Granular bacteria gastroprotected with a coating matrix in crystalline form, method for the production thereof and compositions thereof

Abstract

To provide a bacterial strain for both probiotic products and live biotherapeutic products (LBP) gastroprotected with a coating matrix that maintains the viability and functionality it possessed prior to the gastroprotection process.SOLUTION: There is provided a granular bacterium gastroprotected with a coating matrix having a crystalline structure, wherein the coating matrix comprises or consists of at least one lipid, wherein the at least one lipid has a lamellar arrangement having a crystalline structure, preferably a multi-lamellar arrangement.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to crystalline gastroprotective granular bacteria, such as bacterial strains in granular form protected with a preferably reduced amount of a lipid coating matrix, wherein the lipid coating matrix has a crystalline morphology. The present invention also relates to methods for producing the crystalline gastroprotective granular bacteria. Finally, the present invention relates to compositions comprising the crystalline gastroprotective granular bacteria. [Background technology]

[0002] Probiotic bacterial strains are defined as "viable microorganisms that, when administered in adequate amounts, confer a health benefit to the host." To perform their beneficial effects, viable bacterial strains present in probiotic products or live biotherapeutic products (LBPs) (medicinal products containing viable bacterial strains) must, upon oral ingestion, pass through the stomach in a viable state and reach the intestine to colonize and function. To protect bacterial strains from the acid pH environment of the stomach, it is known to coat bacterial strains with a variety of gastroprotective matrices, such as lipid coating matrices.

[0003] To perform its beneficial effects, the probiotic or viable bacterial strain must also remain effective throughout the shelf life of the product until consumption. Stability is primarily compromised by temperature and humidity, therefore, optimal control of temperature and protection from humidity can help maintain the probiotic or viable bacterial strain under optimal conditions for full efficacy.

[0004] Marino et al., Journal of Functional Foods, 35 (2017), reported a study on the effect of an emulsion structure containing saturated monoglycerides on the viability of probiotics during cold storage for up to 56 days. This paper describes the preparation of probiotic Lactobacillus rhamnosus by first freeze-drying and then mixing with a liquid mixture containing a lipid phase (sunflower oil), an aqueous phase, and a monoglyceride (MG) cosurfactant (CO). However, although the bacterial strain is freeze-dried, it is not in granular form. Furthermore, the method used involves the use of a liquid mixture that may impair the viability and / or functionality of the bacteria.

[0005] Document CN109480038 describes a method for producing a temperature-resistant product containing probiotics and chocolate. However, this document does not describe the bacteria in granular form being coated with a lamellar crystal structure, nor does it describe a method that makes it possible to obtain such a coating without compromising the viability of the bacteria.

[0006] In any case, the gastroprotective process may result in a reduction in the viability and / or functionality of the coated bacterial strains for various reasons: for example, heat tempering (or fat ripening) of the lipid coating matrix generally reduces the viability and / or functionality of the bacteria given that the bacterial strains are heat-labile. Summary of the Invention [Problem to be solved by the invention]

[0007] The technical problem addressed and solved by the present invention is a method for the preparation of bacterial strains for both probiotic products and live biotherapeutic products (LBPs) (i.e., the method of the present invention) gastroprotected with a coating matrix, which is accompanied by a low mortality rate of the bacterial cells (i.e., maintenance of membrane integrity), and therefore maintains the viability and functionality that the cells had before the gastroprotection process. At the same time, the technical problem addressed by the present invention is the provision, by means of the method of the present invention, of a bacterial strain or a composition (probiotic product or LBP) containing the same, enriched in viable and functional bacteria, wherein the coating matrix provides efficient gastroprotection and / or protection from moisture buildup after opening, which involves unavoidable exposure to moisture and / or environmental humidity due to residual moisture and / or primary packaging that is not completely moisture-impermeable in the finished product form. [Means for solving the problem]

[0008] In light of the above technical challenges and subsequent intensive research and development, in the present invention, the applicant provides a method (gastroprotection method, briefly the method of the present invention), as described below and claimed in the claims, which essentially comprises the steps of (I) granulating a "naked" (uncoated) bacterial strain to obtain a granular bacterial strain, (II) coating the granular bacterial strain with a preferably reduced amount of a lipid coating matrix to obtain a gastroprotective granular bacterial strain per se (or naked or uncoated), and (III) tempering (or maturing) the gastroprotective granular bacterial strain per se to obtain a granular bacterium gastroprotected with a coating matrix in crystalline form.

[0009] In the context of the present invention, the terms "naked", "naked" or "uncoated" in relation to bacterial strains or granular bacterial strains are synonymous in nature and can be used interchangeably. All these terms refer to uncoated or non-microencapsulated bacterial strains or granular bacterial strains.

[0010] The method of the present invention provides bacteria in the form of granules gastroprotected with a lipid coating matrix in crystalline form (low lethal treatment) with a quantity of viable and functional bacterial cells that is approximately the same as that present before the gastroprotection method.

[0011] In other words, the method of the invention makes it possible to granulate, coat and temper bacteria without killing the cells, as assessed—for example—by means of flow cytometry (or flow cytofluorometry).

[0012] Advantageously, the method of the invention performs each step in the absence of a solvent and / or aqueous phase, which allows for almost zero lethality of the bacterial strain.

[0013] The low or near zero lethality of the bacterial strains during the granulation, coating and tempering steps of the coating method of the present invention preserves their viability and functionality, thus allowing for the preparation of products (compositions) containing the crystalline gastroprotective bacterial strains with increased viable and functional bacteria, and therefore allowing for cost-effective methods of producing gastroprotective bacteria and products containing same.

[0014] Advantageously, the crystalline structure of the lipid coating matrix obtained due to the presence of the tempering step of the method of the present invention confers on the gastroprotective granular bacteria and products containing said bacteria a high resistance to delivery of the active ingredient (i.e., probiotic or viable bacteria) when administered by oral route, which equates to a high gastric resistance and a high stability over a long period of time (i.e., quantitatively, a long shelf life and long-term stability).

