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WO2025052240A1 - Microcapsules contenant des souches probiotiques et leur procédé de préparation - Google Patents

Microcapsules contenant des souches probiotiques et leur procédé de préparation Download PDF

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Publication number
WO2025052240A1
WO2025052240A1 PCT/IB2024/058541 IB2024058541W WO2025052240A1 WO 2025052240 A1 WO2025052240 A1 WO 2025052240A1 IB 2024058541 W IB2024058541 W IB 2024058541W WO 2025052240 A1 WO2025052240 A1 WO 2025052240A1
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WO
WIPO (PCT)
Prior art keywords
microcapsules
suspension
shell
constitute
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/IB2024/058541
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English (en)
Inventor
Angela Assunta LOPEDOTA
Nunzio DENORA
Antonio LOPALCO
Maria De Angelis
Vita D'AMICO
Mirco VACCA
Sonya SIRAGUSA
Sergio Fontana
Flavia Maria LA FORGIA
Antonio Pepe
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
FARMALABOR Srl
Universita degli Studi di Bari Aldo Moro
Original Assignee
FARMALABOR Srl
Universita degli Studi di Bari Aldo Moro
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by FARMALABOR Srl, Universita degli Studi di Bari Aldo Moro filed Critical FARMALABOR Srl
Publication of WO2025052240A1 publication Critical patent/WO2025052240A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5026—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates

