Microcapsules containing probiotic strains and process for their preparation

Abstract

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 microcapsides 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.

Description

[0001] “Microcapsules containing probiotic strains and process for their preparation”

[0002] ****

[0003] Summary of the invention

[0004] 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.

[0005] Technical background

[0006] 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. Furthermore, the microbiota competes with non-symbiont microorganisms determining, indeed, a protective action against the development of diseases related to them.

[0007] Precisely because of its complex interaction with the human organism and its responses, in both physiological and pathological settings, the microbiota is one of the most significant topics of interest in the health scenario in the last decades. As a matter of fact 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.

[0008] 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. In particular, 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.

[0009] The effectiveness of 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). A large number of microorganisms belonging to different genera and species could have potential probiotic properties. Lactic acid bacteria, such as Lactobacilli (basionym Lactobacillus), but also Bifidobacteri, have been the bacteria most widely- used as probiotics over the years. The main reason for their wide use is that, both of these genera are considered GRAS (Generally Recognised As Safe). Bifidobacteria are gram -positive, rod-shaped, anaerobic bacteria although with some aero-tolerance exceptions, whereas lactobacilli are gram-positive, non-spore-forming, rod-shaped, acid-tolerant organisms that generally live in a non-aerobic environment, although they are aero-tolerant.

[0010] The well-known and well-accepted knowledge regarding the many benefits of probiotics has led to a growing interest in them, as well as to the development and marketing of a large number of products containing them (such as, for example, drugs, dietary supplements and fortified foods). However, it is not enough to define and study the probiotic activity of a microorganism if the intended use is not compatible with the conditions necessary to promote it. As a matter of fact, 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. Therefore, once 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.

[0011] For all these reasons, careful formulation of probiotics within the pharmaceuticals, dietary supplements and fortified foods containing them is necessary to enable proper administration and effective use. Only in this way said probiotics can remain alive and viable at high cell densities until they reach and colonize the gut, thus promoting proper homeostasis of the gut microbiota. Furthermore, probiotics must also be protected during the production process steps and the subsequent storage of the finished product.

[0012] Several approaches have been developed to address these needs, including one that involves the micro-encapsulation of said probiotics inside 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

[0013] For example, 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. 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.

[0014] Micro-encapsulated probiotics can constitute, alone or in mixtures with other substances, products classified as dietary supplements, but also become part of a new class of products called functional foods that has been gaining great resonance and attention in recent years. Certain foods may already, by their nature, contain probiotics, see foods such as yogurt, cheese or other daily products, or they may be foods that do not normally include them but to which the probiotics can be added, such as, for example, juices, sauces, cookies, chocolate and others. Fermented milk products normally include a fermentative bacterial culture of one or more lactic acid bacteria, including, for example, Lactobacillus, Lactococcus and Streptococcus, which are able to impart distinctive sensory characteristics to the product.

[0015] On the other hand, some bacterial strains can be selected and added to foods, thus making them "functional foods", for their positive activity on human health in order to obtain a final product that can show specific benefits.

[0016] One of the problems to be solved, which accompanies the preparation of functional foods enriched / fortified with probiotics, is linked to the low7viability of microbial strains related to the production technologies and during the storage period of the food, i.e., leading to a reduction in its shelf life (expiration date close to the production date). This phenomenon not only brings with it problems of increased costs and inventory management, but also negatively impacts the sustainability of the food production.

[0017] Another problem related to the introduction of probiotic substances into food is that, in some cases, it is the technological process of food preparation itself that can be harmful to the survival of the probiotics introduced into it.

[0018] Finally, although consumers are increasingly oriented toward health products, the sensory characteristics of a food remain the main criterion for choosing these products. Therefore, it is necessary to take into account the impact that the introduction of probiotics may have with respect to food preparations that contain them because they should not affect their taste or any other quality aspect, with the aim of not diminishing their appreciation by the consumer.

[0019] Since the production and development of functional food products enriched with probiotics is a relatively recent approach, the resulting studies related to the making of micro-encapsulated probiotic additives that are resistant to possible conditions present during the preparation and storage of said functional foods (e.g., high temperatures, non-optimal pH values, oxidizing conditions) are also still not completely satisfactory. For example, the dairy industry to date is constantly seeking the possibility of producing functional foods of various kinds containing probiotics, starting from classic yogurt but also from milk, daily products and cheese, so as to have a range of new enriched / fortified foods to offer to consumers, who are increasingly attentive and looking for innovative and specific diets that are capable of improving the quality of life and physical well-being.

