Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1682—Processes
  • A61K9/1694—Processes resulting in granules or microspheres of the matrix type containing more than 5% of excipient
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
  • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
  • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/111—Aromatic compounds
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/142—Amino acids; Derivatives thereof
  • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/163—Sugars; Polysaccharides
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/20—Inorganic substances, e.g. oligoelements
  • A23K20/22—Compounds of alkali metals
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/20—Inorganic substances, e.g. oligoelements
  • A23K20/26—Compounds containing phosphorus
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K40/00—Shaping or working-up of animal feeding-stuffs
  • A23K40/10—Shaping or working-up of animal feeding-stuffs by agglomeration; by granulation, e.g. making powders
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/745—Bifidobacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1605—Excipients; Inactive ingredients
  • A61K9/1617—Organic compounds, e.g. phospholipids, fats
  • A61K9/1623—Sugars or sugar alcohols, e.g. lactose; Derivatives thereof; Homeopathic globules
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1605—Excipients; Inactive ingredients
  • A61K9/1629—Organic macromolecular compounds
  • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1605—Excipients; Inactive ingredients
  • A61K9/1629—Organic macromolecular compounds
  • A61K9/1658—Proteins, e.g. albumin, gelatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1682—Processes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2200/00—Function of food ingredients
  • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
  • A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system