Definitions

  • Microcapsules containing probiotic strains and process for their preparation are described in detail below.
  • the present invention relates to microcapsules containing probiotic strains and a process for their preparation that uses a polymer mixture capable of imparting to said microcapsules high resistance to agents potentially adverse to the viability of probiotics such as, for example, temperature, pH, gastric and small intestinal enzymes and bile salts.
  • the human microbiota is constituted by the set of symbiotic microorganisms that coexist with the human organism. Each individual has its own, specific microbiota constituted by hundreds of different species of bacteria and other microorganisms.
  • the functions performed by the microbiota are multiple and range from the metabolism of compounds that humans are not independently able to digest, to the synthesis of certain essential substances, such as, for example, vitamin K.
  • the microbiota competes with non-symbiont microorganisms determining, indeed, a protective action against the development of diseases related to them.
  • the microbiota is one of the most significant topics of interest in the health scenario in the last decades.
  • various studies have shown how many aspects of our body's physiology are conditioned by the microbiota; not only immune functions (the first functions that have been attributed to the microbiota in an "external" setting compared to its normal location and, in particular, to the interaction between the gut microbiota and the lymphatic organs), but even some aspects related to brain function. This relationship is the basis of what is called the gut-brain axis.
  • Probiotics are, as defined by the FAO and WHO, "live microorganisms that, when administered in suitable amounts, are capable of conferring benefits to the host organism”.
  • the benefits they provide as a result of their ingestion relate to a relevant improvement in gut health, acting, for example, through mechanisms of cooperation with the already resident microbiota.
  • probiotics are known to stimulate and develop the immune system, synthesize and improve the absorption of food nutrients, as well as reduce symptoms resulting from the lactose intolerance and the risk of occurrence of certain diseases.
  • probiotics in interacting with the body and in counteracting various clinical conditions for which they are usually used, depends on the enzymatic properties of the individual strains.
  • the "ideal" probiotic must be well tolerated by the human organism, must be able to survive in the various physiological conditions of the gastrointestinal tract, must be able to adhere to the epithelium, actively replicate and, finally, must exert beneficial effects on the host in terms of competing with other microorganisms of the microbiota, regulating physiological functions and stimulating the immunological properties of gut associated ly mphoid tissue (GALT).
  • GALT gut associated ly mphoid tissue
  • probiotic microorganisms are particularly sensitive to different factors such as temperature, humidity, presence of water, oxygen, acidity of the gastric environment, and presence of bile salts and enzymes at intestinal level.
  • probiotics are included in a formulation or dosage form for oral use, it is necessary’ to preserve their viability during gastrointestinal transit such that their functionality is ensured after their consumption, so that they can reach the target site, e.g. the colon, in sufficient cell densities. Furthermore, it must be ensured that they remain metabolically active within the formulation at least until the expiration date of the formulation.
  • probiotics must also be protected during the production process steps and the subsequent storage of the finished product.
  • microparticles which can be constituted by different types of materials and/or polymers and produced by various techniques, that constitute a kind of "protective shell” inside which the active ingredients/probiotics are enclosed. Delivery /release of the content from microparticles can occur according to various mechanisms, depending on the type, thickness and general characteristics of the microparticle itself
  • US9089152 describes the use of denatured whey proteins for the production of microparticles in which to encapsulate active ingredients, specifically probiotic bacteria, in order to protect them from the acidic environment of the stomach thanks to the formation of a gelled polymer matrix.
  • active ingredients specifically probiotic bacteria
  • the components contained in the microparticles are then released at the intestinal level in a controlled manner thanks to the increase of the pH values of the micro-environment.
  • Another object of the present invention is to provide a formulation constituted by microcapsules containing at least one probiotic microorganism and at least one prebiotic substrate.
  • Figure 5 degree of viability, expressed as Log CFU/mL, of lactobacilli strain LB931, free (probiotics) or micro-encapsulated (microcapsules), following heat stress treatment for 30 minutes at 60°C (gray histograms on the left) or heat stress treatment for 1 minute at 90°C (black histograms on the right).
  • Object of the present invention are micro-encapsulated probiotics and a process for their production that involves the use of a polymer mixture based on a copolymer of methacrylic acid and alginic acid or its salt, and the application of the prilling/vibration technique.
  • microcapsule and “microparticle” are to be considered synonymous and refer to micrometer-sized particles, for example, between 100 and 1,500 microns in size, preferably between 500 and 1,000 microns, constituted by an inner core, also referred to as a ’’core”, and an outer shell, also referred to as a “shell”, with different composition.
  • the microcapsules that are object of the present invention are constituted by a core containing at least one polymer and at least one probiotic microorganism coated by a shell comprising at least one polymer and at least one prebiotic component.
  • a food ingredient To be classified as a prebiotic, a food ingredient must be neither hydrolyzed nor absorbed in the upper part of the gastrointestinal tract, it must act as a selective substrate for only one, or at least a limited number, of potentially beneficial commensal bacteria in the colon, by stimulating their growth or activating their metabolism, and finally it must be capable of modifying the colonic microbiota to a composition that is presumably more favorable in terms of host health. Thanks to their chemical structure and the host’s consequent inability to digest them directly, prebiotics are only fermented at the colon level by the commensal bacteria, thus leading to the formation of short-chain fatty acids resulting in decreasing the pH.
  • microcapsules that have, among others, the advantage of cartying at least one probiotic microorganism and at least one prebiotic component in a single microparticle, without compromising the activity and stability of each of the two components.
  • the coating shell, or shell, of the microcapsules according to the present invention comprises or alternatively consists of i. a neutral, or positively or negatively charged, copolymer of methacrylic acid, ii. alginic acid or a salt thereof; and iii. at least one prebiotic.
  • said coating shell may also include other accessory components/ excipients that can enhance some characteristics such as, for example but not limited to, stabilizers, dyes, flavorings, antioxidants, etc.
  • the alginic acid or its salt (ii) is sodium alginate.
  • Said alginic acid or a salt (ii) thereof can be at various degrees of viscosity, i.e., be low, medium or high viscosity, for example, from 100 to 1000 cPs (1% w/v in water at 25°C), and at various molecular weights, for example from 50,000 to 500,000 Daltons.
  • it is sodium alginate with medium or high viscosity, between 500 and 650 cPs (1% w/v in water at 25°C).
  • the at least one prebiotic (iii) belongs to the category of the fructose polymers also known as fructans or fructo- oligosaccharides (FOS), such as, for example but not limited to, inulin, which can come from various sources, including chicory root but also white truffle and dahlia tubers.
  • FOS fructo- oligosaccharides
  • the microcapsules according to the present invention have a shell constituted by neutral acrylic acid copolymer El 206, high viscosity sodium alginate and inulin.
  • the core of the microcapsules according to the present invention comprises or alternatively consists of: iv. alginic acid or a salt thereof; v. a cryoprotectant; and vi. at least one probiotic microorganism.
  • said core may also include other accessory components/ excipients that can enhance some characteristics such as, for example but not limited to, stabilizers, dyes, flavorings, antioxidants, etc.
  • the alginic acid or its salt (iv) is sodium alginate.
  • Said alginic acid or a salt (iv) thereof can be at various degrees of viscosity , that is, be low, medium or high viscosity.
  • it has a viscosity of 4 to 150 cPs (1% w/v in water at 25°C) and has a molecular weight between 10,000 and 100,000 Daltons.