[0015] Advantageously, the crystalline lamellar structure of the lipid coating makes it possible to obtain a coating structure with a more stable structure in terms of gastroresistance and resistance to humidity and temperature during storage (shelf life).

[0016] Furthermore, the crystalline structure of the lipid coating matrix, which confers greater resistance to delivery of the active ingredient (ie, probiotic or viable bacteria), enhances the prolonged delivery of the active ingredient to the intestine over time.

[0017] In this specification, the expression lamellar structure is used to denote a spatial arrangement of lipids that corresponds to a molecular structure of lamellae, with the lipid chains being more or less perpendicular to the plane of the lamellae.

[0018] As used herein, lateral structure refers to the 2D structure of the molecules within the lamella.

[0019] The presence of a Bragg peak indicates a long lamellar structure and allows the lamellar pitch to be calculated.

[0020] Various types of lateral ordering, such as fluid and crystals with more than one type of packing, can coexist within a lamellar structure.

[0021] Advantageously, the method of the invention makes it possible to obtain lipid coating structures with a crystalline lamellar structure, which is reflected in a more stable structure in terms of gastroresistance and resistance to humidity and temperature.

[0022] Finally, the method of the present invention is preferably carried out by providing gastroprotective bacteria and products comprising said gastroprotective bacteria, which can be carried out using a coating matrix comprising reduced amounts of lipids, preferably lipids of vegetable origin, as defined in the present invention, and which falls within the limits set by authorities for the regulation of human consumption of products, in particular pediatric products.

[0023] Finally, the bacteria, compositions, mixtures and processes of the present invention are easy to source and cost effective.

[0024] These and other objects that will become apparent from the following detailed description are achieved by the bacterial strains, compositions and mixtures of the present invention by virtue of the technical features claimed in the accompanying claims. [Brief explanation of the drawings]

[0025] [Figure 1] Schematic of X-ray diffraction analysis; [Figure 2] WAXS images of the tested samples; [Figure 3] SAXS images of the tested samples; [Figure 4] WAXS and SAXS analysis pattern charts of the tested samples; [Figure 5] WAXS and SAXS analysis pattern charts of the tested samples; [Figure 6] Showing the spatial arrangement of lipids in the "lamellar phase"; [Figure 7] exhibiting lipid lamellae with a fluid or crystalline structure; [Figure 8] 8 are SEM images of the samples tested; in particular, FIG. 8 is an SEM image of Sample 1 at various magnifications. [Figure 9] 9 are SEM images of the samples tested; in particular, FIG. 9 is an SEM image of Sample 1 at various magnifications. [Figure 10] 10 are SEM images of the samples tested; in particular, FIG. 10 is an SEM image of Sample 4 at various magnifications. [Figure 11] 11 are SEM images of the samples tested; in particular, FIG. 11 is an SEM image of Sample 4 at various magnifications. [Figure 12] 12 are SEM images of the samples tested; in particular, FIG. 12 is an SEM image of Sample 5 at various magnifications. [Figure 13] 13 are SEM images of the samples tested; in particular, FIG. 13 is an SEM image of Sample 5 at various magnifications. [Figure 14] 14 are SEM images of the samples tested; in particular, FIG. 14 is an SEM image of Sample 6 at various magnifications. [Figure 15] 15 are SEM images of the samples tested; in particular, FIG. 15 is an SEM image of Sample 6 at various magnifications. [Figure 16] shows the measurement of the viability of naked bacterial strains and coated bacterial strains of the present invention in vegetable oil at controlled temperature and humidity (30°C-75%RH) for a time range including 0-12 months; [Figure 17] Figure 1 shows the measurement of viability of naked bacterial strains in vegetable oil and coated bacterial strains of the invention in vegetable oil at controlled temperature and humidity (40°C-75% RH) for a time range encompassing 0-60 days. DETAILED DESCRIPTION OF THE INVENTION

[0026] Detailed Description of the Invention Forming the object of the present invention are granular bacteria gastroprotected with a coating matrix having a crystalline structure (briefly crystalline gastroprotected granular bacteria of the invention or bacteria of the invention), wherein said bacteria belong to at least one strain or mixture of strains of bacterial cells (probiotic or viable bacteria) belonging to the genera and species described in the present invention, and wherein said coating matrix comprises or consists of at least one lipid, preferably of plant origin, as described in the present invention, wherein said at least one lipid preferably has a lamellar arrangement with a crystalline structure, more preferably a multilayered crystalline structure-like lamellar arrangement.

[0027] Furthermore, forming an object of the present invention are granular bacteria gastroprotected with a coating matrix having a crystalline structure, wherein said coating matrix comprises or consists of at least one lipid, wherein said at least one lipid has a lamellar arrangement with a crystalline structure, preferably a multilayered crystalline-like lamellar arrangement.

[0028] In the context of the present invention, the terms bacteria, bacterial strain, bacterial cell and bacterial strain cell are synonymous and are used interchangeably.

[0029] In the context of the present invention, the terms gastroprotective, coating and covering are synonymous and are used interchangeably. These terms indicate that the bacteria are coated with a covering that makes it possible to protect the bacterial strain from the acidic environment of the stomach.

[0030] The at least one lipid, preferably of plant origin, - mono-, di- or triglycerols (i.e. monoglycerides, diglycerides and / or triglycerides) esterified with saturated or unsaturated fatty acids (preferably monounsaturated), preferably esterified with saturated fatty acids, more preferably mono- and diglycerols esterified with saturated fatty acids having a carbon number ranging from C16 to C18 and / or mono-, di- or triglycerols esterified with saturated fatty acids having a carbon number ranging from C16 to C22; - free saturated fatty acids; - free unsaturated fatty acids, preferably monounsaturated; - mono-alcohols esterified with saturated or unsaturated fatty acids (preferably monounsaturated), preferably with saturated fatty acids; - di-alcohols esterified with saturated or unsaturated fatty acids (preferably monounsaturated), preferably esterified with saturated fatty acids; - sucrose fatty acid esters (also known as sugar esters), preferably mixtures of mono-, di- or tri-sucrose fatty acid esters, more preferably mainly sucrose esters of stearic and / or palmitic acid; wherein the saturated or unsaturated fatty acids, both free and esterified with glycerol or mono- or di-alcohols or sucrose, have a number of carbon atoms in the range of C6 to C32, preferably C12 to C28, more preferably C14 to C24, for example C16, C18, C20 and / or C22.