Definitions

• the present invention is part of the field of food, nutraceutical, cosmeceutical and pharmaceutical technology.
• it refers to microparticles comprising a matrix of casein and chitosan and probiotic bacteria, a method for obtaining them and their applications.
• the intestinal microbiota of a healthy adult is relatively stable and contains several beneficial bacterial populations, consisting mainly of species of lactobacilli and bifidobacteria, which play an important role in host health.
• the imbalance of the beneficial colonic microbiota can contribute to the development of different disorders, such as infections of the gastrointestinal tract, constipation, irritable bowel syndrome, inflammatory bowel disease, allergies, heart disease and colon cancer.
• WHO World Health Organization
• Probiotics are defined as living microorganisms that, administered in sufficient quantities, provide beneficial physiological effects on the host (Pérez-Luyo, 2008). In this sense, they are attributed: aid in the digestion of lactose, prevention of intestinal infections, immunomodulatory action, prevention of cancer and cardiovascular diseases. Additionally, the possible role of probiotics in the prevention of dental caries is being investigated.
• probiotic consumption There are four basic forms of probiotic consumption: as a concentrated culture added to any beverage (eg, fruit juice, etc.), inoculated into prebiotic fibers, as a dietary supplement in pharmaceutical forms of lyophilized cells (eg, powder, capsules , tablets, etc.) and inoculated in dairy foods.
• Probiotic bacteria have been incorporated into a wide range of foods, primarily in dairy products (yogurt, cheese, ice cream, dairy desserts, etc.), but also in other foods such as cereals, juices, chocolate, etc.
• probiotics in these products are very low (de Vos et al., 2010), and for these microorganisms to produce the aforementioned beneficial effects they must remain viable and in the concentration adequate at the time of consumption.
• factors responsible for reducing the viability of probiotic cultures such as, for example, acidity at the end of food processing / manufacturing, acidity produced throughout its shelf life or post-acidification, inhibition by fermentation metabolites , lack of nutrients, permeability of the container, osmotic pressure, storage temperature, interaction with other microbial species, etc.
• the more acidic the product is throughout its useful life the lower the viability of probiotic bacteria such as Bifidobacteria and L acidophilus.
• probiotics include the selection of acid-resistant strains, the increase in the initial concentration of probiotic microorganisms, or the addition of a suitable prebiotic that maintains an active metabolism in their lives, a low level of post -acidification, and avoid the formation of unwanted fermentation metabolites.
• probiotic concentrates are often stored for long periods of time before use and after their incorporation into food and / or nutraceutical products, it is very important to find a system that allows to maintain bacterial viability throughout of all this time and thus extend the life of the product, if possible, without using specific temperature and humidity conditions to avoid additional economic costs.
• probiotics are used in order for them to produce the aforementioned beneficial effects on human health, they must reach the colon in a viable way, so they need to overcome the upper gastrointestinal tract barrier, that is, acidity and digestive enzymes of the stomach and bile salts of the small intestine.
• probiotic bacteria are exposed to various stress factors during their industrial production such as freezing, drying, oxygen exposure, temperatures, large concentrations of lactic acid in the culture medium, etc.
• microcapsules most suitable for encapsulation of probiotics should meet the following requirements (Heidebach et al., 2011):
• casein a conjugated protein that constitutes approximately 80% of the total milk protein. Studies have been developed using this protein, alone or in combination with other polymers, including polysaccharides, to encapsulate probiotic bacteria (Heidebach et al., 2009, Heidebach et al., 2010, Oliveira et al., 2007), obtaining good results of encapsulation efficacy without compromising bacterial viability. Of the three works mentioned, the first two are based on a poorly scalable emulsification system, which results in microcapsules of excessively large sizes (greater than 100 ⁇ ).
• Lactobacillus plantarum is one of the most commonly used lactic acid bacteria; This bacterium is considered a GRAS organism ("Generally Recognized as Safe") capable of healthy colonizing the human gastrointestinal tract. Many strains of L plantarum are currently marketed as probiotics. However, it is a bacterium very sensitive to the conditions of the gastrointestinal environment, which can be kept for a very short time in storage, even in refrigeration, since in a few days there is a very significant reduction in its counts (Ayub and Brinques , 2011). Recently two works have been published that deal with the encapsulation of these bacteria to protect them both during storage and during their passage through the gastrointestinal barrier.
• Lactobacillus casei Another of the most commonly used lactic acid bacteria is Lactobacillus casei, whose potential beneficial to health has been widely reported. It can be found in various products distributed worldwide, including traditional fermented milks such as yakult, kefier, actimel, gefilus and vifit, and in cheeses such as Parmesan and Manchego among others.
• this bacterium has the same limitations as the previous one (L plantarum), that is, it is sensitive to the conditions of the upper gastrointestinal tract and its stability during storage periods is very limited.
• said systems will be microparticles containing the probiotic bacteria, of uniform size and that do not interfere with the organoleptic characteristics of the product in which they are eventually incorporated, and capable of protecting the probiotic bacteria both during their processing and storage under controlled conditions or environmental as during its transit through the gastrointestinal tract.
• microparticles that solve the aforementioned problems, that is, microparticles that have the ability to encapsulate probiotic bacteria for incorporation into food and nutraceutical, cosmeceutical and pharmaceutical products.
• These microparticles protect the probiotic bacteria from inactivation by external agents, both during the processing of the food or nutraceutical, cosmeceutical or pharmaceutical product in which they are incorporated, and during their storage over prolonged periods under environmental or controlled conditions, increasing The shelf life of these foods or nutraceuticals.
• they facilitate the release of probiotic bacteria in the desired place, protecting them from the "peptic acid" conditions of the upper gastrointestinal tract, particularly the stomach.
• microparticles are stable and inert in the food or in the nutraceutical, cosmeceutical or pharmaceutical formulation in which they are incorporated, preventing the food, nutraceutical, cosmeceutical or pharmaceutical matrix from compromising the viability of the bacteria.
• the inventors have developed a simple method of obtaining these microparticles that is applicable on an industrial scale. This method does not include the use of surfactants or emulsifiers, synthetic polymers, or any reagent that is not approved as a food additive. In addition, this method allows controlling that the size of the microparticles obtained is less than 100 ⁇ to prevent it from being perceived by the consumer or that negatively affects the organoleptic characteristics of the product in which they are incorporated.
• microparticles can be resuspended, not dissolved, easily in aqueous medium, protecting the probiotic bacteria they contain from the medium.
• the microparticles of the invention remain stable in the product in which they are incorporated, thereby avoiding a significant decrease in the count of viable bacteria after long periods of storage under environmental and / or controlled conditions.
• these microparticles are applicable to different types of foods, from beverages and dairy products to solid foods, and in nutraceutical products.
• said microparticles can be formulated in cosmeceutical and pharmaceutical formulations.
• microparticles have a potent immunomodulatory effect and favor the induction of a T helper 1 (Th1) response and / or shift the immune response towards Th1, so they can be used in the preparation of a modulating composition of the immune system (immunomodulatory) for the prevention and / or treatment of an alteration of the immune system, for example, in the prevention and / or treatment of rejection of a Th2-mediated transplant, allergies and diseases associated with allergies, immunodeficiencies and derived pathologies of said immunodeficiencies, infections caused by intracellular pathogens and / or mucosal infections.
• Th1 T helper 1
• the microparticles of the invention provide a new system for encapsulation and stabilization of probiotic microorganisms.
• casein in combination with chitosan, is used as a vehicle to protect probiotic bacteria from environmental conditions during long storage periods and from gastric conditions, thereby increasing their shelf life and facilitating their release in the intestine, thus improving its probiotic effect.
• casein per se has proven nutritional properties that complement the beneficial effects of encapsulated probiotic bacteria itself.
• the microparticles of the invention have the ability to protect probiotic bacteria during (i) processing, (ii) storage and (iii) transit through the gastrointestinal tract.
• the invention relates to microparticles comprising a matrix and a probiotic bacterium, wherein said matrix is constituted by casein and chitosan.
• the invention relates to a method of obtaining the microparticles provided by this invention, which comprises mixing a casein or a source of casein, probiotic bacteria and chitosan.
• the invention in another aspect, relates to a composition consisting of a plurality of microparticles provided by this invention, or comprising at least one microparticle provided by this invention and a vehicle acceptable in food, nutraceutical, cosmeceutical or pharmacy.
• the invention relates to a food, nutraceutical, cosmeceutical or pharmaceutical product comprising the microparticles provided by this invention.
• the invention relates to the use of said microparticle, composition or product provided by this invention in the preparation of a modulating composition of the immune system, or, expressed in another way, the invention relates to said microparticle, composition or product provided by this invention for use in a modulating composition of the immune system.
• Said immunomodulatory composition favors the induction of a Th1 response and / or shifts the immune response towards Th1, preferably Th2 towards Th1, and can be used for the prevention and / or treatment of immune system disorders, for example, in the prevention and / or treatment of (i) rejection of a Th2-mediated transplant, (ii) allergies and diseases associated with allergies, (iii) immunodeficiencies and pathologies derived from said immunodeficiencies, (iv) infections caused by intracellular pathogens and / or infections of mucous
• the invention also relates to a microparticle, composition or product provided by this invention for use in the prevention and / or treatment of an oral immune system alteration; as well as with a microparticle, composition or product provided by this invention for use in the prevention and / or oral treatment of rejection of a Th2-mediated transplant; allergies and diseases associated with allergy; immunodeficiencies and pathologies derived from said immunodeficiencies; infections caused by intracellular pathogens, or mucosal infections.
• Figure 1 Optical microscopy images of chitosan modified casein microparticles obtained by spray-drying: A) empty (x 20); B) with L encapsulated plantarum (x 20). The horizontal line in the lower right represents 100 ⁇ .
• Figure 2 Fluorescence optical microscopy images of: A) L plantarum stained with fluorescent marker (x 20); B) Chitosan modified casein microparticles obtained by spray-drying with L plantarum (stained with fluorescent marker) encapsulated (x 20).
• the horizontal line in the lower right represents 100 ⁇ .
• Figure 3 Fluorescence optical microscopy image of casein microparticles modified with chitosan and in the presence of calcium salts obtained by spray-drying with L plantarum (stained with fluorescent marker) encapsulated (x 20).
• the horizontal line in the lower right represents 100 ⁇ .
• Figure 4 Graph showing the survival of L plantarum in environmental conditions (25 ° C) over time: fresh suspension of L plantarum; L. lyophilized plantarum without encapsulation; L. plantarum encapsulated in casein microparticles modified with chitosan (Ap formulation); L. plantarum encapsulated in casein microparticles modified with chitosan in the presence of vanillin (Bp formulation); L plantarum encapsulated in casein microparticles modified with chitosan in the presence of TPP (Cp formulation); and L plantarum encapsulated in casein microparticles modified with chitosan in the presence of calcium salts (Dp formulation).
• Figure 5 Graph showing the survival of L. plantarum in simulated gastrointestinal medium (0 to 2 hours: simulated gastric medium; 2.1 to 8 hours: simulated intestinal medium): fresh suspension of L plantarum; L lyophilized plantarum without encapsulation; L. plantarum encapsulated in casein microparticles modified with chitosan (Ap formulation); L plantarum encapsulated in casein microparticles modified with chitosan in the presence of vanillin (Bp formulation); L plantarum encapsulated in casein microparticles modified with chitosan in the presence of TPP (Cp formulation); and L. plantarum encapsulated in casein microparticles modified with chitosan in the presence of calcium salts (Dp formulation). * Indicates significant differences in the microparticle counts with respect to the lyophil (P ⁇ 0.05).
• Figure 6 Scanning electron microscopy images showing casein microparticles modified with chitosan in the presence of vanillin (Bp formulation), in the process of degradation, with encapsulated L. plantarum, after being subjected to treatment in simulated gastrointestinal medium.
• Figure 7 Graph showing the survival of L. casei in environmental conditions (25 ° C) over time: fresh suspension of L casei; L. casei lyophilized without encapsulation; L. casei encapsulated in casein microparticles modified with chitosan (Ac formulation); L. casei encapsulated in casein microparticles modified with chitosan in the presence of vanillin (Ce formulation); and L. casei encapsulated in casein microparticles modified with chitosan in the presence of tripolyphosphate (TPP) (formulation De).
• Ac formulation L. casei encapsulated in casein microparticles modified with chitosan
• Ce formulation vanillin
• TPP tripolyphosphate
