: Flavourzyme® Β-Lg fragment Amino Acid Sequence Β-Lg fragment Amino Acid Sequence Β-Lg fragment Sequence of Amino Acids 45-55 ELKPTPEGDLE 20-25 YSLAMA 8-20 KGLDIQKVAGTWY 46-55 LKPTPEGDLE 47-55 KPTPEGDLE 16-27 AGTWYSLAMAAS 46-53 LKPTPEGD 42-55 YVEELKPTPE GDLE 47-54 KPTPEGDL 43-55 VEELKPTPEG DLE 43-53 VEELKPTPEG D 47-55 KPTPEGDLE 46-55 LKPTPEGDLE 43-54 VEELKPTPEG DL 42-58 YVEELKPTPEGD LEILL 75-82 KTKIPAVF 43-55 VEELKPTPEG DLE 43-54 VEELKPTPEGDL 96-102 DTDYKKY 43-56 VEELKPTPEG DLEI 43-55 VEELKPTPEGDLE 99-102 YKKY 43-57 VEELKPTPEG DLEIL 43-56 VEELKPTPEGDLEI 122 -127 LVRTPE 47-56 KPTPEGDLEI 43-57 VEELKPTPEGDLEIL 124-129 RTPEVD 49-59 TPEGDLEILLQ 43-58 VEELKPTPEGD LEILL 128-134 VDDEALE 125-135 TPEVDDEALEK 44-54 EELKPTPEGDL 123 - 129 VRTPEVD 125-134 TPEVDDEALE 44-56 EELKPTPEGDLEI 122 - 129 LVRTPEVD 125-132 TPEVDDEA 44-57 EELKPTPEGDLEIL 122-131 LVRTPEVDDE 125-131 TPEVDDE 44-58 EELKPTPEGDLEILL 135 - 139 KFDKA 125-129 TPEVD 46-55 LKPTPEGDLE 146-149 HIRL 130-135 DEALEK 47-56 KPTPEGDLEI 148-159 RLSFNPTQLE EQ 47-57 KPTPEGDLEIL 47-58 KPTPEGDLEILL 49-59 TPEGDLEILLQ 73-82 AEKTKIPAVF 73-80 AEKTKIPA 74-80 EKTKIPA 73-84 AEKTKIPAVFKI 73-85 AEKTKIPAVFKID

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97-102 TDYKKY 96-102 DTDYKKY 124-135 RTPEVDDEALEK 125-136 TPEVDDEALEKF 125-132 TPEVDDEA 125-135 TPEVDDEALEK

[0051] The flowchart describing the process of the IMAC technique and isolation of peptides capable of binding to Iron is shown in Figure 2. The FPLC equipment with UV detection and fraction collector, highlighting the XK 16/20 column containing 10ml_ High Performance Sepharose (IMAC-HP).

[0052] With this technique it is possible to isolate the peptides to characterize the isolated material as to the size and sequence of amino acids that formed the peptides. This analysis was repeated for hydrolysates less than 5 kDa obtained with the different proteolytic enzymes. Alcalase® (Subtilisin) was less specific and cut the peptides in different regions of the protein, for example, it was found that pancreatin and Flavourzyme® (peptidase prepared from Aspergillus oryzae) has an affinity for cutting the β-lactoglobulin binding 125, while Alcalase® in this region has less specificity by cutting at different positions and originating peptides that start at position 122, 123, 124 and 128 of the β-lactoglobulin protein.

[0053] In another embodiment of the first object, the protease used is selected from Alcalase®, Pancreatina and Flavourzyme®. Preferably, the enzyme used in step (a) is Alcalase®, in which said Alcalase® must be maintained at pH 8.0 and at a temperature of 60 ^Q C, in which the pH is adjusted by adding NaOH ( 1.0 or 0.25 mol / L) and where Alcalase® is inactivated by cooling to the ultrafiltration stage and the hydrolysis time of 120 min, I / O ratio of 1: 100; where pancreatin must be maintained at pH 7.2 and at a temperature of 40 ^Q C, where the pH is adjusted by adding NaOH (1.0 or 0.25 mol / L) in which the hydrolysis time is

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18/32 preferably 180 minutes, the I / O ratio of 4: 100; where Flavouryzyme must be kept at pH 6.7 and at a temperature of 55 ° C, where the pH is adjusted by adding NaOH (1.0 or 0.25 mol / L), where the hydrolysis is preferably 180 minutes and the I / O ratio 4: 100.