[0031] In the present invention, the terms sucrose fatty acid ester or saccharide ester or sucrose ester are synonymous and are used interchangeably. The sucrose ester is preferably a sucrose fatty acid ester of stearic acid (C 18 H 36 OR2) and / or palmitic acid (C 16 H 32 The sucrose esters are preferably mixtures of mono-, di-, and / or tri-fatty acid esters of fatty acids having carbon atoms falling within the range of C6 to C32, preferably C12 to C28, more preferably C16 to C18, such as sucrose esters (OR2). Sucrose esters are obtained by esterification of fatty acids or by transesterification of methyl fatty acid esters with sucrose. The chemical-physical properties of the sucrose esters depend on the number and type of esterified fatty acids.

[0032] In an embodiment of the present invention, the at least one lipid is a free saturated fatty acid, preferably of vegetable origin, selected from saturated fatty acids having a melting point comprised between 35°C and 85°C, preferably between 45°C and 70°C, more preferably between 50°C and 60°C.

[0033] In a preferred embodiment of the invention, the at least one lipid is - lipids (I): for example, glyceryl dipalmitostearate E471, related to CAS No. 85251-77-0 (or 1323-83-7), EINECS: 286-490-9 (or 215-359-0), REACh (EC) no. 1907 / 2006: Exemption (food), IUPAC name "Glycerides, C16-C18 mono-di-", INCI (PCPC): Glyceryl distearate; commercial example: Biogapress Vegetal BM297 ATO (E471) manufactured by Gattefosse SAS; physical state: powder; melting point range: 53.00-58.00 °C; boiling point: >250.0 °C; flash point: >200.0 °C; ignition temperature (autoignition): >350.00 °C; - lipids (ii): for example glyceryl palmitostearate E471 / gras, related to CAS No. 85251-77-0 (or 31566-31-1 or 123-94-4); EINECS: 286-490-9 (or 250-705-4 or 204-664-4); REACh (EC) no. 1907 / 2006 01-2119495562-30-0014, IUPAC name "Glycerides, C16-C18 mono-, di-", INCI (CTFA): glyceryl stearate; commercial example: GELEOL N MB, manufactured by Gattefosse SAS; physical state: solid; flash point: >200.0 °C DIN 51376; Ignition temperature (autoignition): >350.00°C; Vapor pressure: 0.0100 mbar at 20.00°C (briefly lipid(ii)); - Lipids (iii): Glyceryl dibehenate E471 / GRAS, related to CAS No: 77538-19-3 (or 91052-55-0) (or 30233-64-8) (or 94201-62-4), EINECS: 278-717-5 (or 293-216-1) (or 250-097-0) (or 303-650-6), REACh (EC) no. 1907 / 2006: Exemption (food); IUPAC name "Glycerides, C16-22 mono-, di- and tri-", INCI (PCPC): Glyceryl behenate; Physical state: powder; Flash point: >200.0°C; Ignition temperature (autoignition): >350.00°C; Vapor pressure: 0.0100 mbar at 20.00°C; Examples of commercially available products: COMPRITOL E ATO or COMPRITOL E ATO FPF manufactured by Gattefosse SAS; Lipid (iv): sucrose esters or a mixture of sucrose fatty acid esters E-473 having the following composition: at least 80.0% mono-, di-, and tri-esters (of which sucrose monopalmitate is about 55%, sucrose dipalmitate is about 20%, sucrose monostearate is about 13%, sucrose distearate is about 5%, and others are less than 10%), free sugars do not exceed 4.0%, free fatty acids do not exceed 3% (of which palmitic acid is about 75%, stearic acid is about 20%, and other fatty acids are about 5%), fatty acid / carbohydrate composition is about 1 / 1, preferably about 52 / 48, % by weight relative to the total weight of lipid (iv); an example of a commercially available product is Ryoto Sugar Ester P-1570 manufactured by Mitsubishi Chemical Foods Corporation, Japan; Lipids (v): sucrose esters or mixtures of sucrose fatty acid esters having the following composition: mono-, di-, and tri-esters at least 80.0% (of which sucrose monostearate is about 15%, sucrose distearate is about 22%, sucrose tristearate is about 20%, sucrose dipalmitate is about 10%, sucrose polystearate is about 30%), and other suitable amounts, which are identified as CAS Nos. 25168-73-4, 27195-16-0, 27923-63-3, and 25637-97-2, respectively, and EINECS: 246-705-9, 248-317-5, 248-731-6 and 247-147-9), free sugars not exceeding 4.0%, free fatty acids not exceeding 3% (of which stearic acid is about 90% and other fatty acids about 10%), fatty acid / carbohydrate composition about 60 / 40, weight % of lipids (v) relative to the total weight; an example of a commercially available product is Ryoto Sugar Ester-370 manufactured by Mitsubishi Chemical Foods Corporation, Japan, which has the following characteristics: physical state: powder; melting point: 51°C (onset) to 58°C and 69°C (max) (DSC); decomposition point: 238°C; ignition temperature (autoignition): about 392°C / 200°C (SETA); specific gravity: about 0.46; or An example of a commercially available product is SURFHOPE SE COSME C-1803 manufactured by Mitsubishi Chemical Foods Corporation, Japan, which has the following characteristics: physical state: powder; melting point: 51°C (onset) to 61°C (maximum) (DSC); decomposition point: 260°C; ignition temperature (autoignition): approximately 224°C; specific gravity: approximately 0.46; - Lipids (vi) Polyglyceryl-6-distearate (or hexaglycerol diester and stearic acid) E475, identified by CAS No. 34424-97-0, INCI name: Polyglyceryl-6-distearate, molecular formula C 54 H 106 O 15 a commercial example is Plurol® Stearique WL 1009 (briefly lipid (vi)) manufactured by Gattefosse SAS; and mixtures thereof Group B (a subgroup of Group A) comprising or consisting of:

[0034] In another embodiment, said lipid group B comprises or consists of said lipid (i), lipid (ii), lipid (iii), lipid (iv) and lipid (v).