• Figure 8 Graph showing the survival of L. casei in simulated gastrointestinal medium (0 to 2 hours: simulated gastric medium; 2, 1 to 8 hours: simulated intestinal medium): Lyophilized L casei without encapsulation; L casei encapsulated in casein microparticles modified with chitosan (Ac formulation); L. casei encapsulated in casein microparticles modified with chitosan in the presence of vanillin (Ce formulation); and L casei encapsulated in casein microparticles modified with chitosan in the presence of tripol phosphate (TPP) (formulation De).
• Ac formulation L casei encapsulated in casein microparticles modified with chitosan
• Ce formulation vanillin
• TPP tripol phosphate
• * Indicates significant differences in the microparticle counts with respect to the lyophil (P ⁇ 0.05).
• Figure 9 Bar chart showing the results of the immunophenotypic analysis of peripheral lymphocytes obtained from mice treated with L. plantarum in its non-encapsulated form (free LP), L encapsulated plantarum (Bp) or with the physical mixture of bacteria (L .plantarum) and particles (Physical Mix). The dotted line shows the ratio obtained in the untreated control group.
• Figure 10 Bar chart showing the results of the Th1 / Th2 ratio index after stimulating in vitro peripheral lymphocytes obtained from mice treated with L plantarum in its non-encapsulated form (free LP), L. plantarum encapsulated (Bp) or with the physical mixture of bacteria and particles (Physical Mix).
• the present invention relates to the manufacture of microparticles for the encapsulation of probiotic bacteria, with the aim of preventing their inactivation after incorporation into food, nutraceutical, pharmaceutical or cosmeceutical matrices or, protecting them during their processing and storage over prolonged periods. storage under controlled or environmental conditions and, in addition, protect them from "peptic acid” conditions during transit through the gastrointestinal tract once ingested.
• microparticles of the invention comprising a matrix and a probiotic bacterium, wherein said matrix is constituted by casein and chitosan.
• microparticles is used to designate colloidal systems of type spheres or similar shapes smaller than 1 mm (mm), generally of the order of 0.5 to 999 micrometers ( ⁇ ), typically of the order of 1 to 900 ⁇ .
• the microparticles of the invention have a size of less than 1 mm, generally comprised between 0.1 and 999 ⁇ , typically between 0.2 and 900 ⁇ , advantageously between 0.3 and 500 ⁇ , preferably between 0, 4 and 250 ⁇ , more preferably between 0.5 and 125 ⁇ , even more preferably between 0.7 and 50 ⁇ , still more preferably between 1 and 40 ⁇ , still more preferably between approximately 2 and 12 ⁇ .
• matrix in the scope of the present invention refers to the coating composition or agent (s).
• said matrix is constituted by casein and chitosan and covers, totally or partially, probiotic bacteria.
• probiotic is defined as a living microorganism that, when administered in adequate amounts, exerts a beneficial physiological action on the health of the host (FAO / WHO 2002. Guidelines for the evaluation of probiotics in food, London).
• the probiotics used in the present invention are "probiotic bacteria,” that is, live bacteria that, when administered in adequate amounts, exert a beneficial physiological action on the health of the host.
• said probiotic bacterium is a bacterium of the genus Bifidobacterium or Lactobacillus.
• said probiotic bacteria is selected from L plantarum and L. casei.
• the probiotic bacteria are L plantarum CECT 220 and L casei CECT 475 T isolated from corn and cheese forage, respectively.
• said probiotic bacterium is a strain of Bifidobacterium animalis subsp. lactis, such as that sold under the BB-12® brand.
• casein refers to a conjugate protein that constitutes approximately 80% of the total milk protein. It is a phosphoprotein-like protein that falls within the definition of globulins; it is soluble; It has a high water retention capacity and precipitates at an approximate pH of 4.6 at 20 ° C. It consists of four fundamental fractions (as1-casein, as2-casein, ⁇ -casein and ⁇ -casein) differentiated from each other by their composition in amino acids, their distribution of charges and their tendency to form aggregates in the presence of calcium. In milk, caseins are forming colloidal micelles, stable, between 50 and 600 nm in diameter (approximately 150 nm on average).
• Chitosan is a naturally occurring polymer derived from the N-deacetylation of chitin (poly-N-acetyl-D-glucosamine).
• the deacetylation process involves the removal of acetyl groups from the chitin molecular chain, leaving behind a complete amino group (-NH 2 ).
• the degree of deacetylation in a chitosan sample thus refers to the content of free amino groups in the subunits of the polysaccharide and can be determined, for example, according to the method described by Hidalgo et al.
• the degree of deacetylation of commercial chitosan is equal to or greater than 40%, preferably equal to or greater than 60%.
• the degree of chitosan deacetylation is between 60% and 100%, typically between 75% and 95%, or higher.
• the chitosan has an aminopolysaccharide structure based on the repetition of monomer units of formula (I):
• Chitosan at acidic pH is mostly protonated, so at acidic pH it is a positively charged polysaccharide.
• the molecular weight of chitosan can vary within a wide range; however, in a particular embodiment, the molecular weight of the chitosan used to obtain the microparticles of the invention is between 5 and 850 kDa, typically between 25 and 300, preferably between 40 and 200 kDa, more preferably between 50 and 150 kDa.
• the molecular weight of the chitosan used to obtain the microparticles of the invention is between 5 and 850 kDa, typically between 25 and 300, preferably between 40 and 200 kDa, more preferably between 50 and 150 kDa.
• a derivative thereof can be used, with chitosan being understood as such in which one or more hydroxyl groups and / or one or more amino groups have been modified.
• derivatives include, among others, acetylated, alkylated or sulphonated chitosans, as well as thiolated derivatives, as described in Roberts, Chitin Chemistry, Macmillan, 1992, 166.
• a derivative is selected from O-alkylenes of chitosan, chitosan O-acyl esters, trimethylchitosan, polyethylene glycol modified chitosans, etc.
• Other possible derivatives include salts, such as citrate, nitrate, lactate, phosphate, glutamate, etc.
• the chitosan derivative is a hydrophilically modified chitosan; as used herein a "hydrophilically modified chitosan” is a chitosan modified with a hydrophilic group, such as a group that increases the solubility of chitosan in an aqueous medium, for example, an alkylated chitosan (eg, trimethylchitosan, etc.), a sulfonated chitosan, a thiolated chitosan, a chitosan salt (eg, glutamate, chloride, lactate, acetate, etc.), a chito-oligosaccharide, etc.
• a hydrophilically modified chitosan is a chitosan modified with a hydrophilic group, such as a group that increases the solubility of chitosan in an aqueous medium, for example, an alkylated chitosan (eg, trimethylchitosan
• the chitosan derivative is not a hydrophobically modified chitosan; as used herein, a "hydrophobically modified chitosan"; it is a chitosan modified with a hydrophobic group, that is, with a group that reduces the solubility of the chitosan in an aqueous medium, for example, an alkyl or aryl group of sufficient size to confer an increased hydrophobicity to the chitosan, for example, residues of aldehydes or fatty acids, preferably fatty acids of 3 to 18 carbon atoms, saturated or unsaturated, such as, for example, palmitic, lauric, oleic, linoleic, linolenic, caproic, caprylic, stearic, propionic or butyric acid.
• the microparticle matrix of the invention when the microparticle matrix of the invention consists of casein and a chitosan derivative, such as a hydrophobically modified chitosan, the microparticle of the invention lacks an external coating of hydrophobically modified alginate or alginate (modified alginate with a hydrophobic group, as previously defined in relation to hydrophobically modified chitosan).
• Chitosan and casein constitute the matrix that is part of the microparticles of the invention.
• the chitosan: casein ratio, by weight may vary within a wide range; however, in a particular embodiment, said chitosan: casein ratio, by weight, is 1: 1-150, preferably 1: 5-100, more preferably about 1: 14-40.
• the amount of probiotic bacteria per unit weight of the matrix that may be present in the microparticles of the invention may vary within a wide range; however, in a particular embodiment, the microparticles of the invention comprise at least 10 6 colony forming units per milligram (CFU / mg) of matrix, generally between 10 6 CFU / mg and 5x10 13 CFU / mg, preferably, between 10 8 CFU / mg and 10 12 CFU / mg.
• CFU / mg colony forming units per milligram
• the microparticles of the invention further comprise a crosslinking agent.
• crosslinking agents include pharmaceutically or cosmetically acceptable divalent metal cations, or suitable for use in food, human or animal; tripolyphosphates; and, in general, any substance capable of establishing a chemical interaction with casein and / or chitosan.
• a "pharmaceutically or cosmetically acceptable divalent metal cation, or suitable for use in food, human or animal” is a cation from any metallic element whose valence is 2, such as an alkaline earth metal, for example, calcium, magnesium, zinc, etc., or, if it has several valences, one of them is 2, for example, iron, etc., with the condition that it is pharmaceutically or cosmetically acceptable, or suitable for use in feeding;
• said divalent metal cation is selected from the group consisting of Ca 2+ , Mg 2+ , Zn 2+ , Fe 2+ and combinations thereof.
• the divalent metal cation useful as a crosslinking agent can be provided by a source of said appropriate metal cation, such as a compound that in aqueous solution originates said divalent metal cation, for example, calcium chloride, calcium acetate. , calcium gluconate, calcium lactate, calcium sorbate, calcium ascorbate, calcium citrate, calcium propionate, calcium sulfate, etc., or mixtures of said compounds.
• a source of said appropriate metal cation such as a compound that in aqueous solution originates said divalent metal cation, for example, calcium chloride, calcium acetate. , calcium gluconate, calcium lactate, calcium sorbate, calcium ascorbate, calcium citrate, calcium propionate, calcium sulfate, etc., or mixtures of said compounds.
• tripol phosphate is a compound comprising pentanion polyphosphate, which is the conjugate base of triphosphoric acid, for example, tripol sodium phosphate, usually identified as STPP (from English “sodium tripolyphosphate ”) or simply as” TPP ".
• substances capable of establishing a chemical interaction with casein and / or chitosan which can be used as crosslinking agents within the present invention include vanillin [3-methoxy-4-hydroxybenzaldehyde], genipin [(1 f ?, 2f?, 6S) -2-hydroxy-9- (hydroxymethyl) -3-oxabicyclo [4.3.0] nona-4,8-diene-5- methyl carboxylate], etc.
• the crosslinking agent is a divalent metal cation selected from the group consisting of Ca 2+ , Mg 2+ , Zn 2+ , Fe 2+ and combinations thereof; a tripolyphosphate; vanillin; genipin; or any combination thereof.
• the crosslinking agent is calcium cation (Ca 2+ ), TPP or vanillin.
• the crosslinking agent is Ca 2+ and the crosslinking further comprises subjecting the mixture containing the casein and chitosan matrix, the probiotic bacteria and the crosslinking agent to a pressure treatment, such as a cycle of hydrostatic pressure, at a pressure between 100 and 800 MPa.
• the micro particles of the invention comprise two or more crosslinking agents, preferably two different crosslinking agents;
• Illustrative examples of said combinations of two different crosslinking agents possibly present in the microparticles of the invention include the binary combinations of:
• tripolyphosphate for example, TPP, and vanillin
• tripolyphosphate for example, TPP, and genipin
• tripolyphosphate for example, TPP
• a pharmaceutically or cosmetically acceptable divalent metal cation or suitable for use in food, human or animal, such as, for example, a divalent metal selected from the group consisting of Ca 2+ , Mg 2+ , Zn 2+ , Fe 2+ and combinations thereof;
• - vanillin and genipin - vanillin and genipin
• - vanillin and a pharmaceutically or cosmetically acceptable divalent metal cation or suitable for use in food, human or animal, such as, for example, a divalent metal selected from the group consisting of Ca 2+ , Mg 2+ , Zn 2+ , Faith 2+ and combinations thereof;
• a pharmaceutically or cosmetically acceptable divalent metal cation or suitable for use in food, human or animal, such as, for example, a divalent metal selected from the group consisting of Ca 2+ , Mg 2+ , Zn 2+ , Faith 2+ and combinations thereof.
• the weight ratio between the crosslinking agent and the casein and chitosan matrix may vary over a wide range, depending on the type of crosslinking agent.
• the crosslinking agent when the crosslinking agent is TPP, the crosslinking agent (TPP): matrix (casein and chitosan) ratio is 1: 0, 1-800, advantageously 1: 1-500, preferably about 1: 100-300 approximately.
• the ratio of crosslinking agent (vanillin): matrix (casein and chitosan) is 1: 0, 1-500, advantageously 1: 1-250, preferably about 1: 50-100 approximately.
• the crosslinking agent ratio (Ca 2+ or source of Ca 2+ : matrix (casein and chitosan) is 1: 0, 1-50, advantageously 1: 1 -25, preferably about 1: 6-16 approximately.
• the microparticles of the invention further comprise a compound that protects the matrix and probiotic bacteria during the process of drying them, or dewatering the suspension containing the microparticles of the invention, by conventional methods, for example, by spray drying ("spray-drying"), hereinafter, "protective agent”.
• a protective agent Virtually any compound that meets these characteristics can be used as a protective agent.
• said protective agent is a saccharide or, in general, a suitable food additive, which, in addition to the protective paper, acts as a prebiotic.
• prebiotic refers to a non-digestible food ingredient that stimulates the growth and / or activity of probiotics.
• the protective agent is mannitol or sucrose.
• the weight ratio between the casein matrix and chitosan and the protective agent may vary within a wide range; however, in a particular embodiment, the weight ratio between the matrix (casein and chitosan): protective agent is 1: 0.1-5, typically 1: 0.5-4, preferably about 1: 1.
• the invention relates to a method, hereinafter "method of the invention", for obtaining microparticles comprising a matrix and a probiotic bacterium, wherein said matrix is constituted by casein and chitosan (microparticles of the invention) , which comprises mixing casein or a source of casein, probiotic bacteria and chitosan.
• method of the invention for obtaining microparticles comprising a matrix and a probiotic bacterium, wherein said matrix is constituted by casein and chitosan (microparticles of the invention) , which comprises mixing casein or a source of casein, probiotic bacteria and chitosan.
• Casein can be incorporated as is or can be provided by a casein source.
• casein and “source of casein” are used interchangeably in this description.
• Virtually any source of casein can be used in the practice of the method of the invention.
• the origin of the casein source can be very diverse, for example, milk, beans, etc.