[0054] In one embodiment of the process the enzyme used in step (iv) is subtilisin, and that said subtilisin must be maintained at pH 8.0 and at a temperature of 60 ° C, in which the pH is adjusted through addition of NaOH (0.25 or 1.0 mol / L), in which the subtilisin is inactivated in an ice bath.

[0055] In another embodiment, step (b) consists of the filtration being by an ultrafiltration process in tangential flow membranes with a 5 kDa cut, in which the particles obtained are less than 2.5 kDa.

[0056] The wall-forming polymers for placement and stability of the dry asset were selected based on yield, percentage of asset retention, cost and accessibility of iron through in vitro tests. Preferably, the polymer is selected from Maltodextrin, Hicap, Wallocel, Pectin, Gum arabic, Chitosan, polydextrose, Methocel and a combination thereof.

[0057] More preferably, the wall-forming polymer is selected from 11 different polymer mixtures. Therefore, other polymers that can be used.

Table 4 - Mixtures of wall-forming polymers.

TEST POLYMERS PROPORTION(%) 1 Maltodextrin 100 2 Maltodextrin + Hicap 80 + 20 3 Maltodextrin + Polydextrose 80 + 20 4 Maltodextrin + Hicap 50 + 50 5 Maltodextrin + Wallocel 99.8 + 0.2 6 Hicap 100 7 Pectin + Hicap 19 + 81 8 Methocel + Gum arabic 2 + 98 9 Chitosan + gum arabic 2 + 98 10 Methocel + Hicap 2 + 98 11 Chitosan + Hicap 2 + 98

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19/32 [0058] The formulations were composed of 20% of active (peptides, ferrous sulfate and ascorbic acid in the proportion of 1.6 times the amount of iron used) and the other 80% of wall material.

[0059] The Active: polymer ratio can vary from ”20:80 to 25:75”, depending on the desired concentration or iron content in the microparticles after drying. This is not a critical parameter in the process of obtaining microparticles, and the preferred proportion would be 20:80.

[0060] In another embodiment of the invention the wall-forming polymer, wall agent, used in step f) is selected from maltodextrin, polydextrose, modified starch, hicap, wallocel, pectin, gum arabic, chitosan, methocel or a mixture thereof , in which in a mixture, the Fe-Peptide ratio and the wall agent is between 20:80 to 25:75 (w / w), preferably 20:80 (w / w).

[0061] In another embodiment, step h) of drying is carried out using a spray dryer equipment in the following operating conditions: inlet temperature of 140 ° C and outlet temperature of 78 ° C; air pressure of approximately 50 mbar; aspiration of 100% of the power of the vacuum cleaner; air flow of 500 L / h.

Microparticle containing Fe-Peptide of high bioavailability [0062] The invention also relates to a microparticle produced according to the process of the first object. Preferably, the microparticle is obtained from peptides smaller than 5kDa (produced by the hydrolysis of whey protein, followed by ultrafiltration) reacted with iron and added with ascorbic acid and wall agent for encapsulation.

[0063] The particle comprises an active component, ascorbic acid and a polymer encapsulating the active, composed of a wall agent. More precisely, the active component refers to a Fe-peptide, in which the peptides were used in the synthesis of chelates and or complexes by association with iron under controlled conditions and added with acid

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Ascorbic 20/32 served as spray drying assets. The size of the chelated and / or complexed particles may be larger due to the process of binding to the metal ion inside the peptides.

[0064] Figure 4 does not exhaustively show examples of microparticles that can be characterized by being produced by spray drying, and by varying sizes, with a diameter between 5.96 to 6.33 pm, approximately.

[0065] Thus, in a second object, the present invention describes a microparticle, produced according to the first object, comprising:

I. a Fe-peptide composition, comprising at least the following components:

- peptides smaller than 5kDa, in which said peptides are obtained through an enzymatic hydrolysis reaction of whey proteins and fractionation of the hydrolyzate by an ultrafiltration process,

- iron salt, where the iron salt is ferrous sulphate,

- Ascorbic acid;

in which peptides smaller than 5kDa and the iron salt are complexed or chelated to form an active component (Fe-peptides), in which the proportion of peptides less than 5kDa and the iron salt varies between 10: 1 to 40: 1 ( w / w);

in the amount of ascorbic acid is 1.6 times the amount of iron used;

II. a capsule encompassing the composition (I) comprising at least one wall agent, wherein said wall agent is selected from the group maltodextrin, hicap, wallocel, pectin, gum arabic, chitosan, polydextrose, methocel and combination thereof.