  • it is sodium alginate with low viscosity, between 4 and 100 cPs (1% w/v in water at 25°C).
  • said cryoprotectant (v) is selected from the compounds known to the skilled in the art such as, for example but not limited to, trehalose, mannitol and sorbitol.
  • said at least one probiotic microorganism (vi) is selected from one or more microorganisms belonging to the LM.ctobaci.llus and Bifidobacterium families, and mixtures thereof.
  • the microcapsules according to the present invention have a core constituted by low-viscosity sodium alginate, mannitol and Lactiplantibacillus plantarum (basionym Lactobacillus plantarum).
  • the preparation method of the microcapsules according to the present invention involves the use of two different aqueous solutions and/or suspensions, one for core formation and one for shell formation, to be used by the prilling/vibration technique.
  • Said solutions and/or suspensions are prepared by dissolving or suspending in water, for that which will constitute the shell, at least the components (i)-(iii) and, for that which will constitute the core, the components (iv)-(vi), together with any further accessory components/excipients, according to techniques known to the skilled in the art.
  • the components (i) and (ii) are present in said suspension and/or coating solution that will constitute the shell of the microcapsule in a ratio by weight to each other between 0.5: 1 and 3: 1, respectively, and in a total amount of mixture (i)+(ii) between 2% and 8% by weight to the volume of the solution and/or suspension.
  • the at least one prebiotic (iii), or mixture of prebiotics is present in said suspension and/or coating solution that will constitute the shell of the microcapsule in a volume percent, to the total volume of the solution and/or coating suspension that will constitute the shell of the microcapsule, from 10% w/v to 90% w/v, preferably from 30% w/v to 60% w/v.
  • the alginic acid or its salt (iv) of the solution and/or suspension that, will constitute the core of the microcapsules is present in a weight to volume percent, to the total volume of the solution used to constitute the core of the microcapsule, between 0.05% w/v and 1% w/v.
  • cryoprotectant (v) of the solution and/or suspension that will constitute the core of the microcapsules is present in a. weight to volume percent, to the total volume of the solution used to constitute the core of the microcapsule, between 2% w/v and 5% w/v, preferably 3% w/v.
  • the at least one probiotic microorganism (vi) of the solution and/or suspension that will constitute the core of the microcapsules is present in an amount between about 5xl0 6 and 5xl0 8 CFU (colony-forming units) per mL of solution and/or suspension.
  • the two solutions and/or suspensions that contain all the components that constitute the core and coating shell of the microcapsules of the present invention are processed with an apparatus for the prilling/vibration technique (scheme in Figure I) and in particular through the use of an apparatus equipped with a concentric nozzle (3), which has an inner and an outer nozzle with different diameters, suitable for the formation of microparticles.
  • Such technique is based on the principle of the controlled rupture of a laminar liquid jet into one-dimensional droplets (prills') by applying mechanical vibration frequencies (via the vibration unit (2)).
  • a controlled vibration frequency causes the liquid in a chamber (called sphere-producing unit (1)) to vibrate before it is extruded through a concentric nozzle (3) and causes it to break into equal-sized droplets.
  • a charge is induced on the droplet surface by using an electrostatic voltage system (4).
  • a gelling (or consolidation) bath (8) that allows them to consolidate with the formation of the aforementioned microcapsules.
  • a consolidation bath (8) consists of a suitable gelling solution, for example but not limited to, an aqueous solution of a salt selected from calcium chloride, barium chloride, zinc chloride, strontium chloride and calcium gluconate, at a concentration between 0.1 M and 0.8 M, preferably 0.3 M.
  • Said consolidation bath and related gelling solution may vary' depending on the type of polymers and components used.
  • the consolidation occurs under mechanical stirring (for example, by means of a magnetic stirrer (9)) for a sufficient time, for example for 10 minutes.
  • the microcapsules obtained can be separated from the gelling solution by filtration according to any suitable method known to the skilled in the art, for example by the use of paper filters, and washed with distilled water.
  • said filtered and washed microcapsules are finally frozen at -20 °C and freeze-dried in order to obtain a final product in the form of a solid powder that is easily dosed and more stable over time.
  • the prilling/vibration technique can be directly associated with the freezing of the droplets produced (also referred to as the prilling/congealing technique); according to this alternative, the consolidation bath is constituted by liquid nitrogen in which the droplets are directly immersed, resulting in instantaneously frozen microcapsules, which are then freeze-dried.
  • the preparation method of the microcapsule according to the present invention comprises the following steps: a. preparing two aqueous suspensions and/or solutions by dissolving or suspending in water, for the one that wall constitute the shell, at least the components (i)-(iii) and, for the one that will constitute the core, at least the components (iv)-(vi), b.
  • step (a) introducing the two solutions prepared in step (a) into a prilling/vibration apparatus equipped with a concentric nozzle in which the inner nozzle (with the smaller diameter) with the solution/suspension that wall constitute the core of the microcapsule is fed, via an air flow that minimizes the dead space, and the outer nozzle (with the larger diameter) with the suspension/solution that will constitute the shell of it, thus obtaining droplets with different internal/ external composition due to the application of a specific vibration frequency and a specific electric field; c. immersing the drops produced in step (b) in a consolidation bath under mechanical stirring, filtering them, washing them with distilled water, thus obtaining the microcapsules; d. optionally, freezing the washed microcapsules obtained in step (c) by bringing them to -20°C and subjecting them to a freeze-drying process.
  • the "droplets” are microcapsules that have not yet been consolidated.
  • - diameter size of the inner nozzle forming the core from 80 to 750 pm, preferably 450 um;
  • - diameter size of the outer nozzle forming the shell from 200 to 900 um, preferably 700 pm;
  • - flow rate of the solution and/or suspension forming the core from 1.5 mL/min to 6 mL/min, preferably 3.15 mL/min;
  • - flow rate of the solution and/or suspension forming the shell from 14 mL/min to 30 mL/min, preferably 22.5 mL/min;
  • - vibration frequency from 40 Hz to 6,000 Hz, preferably 1,000 Hz;
  • - electrode potential from 250 V to 2,500 V, preferably 1,500 V.
  • the preparation method of the microcapsules according to the present invention allows the microcapsules of spherical shape and homogeneous size (within a narrow particle size range) to be obtained reproducibly and with high yields and efficiency. Furthermore, processing by the prilling technique makes it feasible to encapsulate the microorganisms in a sterile manner, thanks to the ability to introduce and remove the incoming fluids and the obtained products from the reaction vessel in a totally aseptic manner.
  • the selected technique can be also easily integrated into an industrial production process, also in accordance with the Good Manufacturing Practice (GMP) standards, compliance with which is necessary when working in the pharmaceutical field.
  • GMP Good Manufacturing Practice
  • the microcapsules of the invention containing probiotic microorganisms, are prepared easily, reproducibly and at low 7 cost.
  • the final product, obtained after freeze-drying, is constituted by a powder with a narrow size range.
  • the micro-encapsulated microorganisms are protected from the acidic environment determined by the gastric pH as well as by the digestive enzymes, allowing the maintenance of high viability of the probiotics until they reach the identified target site, that is the colon, where the probiotics are released thanks also to the degradation by the resident microbiota of the prebiotic component contained at the level of the coating shell.
  • probiotic-containing microcapsules were found to be stable during all the steps of production, transportation, storage and use, also ensuring a prolonged shelf life, both when considered as is and when formulated within more complex products such as, for example, fortified foods.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Nutrition Science (AREA)
  • Medicinal Chemistry (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • Polymers & Plastics (AREA)
  • Food Science & Technology (AREA)
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Abstract