• the casein source is in the form of an aqueous solution or suspension; in this case, the casein may be in the form of caseinic acid or caseinate, for example, sodium caseinate, etc., or any other soluble form of casein.
• caseinates can be used, for example, calcium or phosphocalcium caseinate, in practice it is more advantageous to use sodium caseinate.
• the aqueous solution or suspension containing the casein source can be obtained by conventional methods known to those skilled in the art, for example, by adding the casein source to an aqueous medium.
• an "aqueous medium” is a medium comprising water, preferably, a medium containing mostly water, more preferably, the aqueous medium consists essentially of water.
• the amount of casein that can contain aqueous solution may vary within a wide range; however, in a particular embodiment, the amount of casein contained in said aqueous solution is comprised between 0.1% and 10% (w / v), preferably between 0.5% and 5%, more preferably between 1% and 2 %.
• said aqueous casein solution does not contain any organic solvent.
• a suspension of probiotic bacteria is advantageously prepared.
• said probiotic bacteria is a bacterium of the genus Bifidobacterium or Lactobacillus.
• said probiotic bacteria is L plantarum or L. casei.
• the probiotic bacteria are L plantarum CECT 220 and L casei CECT 475 T.
• said probiotic bacteria is a strain of Bifidobacterium animalis subsp. lactis, such as that sold under the BB-12® brand.
• the bacterial suspension comprises, in addition to the probiotic bacteria, a suitable medium for the corresponding probiotic bacteria.
• said medium comprises broth for Lactobacillus according to De Man, Rogosa and Sharpe, such as that identified as 110661 MRS Broth (Merck) [broth] MRS]; said medium allows a good growth of lactobacilli and other lactic acid bacteria and is usually used for the cultivation and enrichment of lactobacilli from clinical samples and foods, especially dairy.
• the MRS medium comprises (in g / L): polypeptone, 10 g; meat extract, 10 g; yeast extract, 5 g; glucose, 20 g; Tween® 80 (polyethoxylated sorbitan monoloelate or polysorbate 80), 1, 08 ml; potassium phosphate, 2 g; sodium acetate, 5 g; ammonium citrate, 2 g; magnesium sulfate, 0.2 g; manganese sulfate, 0.05 g.
• the pH of the medium at a temperature of 25 ° C is 6.4 ⁇ 0.2. This culture medium allows an abundant development of all lactobacillus species.
• Peptone and glucose are the source of nitrogen, carbon and other elements necessary for bacterial growth.
• Polyethoxylated sorbitan monoleate, magnesium, manganese and acetate provide cofactors and can inhibit the development of some microorganisms.
• Ammonium citrate acts as a growth inhibitor agent for Gram negative bacteria.
• the amount of probiotic bacteria that may be present in the bacterial suspension can vary within a wide range; however, in a particular embodiment, the amount of probiotic bacteria present in the bacterial suspension is at least 10 6 CFU / ml, generally between 10 6 and 5x10 12 CFU / ml, preferably between 10 8 and 10 12 CFU / ml.
• the bacterial suspension also contains a saccharide, such as sucrose or other suitable disaccharide such as, for example, maltose or trehalose;
• a saccharide such as sucrose or other suitable disaccharide such as, for example, maltose or trehalose
• these compounds play an important role during the drying process of the microparticles as they protect both the cell membrane and the proteins. Disaccharides form hydrogen bonds with proteins when dehydration occurs, which allows maintaining the structure of the protein and preventing denaturation. On the other hand, it seems that sugars could act as substitutes for water molecules during dehydration, surrounding the polar groups of both phospholipid bilayers and membranes, maintaining the structural integrity of the membrane and proteins.
• said bacterial suspension contains a disaccharide for the indicated purposes, for example, sucrose
• the amount of disaccharide (eg, sucrose) present in said bacterial suspension will be between 0.1% and 10% (w / v) of disaccharide (eg, sucrose), preferably between 1% and 3% (w / v).
• chitosan virtually any chitosan, or derivative thereof, may be used in the practice of the method of the invention; however, in a particular embodiment, said chitosan has a degree of deacetylation between 60 and 100%, preferably between 75% and 95%, and a molecular weight between 5 and 850 kDa, typically between 25 and 300 kDa, preferably between 40 and 200 kDa, more preferably between 50 and 150 kDa. In a particular embodiment, the chitosan is in the form of an aqueous solution or suspension.
• the aqueous solution or suspension containing chitosan can be obtained by conventional methods known to those skilled in the art, for example, by adding chitosan to an aqueous medium, for example, water or a medium containing mostly water.
• the amount of chitosan that said aqueous solution or suspension may contain may vary within a wide range; however, in a particular embodiment, the amount of chitosan contained in said aqueous solution or suspension is between 0.05% and 1% (w / v), preferably between 0.1% and 0.3%, more preferably between 1 and 2%.
• said aqueous chitosan solution or suspension does not contain any organic solvent.
• casein, probiotic bacteria and chitosan are mixed in the mixing stage of the method of the invention.
• casein and probiotic bacteria are mixed first, and then chitosan is added; in another particular embodiment, casein and chitosan are mixed first, and subsequently, probiotic bacteria are added; in another particular embodiment, the probiotic bacteria and the chitosan are mixed first, and then the casein is added; and, in another particular embodiment, casein, probiotic bacteria and chitosan are added and mixed.
• said components are added, as previously mentioned, in the form of an aqueous casein solution, a suspension of probiotic bacteria and an aqueous chitosan solution.
• the mixture of casein, probiotic bacteria and chitosan is carried out, preferably at room temperature, that is, at a temperature between 18 ° C and 25 ° C, preferably between 20 ° C and 22 ° C, in order to do not affect the viability of probiotic bacteria, advantageously with agitation.
• casein and chitosan ratio by weight, is 1: 0.01-0.5, preferably 1: 0.01-0.1, more preferably about 1: 0, 02-0.07, or, expressed in another way, the ratio chitosan: casein, by weight, present in the mixture prior to the formation of the microparticles of the invention is 1: 1-150, preferably 1: 5- 100, more preferably about 1: 14-40.
• probiotic bacteria and the matrix components present in the mixture prior to the formation of the microparticles of the invention can vary within a wide range; however, in a particular embodiment, said probiotic bacteria / matrix ratio is at least 10 6 CFU per mg of matrix, generally between 10 6 CFU / mg and 10 13 CFU / mg, preferably between 10 9 CFU / mg and 10 12 CFU / mg.
• the microparticles of the invention further comprise a crosslinking agent, such as, for example, a tripol phosphate (eg, sodium tripol phosphate (TPP)); vanillin; genipin; a pharmaceutically or cosmetically acceptable divalent metal cation, or suitable for use in food, human or animal, such as, for example, a divalent metal selected from the group consisting of Ca 2+ , Mg 2+ , Zn 2+ , Fe 2+ and combinations thereof; or any combination thereof, or any other substance capable of establishing a chemical interaction with casein and / or chitosan.
• the microparticles of the invention comprise two or more crosslinking agents, preferably combinations of two different crosslinking agents previously mentioned in relation to the microparticles of the invention.
• the method of the invention comprises adding said at least one crosslinking agent to the mixture of casein, probiotic bacteria and chitosan.
• the crosslinking agent (or agents), in a particular embodiment, can be added to said mixture in the form of an aqueous solution.
• the crosslinking agent is the calcium cation (Ca 2+ )
• it can be provided by a source of said appropriate cation, such as a compound that in the aqueous solution originates said divalent cation, for example, calcium chloride, calcium acetate, calcium gluconate , calcium lactate, calcium sorbate, calcium ascorbate, calcium citrate, calcium propionate, calcium sulfate, etc., or mixtures of said compounds.
• a source of said appropriate cation such as a compound that in the aqueous solution originates said divalent cation, for example, calcium chloride, calcium acetate, calcium gluconate , calcium lactate, calcium sorbate, calcium ascorbate, calcium citrate, calcium propionate, calcium sulfate, etc., or mixtures of said compounds.
• the microparticles of the invention include a crosslinking agent
• the amount of crosslinking agent to be added depends on the nature of the crosslinking agent, as previously mentioned in
• said crosslinking agent will be added in sufficient quantity so that, when the crosslinking agent is TPP, the ratio of crosslinking agent (TPP): matrix (casein and chitosan) is 1: 0, 1-800, advantageously 1 : 1-500, preferably about 1: 100-300 approximately; when the crosslinking agent is vanillin, the ratio of crosslinking agent (vanillin): matrix (casein and chitosan) is 1: 0.1-500, advantageously 1: 1-250, preferably about 1: 50-100 approximately; and when the crosslinking agent is Ca 2+ , the crosslinking agent ratio (Ca 2+ or calcium source: matrix (casein and chitosan) is 1: 0, 1-50, advantageously 1: 1-25, preferably around from 1: 6-16 approximately.
• the microparticles of the invention After mixing the casein, the probiotic bacteria and the chitosan, under the conditions mentioned above, that is, at room temperature and with stirring, the microparticles of the invention are formed, comprising a matrix consisting of casein and chitosan, and a probiotic bacteria Said microparticles of the invention are, in a particular embodiment, suspended in the medium in which they have been formed.
• the suspension resulting from the mixture of casein, probiotic bacteria and chitosan, which contains the microparticles of the invention is subjected to a drying treatment by conventional methods, advantageously by spray drying (“spray-drying” ") or by lyophilization, in order to dry out the microparticles of the invention;
• This drying treatment makes it possible to obtain the microparticles of the invention in the form of a powder, which contributes to increasing their stability.
• this drying treatment in particular, when performed by spray drying ("spray-drying") or by lyophilization, comprises the addition of a protective agent, as previously mentioned in relation to the microparticles of the invention, which protects the matrix and probiotic bacteria during the drying process thereof, such as, for example, a saccharide or, in general, a suitable food additive, which, in addition to the protective paper, acts as a prebiotic .
• a protective agent as previously mentioned in relation to the microparticles of the invention, which protects the matrix and probiotic bacteria during the drying process thereof, such as, for example, a saccharide or, in general, a suitable food additive, which, in addition to the protective paper, acts as a prebiotic .
• saccharides that can be used as protective agents within the context of the present invention include lactose, mannitol, sucrose, maltodextrin, glucose, sorbitol, etc., as well as polysaccharides with prebiotic characteristics, such as, for example.
• the protective agent is mannitol. If the microparticles of the invention include a protective agent, this is added in the appropriate amount;
• the weight ratio between the casein matrix and chitosan and the protective agent can vary over a wide range, in a particular embodiment, the weight ratio between the matrix (casein and chitosan): protective agent is 1: 0, 1-5, typically 1: 0.5-4, preferably about 1: 1.
• the drying of said suspension and The drying of the microparticles of the invention is carried out by spray drying ("spray-drying"); for this purpose, the suspension containing the microparticles of the invention and / or the mixture of casein, probiotic bacteria and chitosan, and, optionally, a crosslinking agent and / or a protective agent, is introduced into a spray-dryer and the conditions are controlled.
• processing air inlet temperature); outlet air temperature (outlet temperature); air pressure (a / r pressure); sample pumping rate; aspiration; and air flow (a / r flow)].
• the person skilled in the art can set the most appropriate processing conditions for each case.
• the method of the invention may include some additional step to stabilize the microparticles of the invention.
• a cross-linking agent for example, a divalent metal cation, such as Ca 2+
• the method of the invention comprises introducing the suspension containing the microparticles of the invention, further comprising a crosslinking agent, and / or the mixture comprising casein, probiotic bacteria, chitosan and crosslinking agent, in a suitable container, for example, a plastic bag, which is seals and is subjected to at least one cycle of hydrostatic pressure, at a pressure between 100 and 800 MPa, preferably between 100 and 400 MPa, for a period of time between 1 and 30 min, preferably between 2 and 10 min .
• said high hydrostatic pressure treatment comprises the application on said mixture comprising casein, probiotic bacteria, chitosan and crosslinking agent, 1 cycle of 5 minutes at 100 MPa, or 1 cycle of 2 minutes at 300 MPa.
• the mixture comprising casein, probiotic bacteria, chitosan and crosslinking agent (Ca 2+ ) is subjected to 1 cycle of 5 minutes at 100 MPa.
• This high pressure treatment is applied to the mixture comprising casein, probiotic bacteria, chitosan and crosslinking agent before being subjected to the spray drying process.
• high pressure treatment is a treatment that allows the microparticles to be crosslinked per se without the need for the incorporation of a crosslinking agent, whereby the microparticles of the invention could be crosslinked by subjecting them to a treatment with high pressures, in the absence of crosslinking agent.
• the method of the invention allows obtaining the microparticles of the invention in the form of a dry powder, which contributes to the stability of the microparticles of the invention during long periods of storage under controlled or environmental conditions and, in addition, can be easily incorporated into the different products (eg, food, etc.) to which it is intended, both solid and liquid.
• microparticles obtainable by the method of the invention have the characteristics of the microparticles of the invention, and constitute an additional aspect of the present invention.
• microparticles of the invention have the ability to encapsulate probiotic bacteria and protect them during processing (obtaining microparticles comprising a matrix consisting of casein and chitosan, loaded with said probiotic bacteria) and storage over prolonged periods of storage under controlled conditions. or environmental and, in addition, protect them from the "acid-peptic" conditions during their transit through the gastrointestinal tract once ingested; in this way, the inactivation of probiotic bacteria is avoided, or substantially reduced after their incorporation into the different products (e.g., foodstuffs, etc.) to which they are intended.