[0066] In an embodiment of the second aspect, the microparticle has a size ranging between 5.96 to 6.33 microns and;

- the wall agent used is selected from maltodextrin,

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21/32 polydextrose, modified starch, or a mixture thereof, where in a mixture, the proportion of maltodextrin is at least 80%.

Uses of the microparticle [0067] After drying the microparticles were collected and iron accessibility tests were carried out in vitro, always using ferrous sulfate as a reference standard. Iron solubility and in vitro dialysability tests were performed. The latter uses dialysis membranes with pores ranging from 6 to 8 kDa, to simulate the conditions of gastrointestinal digestion, followed by passage through the intestinal mucosa. In this case the dialysis membrane simulates the intestinal membrane. The iron was always dosed with the ICP-OS equipment, which has very high sensitivity and precision. The chelation or complexation of peptides with iron increased the dialysability of iron by approximately 2 times and the wall covering (microparticles) did not interfere in the process of making iron available.

[0068] Therefore, a dry product was obtained to be used as a mineral supplement with high availability of iron with high solubility, in powder, to be incorporated in water-soluble drinks, intended for the anemic population (Step h, figure 1).

[0069] As demonstrated in the tests carried out, the microparticles demonstrate surprising stability and bioavailability of iron, in addition to contributing to the flavor and aroma of the drinks that can be incorporated.

[0070] Thus, in a third aspect, the present invention relates to the use of a microparticle, obtained according to the process of the first object, for the production of drinks with high bioavailability of iron.

[0071] In a fourth aspect, the invention relates to the use of microparticles, for the preparation of drinks with high bioavailability of iron and for the production of food supplements with high bioavailability of iron.

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22/32 [0072] In one embodiment of the use of microparticles it is used as dietary supplements, beverages and medicines with high bioavailability of iron for the treatment of iron deficiency anemia.

Examples - Embodiments [0073] The examples shown here are intended only to exemplify one of the countless ways to carry out the invention, however without limiting the scope of it.

Example I - Process [0074] With reference to Figure 1, it can be seen that the process begins with the dilution of proteins isolated from whey in water, homogenization of the sample at room temperature (22 to 25 ° C) in the proportion of 8-12% substrate. The pH and temperature were adjusted according to the specifications of the enzyme used (Step a, figure 1). In the case of Alcalase® the temperature was maintained at 60 ° C, through a jacketed reactor. The pH was initially adjusted to 8.0 with a constant stirring system. The Alcalase® enzyme was added (Step b, figure 1) and the pH was kept constant through the addition of 0.25 mol / L NaOH, with base consumption being constantly monitored. For monitoring, the pH-stat equipment (automatic titrator model DL 50 Graphics from Mettler Toledo, Scwerzenbach, Switzerland) can be used.

[0075] After the hydrolysis time between 120 and 340 minutes, preferably 180 minutes (Step c, figure 1), the hydrolysis reaction was interrupted by cooling in an ice bath (Step d, figure 1). The degree of hydrolysis obtained is in the range of 17 to 23%, generating many low molecular weight peptides and free amino acids. After cooling the enzyme inactivation (Step d, figure 1), the hydrolysates were ultrafiltered in a tangential flow membrane with a 5 kDa cut (Step e, figure 1). Then the protein content was measured and in the liquid material the pH was adjusted to pH values between 5.0 and 5.5 and the iron salt (ferrous sulfate) was added to the

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23/32 ratio of peptide: iron ranging from 10: 1 to 40: 1 (w / w) (Step f, figure 1). After the reaction of 30 minutes at 30 - 35 ° C with the salt (ferrous sulfate) and preferably with ascorbic acid in the proportion of 1.6 times the amount of iron used, the samples were added with polymers in the active proportion: 20:80 (w / w).