[0020] Therefore, there is stiil a great need for micro-encapsulated probiotics and a process for their preparation that, on the one hand, allows for the protection of said probiotic bacteria during the production and storage steps of the final formulations, such as, for example, but not only, functional foods, drugs, food supplements, etc., and, on the other hand, is able to carry them alive and viable at high cell densities to the intestinal level, where they wall be released to colonize the environment and cany out their beneficial action.

[0021] Objects of the invention

[0022] An object of the present invention is to provide micro-encapsulated probiotics for beneficial use in the preparation of pharmaceutical and non-pharmaceutical formulations, for example, in the preparation of functional foods containing them, that have the ability to withstand the stressful conditions that may possibly be present in the preparation and / or storage of said formulations.

[0023] 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.

[0024] Further object of the present invention is to provide a process for the preparation of micro-encapsulated probiotics that are able to withstand stress conditions and that can be released alive and viable at high cell densities at the intestinal level, preferably at the colon level, following their intake.

[0025] Another object of the present invention is to provide micro-encapsulated probiotics for specific release to the colon.

[0026] These and other purposes are achieved by the subject matter of the present invention, which relates to microcapsules containing probiotic bacteria and a new process for obtaining micro-encapsulated probiotic bacteria.

[0027] Brief description of the Figures

[0028] Figure 1 : schematic diagram of the apparatus and process for producing microparticles according to the present invention by the prilling / v ibration technique. Figure 2: images of the microparticles according to the present invention by optical microscopy (A), before and after the freeze-drying process, and SEM (B) at different magnifications in which the distinctions between the outer layer (shell) and the inner core (core) are also highlighted.

[0029] Figure 3: degree of viability, expressed as Log CFU / mL, of LB931 and 4S6R lactobacilli strains, free (top graph) and micro-encapsulated (bottom graph) in simulated fluids: gastric pH 1.2 for two hours; intestinal pH 6.8 in the presence of bile salts for the next three hours.

[0030] Figure 4: release profile, expressed as Log CFU / mL, of LB931 and 4S6R lactobacilli strains micro-encapsulated in simulated fluids: gastric pH 1.2 for 2 hours; intestinal pH 6.8 in the presence of bile salts for the next four hours and colonic in the presence of microbiota up to 24 hours.

[0031] 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).

[0032] Description of the invention

[0033] 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.

[0034] The technique referred to as prilling / vibration, or simply prilling, is a microencapsulation technique by which microparticles are obtained as a result of a rupture of a laminar flow of a polymer solution into droplets generated thanks to the presence of a device with a vibrating nozzle. This technique allows to produce homogeneously sized microparticles with high encapsulation efficiency, and because it does not involve the use of high temperatures or mechanical stress, its use is also suitable for processes involving the need to micro-encapsulate sensitive active ingredients, such as, for example, the probiotic microorganisms.

[0035] In the present document, the terms "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.

[0036] According to a preferred aspect, 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.

[0037] By the term "prebiotic" or "prebiotic component" is meant a food component that is not digestible by humans and that, upon metabolization by the gut microbiota, is able to result in selective stimulation of the growth and activity of one or more bacterial genera / species of said microbiota that are found to have beneficial properties for the host organism. Therefore, prebiotics are essential elements for the microbiota balance because they act as nutrients for one or more "good bacteria" already present in the gut. Such "good bacteria", which are able to ferment said prebiotics, derive energy from them for their own metabolism and growth, thus secondarily resulting in favorable effects for the human organism that hosts them.

[0038] 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. By the activation of this process, prebiotics can exert anti-inflammatory effects by, for example, stimulating the increase of the regulatory' T cells and the reduction of interferon. Furthermore, prebiotics can help to inhibit the adherence of pathogenic microorganisms to the intestinal epithelium, thus also preventing their passage through said epithelium.

[0039] Thanks to extensive studies and research, the applicant has developed 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.

[0040] According to a preferred aspect, 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.

[0041] If desired or necessary, 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.

[0042] According to a preferred aspect, the methacrylic acid copolymer (i) is a neutral acrylate copolymer, for example, the component identified as a food additive by the international abbreviation E1206.

[0043] According to another preferred aspect, 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. Preferably it is sodium alginate with medium or high viscosity, between 500 and 650 cPs (1% w / v in water at 25°C).