• microparticles of the invention have a potent immunomodulatory effect and can therefore be used in the preparation of a modulating composition of the immune system for the prevention and / or treatment of immune system alterations.
• composition of the invention selected from:
• composition consisting of a plurality of microparticles of the invention, or a plurality of microparticles obtainable by the method of the invention, or by a plurality of microparticles of the invention and microparticles obtainable by the method of the invention; Y (ii) a composition comprising at least one microparticle of the invention, and / or a microparticle obtainable by the method of the invention, and a vehicle acceptable in food, nutraceutical, cosmeceutical or pharmacy.
• the average size of the microparticles of the invention is between 0.5 and 125 ⁇ , preferably between 1 and 40 ⁇ , more preferably, between 2 and 12 ⁇ .
• average size is meant the average diameter of the population of microparticles, which moves together in an aqueous medium.
• the average size of these systems can be measured by standard procedures known to the person skilled in the art, and which are described, for example, in the experimental part described below.
• the probiotic bacteria present in the microparticles of the invention are selected from bacteria of the genus of Bifidobacterium and Lactobacillus; In a more particular embodiment, said probiotic bacteria is selected from L plantarum and L. casei. In a specific embodiment, the probiotic bacteria are L. plantarum CECT 220 and L. casei CECT 475 T. In another particular embodiment, said probiotic bacteria is a strain of Bifidobacterium animalis subsp. lactis, such as that sold under the BB-12® brand.
• the microparticles of the invention comprise a crosslinking agent, as previously mentioned, for example, TPP, vanillin or a divalent metal cation, for example, Ca 2+ .
• the microparticles of the invention comprise two or more crosslinking agents, preferably combinations of two different crosslinking agents previously mentioned in relation to the microparticles of the invention.
• the microparticles of the invention comprise a protective agent, such as a saccharide, for example, mannitol.
• the microparticles of the invention are in the form of dry powder.
• composition of the invention (i) consists solely and exclusively of microparticles of the invention and / or microparticles obtainable by the method of the invention.
• said composition of the invention (i) is selected from: a composition A, comprising: casein, between 40% and 60% by weight,
• chitosan between 0.1% and 3.5% by weight
• probiotic bacteria between 10 9 CFU / g and 5 x 10 12 CFU / g, tripol sodium phosphate, between 0% and 0.15% by weight, and
• composition B comprising: casein, between 40% and 60% by weight,
• chitosan between 0.1% and 3.5% by weight
• probiotic bacteria between 10 9 CFU / g and 5 x 10 12 CFU / g, vanillin, between 0% and 0.6% by weight, and
• composition C comprising: casein, between 40% and 60% by weight
• chitosan between 0.1% and 3.5% by weight
• probiotic bacteria between 10 9 CFU / g and 5 x 10 12 CFU / g,
• the composition of the invention comprises, at least, a microparticle of the invention and / or a microparticle obtainable by the method of the invention, and a vehicle acceptable in food, nutraceutical, cosmeceutical or pharmacy.
• the composition of the invention is a food or feed comprising at least one microparticle of the invention and / or a microparticle obtainable by the method of the invention, or a composition comprising a plurality of microparticles of the invention and / or my ero particles obtainable by the method of the invention.
• the term "food” is any substance or product of any nature, solid or liquid, natural or transformed, which, due to its characteristics, applications, components, preparation and conservation status, is likely to be habitual or ideally used for any of the following purposes: a) for normal human or animal nutrition or as fruitive; or b) as dietary products, in special cases of human or animal feed.
• feed includes all natural materials and processed products, of any origin, which, separately or conveniently mixed together, are suitable for animal feed.
• a ready-to-eat food is one that does not need to be diluted by, for example, an aqueous solution suitable for consumption.
• a concentrated food is one in which one or more ingredients are present in greater concentration than in a ready-to-eat food, so for use, it is necessary to dilute it by, for example, an aqueous solution suitable for consumption.
• Illustrative, non-limiting examples of foods provided by this invention include both dairy products and derivatives, for example, fermented milks, yogurt, kefir, curd, cheeses, butters, ice cream, dairy desserts, etc., as non-dairy products, such as bakery products, pastries and pastries, cereals, chocolates, jams, juices, other derivatives of fruits, oils and margarines, prepared dishes, etc.
• dairy products and derivatives for example, fermented milks, yogurt, kefir, curd, cheeses, butters, ice cream, dairy desserts, etc.
• non-dairy products such as bakery products, pastries and pastries, cereals, chocolates, jams, juices, other derivatives of fruits, oils and margarines, prepared dishes, etc.
• the composition of the invention is a nutraceutical composition comprising at least one microparticle of the invention and / or a microparticle obtainable by the method of the invention, or a composition comprising a plurality of microparticles of the invention. and / or microparticles obtainable by the method of the invention.
• the term "nutraceutical composition” refers to a composition suitable for use in humans or animals, comprising one or more natural products with therapeutic action or that provide a health benefit or that have been associated with the prevention or reduction of diseases, for example, probiotic bacteria, etc., and includes dietary supplements presented in a non-food matrix (eg, capsules, powder, etc.) of a concentrated natural bioactive product usually present (or not) in foods and which, taken in a dose higher than that existing in those foods exerts a favorable effect on health, greater than what normal food could have .
• a non-food matrix eg, capsules, powder, etc.
• nutraceutical composition thus includes isolated or purified food products as well as food additives or supplements, which, in general, are presented in pharmaceutical forms normally used orally, for example, capsules, tablets, sachets, drinking blisters, etc .; such products provide a physiological benefit or protection against diseases, generally against chronic diseases.
• the nutraceutical composition provided by the invention may contain, in addition to probiotic bacteria, one or more nutraceuticals (products or substances associated with disease prevention or reduction), for example, flavonoids, omega-3 fatty acids, etc.
• prebiotics non-digestible food ingredients that stimulate the growth and / or activity of probiotics
• prebiotics non-digestible food ingredients that stimulate the growth and / or activity of probiotics
• oligofructose for example, pectin, inulin, galacto-oligosaccharides, lactulose, breast milk oligosaccharides, food fiber , etc.
• the composition of the invention is a pharmaceutical composition comprising at least one microparticle of the invention and / or a microparticle obtainable by the method of the invention, or a composition comprising a plurality of microparticles of the invention. and / or my ero particles obtainable by the method of the invention, suitable for oral, topical, rectal or vaginal administration; for this, said composition comprises a pharmaceutically acceptable carrier comprising one or more excipients suitable for oral administration, for example, in the form of a capsule, powder, granulate, tablet (coated or not), on, matrix, suspension, etc.
• a pharmaceutically acceptable carrier comprising one or more excipients suitable for topical administration, for example, in the form of cream, ointment, ointment, etc., or a pharmaceutically acceptable carrier comprising one or more excipients suitable for its rectal administration, for example, in the form of a suppository, etc., or a pharmaceutically acceptable carrier comprising one or more excipients suitable for vaginal administration, for example, in Bolus form, suppository, etc.
• Information on excipients suitable for the formulation of pharmaceutical compositions intended for administration orally, topically, rectally or vaginally, as well as on the production of said pharmaceutical compositions can be found in the book "Treaty of Galenic Pharmacy", by C. Faul ⁇ i Trillo, 10 Edition, 1993, Luzán 5, SA de Ediations.
• the composition of the invention is a cosmetic composition comprising at least one microparticle of the invention and / or a microparticle obtainable by the method of the invention, or a composition comprising a plurality of microparticles of the invention. and / or my ero particles obtainable by the method of the invention.
• cosmetic composition refers to a composition suitable for use in the body hygiene of humans or animals, or in order to improve beauty, or alter body appearance without affecting the structure or functions of the human or animal body, which comprises one or more products that provide such effects.
• the cosmetic composition provided by the invention may contain, in addition to the probiotic bacteria, one or more cosmetic products, that is, substances or mixtures intended to be brought into contact with the surface parts of the human or animal body (epidermis, hair and hair system, nails, lips and external genital organs) or with teeth and oral mucous membranes, with the exclusive or main purpose of cleaning, perfuming, modifying their appearance, protecting them, keeping them in good condition or correcting body odors.
• cosmetic products include the products contained in the INCI list ("International Nomenclature of Cosmetic Ingredients").
• the composition of the invention is a cosmeceutical composition comprising at least one microparticle of the invention and / or a microparticle obtainable by the method of the invention, or a composition comprising a plurality of microparticles of the invention. and / or microparticles obtainable by the method of the invention.
• the term "cosmeceutical composition” refers to a composition suitable for use in the human or animal body comprising one or more cosmeceutical products ("functional cosmetics", “dermoceuticals” or “active cosmetics” ), that is, hybrid topical products with cosmetic-pharmaceutical characteristics that contain active ingredients with effect on the skin, hair and / or the user's nails, in higher and more effective concentrations, so that they are located at an intermediate level between the cosmetic and the drug.
• cosmeceutical products include essential oils, ceramides, enzymes, minerals, peptides, vitamins, etc.
• micro particles of the invention may be part of a food or feed, or of a nutraceutical, pharmaceutical, or cosmeceutical product, which constitute an additional aspect of the present invention
• Such products may be in liquid, semi-solid or solid form.
• the micro particles of the invention have a potent immunomodulatory effect and favor the induction of a Th1 response and / or shift the immune response towards Th1, preferably Th2 towards Th1 (Example 7), so they can be used in the development of a modulating composition of the immune system for the prevention and / or treatment of an alteration of the immune system, for example, in the prevention and / or treatment of rejection of a Th2-mediated transplant, allergies and diseases associated with allergies, immunodeficiencies and pathologies derived from said immunodeficiencies, infections caused by intracellular pathogens and / or mucosal infections.
• the invention relates to the use of a microparticle of the invention, or of a composition of the invention, or of a food, pharmaceutical, cosmeceutical or nutraceutical product comprising at least one microparticle of the invention or said composition of the invention, hereinafter "product of the invention", in the preparation of a modulating composition of the immune system.
• product of the invention in the preparation of a modulating composition of the immune system.
• the invention relates to a microparticle of the invention, or a composition of the invention, or a product of the invention for use in a modulating composition of the immune system.
• a “immune system modulating composition” is a composition that is capable of stimulating certain immune system responses by making it more reactive, for example, by intervening, to through the production of specific cytokines, in the development of the cells involved in the immune response.
• composition includes Any pharmaceutical, food (feed or feed), nutraceutical, etc. composition comprising the microparticles of the invention, the composition of the invention, or the product of the invention, described above.
• Example 7 show that oral administration of my ero particles of the invention containing L plantarum to CD1 mice, on the one hand, induces a slight increase in the number of cytotoxic lymphocytes (evidenced by a decrease in the CD4 + / CD8 + ratio) (Figure 9), and, on the other hand, causes an increase in the synthesis of interferon-gamma (IFN-g), compared to the production of interleukin 6 (IL-6), displacing from that mode the immune response towards a Th1 profile.
• IFN-g interferon-gamma
• IL-6 interleukin 6
• the immunomodulatory composition of the invention is a composition that preferentially induces a Th1 response and / or shifts the immune response towards Th1, preferably Th2 towards Th1.
• the immunomodulatory composition of the invention stimulates or induces the majority or preferably the Th1 response of the immune system making it more reactive, through the production of specific cytokines, such as, for example, IFN-g, interferon alpha (IFN-a), interleukin 12 (IL-12), interleukin 18 (IL-18), etc., in the development of cells involved in the Th1 immune response.
• specific cytokines such as, for example, IFN-g, interferon alpha (IFN-a), interleukin 12 (IL-12), interleukin 18 (IL-18), etc.
• microparticles of the invention preferably induces a Th1 response and / or shifts the Th2 immune response to Th1, by conventional methods, for example, by methods based on the quantification of specific cytokines. of the Th1 response and, optionally, Th2, such as, for example, the assay described in Example 7.
• the immunomodulatory composition of the invention is a composition suitable for oral administration (sometimes referred to in this description, for simplicity, as "oral composition") and will be presented in a solid administration form, liquid or semi-solid.
• said immunomodulatory composition of the invention will include, together with the microparticles of the invention, or the composition of the invention, or the product of the invention, a pharmaceutically acceptable carrier; said pharmaceutically acceptable carrier comprises one or more excipients suitable for oral administration, for example, in the form of a capsule, powder, granulate, suspension, etc.
• a pharmaceutically acceptable carrier comprises one or more excipients suitable for oral administration, for example, in the form of a capsule, powder, granulate, suspension, etc.