[0076] The polymers used as wall were maltodextrin, polydextrose and modified starch mixed in the majority proportion of maltodextrin (approximately 80%). For drying, a laboratory scale spray dryer (BÜCHI, model B-290) was used with a hole for the passage of the sample in the 0.7 mm atomization. Operating conditions: inlet temperature of 140 ° C and outlet around 78 ° C; air pressure of approximately 50 mbar; aspiration of 100% of the power of the vacuum cleaner; air flow of 500 L / h (Step g, figure 1).

[0077] The capsules were evaluated for the percentage of recovery of the asset in the dry material, mass recovery and release of the asset for absorption. The particles had different sizes, with a diameter between 5.96 to 6.33 pm, the microencapsulation efficiency (recovery of the asset) was around 90% and process yield between 82 to 85%. After drying, microparticles were collected and iron accessibility tests were carried out in vitro, always using ferrous sulfate as a reference standard. Iron solubility and in vitro dialysability tests were performed on dry samples.

[0078] The latter uses dialysis membranes with pores ranging from 6 to 8 kDa, to simulate the conditions of gastrointestinal digestion, followed by passage through the intestinal mucosa. In this case the dialysis membrane simulates the intestinal membrane. The iron was always dosed with the ICP-OS equipment, which has very high sensitivity and precision. The chelation or complexation of peptides with iron increased the dialysability of iron by 2 times and the coating of the active with a wall (microcapsules) did not interfere in the process of making iron available. Being

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Thus, a dry product was obtained to be used as an ingredient for the formulation of a mineral supplement with high availability of iron with high solubility, in powder, to be incorporated in water-soluble beverages, foods and medicines, intended for the anemic population (Step h , figure 1).

[0079] The best hydrolytic conditions for the different enzymes were selected by a central rotational composite design (CCRD) 2 ² , with 11 assays, with 3 central points. The independent variables were pH and enzyme: substrate (I / O) ratio. The dependent variables were degree of hydrolysis and protein recovery. For whey proteins, the conditions selected for the enzymes Alcalase®, pancreatin and Flavourzyme® are shown in Table 8. The normality of the base used for pH adjustments was 1.0 mol / L to 0.25 mol / L and from 1.0 mol / L to 0.25 mol / L HCL. As the total hydrolyzate of the respective enzymes was ultrafiltered and only the low molecular weight fraction (<5kDa) was collected, it was not necessary to inactivate the enzyme by elevated temperature or lowering the pH, as the enzyme remains in the high molecular weight fraction.

Table 8. Optimum conditions for hydrolysis of whey proteins, for the different enzymes, obtained by rotational central compound design (CCRD).

Enzyme

S / E Ratio

Alcalase® 8.0 100: 1 60 Pancreatin 7.2 100: 3 40 Flavourzyme® 6.7 100: 2.6 55

Temperature (° C) [0080] The fractionated hydrolysates (<5kDa) with the different enzymes, presented the amino acid composition, given below:

Table 9. Amino acidic profile of fractions <5kDa obtained in ultrafiltration for the Whey Protein Hydrolyzate with different enzymes (Alcalase®, pancreatin, Flavorzyme®).

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25/32

Amino acid (g / 100g of protein) Alcalase® Pancreatin Flavourzyme® Hydrolyzate <5 kDa Threonine 6.07 5.73 7.52 Methionine 2.44 2.22 2.36 Cystine 1.46 0.75 1.83 Valina 4.72 4.62 5.26 Leucine 10.36 10.88 8.38 Isoleucine 6.67 7.97 8.38 <0 Phenylalanine 2.60 2.76 1.93 .ss ü Lysine 8.41 9.36 6.77 ç Φ Histidine 1.25 1.34 0.97 <0 LU Tryptophan 3.15 1.94 3.33 Tyrosine 2.49 4.24 2.90 B.C. aspartic 8.41 8.89 7.20 Serina 4.01 4.02 3.76 ω dog B.C. glutamic 24.57 21.08 25.78 O ç Proline 4.56 5.18 4.83 Φ <0 Glycine 1.36 1.34 1.18 Φ Q Alanine 5.80 5.45 4.73 ! «J z Arginine 1.68 2.22 2.90 * <5 = molecular weight < 5KDa. Table 10. Amino acid content free from protein hydrolysates of

whey proteins with different enzymes (Alcalase®, pancreatin, Flavorzyme®).