[0044] In another preferred aspect of the present invention, 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. Other possible prebiotics, according to the present invention, are galactooligosaccharides (GOS), polymers constituted by galactose and glucose, which are present mainly in the legumes, including beans, lentils, chickpeas and peas; betaglucans, compounds which are present in the cell walls of the endosperm of some cereals (in particular barley and oats), fungi and algae; and resistant starches, namely starches capable of resisting to the action of amylase, which are obtained with the help of various chemical / physical approaches knowm to the skilled in the art. Advantageously, the microcapsules according to the present invention have a shell constituted by neutral acrylic acid copolymer El 206, high viscosity sodium alginate and inulin.

[0045] According to a preferred aspect, 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.

[0046] If desired or necessary, 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.

[0047] According to a preferred aspect, 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. For example, 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. Preferably it is sodium alginate with low viscosity, between 4 and 100 cPs (1% w / v in water at 25°C).

[0048] According to another preferred aspect, 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.

[0049] According to another preferred aspect, 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.

[0050] Advantageously, the microcapsules according to the present invention have a core constituted by low-viscosity sodium alginate, mannitol and Lactiplantibacillus plantarum (basionym Lactobacillus plantarum).

[0051] 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. Some non-limiting examples regarding the qualitative / quantitative composition of said solutions and / or suspensions will be shown in the Experimental Section below.

[0052] According to a preferred aspect of the present invention, 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.

[0053] According to another preferred aspect 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.

[0054] According to a preferred aspect of the present invention, 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.

[0055] According to a further preferred aspect, the 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.

[0056] According to a preferred aspect of the present invention, 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 5xl06and 5xl08CFU (colony-forming units) per mL of solution and / or suspension.

[0057] 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.

[0058] 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)). The polymer liquid containing the material to be encapsulated (core) and the coating liquid (shell), going through the nozzle (3) of the encapsulator, forms a laminar liquid jet. 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. To prevent coalescence of the droplets during the flow rupture and / or when they enter the consolidation bath (8), a charge is induced on the droplet surface by using an electrostatic voltage system (4). Once formed at the nozzle outlet, the droplets are immersed in a gelling (or consolidation) bath (8) that allows them to consolidate with the formation of the aforementioned microcapsules. Such 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.

[0059] The consolidation occurs under mechanical stirring (for example, by means of a magnetic stirrer (9)) for a sufficient time, for example for 10 minutes. Subsequently, 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. Optionally and preferably, 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.

[0060] According to a possible alternative aspect of the present invention, 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.

[0061] In summary, 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. 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.

[0062] The “droplets" with different intern al / extemal composition that are obtained in step (b), exiting the concentric nozzle previously described, constitute the microcapsules according to the invention following the consolidation in the next step (c) of the process. In other words, the "droplets" are microcapsules that have not yet been consolidated.

[0063] The main and preferred process parameters, according to the present invention, are listed herein below:

[0064] - diameter size of the inner nozzle forming the core: from 80 to 750 pm, preferably 450 um;

[0065] - 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;

[0066] - flow rate of the solution and / or suspension forming the shell: from 14 mL / min to 30 mL / min, preferably 22.5 mL / min;

[0067] - vibration frequency: from 40 Hz to 6,000 Hz, preferably 1,000 Hz;

[0068] - electrode potential: from 250 V to 2,500 V, preferably 1,500 V.

[0069] 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.

[0070] The present invention, as will also be demonstrated by the Experimental Section that follows, has a number of advantages over the known art.

[0071] In particular, the microcapsules of the invention, containing probiotic microorganisms, are prepared easily, reproducibly and at low7cost. 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.

[0072] Furthermore, the 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. Experimental section

[0073] / . Formulations according to the present invention

[0074] Example 1.1

[0075] Composition of the suspension that will constitute the coating sheik

[0076] (i) E1206 (trade name Eudraguard®), 30% w / v aqueous suspension;

[0077] (ii) sodium alginate: viscosity 500-650 cPs 1% w / v in water at 25°C, with a mannuronic acid to glucuronic acid (M / G) ratio between 1.8 and 2.2, 2% w / v aqueous solution;

[0078] (iii) inulin from chicory.

[0079] The components (i) and (ii) are mixed together in a 2: 1 weight ratio to obtain 3 grams in total dispersed in a volume of about 57 mL of water.

[0080] To this suspension the component (iii) equal to 40% by weight of components (i) and (ii), i.e. 1 .2 g is added.