• the immunomodulatory composition of the invention is an oral composition for the prevention and / or treatment of an immune system disorder; said alteration of the immune system can be a natural (genetic) alteration or an induced alteration, such as an alteration induced by an infectious process, stress, etc.
• the immunomodulatory composition of the invention is an oral composition for the prevention and / or treatment of:
• Transplant rejection is a process in which the transplant recipient's immune system attacks the transplanted organ or tissue. Due to its characteristics, the immunomodulatory composition of the invention can be particularly useful in the prevention and / or treatment of transplant rejection (e.g., an organ, tissue, etc.) mediated by a Th2 response.
• transplant rejection e.g., an organ, tissue, etc.
• An allergy is a hypersensitivity disorder of the immune system. Allergic reactions occur when a person's immune system reacts to substances normally harmless in the environment. A substance that causes an immune (allergic) reaction in a subject sensitive to that substance is known as an "allergen.” When an allergen enters the body of a subject who is allergic to it, its immune system responds by producing a large amount of antibodies (IgE); successive exposure to the same allergen causes the release of chemical mediators, in particular histamine, which will produce the typical symptoms of the allergic reaction.
• IgE antibodies
• allergens there are many allergens; by way of illustration, not limitation, said allergens may be allergenic extracts of pollens, allergenic extracts of animals, including domestic animals, insects, mites, etc., allergenic extracts of food or food products, metals, components present in saliva, tweezers or stingers of insects that induce a sensitivity reaction in a subject, components present in plants that induce a sensitivity reaction in a subject, etc.
• allergies to animals including allergies to epithelium, dander or feathers of animals (eg, dog, cat, horse, birds, etc.), insect allergies, for example, allergy to components present in saliva, tweezers or insect stingers that induce a sensitivity reaction in a subject (eg, bees, wasps, mosquitoes, horseflies, etc.), dust mite allergies, for example, dust mites (eg, Dermatophagoides pteronyssimus, Dermatophagoides farinae, Acaros Siró, Blomia tropicalis, Euroglyphus maynei, Glyciphagus domesticus, Lepidoglyphus destructor, Tyrophagus putrescentiae, etc.);
• - fungal allergies e.g., Alternar ⁇ a alternata, Cladosporium herbarum, etc.
• the immunomodulatory composition of the invention can be used for the prevention and / or oral treatment of allergies in general; in a particular embodiment, said allergy is selected from the allergy group indicated above, that is, from the allergy group formed by allergies to plant pollens, insect allergies, mite allergies, fungal allergies; animal allergies, allergies to food components present in food, metal allergies, dust allergies, etc., and combinations thereof.
• the prevention and / or treatment of allergies by using the immunomodulatory composition of the invention may be favored by the administration of the allergen causing the allergy.
• said allergen can be administered to the subject together with the immunomodulatory composition of the invention (simultaneous administration of the immunomodulatory composition of the invention and the allergen) including the allergen in the formulation of the immunomodulatory composition of the invention itself or by administering the allergen in an independent formulation but simultaneously with the administration of the immunomodulatory composition of the invention.
• the allergen may be administered to the subject in a period of time prior to, or after, the administration of the immunomodulatory composition of the invention (sequential administration of the immunomodulatory composition of the invention and the allergen); In this case, the allergen would be formulated in its own formulation.
• said allergen is an allergen causing the allergies referred to in the preceding paragraphs.
• Said allergens can be obtained by conventional methods known to those skilled in the art or they can be purchased commercially.
• the immunomodulatory composition of the invention can be used for the prevention and / or oral treatment of diseases associated with allergies.
• the matera expert knows the diseases generally associated with allergies
• the diseases associated with the most common allergies are selected from asthma and atopic dermatitis.
• Immunodeficiency is a pathological state in which the ability of the immune system to fight infectious diseases is compromised or absent; Under these conditions, the immune system does not fulfill its protective role, leaving the organism vulnerable to infection.
• immunodeficiencies cause affected people a high susceptibility to infections.
• most immunodeficiencies are acquired (“secondary immunodeficiency”); however, some people are born with defects in their immune system (“primary immunodeficiency”).
• secondary immunodeficiency Among the subjects that may have an immunodeficiency are transplanted patients who take medications to suppress their immune system as an anti-transplant rejection measure, as do some patients who suffer from an overly active immune system.
• people with immunodeficiency are usually particularly vulnerable to opportunistic infections, in addition to normal infections that can affect everyone.
• the immunodeficiency can be physiological, congenital, or acquired.
• physiological immunodeficiencies eg, in newborn babies, during pregnancy, etc.
• primary or congenital immunodeficiencies eg, genetic diseases, for example, such as agammaglobulinemia in Di George's syndrome, etc.
• acquired or secondary immunodeficiencies eg, acquired immunodeficiencies as a result of malnutrition, aging, treatment with certain medications, such as chemotherapeutic agents, ntirreum attics, immunosuppressants (administered after organ transplantation), glucocorticoids, etc .; acquired immunodeficiency syndrome (AIDS), autoimmune diseases, etc.).
• the immunomodulatory composition of the invention is useful for supporting the body's natural immune defenses, for example in certain stress states such as psychophysical stress that, if it is excessively intense or prolonged, It can lead to an immunodeficiency situation, clinically manifested by vulnerability to infections of varying intensity.
• the immunomodulatory composition of the invention by modulating the immune system, for example, preferably inducing a Th1 response and / or shifting the immune response towards Th1 (for example from Th2 to Th1), can also be used for prevention and / or oral treatment of pathologies derived or resulting from immunodeficiencies.
• the person skilled in the art knows the pathologies derived from immunodeficiencies, for example, infections, etc.
• the immunomodulatory composition of the invention may be useful in the prevention and / or treatment of immunodeficiencies of any origin and resulting pathologies, for example, in the prevention and / or oral treatment of infections caused by intracellular pathogens.
• infections caused by intracellular pathogens.
• bacteria, protozoa, viruses, etc. as well as mucosal infections (eg, oral cavity infections, respiratory tract infections, gastrointestinal tract infections, genitourinary tract infections, mucous membrane infections, skin infections, etc.) and, in general, all infections that result from immunodeficiency states.
• the immunomodulatory composition of the invention can be used in the treatment and / or prevention of infections caused by intracellular pathogens.
• said intracellular pathogen is a eukaryotic pathogen, such as, for example, a protozoan (eg, Plasmodium vivax (which causes malaria), Leishmania sp. (Associated with Leishmaniasis), Entamoeba sp., Cryptosporidium sp., Etc.
• a protozoan eg, Plasmodium vivax (which causes malaria), Leishmania sp. (Associated with Leishmaniasis), Entamoeba sp., Cryptosporidium sp., Etc.
• a prokaryotic pathogen such as a bacterium (eg, Escherichia coli, Salmonella sp., Shigella sp., Campylobacter sp., Yersinia sp., Vibrio sp., Mycobacterium tuberculosis, M. leprae ,, Listeria sp.
• bacterium eg, Escherichia coli, Salmonella sp., Shigella sp., Campylobacter sp., Yersinia sp., Vibrio sp., Mycobacterium tuberculosis, M. leprae ,, Listeria sp.
• a virus eg, double stranded DNA virus (dsDNA), for example, adenovirus, herpesvirus, poxvirus, etc.); single stranded DNA virus (ssDNA), for example, parvovirus, etc .; double stranded RNA virus (dsRNA), for example, reovirus, etc .; single stranded positive RNA virus ((+) ssRNA), for example, picornavirus, togavirus, etc .; single stranded negative RNA virus [(-) ssRNA], for example, orthomyxovirus, rhabdovirus, etc .; retrotranscribed single stranded RNA virus (ssRNA-RT), for example, retrovirus, etc .; or bic RNA virus retrotranscribed attendee (dsRNA-RT), for example, hepadnavirus, etc.).
• dsDNA double stranded DNA virus
• ssDNA single stranded DNA virus
• ssDNA for
• the immunomodulatory composition of the invention can be used in the treatment and / or prevention of mucosal infections; by way of illustration, not limitation, said mucosa may be the mucosa of the oral cavity, the mucosa of the gastrointestinal tract, the mucosa of the genitourinary tract and the mucosa of the respiratory tract, etc.
• these infections can be caused by intracellular pathogens.
• mucosal infections are caused by enterobacteria (eg, Escherichia coli, Salmonella sp., Shigella sp., Campylobacter sp., Yersinia sp., Vibrio sp., Etc.), enterovirus (eg, calicivirus, rotavirus, adenovirus, astrovirus, etc.) or protozoa (eg, Entamoeba sp., Cryptosporidium sp., Leishmania sp., etc.).
• enterobacteria eg, Escherichia coli, Salmonella sp., Shigella sp., Campylobacter sp., Yersinia sp., Vibrio sp., Etc.
• enterovirus eg, calicivirus, rotavirus, adenovirus, astrovirus, etc.
• protozoa eg, Entam
• the immunomodulatory composition of the invention preferably suitable for oral administration, can be prepared by methods known to those skilled in the art taking into account the particular nature of the active ingredients present therein, which include live material, namely, probiotic bacteria, and, preferably, by the method provided by this invention since the microparticles thus produced protect said probiotic bacteria during their processing, storage and administration, in particular, during their transit through the gastrointestinal tract (oral administration) . Additionally, after being ingested, they facilitate the release of probiotic bacteria in the desired place, protecting them from the "peptic acid" conditions of the upper gastrointestinal tract, particularly the stomach.
• the immunomodulatory composition of the invention is in the unit dosage form for administration once or several times a day, according to the type and severity of the pathology to be treated and the age and weight of the subject.
• the microparticles of the invention present in the immunomodulatory composition of the invention are in the form of a dry powder or a lyophilisate, optionally present in a vehicle suitable for administration to a subject.
• the active ingredients are included in the appropriate compositions.
• the imunomodulatory composition of the invention comprises a vehicle acceptable in food, pharmacy or nutraceutical.
• the pharmaceutical, nutraceutical compositions or food products provided by this invention provide a suitable vehicle for probiotic bacteria.
• the immunomodulatory composition of the invention comprises a pharmaceutical, nutraceutical composition or is comprised in a food product.
• Illustrative, non-limiting examples include medications, dietary products, milk products, such as yogurt, cheese, cream, confectionery, fruit juices, etc., and may include, if desired, as mentioned. previously, other beneficial substances for the organism, such as, for example, vitamins, mineral salts, other compatible active ingredients, such as, for example, prebiotic agents, fibers, etc.
• this inventive aspect may alternatively be expressed as a microparticle of the invention, or a composition of the invention, or a product of the invention for use in a modulating composition of the immune system (immunomodulating composition of the invention).
• immunomodulating composition of the invention preferably induces a Th1 response and / or shifts the immune response towards Th1; preferably from Th2 to Th1.
• said immune system modulating composition comprises, in addition to the microparticle of the invention, composition of the invention, or product of the invention, a vehicle acceptable in food, pharmacy or nutraceuticals.
• said immune system modulating composition comprising microparticles of the invention, a composition of the invention, or a product of the invention, is in the form of a pharmaceutical, nutraceutical composition or, alternatively, is comprised in A food product.
• the microparticles present in said immune system modulating composition are in the form of a dry powder.
• the invention also relates to a microparticle of the invention, or a composition of the invention, or a product of the invention for use in the prevention and / or treatment of an alteration of the immune system (eg, a natural or induced alteration of the immune system) orally.
• said microparticle of the invention is in a pharmaceutical composition formulated for oral administration.
• said composition of the invention is a pharmaceutical composition formulated for oral administration.
• said product of the invention is a pharmaceutical product suitable for oral administration.
• the invention also relates to a microparticle of the invention, composition of the invention, or product of the invention for use in the prevention and / or oral treatment of rejection of a Th2-mediated transplant; allergies and diseases associated with allergy; immunodeficiencies and pathologies derived from said immunodeficiencies; infections caused by intracellular pathogens, or mucosal infections [the characteristics of Th2-mediated transplant rejection, allergies and allergy-associated diseases; immunodeficiencies and pathologies derived from said immunodeficiencies; infections caused by intracellular pathogens, or mucosal infections have already been mentioned previously and are incorporated by reference].
• said microparticle of the invention is in a pharmaceutical composition formulated for oral administration.
• said composition of the invention is a pharmaceutical composition formulated for oral administration.
• said product of the invention is a pharmaceutical product suitable for oral administration.
• the invention in another aspect, relates to a method for the prevention and treatment of an alteration or pathology of the immune system of a subject, which comprises administering by mouth to a subject in need of treatment, an effective amount of an immunomodulating composition. of the invention, or of microparticles of the invention, or of a composition of the invention, or of a product of the invention.