[0035] In a preferred embodiment of the present invention, the at least one lipid, preferably of plant origin, is selected from Group B1 (a subgroup of Group B) comprising or consisting of the lipid (i), the lipid (ii), the lipid (iii), and a mixture thereof. Alternatively, Group B1 comprises or consists of the lipid (i), the lipid (iii), and a mixture thereof; or, Group B1 comprises or consists of the lipid (ii), the lipid (iii), and a mixture thereof.

[0036] In a preferred embodiment of the present invention, the at least one lipid, preferably of plant origin, is selected from group B2 (a subgroup of group B), which comprises or consists of lipid (iv), lipid (v) and mixtures thereof.

[0037] In an embodiment of the invention, the at least one lipid comprises at least one first lipid, wherein the first lipid is a mono-, di- or triglycerol esterified with a saturated or unsaturated fatty acid (e.g., monounsaturated), preferably a saturated fatty acid (i.e., a monoglyceride, diglyceride or triglyceride), more preferably a saturated fatty acid having a carbon number between C6 and C32, preferably between C14 and C24, more preferably between C16, C18, C20 and / or C22; and further comprises at least one second lipid, wherein the second lipid is a sucrose fatty acid ester (sucrose ester) as defined herein, preferably a mixture of mono-, di- or tri-sucrose fatty acid esters; wherein the fatty acid esterified with glycerol or sucrose has a carbon number between C6 and C32, preferably between C14 and C24, more preferably between C16, C18, C20 and / or C22.

[0038] Advantageously, the at least one lipid comprises at least one first lipid selected from Group B1, which comprises or consists of the lipid (i), the lipid (ii), the lipid (iii) and a mixture thereof; and further comprises at least one second lipid selected from Group B2, which comprises or consists of the lipid (iv), the lipid (v) and a mixture thereof. For example, the lipid comprises the following lipids: (i) and (iv) or (i) and (v) or (ii) and (iv) or (ii) and (v) or (iii) and (iv) or (iii) and (v) or (i) and (ii) and (iv) or (i) and (ii) and (v) or (i) and (iii) and (iv) or (i) and (iii) and (v) or (ii) and (iii) and (iv) or (ii) and (iii) and (v) or (i) and (iv) and (v) or (ii) and (iii) and (iv) or (ii) and (iii) and (v) or (i) and (iv) and (v) or (ii) and (iv) and (v) or (ii) and (iii) and (iv) or (ii) and (iii) and (v) or (i) and (iv) and (v) or (ii) and (iv) and (v) or (ii) and (iv) and (v).

[0039] The initials E471, E473 and E476 indicate that the respective glycerides or sucrose esters are food additives approved by European Union law and regulated by Italian legislative decree (DM 1996).

[0040] It is understood that the lipid, preferably of plant origin, contained in the coating matrix is ​​selected according to the intended use of the bacterium or composition of the invention, the chemical-physical properties of further components optionally contained in the coating matrix and additives and / or excipients optionally contained in the composition of the invention, and the physical state of the composition of the invention.

[0041] Advantageously, the gastroprotected granular bacteria with a coating matrix having a crystalline structure according to the invention comprise or consist of (a) a weight percentage of bacteria (as such or naked or uncoated) comprised between 60% and 90% and (b) a coating matrix comprising or consisting of at least one lipid according to the various embodiments described herein (a lipid of group A, preferably a lipid of group B, more preferably a lipid of group B1 or a lipid of group B1 in combination with a lipid of group B2 according to the examples reported in the present invention) comprised between 10% and 40% by weight relative to the total weight of the gastroprotected granular bacteria; preferably 65% ​​to 85% bacteria and 15% to 35% coating matrix; more preferably 70% to 80% bacteria and 20% to 30% coating matrix.

[0042] The subject bacteria of the present invention, such as the bacteria of the present invention, the bacteria of the present invention contained in the compositions of the present invention and the bacteria of the present invention obtained by the methods of the present invention, comprise or consist of at least one bacterial cell strain or a mixture of different bacterial cell strains, said at least one strain or mixture of bacterial cell strains belonging to one or more families selected from the group comprising or consisting of Firmicutes, Actibacteria, Bacteroidetes, Proteobacteria and mixtures thereof. The at least one strain or mixture of bacterial cell lines may belong to one or more genera selected from the group consisting of Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, Akkermansia, Intestinimonas, Eubacterium, Faecalibacterium, Neisseria, Roseburia, Cutibacterium, and mixtures thereof. The at least one strain or mixture of bacterial cell lines may belong to one or more genera selected from the group consisting of Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus diffus ... buchneri, Lactobacillus fermentum, Lactobacillus salivarius subsp. salivarius, Lactobacillus crispatus, Lactobacillus paracasei subsp. paracaseiparacasei, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus rhamnosus, Lactobacillus pentosus, Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus casei, Lactobacillus reuteri reuteri, Lactobacillus johnsonii, Bifidobacterium adolescentis, Bifidobacterium animalis subsp. lactislactis, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacterium longum, Akkermansia munichipila, Intestinimonas butyriciproducens, Eubacterium hallii The bacteria belong to one or more species selected from the group consisting of or including: Faecalobacterium prausnitzii, Neisseria lactamica, Roseburia hominis, Cutibacterium acnes, and mixtures thereof.

[0043] The crystalline gastroprotective granular bacteria of the present invention may comprise or consist of a single bacterial strain or a mixture of bacterial strains belonging to the same or different species and / or genera described in the present invention; in particular it may be a mixture of 2, 3, 4, 5 or 6 different bacterial strains.

[0044] The crystalline gastroprotective granular bacteria of the present invention may contain a small percentage of bacteria that are not coated with the coating matrix.