Amino acid (g / 100g sample) Peptide fraction <5kDa Alcalase Pancreatin Flavorourme B.C. Aspartic 0.807 0.012 0.371 B.C. Glutamic 0.229 0.021 0.322 Serina 0.022 0.085 0.566 Glycine 1.820 1,493 1,839 Histidine 0.043 0.067 0.232 Arginine 0.157 1,242 0.636 Threonine 0.187 0.194 1,434 Alanine 0.208 0.071 0.891 Proline 0.259 0.030 0.077 Tyrosine 2,797 1,122 0.341 Valina 0.599 0.737 1.814 Methionine 1,286 0.761 0.690 Cystine 1,553 0.947 0.509 Isoleucine 1.803 1,151 2,154 Leucine 1,635 2,045 2,232

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26/32

Phenylalanine 1,572 1,224 0.688 Lysine 0.323 4,234 1.507

Example II - Characterization of peptides <5kDa [0081] The microparticles were obtained from peptides smaller than 5kDa related to iron and added with ascorbic acid and wall material for encapsulation. The peptides of the hydrolysates showed the size measured by scattering the light.

[0082] The measurements of the hydrodynamic diameter of the samples show that the peptides in the hydrolyzates of IPS proteins, followed an order of relative size following: hydrolyzate IPS / pancreatin <hydrolyzate IPS / Flavourzyme® <hydrolyzate IPS / Alcalase®.

[0083] The hydrolysates had a light scattering distribution, where the greatest intensity of distribution by quantity (volume%) of sample is concentrated in a range of close size. Table 5 shows that the hydrodynamic diameter (nm) was closer among the hydrolysates of the same origin, independent of the enzyme, whereas the whey protein hydrolysates (HPS) were between 2.0 to 2.30 nm.

Table 5. Hydrodynamic diameter (nm) referring to the size of the particles obtained from the different hydrolysates <5KDa

Record Sample Name * T (° C) Size (d.nm) ** Red HPSL / Flavourzime <5 kDa 25 2.12 Green HSPL / Alcalase® <5 kDa 25 2.27 Blue HPSL / Pancreatin <5 kDa 25 2.04

* HPSL = protein hydrolyzate of whey proteins with the respective enzymes; ** nanometer diameter [0084] The peptides were used in the synthesis of chelates and or complexes by association with iron under controlled conditions and later served as spray drying assets.

[0085] The size of the chelated and / or complexed particles may be larger due to the process of binding to the metal ion inside the peptides.

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27/32 [0086] The degree of solubility of iron in the peptide medium is an indicator of the iron binding capacity, as they were able to keep iron soluble in pH conditions unfavorable to the solubility of the iron salt (ferrous sulfate). The results obtained for fractions less than 5 kDa of the different hydrolysates are shown in Table 6.

Table 6. Values of% solubility of iron in whey protein hydrolyzate solutions with different enzymes, less than 5 KDa

Hydrolyzed Sample * % Soluble Iron Content ± SD (as an index of binding capacity) HPSL / Alcalase® 96.2 ± 0.3 HPSL / Pancreatin 97.6 ± 0.4 HPSLFlavourzyme® 99.7 ± 1.1

* HPSL = protein hydrolyzate of whey proteins with the respective enzymes [0087] The results of the studies of iron dialysability, using fractions less than 5KDa chelated, for the different enzymes are shown in Table 7.

Table 7. Iron dialysability (%) of the <5KDa fraction of the whey protein hydrolysates with different enzymes added with ferrous sulfate in 0.2mM Fe content.

Peptide fractions> 5kDa chelated to Fe % dialysability Fe ± DP Alcalase® 42.4 ± 4.7 Pancreatin 38.6 ± 4.2 Flavorzyme® 40.2 ± 0.9 0.075 M PIPES (FeSO ₄ control) 24.1 ± 2.4

[0088] In this experiment the positive control was PIPES plus ferrous sulfate, where the pH conditions of the medium are maintained, simulating the conditions of the beginning of the small intestine. In general, the dialysability values of the HPSL peptides showed higher control values and similar to each other, being those resulting from the hydrolysis with Alcalase® of greater value (42.4%). Ferrous sulfate was selected as a control because it is the most soluble compound and used in therapies for people with iron deficiency.