• alteration or pathology of the immune system of a subject comprises alterations of the immune system, both natural and induced of the immune system, such as diseases in which a treatment based on a Th1 response may result beneficial, for example, rejection of a Th2-mediated transplant; allergies and diseases associated with allergy; immunodeficiencies and pathologies derived from said immunodeficiencies; infections caused by intracellular pathogens, or mucosal infections.
• the characteristics of transplant rejection. mediated by Th2, allergies and allergy-associated diseases; immunodeficiencies and pathologies derived from said immunodeficiencies; Infections caused by intracellular pathogens, or mucosal infections have already been mentioned previously and are incorporated by reference.
• the term "subject” includes any mammalian animal, including humans.
• the immunomodulatory composition of the invention, the microparticles of the invention, the composition of the invention, or the product of the invention can be found in a vehicle acceptable in food, pharmacy or nutraceuticals, or it can be be present in a pharmaceutical composition, nutraceutical or included in a food product.
• the method of producing casein and chitosan microparticles comprises the dissolution of sodium caseinate (ANVISA, Madrid, Spain) in aqueous medium followed by the addition, under magnetic stirring and with constant flow, of a given volume of a chitosan solution and optionally a certain amount of a crosslinking agent.
• the microparticles formed are dried after passing the suspension containing them through a spray-dryer after adding a protective agent, such as mannitol.
• the chitosan used in these examples was Chitosan Characterized, with a degree of deacetylation of 90.2% and a molecular weight of 105 + 0.01 kDa, from Guinama (Valencia, Spain).
• the spray-dryer used in these examples was the Büchi Mini Spray Dryer B-290 spray dryer with Büchi Inert Loop B-295 and Dehumidifier B-296 accessories. Büchi Switzerland, Flawil (Switzerland).
• the mannitol used in these examples was D-mannitol, E-421, wealth 99.4%, from Guinama (Valencia, Spain), although sometimes it also used Sigma-Aldrich D-mannitol.
• the size of the microparticles was determined by optical microscopy using an Olympus CH40 microscope with Color Camera view-soft imaging Systems.
• microparticles The morphology of the microparticles was also observed by scanning electron microscopy (Zeiss, DSM 940A Germany). To do this, the microparticles were covered with a layer of molecular gold of about 9 nm (Team Emitech K550, Sputter-Coater, United Kingdom) and the photographs were taken with a Zeiss DMS 940 A microscope (United States).
• the probiotic bacteria used in the realization of these examples were Lactobacillus plantarum CECT 220 and Lactobacillus casei CECT 475 T isolated from corn and cheese forage, respectively.
• the lyophiles of both microorganisms were revitalized in MRS broth (Merck, Barcelona) at 37 ° C in anaerobic atmosphere (85% nitrogen, 10% hydrogen, 5% carbon dioxide) in anaerobic booth (MACS 500 AIRLOCK, AES Chemunex, Spain ). From these revitalized cultures, aliquots of 500 stock suspensions were prepared and kept frozen at -85 ° C until they were used.
• the general method of producing casein and chitosan microparticles containing encapsulated probiotic bacteria comprises the dissolution of sodium caseinate (ANVISA, Madrid, Spain) in aqueous medium followed by the addition, under stirring and with a constant flow of a given volume of bacterial suspension, obtained according to the method described in section III above and after being centrifuged and resuspended in a given volume of 2% sucrose solution (w / v). Subsequently, a certain volume of a chitosan solution is added, and optionally a certain volume of a crosslinking agent.
• This procedure was carried out to qualitatively confirm, through fluorescence optical microscopy, that the bacteria are trapped inside the microparticles, that is, that the casein and chitosan matrix covers the probiotic bacteria.
• the bacterial staining process comprises the preparation of a saturated solution of rhodamine B isothiocyanate in phosphate buffer (pH 7.4), its filtration through a 0.2 ⁇ membrane and its addition over a given volume of bacterial suspension , obtained according to the method described in previous sections. Once the mixture is centrifuged at 3,000 rpm for 15 minutes to remove excess rhodamine in the supernatant, the stained bacteria are resuspended in a given volume of 2% sucrose solution (w / v). Encapsulation of stained bacteria is performed according to the method described in the previous section.
• the gastric simulant was prepared according to pharmacopoeia and had the following composition for 1 liter of solution:
• the intestinal simulant also prepared from a pharmacopoeia prescription, was composed of:
• the 5 tubes were kept at 37 ° C in orbital shaker (150 rpm) until the moment of sample extraction and evaluation of survivors. After the treatment time in gastric simulant (2 hours), the PVC tubes were centrifuged (13,000 rpm / 10 minutes) and the supernatant was discarded. To evaluate the time 2 hours, the pellet of one of the tubes was subjected to the treatment of rupture of the microcapsules with 1% NaOH (pH 10) described above. The pellets of the remaining tubes were resuspended in 0.99 mL of intestinal simulant to assess resistance in this medium at time 0, 3 and 6 hours (2, 5 and 8 hours from the start of the study). After these times, the tubes were centrifuged, the supernatants were discarded and the pellets were treated with 1% NaOH (microcapsule rupture treatment) to proceed to evaluate the remaining survivors.
• 1% NaOH microcapsule rupture treatment
• Viable bacteria counting was performed by the MRS agar plate counting method described above. Plates were incubated at 37 ° C for 24 to 48 h under anaerobic conditions determining the number of colony forming units. The fraction of surviving bacteria was calculated according to the following equation:
• Log Fraction Survivor Lo ⁇ ⁇ 77- where N t represents the total viable lactic acid bacteria after each treatment time, and N 0 represents the initial number of inoculated lactic acid bacteria (BAL) (Bao et al., 2010 ). VIII. Procedure for assessing the stability of microencapsulated lactic bacteria over storage time in environmental conditions
• the stability study of the microencapsulated bacteria was carried out by evaluating the bacterial viability throughout the storage time at room temperature (25 ° C).
• microparticles containing bacteria were prepared, all with casein as chitosan modified base polymer.
• the method of preparing said microparticles depended on the presence or absence of crosslinker and the type of crosslinker used.
• Air Pressure 6 bar (6 x 10 5 Pa)
• Figure 1 shows the optical microscopy images obtained for the particles, both in the presence and absence of the probiotics. In them it can be verified that the size of the particles is not affected by the presence of encapsulated bacteria.
• Figure 2 shows the fluorescence optical microscopy images of both free stained bacteria (A) and encapsulated ones (B).
• the fluorescence observed in the microparticles (A) is due exclusively to bacteria. When practically no presence of bacteria is observed outside the microparticles, it is confirmed that they are encapsulated.
• microparticles obtained in powder form were collected for characterization and quantification.
• an aqueous solution of 10 mg / mL of sodium caseinate was added 1.5 mL of the bacterial suspension (4.7 x 10 11 CFU / mL) described in section III of the "General Methods", after being centrifuged and resuspended in a 2% sucrose solution (w / v). Subsequently, on the mixture, 10 mL of a chitosan solution of concentration 1.6 mg / mL prepared in aqueous medium with pH 5.5-6 was added. After five minutes of incubation, 0.8 mL of a 1 mg / mL TPP solution was added.
• microparticles obtained in powder form were collected for characterization and quantification.
• the only particles obtained in powder form were collected for characterization and quantification.
• Figure 3 shows the fluorescence optical microscopy image obtained for the microparticles, in which no presence of free bacteria is observed.
• Table 1 summarizes the death cycles of L plantarum derived from the encapsulation process in the casein and chitosan microparticles.
• microparticles obtained are similar in all formulations and range around 7 ⁇ 4 ⁇ . However, the cycles of bacterial death are lower when vanillin or TPP is used as crosslinking agents.
• the Bp and Cp formulations offer the greatest protection to probiotics during the process of obtaining. So they were chosen both formulations to proceed with the studies of gastrointestinal resistance and viability during storage.
• microparticles were characterized to assess their status during the process of degradation over time.
• Figure 6 shows that the bacteria are housed inside the microparticles and are released to the medium when said microparticles degrade over time.
• microparticles containing bacteria were prepared, all with casein as the base polymer and chitosan.
• the method of preparing said microparticles depended on the type of crosslinker used.
• microparticles obtained in powder form were collected for characterization and quantification. This same study was conducted in the absence of bacteria to verify how the presence of these probiotics affects the physicochemical characteristics of the particles.
• Air Pressure 6 bar (6 x 10 5 Pa)
• the only particles obtained in powder form were collected for characterization and quantification.
• microparticles obtained in powder form were collected for characterization and quantification.
• microparticles obtained in powder form were collected for characterization and quantification.
• Table 2 summarizes the physicochemical characteristics of the casein and chitosan microparticles containing encapsulated L casei, as well as the cycles of bacterial death derived from the manufacturing process of the particles.
• the sizes of the microparticles obtained are similar in all cases and they range around 7 ⁇ 4 ⁇ .
• the data reveal that, in general terms, the formulations developed better protect L casei than L. plantarum throughout the process, and, in addition, the Ce and De formulations, are the ones that provide the most protection confer
• the method provided by the present invention can be considered as a suitable method since it maintains bacterial counts of the order of 10 9 CFU / g (Table 2), which allows, for example, its formulation in foods in proportions of the order 1% maintaining the necessary concentration of probiotic bacteria of 10 7 CFU / g.
• the casein microparticles modified with chitosan were used in the presence of vanillin described in Example 1 (reference Bp).
• vanillin 5 mg / mL
• aqueous solution of 10 mg / mL of sodium caseinate 10 mg / mL of sodium caseinate.
• 1 mL of the bacterial suspension 4 x 10 10 CFU / mL
• microparticles obtained in powder form were collected for characterization and quantification.
• the average size of the microparticles obtained was 7 ⁇ 4 ⁇ .
• the bacterial count gave a titre of 5, 1x10 10 CFU per gram of microparticles
• the first group was administered orally 0.1 ml_ of PBS (phosphate buffered saline pH 7.4) (control).
• a second group was treated with a suspension of Lactobacillus plantarum in 2% sucrose with a dose of 10 7 CFU / mouse (free LP).
• the third group was treated with a physical mixture in the form of a suspension formed by L plantarum in 2% sucrose (10 7 CFU / mouse) mixed with empty casein microparticles modified with chitosan and crosslinked with vanillin (100 ⁇ g / mouse) (mixture physics, MF).
• the fourth group received the above described formulation of L. plantarum incorporated into casein microparticles modified with chitosan and cross-linked with vanillin (10 7 CFU / mouse) (Bp).
• a blood volume of approximately 250 ⁇ was extracted using serum separation tubes (SARSTEDT Microtube 1, 1 ml_ Z-Gel). Subsequently, the animals were sacrificed and the spleens were removed, whose cells disintegrated in RPMI 1640 medium with glycine at 4 ° C. The erythrocytes were lysed and the splenocytes were counted, whose concentration was adjusted in complete RPMI medium. About 100 replicates of the cell suspension was added as stimulus L plantarum (10: 1 ratio with respect to splenocytes).
• cytokines were captured using the "BD cytometric bead array Th1 / Th2 / Th17 CBA" kit (BD, USA) and determined using a flow cytometer (Attune® Acoustic Focusing Cytometer).
• Figure 9 shows how oral administration of L plantarum (in free, encapsulated or physical mixture form) induces a slight increase in the number of cytotoxic lymphocytes that is manifested by a decrease in the CD4 + / CD8 + ratio. This effect is consistent with the previous data described in the literature that correlate this increase with a colonizing effect of the intestine by bacteria [Her ⁇ as et al., 1999; Smelt et al., 2012]. On the other hand, it is observed that encapsulation did not affect the ability of bacteria to alter the CD4 + / CD8 + ratio.
• Figure 10 shows the interferon-gamma / interleukin 6 (IL-6) ratio as a function of the treatment received.
• IL-6 interferon-gamma / interleukin 6
• BORGOGNA M., BELLICH, B., ZORZIN, L, LAPASIN, R. & CESARO, A. 2010. Food microencapsulation of bioactive compounds: Rheological and thermal characterization of non-conventional gelling system. Food Chemistry, 122, 416-423.
• HEIDEBACH T., LEEB, E., FOERST, P. & KULOZIK, U. 201 1. Microencapsulation of probiotic cells.
• MONZER FANUN C.-P. (ed.) Colloids in Biotechnology.
• HERIAS M.V. HESSLE C
• TELEMO E. MIDTVEDT T.
• HANSON LA. WOLD A.E.
• LACROIX C. & PICOT, A. 2004. Encapsulation of bifidobacteria in whey protein-based microcapsules and survival in simulated gastrointestinal conditions and in yoghurt. International Dairy Journal, 14, 505-515. MATTILA-SANDHOLM, T. & SAARELA, M. (eds.) 2003. Functional Dairy Products. , Boca Raton: Woodhead Publishing Limited Abington Cambidge England CRC. Press LLC.
• MOHAMMADI R. A. M. MORTAZAVIAN
• Probiotic ice cream viability of probiotic bacteria and sensory properties.
• MORTAZAVIAN A.
• RAZAVI S. H.
• EHSANI M. R. & SOHRABVANDI, S. 2007.
• VINDEROLA C. G., W. PROSELLO, T. D. GHIBERTO AND J. A. REINHEIMER (2000a). Viability of probiotic (Bifidobacterium, Lactobacillus acidophilus and Lactobacillus casei) and non probiotic microflora in Argentinean fresh cheese. J Dairy Sci 83: 1905-191 1.
• Lactic acid starter and probiotic bacteria A comparative "in vitro" study of probiotic characteristics and biological barrier resistance. Food Research International, 36, 895-904.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Polymers & Plastics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Microbiology (AREA)
• Epidemiology (AREA)
• Mycology (AREA)
• Food Science & Technology (AREA)
• Zoology (AREA)
• Animal Husbandry (AREA)
• Molecular Biology (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Organic Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Immunology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Inorganic Chemistry (AREA)
• Oncology (AREA)
• Communicable Diseases (AREA)
• Nutrition Science (AREA)
• Pulmonology (AREA)
• Physiology (AREA)
• Biochemistry (AREA)
• Biomedical Technology (AREA)
• Biotechnology (AREA)
• Biophysics (AREA)
• Transplantation (AREA)
• Dermatology (AREA)
• Virology (AREA)
• Medicinal Preparation (AREA)
• Coloring Foods And Improving Nutritive Qualities (AREA)