La présente invention concerne des microcapsules contenant des souches probiotiques et un procédé pour leur préparation qui fait appel à un mélange polymère capable de conférer auxdits microcapsides une résistance élevée à des agents potentiellement défavorables à la viabilité de probiotiques tels que, par exemple, la température, le pH, les enzymes gastriques et du petit intestin et les sels biliaires.
PCT/IB2024/058541 2023-09-04 2024-09-03 Microcapsules contenant des souches probiotiques et leur procédé de préparation Pending WO2025052240A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT102023000018129A IT202300018129A1 (it) 2023-09-04 2023-09-04 Microcapsule contenenti ceppi probiotici e procedimento per la loro preparazione.
IT102023000018129 2023-09-04

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WO2025052240A1 true WO2025052240A1 (fr) 2025-03-13

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103355656A (zh) * 2012-04-01 2013-10-23 中国科学院大连化学物理研究所 一种益生菌微胶囊产品及其制备与应用
EP3205216A1 (fr) * 2016-02-10 2017-08-16 Fundacíon Tecnalia Research & Innovation Microcapsules probiotiques multicouches
US10631564B2 (en) * 2015-06-19 2020-04-28 University Of Southern California Enterically coated microparticle compositions and methods for modified nutrient delivery

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2564190T3 (es) 2009-04-13 2016-03-18 Agriculture And Food Development Authority (Teagasc) Método para producir microperlas

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103355656A (zh) * 2012-04-01 2013-10-23 中国科学院大连化学物理研究所 一种益生菌微胶囊产品及其制备与应用
US10631564B2 (en) * 2015-06-19 2020-04-28 University Of Southern California Enterically coated microparticle compositions and methods for modified nutrient delivery
EP3205216A1 (fr) * 2016-02-10 2017-08-16 Fundacíon Tecnalia Research & Innovation Microcapsules probiotiques multicouches

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