[0045] The crystalline gastroprotective granular bacteria of the present invention are preferably in solid form, in particular in the form of granules, powder, dry powder or freeze-dried powder.

[0046] Forming an object of the present invention is a composition (briefly the composition of the invention) comprising a mixture of or consisting of granular bacteria gastroprotected with a coating matrix having a crystalline structure according to any of the embodiments of the present invention, optionally comprising at least one food-grade or pharmaceutical or cosmetic additive and / or excipient.

[0047] The composition of the present invention may be a pharmaceutical composition (living biotherapeutic product, LBP) or a medical device composition or a cosmetic composition, a dietary supplement or a food product (probiotic product) or a food for special medical purposes (FSMP) or a novel food.

[0048] Advantageously, the composition of the invention contains the bacterium of the invention at a concentration of 1 x 10 6 AFU / g ~ 1 x 10 14 AFU / g, preferably 1 × 10 7 AFU / g ~ 1 x 10 13 AFU / g, more preferably 1×10 8 AFU / g ~ 1 x 10 12 The composition may contain a concentration in the range of AFU / g, where AFU / g (AFU; active fluorescent units) is measured using a flow cytometry method as defined herein and refers to bacteria with complex cell membranes per gram of composition.

[0049] The composition of the present invention optionally comprises at least one pharmaceutical, food or cosmetic grade additive and / or excipient, i.e., a substance without therapeutic activity that is suitable for pharmaceutical, food or cosmetic use. In the context of the present invention, additives and / or excipients acceptable for pharmaceutical, food or cosmetic use include all auxiliary substances known to those skilled in the art for the preparation of compositions in solid, semi-solid or liquid form, such as, for example, diluents, solvents (including water, glycerin, ethyl alcohol), solubilizers, acidifiers, thickeners, sweeteners, flavor enhancers, colorants, lubricants, surfactants, preservatives, pH-stabilizing buffers and mixtures thereof.

[0050] The composition of the present invention comprising the crystalline gastroprotective granular bacteria of the present invention in various embodiments described herein may be a pharmaceutical composition or a medical device composition or a cosmetic composition or a dietary supplement composition or a food product composition or a food for special medical purposes (FSMP), all of which will be referred to for brevity as the "composition of the present invention."

[0051] In the context of the present invention, the expression "medical device" is used in the sense according to Italian Legislative Decree No. 46 of 24 February 1997 or the new Medical Device Regulation (EU) 2017 / 745 (MDR).

[0052] The compositions of the present invention may be in a solid form such as a chewable solid, granules, flakes or powder, a semi-solid form such as a soft gel or a liquid such as a solution, aqueous or hydroalcoholic or oily suspension, dispersion, emulsion or syrup.

[0053] For example, the composition of the present invention may be a suspension of granular bacteria gastroprotected with a coating matrix of the crystalline form of the present invention in an oily phase, preferably a vegetable oil.

[0054] Preferably, the compositions of the present invention are formulated for oral use.

[0055] Forming an object of the present invention is a method for preparing granular bacteria gastroprotected with a coating matrix having a crystalline structure according to any of the embodiments of the present invention (briefly the method of the present invention), comprising: (I) a step of granulating at least one strain or a mixture of bacterial cell lines (viable bacteria themselves or naked or uncoated) belonging to a bacterial species as defined herein, preferably in freeze-dried form, through a mesh comprised in the range of 50 μm to 900 μm to obtain granular bacteria; (II) coating the granular bacteria with a coating matrix comprising at least one lipid, preferably of vegetable origin, wherein the at least one lipid is selected from lipids of group A, preferably lipids of group B, more preferably lipids of group B1 or lipids of group B1 in combination with lipids of group B2 according to the examples reported in the present invention, to obtain gastroprotective granular bacteria as such; and (III) tempering (or maturing) the gastroprotective granular bacteria itself at a temperature comprised between 2°C and 60°C for a time comprised between 48 hours and 96 hours to obtain gastroprotected granular bacteria with a coating matrix having a crystalline structure, wherein the at least one lipid preferably adopts a lamellar arrangement having a crystalline structure, more preferably a multi-layered crystalline structure-like lamellar arrangement, after the tempering step (III). Includes:

[0056] In one embodiment, the method of the present invention comprising steps (I) to (III) further comprises, after step (III), step (IV) of carrying out a bacterial count on a sample of granular bacteria gastroprotected with a coating matrix having a crystalline structure obtained in step (III) using an analytical method, preferably a flow cytometry method as described below, which allows the detection of the amount of bacterial cells with a complex (and therefore viable) cell membrane.

[0057] Preferably, in the method of the present invention comprising steps (I) to (III) and, optionally, step (VI), in step (I), the bacteria are granulated using a mesh ranging from 100 μm to 600 μm, preferably from 150 μm to 500 μm, more preferably 180 μm or 450 μm.

[0058] Preferably, in the method of the present invention comprising steps (I) to (III) and optionally step (VI), in step (II), the granular bacteria and the coating matrix are treated at a weight ratio within the range of 6:4 to 9:1, preferably 6.5:3.5 to 8.5:1.5, more preferably 7:3 to 8:2.

[0059] Preferably, in the method of the present invention comprising steps (I) to (III) and optionally step (VI), in step (III), the gastroprotective granular bacteria itself is tempered at a temperature in the range of 30°C to 40°C, preferably at about 35±1°C.

[0060] In one embodiment of the method of the present invention, in step (I), the bacteria are granulated through a mesh ranging from 100 μm to 600 μm, preferably from 150 μm to 500 μm, more preferably from 180 μm or 450 μm; in step (ii), the granulated bacteria and the coating matrix are preferably coated with at least one lipid of group A, preferably at least one lipid of group B, more preferably at least one lipid of group B1 or at least one lipid of group B2 according to the examples reported in the present invention. The coating matrix containing at least one group B1 lipid is treated in a weight ratio ranging from 6:4 to 9:1, preferably from 6.5:3.5 to 8.5:1.5, more preferably from 7:3 to 8:2; in step (III), the microencapsulated bacteria are tempered at a temperature ranging from 30°C to 40°C, preferably at a temperature of about 35±1°C, for a time ranging from 60 hours to 84 hours, preferably about 72 hours; and, if desired, in step (IV), bacterial counting is performed using a flow cytofluorometry method.