[0089] The correlation of the dialysability of iron in different foods was

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28/32 elevated when compared to the absorption values in humans in a study by Argyri et al. (2011). Eighteen types of food of different matrices, textures and complexity were used in studies where the results of this technique were compared with other studies. The authors concluded that this method is a convenient option for screening a large number of samples to check the bioavailability of iron, before following studies with cell cultures or with humans. Microparticles: Spray drying process conditions [0090] The operating conditions of the equipment were kept constant for all samples such as: A) inlet temperature of 150 ° C, B) flow rate adequate to generate an outlet temperature around 80 ° C, C) 100% suction approximately and D) 50 mbar air pressure.

[0091] The final samples were collected and stored in plastic bottles, for further characterization. The relative humidity of the processing area was maintained to values close to 40% with the use of a dehumidifier and the ambient temperature will be maintained at 23 ± 3 ° C with the use of air conditioning. Table 11 shows the results obtained for the different polymers.

Table 11. Yield and efficiency of microencapsulation of different wall materials used for drying the asset.

Formulations Active encapsulating material Yield (%) Efficiency microencapsulation * (%) 1 Maltodextrin 91.0 81.69 2 Maltodextrin: Hicap 89.5 84.61 3 Maltodextrin: Polydextrose 90.45 85.52 4 Maltodextrin: Hicap 88.0 80.51 5 Maltodextrin: Wallocel 95.0 77.94 6 Hicap 97.0 80.17 7 Pectin: Hicap 48.0 74.39 8 Gum arabic: Methocel 83.0 82.53 9 Gum arabic: Chitosan 89.0 86.58 10 Hicap: Methocel 78.0 81.23 11 Hicap: Chitosan 81.0 78.92

* Expresses the percentage of asset retention.

Selection of formulations with different encapsulating materials

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29/32 [0092] The encapsulation efficiency that comprises the amount of iron retained in the particle in relation to the iron content before drying (in solution). Equation (a) is used to calculate% EE.

Asset determined in the particles after processing x 100% EE = ------- (a)

Asset present in solution before processing [0093] The efficiency of the encapsulation process, shown by the percentage of asset retention (Fe-peptide complex) in polymeric matrices, was 81.68% for the sample composed of 100% maltodextrin of 85 , 51% for the maltodextrin and Hicap® sample and 84.60% for the maltodextrin and polydextrose sample.

[0094] The wall polymers were selected according to the yield, percentage of asset retention, cost and accessibility of iron through in vitro dialysability tests and also due to the pro-biotic claim of the polymers. Thus, maltodextrin (MA samples), maltodextrin combined with Hicap® (MH sample) and maltodextrin with polydextrose (MP sample) were defined as wall material.

Microparticle solubility [0095] Solubility was assessed by the hydration time. Some gums of larger chain size impair the bioaccessibility of iron after in vitro digestion. Samples that had satisfactory results for dialysability were observed in relation to the hydration time, timed during optical microscopy. The microparticles showed instant hydration, with no differences between the samples analyzed, probably due to the fact that maltodextrin is the major component of the wall.

Characterization of the microparticles of the selected formulations [0096] Figure 4 shows the size distribution of the microparticles

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30/32 with different combinations of wall material The size distributions of the dry microparticles were determined by laser diffraction on a LV950-V2 Particle Size Analyzer (Horiba, Kyoto, Japan). For this analysis, a small amount of the powdered product was dispersed in absolute ethanol, added to the equipment's sample chamber, containing the same dispersion medium, until reaching the appropriate transmittance rates for reading (n = 6).

[0097] The average particle size was expressed with the average diameter (D50) and the polydispersity was obtained by the calculated span index, by the equation below. The microparticles had different sizes, with a diameter between 5.96 to 6.33 pm, approximately.

(D90-D10)

Span = (b)

D50 [0098] The polydispersity measures of the microparticles were obtained by the “span index” with the following results: sample (1) with 100% maltodextrin was 1.22 (the microparticles containing only malto showed a lower span index), sample (3) 80% maltodextrin and 20% polydextrose was similar to 1.37 and for sample (2) with 80% maltodextrin and 20% hicap it was 1.47%.