Priority Applications (12)

Applications Claiming Priority (2)

Publications (2)

Family

ID=49162167

Family Applications (1)

Country Status (15)

Cited By (5)

Families Citing this family (28)

Family Cites Families (10)

• 2013
  • 2013-07-05 EP EP13760072.2A patent/EP2868206B1/en active Active
  • 2013-07-05 BR BR112015000041-0A patent/BR112015000041B1/pt active IP Right Grant
  • 2013-07-05 MX MX2015000218A patent/MX366411B/es active IP Right Grant
  • 2013-07-05 US US14/412,146 patent/US10111835B2/en active Active
  • 2013-07-05 DK DK13760072.2T patent/DK2868206T3/en active
  • 2013-07-05 PT PT137600722T patent/PT2868206T/pt unknown
  • 2013-07-05 AU AU2013285330A patent/AU2013285330B2/en active Active
  • 2013-07-05 CA CA2878026A patent/CA2878026C/en active Active
  • 2013-07-05 ES ES13760072.2T patent/ES2606679T3/es active Active
  • 2013-07-05 RU RU2015103826A patent/RU2015103826A/ru unknown
  • 2013-07-05 CN CN201380046390.XA patent/CN104602545B/zh active Active
  • 2013-07-05 WO PCT/ES2013/070477 patent/WO2014006261A2/es not_active Ceased
  • 2013-07-05 JP JP2015519259A patent/JP6442401B2/ja active Active
  • 2013-07-05 KR KR1020157003179A patent/KR102203837B1/ko active Active
  • 2013-07-05 PL PL13760072T patent/PL2868206T3/pl unknown

Non-Patent Citations (30)

Also Published As

Similar Documents

Legal Events