[0061] In a preferred embodiment of the method of the present invention, in step (I), the bacteria are granulated through a 180 μm or 450 μm mesh; in step (II), the granulated bacteria and the coating matrix, preferably comprising at least one Group A lipid, preferably at least one Group B lipid, more preferably at least one Group B1 lipid or at least one Group B1 lipid in combination with at least one Group B2 lipid according to the examples reported in the present invention, are treated in a weight ratio of 7:3 to 8:2; in step (III), the microencapsulated bacteria are tempered at a temperature of about 35±1°C for a time of about 72 hours; and, optionally, in step (IV), bacteria counting is performed using a flow cytofluorometry method.

[0062] The step (II) of coating the granular bacteria with a coating matrix comprising at least one lipid, preferably of vegetable origin, wherein the at least one lipid is selected from lipids of group A, preferably lipids of group B, more preferably lipids of group B1 or lipids of group B1 in combination with lipids of group B2 according to the examples reported in the present invention, and the step of obtaining the gastroprotective granular bacteria themselves can be carried out by techniques known to those skilled in the art, such as, for example, spray coating (spraying the coating matrix onto the granular bacteria).

[0063] In a preferred embodiment of the method of the present invention, in step (II), the granular bacteria are coated with a coating matrix in a fluidized bed chamber (top spray or bottom spray) at a temperature comprised between 40°C and 60°C, more preferably up to 50°C, in a bacteria:coating matrix ratio comprised between 6.5:3.5 and 8.5:1.5, more preferably between 7:3 and 8:2; wherein the coating matrix comprises or consists of at least one Group A lipid, preferably at least one Group B lipid, more preferably at least one Group B1 lipid or at least one Group B1 lipid in combination with at least one Group B2 lipid according to the examples reported in the present invention.

[0064] In a preferred embodiment of the method of the present invention, in step (III), the gastroprotective granular bacteria itself is tempered by methods known to those skilled in the art.

[0065] After tempering the granular bacteria coated with a lipid matrix, also called fat ripening, the lipids adopt a spatial arrangement corresponding to the molecular structure of lamellae (lipid lamellae), in which the lipid chains are more or less perpendicular to the plane of the lamellae, as shown in Figure 6. When the lipids adopt this spatial arrangement, they are defined as "lamellar phase" lipids. In the leaf, there can be a fluid (no ordered structure) or crystalline (packing-type structure) structure, as shown in Figure 7. Furthermore, the lipid lamellae can adopt a stacked structure of lamellar-forming lipid multilayers.

[0066] The process of the present invention, which includes the tempering (or fat maturation) of step (III), results in the arrangement of the lipid coating matrix into lamellae having a crystalline structure, which lamellae preferably stack to form crystalline lipid multilayers, thus resulting in the preparation of gastroprotected granular bacteria with a lipid coating matrix in crystalline form according to the present invention.

[0067] Preferably, the flow cytometry analysis method used in step (IV) of the method of the present invention to detect the amount of bacterial cells with a complex (viable) cell membrane is the analysis method described on page 33, line 24 to page 35, line 17 of patent application IT102019000006056. Briefly, the flow cytometry analysis method comprises: (VI.I) contacting a sample of the gastroprotective granular bacteria having a coating matrix with a crystalline structure obtained from step (III) (briefly, a sample of the bacteria of the present invention) or a sample of the composition of the present invention comprising the bacteria of the present invention obtained from step (III) (briefly, a sample of the composition of the present invention) with two different fluorescent dyes to obtain a fluorescent sample of the gastroprotective bacteria or composition of the present invention; followed by (VI.II) detecting the amount of complex (and therefore viable) cell membranes in fluorescent samples of the gastroprotective bacteria or compositions of the present invention by means of flow cytometry. Includes:

[0068] In particular, in a flow cytometry method according to the method established by the ISO 19344:2015(E) standard, a first membrane-permeable dye (preferably: thiazole orange or SYTO® 24 - a fluorescent dye in the green spectrum) can be shaken into whole bacterial cells to provide total fluorescent units or cells (TFU) of a fluorescent sample of the gastroprotective bacteria or composition of the present invention, and a second dye (preferably: propidium iodide) can only be shaken into bacterial cells with damaged cell membranes to provide non-viable or non-viable fluorescent units or cells (nAFU) of a fluorescent sample of the gastroprotective bacteria or composition of the present invention.

[0069] Therefore, the amount of viable bacterial cells with complex cell membranes can be expressed as active fluorescent units or cells (AFU), i.e., units that are positive only for the first dye (preferably: thiazole orange or SYTO® 24) in the fluorometric assay, for which the following correlation can be applied: TFU = AFU + nAFU [During the ceremony, - TFU is total fluorescent bacterial units or cells; - nAFU are non-active fluorescent bacterial units or cells with uncomplexed or compromised cell membranes (ie, units that are positive for a second dye, preferably propidium iodide).

[0070] According to one embodiment, the flow cytometer is configured and / or calibrated to perform volume determinations on analytical samples containing bacteria of the present invention and to directly calculate cell concentrations (AFU and TFU).

[0071] Advantageously, to obtain AFU and TFU values ​​for a fluorescent sample of the gastroprotective bacteria or composition of the present invention, the flow cytofluorometer uses at least one internal fluorescent standard added to the fluorescent sample of the gastroprotective bacteria or composition of the present invention. In a preferred embodiment, the internal fluorescent standard is in the form of fluorescent spheres or beads and is added at a known concentration to each sample of the gastroprotective bacteria or composition of the present invention to be analyzed. The AFU and TFU of the analyzed fluorescent sample of the gastroprotective bacteria or composition of the present invention can then be calculated as a ratio to the known standard amount.