Microparticle: Evaluation of iron dialysability [0099] Iron dialysability tests were carried out to evaluate whether the wall material selected in the active coating would influence the availability of Fe and the results are shown in Table 12. The encapsulation treatments that showed do not interfere with iron absorption were selected for the work sequence.

[0100] Although the result of these analyzes was shown to be inferior to that obtained previously, according to the test conditions, they demonstrate that there was no difference in the bioaccessibility of iron in the presence of encapsulating agents. There was an increase in the dialysability of iron and probably the addition of ascorbic acid in the formulation contributed to the better performance in the passage of iron through the membrane (bioaccessibility)

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31/32 of the iron.

Table 12. Result of the dialysability of microparticle iron using different wall materials.

Samples Dialysability (% dialyzed iron) Maltodextrin 100% 25.06 (1.14) ^A Matodextrin (80%) + Hi cap (20%) 27.71 (1.91) ^A Maltodextrin (80%) + Polydextrose (20%) 24.35 (0.55) ^A Peptide + Fe solution (control +) A, B W 1 ..- i—, x ..- .. 10.38 (0.60) ^B

A b ^{A, B} Mean values with different letters are significantly different (p <0.05), according to the Tuckey test (n = 3).

[0101] Bioaccessibility was analyzed using the dialysability technique in different microparticles to assess the possible interactions of iron with the wall material, as well as the other ingredients. These tests were carried out to verify the possible risks of the interaction of iron with the other ingredients in the absorption. The presence of ascorbic acid in the microparticles containing the active substance, probably contributed to increase the passage of iron through the dialysis membrane.

[0102] The monitoring of the microparticles during the shelf life for 360 days showed that the microparticles did not show significant morphological changes and maintained their structural integrity, as seen in Figure 5.

In vitro bioaccessibility tests for iron present in microparticles.

[0103] Bioaccessibility is defined as the fraction of a nutrient that, when released from its food matrix to the gastrointestinal region, becomes available to be absorbed, involving the pre-systemic metabolism system (intestinal and hepatic).

[0104] The methodologies used in these studies vary according to the objective, and can be divided into in vitro studies and in vivo studies. In vitro studies are generally used to investigate the effects and interaction

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32/32 of nutrients with issues that involve both the composition of the food matrix and processing.

[0105] In this study, studies on iron solubility and dialysability were carried out using in vitro methodologies (equations b and c). These methodologies are used to predict the behavior of iron in the presence of peptides. Solubility refers to how much of the iron remains soluble, in the presence and absence of the peptides, when submitted to digestion conditions (gastric and intestinal) in vitro, after the centrifugation of the digested product. The dialysability goes further, as it uses a 6 to 8 kDa porosity membrane and quantifies only the iron that managed to pass through the membrane pores, simulating the cell membrane barrier of intestinal enterocytes. According to the equations below:

Iron in the sample after centrifugation of the digested x 100% Soluble iron = --- _:: - = -. . . , Iron from the initial solution (c) [0106] An evaluation of the best peptide-to-iron ratio was performed, aiming at better absorption of this micronutrient. The results of the study of solubility and dialysability are shown in Figure 6 and 7, respectively. The calculation of dialyzed iron was calculated according to the equation below:

% Dialyzed iron =

Iron that passed through the membrane (mg / mL) x100 (d)

Initial iron (mg / mL) [0107] Those skilled in the art will value the knowledge presented here and will be able to reproduce the invention in the modalities presented and in other variants, covered by the scope of the attached claims.

Claims (10)