[0081] The suspension prepared for the shell was sterilized at 121 °C for 15 min. Composition of the suspension that, will constitute the core:

[0082] (a) sodium alginate with low viscosity with a mannuronic acid to glucuronic acid (M / G) ratio of 1.56; 0.5% w / v aqueous solution (viscosity at 1% v / v in water at 25°C: 4-12 cPs); component (a) being sterilized in autoclave at 121°C for 15 minutes;

[0083] (b) mannitol, in amounts such that its final concentration is 3%;

[0084] (c) Lactiplantibacillus plantarum, about I xlO10CFU; to form an aqueous suspension equal to 20 mL.

[0085] Example 1.2

[0086] Composition of the suspension that will constitute the coating shell:

[0087] (i) E1206 (trade name Eudraguard®), 30% w / v aqueous suspension;

[0088] (ii) sodium alginate: viscosity 500-650 cPs 1% w / v in water at 25°C, with a mannuronic acid to glucuronic acid (M / G) ratio between 1.8 and 2.2, 2% w / v aqueous solution;

[0089] (iii) inulin from chicory.

[0090] The components (1) and (ii) are mixed together in a 1 : 1 weight ratio to obtain 3 grams in total in a volume of about 80 mL of water. To this suspension the component (iii) equal to 50% by weight of components (i) and (ii), i.e. 1.5 g is added.

[0091] The suspension prepared for the shell was sterilized at 121 °C for 15 min.

[0092] Composition of the suspension that will constitute the core:

[0093] (a) sodium alginate with low viscosity with a mannuronic acid to glucuronic acid (M / G) ratio of 1.56; 0.5% w / v aqueous solution (viscosity at 1% v / v in water at 25°C: 4-12 cPs); component (a) being sterilized in autoclave at 121 °C for 15 minutes;

[0094] (b) trehalose, in amounts such that its final concentration is 3%;

[0095] (c) Lactiplantibacillus plantarum, about 1 - IO10CFU; to form an aqueous suspension equal to 20 mL.

[0096] Example 1.3

[0097] Composition of the suspension that will constitute the coating shell:

[0098] (i) E1206 (trade name Eudraguard®), 30% w / v aqueous suspension;

[0099] (ii) sodium alginate: viscosity 500-650 cPs 1% w / v in water at 25°C, with a mannuronic acid to glucuronic acid (M / G) ratio between 1.8 and 2.2, 1.5 % w / v aqueous solution;

[0100] (iii) inulin from chicory.

[0101] The components (i) and (ii) are mixed together in a 3: 1 weight ratio to obtain 4 grams in total in a volume of about 77 mL of water.

[0102] To this suspension the component (iii) equal to 50% by weight of components (i) and (ii), i.e. 2 g is added.

[0103] The suspension prepared for the shell was sterilized at 121 °C for 15 min.

[0104] Composition of the suspension that, will constitute the core:

[0105] (a) sodium alginate with medium viscosity with a mannuronic acid to glucuronic acid (M / G) ratio of 1.56, 0.3 % w / v aqueous solution (viscosity at 1% v / v in water at 25°C: 150 cPs); component (a) being sterilized in autoclave at 121°C for 15 minutes;

[0106] (b) trehalose, in amounts such that its final concentration is 3%;

[0107] (c) l.ai iipisiniihncilhss plantarum + Bifidobacteriiim. spp. (lx IO10CFU + 1 x 10sCFU); to form an aqueous suspension equal to 20 mL.

[0108] Example 1.4

[0109] Composition of the suspension that will constitute the coating shell:

[0110] (i) El 206 (trade name Eudraguard^), 30% w / v aqueous suspension;

[0111] (ii) sodium alginate: viscosity 500-650 cPs 1% w / v in water at 25°C, with a mannuronic acid to glucuronic acid (M / G) ratio between 1.8 and 2.2, 3 % w,zv aqueous solution;

[0112] (iii) beta-glucans from oats.

[0113] The components (i) and (ii) are mixed together in a 2: 1 weight ratio to obtain 3 grams in total in a volume of about 40 mL of water.

[0114] To this suspension the component (iii) equal to 40% by weight of components (i) and (ii), i.e. 1.2 g is added.

[0115] The suspension prepared for the shell was sterilized at 121 °C for 15 min. Composition of the suspension that will constitute the core:

[0116] (a) sodium alginate with medium viscosity with a mannuronic acid to glucuronic acid (M / G) ratio of 1.56; 0.5 % w / v aqueous solution (viscosity at 1% v / v in water at 25 °C: 150 cPs), component (a) being sterilized in autoclave at 121 °C for 15 minutes;

[0117] (b) mannitol, in amounts such that its final concentration is 3%;

[0118] (c) Lactiplantibacillus plantarum 1x1010CFU; to form an aqueous suspension equal to 20 mL.