[0072] Forming an object of the present invention is the crystalline gastroprotective granular bacteria of the invention or the composition of the invention for use as a medicament in a subject in need of treatment.

[0073] The present invention relates to a method for preventive or curative or medical treatment comprising administering to a subject in need of treatment an effective amount of the crystalline gastroprotective granular bacteria of the present invention or the composition of the present invention.

[0074] It is the cosmetic use of the bacteria of the invention or of the compositions of the invention that forms the subject of the present invention.

[0075] In the context of the present invention, the expression "subject" is used to refer to a human subject or an animal subject (e.g., a pet such as a dog or cat or other mammal). Preferably, the compositions of the present invention are for use in a method of treating a human subject.

[0076] Unless otherwise specified, the expression "composition" includes components in an amount "between x and y," and the amount is used for compositions including the entire amount of the component present in the range, including both endpoints, even if not specified.

[0077] Unless otherwise specified, expressions that refer to the gastroprotective crystalline granular bacteria of the present invention or the compositions of the present invention contain a certain percentage of an ingredient are expressed as weight percentages based on the total weight of the gastroprotective crystalline granular bacteria or composition.

[0078] Furthermore, the following embodiment (FRan) is an object of the present invention:

[0079] FRa1. A granular bacterium gastroprotected with a coating matrix having a crystalline structure, wherein the coating matrix comprises or consists of at least one lipid, wherein the at least one lipid has a lamellar arrangement with a crystalline structure, preferably a multilayered crystalline-like lamellar arrangement.

[0080] FRa2. The at least one lipid mono-, di- or triglycerol esterified with saturated or unsaturated fatty acids, preferably saturated fatty acids; - free saturated fatty acids; - free unsaturated fatty acids, preferably monounsaturated; monoalcohols esterified with saturated or unsaturated fatty acids, preferably saturated fatty acids; - di-alcohols esterified with saturated or unsaturated fatty acids, preferably saturated fatty acids; - sucrose fatty acid esters (cane sugar esters), preferably mixtures of mono-, di- and / or tri-sucrose fatty acid esters; wherein the saturated or unsaturated fatty acids esterified with free or esterified glycerol or mono- or di-alcohols or sucrose have a number of carbon atoms ranging from C6 to C32, preferably C14 to C24, more preferably C16, C18 and / or C22.

[0081] FRa3. At least one lipid, preferably of plant origin, - at least one first lipid, which is a mono-, di-, or triglycerol esterified with a saturated or unsaturated fatty acid, preferably a saturated fatty acid; and - at least one second lipid, which is a sucrose fatty acid ester (sucrose ester), preferably a mixture of mono-, di- and / or tri-sucrose fatty acid esters; wherein the fatty acids esterified with glycerol or sucrose have a carbon number ranging from C6 to C32, preferably C14 to C24, more preferably C16, C18, C20 and / or C22.

[0082] FRa4. The bacterium of any one of claims 1 to 3, wherein: - the bacteria itself is present in a weight percentage ranging from 60% to 90%; and - a coating matrix comprising or consisting of at least one lipid in a weight percentage comprised between 10% and 40% relative to the total weight of the gastroprotective granular bacteria; preferably between 65% and 85% of the bacteria itself and between 15% and 35% of the coating matrix; more preferably between 70% and 80% of the bacteria itself and between 20% and 30% of the coating matrix.

[0083] FRa5. A composition comprising a mixture of or consisting of the bacteria of any of FRa1 to 4, optionally containing at least one food-grade or pharmaceutical or cosmetic additive and / or excipient.

[0084] FRa6. The bacterium according to any one of claims 1 to 5 is 1 x 10 6 AFU / g ~ 1 x 10 14 AFU / g, preferably 1 × 10 7 AFU / g ~ 1 x 10 13 AFU / g, more preferably 1×10 8AFU / g ~ 1 x 10 12 The composition of FRa5, wherein the composition is present at a concentration in the range of AFU / g, where AFU / g means having viable cells and complex cell membranes on 1 gram of the composition.

[0085] FRa7. A method for producing any one of the bacteria FRa1 to FRa4, (I) granulating at least one bacterial cell strain through a mesh ranging from 50 microns to 900 microns to obtain granular bacteria; (II) coating the granular bacteria with a coating matrix comprising at least one lipid, preferably of plant origin, according to claim 3 or 4 to obtain the gastroprotective granular bacteria per se; and (III) tempering the gastroprotected granular bacteria itself at a temperature comprised between 25°C and 60°C for a time comprised between 48 hours and 96 hours to obtain gastroprotected granular bacteria with a coating matrix having a crystalline structure; preferably, wherein the at least one lipid adopts a lamellar arrangement having a crystalline structure after the tempering step (III).

[0086] FRa8. The method of FRa7, further comprising, following step (III), a step (IV) of performing a bacterial count of the sample of granular bacteria gastroprotected with a coating matrix having a crystalline structure obtained in step (III) using an analytical method, wherein the analytical method allows for the detection of the amount of bacterial cells having a complex cell membrane; preferably, the analytical method is flow cytometry.

[0087] FRa9. The method of FRa7 or 8, wherein in step (I), the bacteria are granulated through a mesh of 100 to 600 microns, preferably 150 to 500 microns, more preferably 180 or 450 microns; wherein in step (II), which is preferably carried out in a fluidized bed chamber, the granulated bacteria and the coating matrix are treated in a weight ratio of 6:4 to 9:1, preferably 6.5:3.5 to 8.5:1.5, more preferably 7:3 to 8:2; and wherein in step (III), the gastroprotective granulated bacteria itself is tempered at a temperature of 30°C to 40°C, preferably about 35+1°C, for a time of 60 to 84 hours, preferably about 72 hours.

[0088] FRa10. Granular bacteria gastroprotected by a coating matrix having a crystalline structure, obtainable by the methods of FRa7-9.

[0089] Furthermore, the following embodiments (FRbn) are objects of the present invention:

[0090] FRb1. A granular bacterium gastroprotected with a coating matrix having a crystalline structure, wherein the coating matrix comprises or consists of at least one lipid, wherein the at least one lipid has a lamellar arrangement with a crystalline structure, preferably a multilayered crystalline-like lamellar arrangement.