1. Process of obtaining a microparticle containing Fe-Peptide of high bioavailability characterized by comprising the steps of: a) obtaining a liquid protein hydrolyzate, from the hydrolysis of whey proteins; b) filtering the liquid protein hydrolyzate obtained in (a), to select peptides smaller than 5 kDa; c) adjustment of the pH of the product obtained in (b) to a pH value between 5.0 to 5.5; d) addition of iron salt in the product obtained at the end of step (c), in which the proportion of peptide and iron salt varies between 10: 1 to 40: 1 (w / w), where the iron salt is Iron Sulfate; e) maintenance of the product obtained in (d) at a temperature of 3035 ° C at pH 5.5 for a period of at least 20 minutes for the synthesis of the Fe-peptides complex to occur; g) addition of polymers as wall agents; h) spray drying to obtain microparticles containing Fe-peptides and ascorbic acid as active; where the final product of the process is a particle with a diameter between 5.96 to 6.33 microns. 2. Process according to claim 2, characterized by obtaining a liquid protein hydrolyzate in step (a) consisting of the following steps: i) dilution of the substrate in water, in the proportion of 8% to 12% of substrate; ii) homogenization of the sample at room temperature (22 to 25 ° C); iii) adjustment of pH and temperature according to the enzyme specification for hydrolysis of the substrate to be used; iv) adding a protease for hydrolysis of the substrate; v) maintenance of the substrate and enzyme for a period of 120 to 340 Petition 870180148219, of 10/22/2018, p. 80 74 2/4 minutes for the hydrolysis reaction to occur; vi) inactivation of the enzymes by cooling in an ice bath and kept cool until the ultrafiltration process. 3. Process according to claim 2, characterized in that the enzyme used in step (iv) is subtilisin or pancreatin or the fungal proteases of Aspergillus oryzae, in which said subtilisin must be maintained at pH 8.0 and at a temperature 60 ° C, where the pH is adjusted by adding NaOH (0.25 or 1.0 mol / L), where the hydrolysis time is preferably 120 minutes and the Enzyme / Substrate ratio is 1: 100; where pancreatin should be maintained at pH 7.2 and at a temperature of 40 ° C, where the pH is adjusted by adding NaOH (0.25 or 1.0 mol / L), where the hydrolysis is preferably 180 minutes and the Enzyme / Substrate ratio is 4: 100; in which the fungal proteases of Aspergillus oryzae must be maintained at pH 6.7 and at a temperature of 55 ° C, where the pH is adjusted by adding NaOH (0.25 or 1.0 mol / L), in that the hydrolysis time is preferably 180 minutes and the Enzyme / Substrate ratio is 4: 100. 4. Process according to claim 1, characterized by step (b) involving filtration by an ultrafiltration process in tangential flow membranes with a 5 kDa cut, in which the particles obtained are less than 2.5 kDa. 5. Process according to claim 1, characterized in that the wall agent used in step f) is selected from maltodextrin, polydextrose, modified starch, hicap, wallocel, pectin, gum arabic, chitosan, methocel or a mixture thereof, and wherein the proportion of Fe-Peptides and the wall agent is between 20:80 to 25:75 (w / w), preferably 20:80 (w / w). 6. Process, according to claim 1, characterized by step h) of drying is carried out with the use of a spray dryer equipment in the Petition 870180143289, of 11/28/2018, p. 50/74 3/4 following operating conditions inlet temperature 140 ° C and outlet temperature 78 ° C; air pressure of approximately 50 mbar; aspiration of 100% of the power of the vacuum cleaner; air flow of 500 L / h. 7. Microparticle, produced as defined in any one of claims 1 to 6, characterized by comprising: I - a Fe-peptide composition comprising: - peptides smaller than 5kDa; - iron salt, where the iron salt is ferrous sulfate; - Ascorbic acid; where peptides smaller than 5kDa and the iron salt are complexed or chelated to form an active Fe-peptide component with a ratio between 20:80 to 25:75 (w / w), preferably 20:80 (w / w), in which the proportion of peptides smaller than 5kDa and the iron salt varies between 10: 1 to 40: 1 (w / w), in which the amount of ascorbic acid is 1.6 times the amount of iron used; II- a capsule comprising the composition (I) comprising at least one wall agent, wherein said wall agent is selected from the group consisting of maltodextrin, hicap, wallocel, pectin, gum arabic, polydextrose, chitosan, methocel and combination of themselves. Microparticle according to claim 7, characterized in that it has a size ranging from 5.96 to 6.33 microns and; - because the wall agent used is selected from maltodextrin, polydextrose, modified starch, or a mixture of them, where in a mixture, the proportion of maltodextrin is 80%. Use of the microparticle, as defined in any one of claims 7 to 8, characterized in that it is for the preparation of beverages with high bioavailability of iron and for the production of food supplements with high bioavailability of iron. 10. Use of the microparticle, as defined in any of the Petition 870180153219, of 10/28/2018, p. 51/58 4/4 claims 7 to 8, characterized in that it is for preparing a supplement of high bioavailability of iron to treat iron deficiency anemia.