[0119] Example 1.5

[0120] Composition of the suspension that will constitute the coating shell:

[0121] (i) El 206 (trade name Eudraguard1®), 30% w / v aqueous suspension,

[0122] (ii) sodium alginate: viscosity 500-650 cPs 1% w / v in water at 25°C, with a mannuronic acid to glucuronic acid (M / G) ratio between 1.8 and 2.2, 2 % w / v aqueous solution;

[0123] (iii) inulin from chicory.

[0124] The components (i) and (ii) are mixed together in a 2: 1 weight ratio to obtain 3 grams in total in a volume of about 57 ml,.

[0125] To this suspension the component (iii) equal to 40% by weight of components (i) and (ii), i.e. 1.2 g is added.

[0126] The suspension prepared for the shell was sterilized at 121°C for 15 min.

[0127] Composition of the suspension that will constitute the core:

[0128] (a) sodium alginate with low viscosity with a mannuronic acid to glucuronic acid (M / G) ratio of 1.56; 0.5% w / v aqueous solution (viscosity at 1% v / v in water at

[0129] 25 °C; 4-12 cPs); component (a) being sterilized in autoclave at 121 °C for 15 minutes;

[0130] (b) mannitol, in amounts such that its final concentration is 3%;

[0131] (c) Lactobacillus 4S6R, circa 1 > < 10i0CFU; to form an aqueous suspension equal to 20 ml...

[0132] 2. Process conditions

[0133] The preparation of the microcapsules having the formulation of the solutions / suspensions according to examples 1.1-1 .5 in the previous section involves the prilling / vibration technique by using the Encapsulator B395-Pro (BUCHI) apparatus with the following process parameters:

[0134] The resulting microcapsules are kept in the consolidation bath under mechanical agitation for 10 minutes, filtered using a paper filter, washed with distilled water, frozen at - 20°C and finally freeze-dried. 5. Characterization of the microcapsules

[0135] The microcapsules prepared according to the paragraphs 1 and 2 above were obtained with high production yield (> 91.5 %) and encapsulation efficiency in the range between 90 and 98%. The viability of the probiotics encapsulated in them remained high, comparable to that of the probiotics before the prilling / vibration treatment.

[0136] The dimensions of the microcapsules were measured by optical microscopy (Inverted Laboratory Microscope Optech IB4 optical microscope equipped with 135 camera and interfaced with Capture 2.1 image analysis program). The average diameter of the microparticles obtained with the 450 / 750 pm nozzle (inner / outer) was 850 ± 95 pm, thus confirming the capability of the technique to produce particles with a narrow particle size range.

[0137] The optical and scanning electron microscope (SEM) images, examples of which are shown in Figure 2, demonstrate that the microcapsules have an essentially spherical shape, and in cross section, the typical core-shell structure is clearly discernible.

[0138] To verify the ability of the microparticles according to the present invention to protect the probiotic microorganisms from the hostile environment of the gastrointestinal tract, and in particular the gastric environment, in vitro tests were performed in simulated fluids (for 2 hours in the gastric environment at pH 1.2 and for the remaining hours in an intestinal environment at pH 6.8 in the presence of bile salts), by using the lactobacilli strains LB931 and 4S6R. The results are shown in the graphs in Figure 3, which demonstrate the ability of the microcapsules to maintain the viability level of the strains they contain at higher levels than the microorganisms in their free condition. As a matter of fact, the viability of the encapsulated strains remains almost unchanged at pH 1.2 for two hours, whereas it drops by one or two logarithmic orders of magnitude when they are in the free form. The same change is found when they are exposed to the simulated intestinal fluid (pH 6.8 in the presence of bile salts).

[0139] Furthermore, a release study carried out in simulated fluids as previously described (i.e., gastric fluid at pH 1.2 for two hours, followed by intestinal fluid at pH 6.8 in the presence of bile salts for the next 4 hours) followed by additional 18 hours, for a total of 24 hours of release in a simulated colonic environment, i.e., in the presence of bacterial strains naturally occurring in this intestinal tract, which are capable of fully degrading the microcapsules of the invention, established how said microcapsules are capable of releasing the probiotics contained inside them at the level of the desired target site: the colon (Figure 4).

[0140] Finally, stability studies were performed on the finished freeze-dried product at 4°C and 25°C, which demonstrated that high viability levels of the probiotic strain LB931 contained in the microcapsules for six months, especially when stored at 4°C, have been maintained.

[0141] Thermal stress studies, the results of which are shown in Figure 5, performed at 65°C for 30 minutes and 90°C for 1 minute, finally demonstrate the ability of the microcapsules according to the present invention to protect the viability of the probiotic strain LB931 contained in them, even during possible necessary' production steps of fortified foods in which said probiotics were to be added.

[0142] The free probiotics show indeed a significant reduction in viability when subjected to a temperature of 65°C for 30 minutes compared with the micro-encapsulated probiotics, whose viability remains virtually unchanged instead. Even more interesting is the result related to the treatment at 90°C following which the free probiotics have zero viability, in contrast to the micro-encapsulated probiotics that maintain the level of viability practically unchanged from that before the heat treatment.

Claims

CLAIMS1. Microcapsules characterized in that they comprise a shell comprising or, alternatively, consisting of: i. a neutral, or positively or negatively charged, copolymer of methacrylic acid; ii. alginic acid or a salt thereof, preferably sodium alginate; and iii. at least one prebiotic; and characterized in that they comprise a core comprising or, alternatively, consisting of: iv. alginic acid or a salt thereof, preferably sodium alginate; v. a cryoprotectant; and vi. at least one probiotic microorganism.

2. Microcapsules according to claim 1, wherein said copolymer of methacrylic acid (i) is a neutral aery late copolymer, preferably is the compound designated by the international food additives abbreviation E1206.

3. Microcapsules according to any one of the preceding claims, wherein said at least one prebiotic (iii) belongs to the category' of fructose polymers, preferably inulin.

4. Microcapsules according to any one of the preceding claims, wherein said cryoprotectant (v) is selected from trehalose, mannitol and sorbitol.

5. Microcapsules according to any one of the preceding claims, wherein said probiotic microorganism (vi) is selected from one or more microorganisms belonging to the genera lactobacillus (basionym), Bifidobacterium, or mixtures thereof.

6. A preparation method for the production of microcapsules, according to any one of the preceding claims, by the prilling / vibration technique, comprising the following steps: a. preparing two aqueous suspensions and / or solutions by dissolving or suspending in water, for the one that will 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. introducing the two solutions prepared in step (a) into a prilling / vibration apparatus equipped with a concentric nozzle to which a specific vibration frequency and a specific electric field are applied, thus obtaining droplets withdifferent internal / external composition; c. immersing the drops produced in step (b) in a consolidation bath under mechanical stirring, filtering them, washing them with distilled water, thus obtaining 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.

7. The method according to claim 6, characterized in that:- the diameter size of the inner nozzle forming the core is from 80 to 1000 gm, preferably 450 pm;- the diameter size of the outer nozzle forming the shell is from 200 to 900 pm, preferably 700 pm;- the flow rate of the solution and / or suspension forming the core is from 1.5 mL / min to 6 mL / min, preferably 3.15 mL / min;- the flow rate of the solution and / or suspension forming the shell is from 14 mL / min to 30 mL / min, preferably 22.5 mL / min;- the vibration frequency is from 40 Hz to 6,000 Hz, preferably 1,000 Hz;- the electrode potential is from 250 V to 2,500 V, preferably 1,500 V;- the consolidation solution is constituted by: 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; or it is constituted by liquid nitrogen.

8. The method according to claim 6 o 7, characterized in that, in said aqueous solution and / or suspension that will constitute the shell of the microcapsule, the components (i) and (ii) are present in a ratio by weight between 0.5: 1 and 3: 1, to each other 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, and that the prebiotic (iii) is present in said aqueous suspension and / or solution that will constitute the shell of the microcapsule in a percentage from 10% w / v to 90% w / v, preferably from 30% w / v to 60% w / v.

9. The method according to any one of claims 6-8, characterized in that, in said aqueous solution and / or suspension that will constitute the core of the microcapsule,alginic acid, or the salt thereof, (iv) is present in an percentage between 0,05% w / v and 1% w / v, the cryoprotectant (v) is present in a percentage between 2% w / v and 5% w / v and the probiotic microorganism (vi) is present in an amount between about 5x106and 5x108CFU (colony-forming units) per ml of solution and / or suspension.

10. Use of the microcapsules according to claim 1 for the preparation of pharmaceutical formulations, nutraceutical formulations, medical devices and / or functional food.

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