[0091] FRb2. The at least one lipid mono-, di- or triglycerol esterified with saturated or unsaturated fatty acids, preferably saturated fatty acids; - free saturated fatty acids; - free unsaturated fatty acids, preferably monounsaturated; monoalcohols esterified with saturated or unsaturated fatty acids, preferably saturated fatty acids; - di-alcohols esterified with saturated or unsaturated fatty acids, preferably saturated fatty acids; - sucrose fatty acid esters (cane sugar esters), preferably mixtures of mono-, di- and / or tri-sucrose fatty acid esters; wherein the saturated or unsaturated fatty acids esterified with free or esterified glycerol or mono- or di-alcohol or sucrose have a number of carbon atoms ranging from C6 to C32, preferably C14 to C24, more preferably C16, C18 and / or C22.

[0092] FRb3. The at least one lipid, preferably of plant origin, - at least one first lipid, which is a mono-, di-, or triglycerol esterified with a saturated or unsaturated fatty acid, preferably a saturated fatty acid; and - at least one second lipid, which is a sucrose fatty acid ester (sucrose ester), preferably a mixture of mono-, di- and / or tri-sucrose fatty acid esters; wherein the fatty acids esterified with glycerol or sucrose have a carbon number ranging from C6 to C32, preferably C14 to C24, more preferably C16, C18, C20 and / or C22.

[0093] FRb4. A bacterium selected from any one of FRb1 to FRb3, - the bacteria itself is present in a weight percentage ranging from 60% to 90%; and - a coating matrix comprising or consisting of at least one lipid in a weight percentage comprised between 10% and 40% relative to the total weight of the gastroprotective granular bacteria; preferably between 65% and 85% of the bacteria itself and between 15% and 35% of the coating matrix; more preferably between 70% and 80% of the bacteria itself and between 20% and 30% of the coating matrix.

[0094] FRb5. A composition comprising a mixture of or containing the bacteria of any of FRb1 to FRb4, optionally containing at least one food-grade or pharmaceutical or cosmetic additive and / or excipient.

[0095] FRb6. The bacterium according to any one of claims 1 to 5 is 1 x 10 6 AFU / g ~ 1 x 10 14 AFU / g, preferably 1 × 10 7 AFU / g ~ 1 x 10 13 AFU / g, more preferably 1×10 8 AFU / g ~ 1 x 10 12 The composition of FRb5, wherein the composition is present at a concentration in the range of AFU / g, where AFU / g means having viable cells and complex cell membranes on 1 gram of the composition.

[0096] FRb7. A method for producing a bacterium according to any one of FRb1 to FRb4, comprising: (I) granulating at least one bacterial cell strain through a mesh in the range of 50 microns to 900 microns to obtain granular bacteria; (II) coating the granular bacteria with a coating matrix comprising at least one lipid, preferably of plant origin, according to claim 3 or 4 to obtain the gastroprotective granular bacteria per se; and (III) tempering the gastroprotected granular bacteria itself at a temperature comprised between 25°C and 60°C for a time comprised between 48 hours and 96 hours to obtain gastroprotected granular bacteria with a coating matrix having a crystalline structure; preferably, wherein the at least one lipid adopts a lamellar arrangement having a crystalline structure after the tempering step (III).

[0097] FRb8. The method of FRb7, further comprising, following step (III), a step (IV) of performing a bacterial count on the sample of granular bacteria gastroprotected with a coating matrix having a crystalline structure obtained in step (III) using an analytical method, wherein the analytical method allows for the detection of the amount of bacterial cells having a complex cell membrane; preferably, the analytical method is flow cytometry.

[0098] FRb9. The method of FRb7 or 8, wherein in step (I), the bacteria are granulated with a mesh of 100 microns to 600 microns, preferably 150 to 500 microns, more preferably 180 microns or 450 microns; wherein in step (II), which is preferably carried out in a fluidized bed chamber, the granulated bacteria and the coating matrix are treated in a weight ratio of 6:4 to 9:1, preferably 6.5:3.5 to 8.5:1.5, more preferably 7:3 to 8:2; and wherein in step (III), the gastroprotective granulated bacteria itself is tempered at a temperature of 30°C to 40°C, preferably about 35+1°C, for a time of 60 hours to 84 hours, preferably about 72 hours.

[0099] FRb10. Granular bacteria gastroprotected by a coating matrix having a crystalline structure, obtainable by the methods of FRb7-9. [Example]

[0100] Experiment Part 1 The following tests (Tests B and C) are intended to examine the morphology of the coating of the sample under analysis and compare it with the crystallographic structure. Furthermore, Test A is intended to analyze the viability of the bacterial strain contained in the sample under analysis.

[0101] material and method Analysis sample - Sample 0 (WBR05018): Non-granular, uncoated and untempered freeze-dried bacterial strain. - Sample 1: GG107-18 granulated to 450 microns (450 μm, bare); uncoated; untempered. - Sample 2: GG107-18 (180 μm, bare) strain granulated to 180 microns; uncoated; untempered. - Sample 3: MCPM-P1 (450 μm, coated) strain granulated to 450 microns; coated with a coating matrix containing lipid (i) Biogapress Vegetal BM297 E471 (briefly Biogapress Vegetal E471) manufactured by Gattefosse SAS; coating method: 80% strain / 20% Biogapress Vegetal E471 in a fluidized bed chamber at 50 °C; not tempered, stored at -20 °C. - Sample 4: MCPM-P2 (180 μm, coated) strain granulated to 180 microns; coated with a coating matrix containing lipid (i) Biogapress Vegetal E471 in a 50 °C fluidized bed chamber at a ratio of 80% strain / 20% Biogapress Vegetal E471; not tempered, stored at -20 °C. - Sample 5: MCPM-P1 / 20 strain granulated to 450 microns (450 μm, coated + 35 °C, matured for 72 hours); coated with a coating matrix containing lipid (i) Biogapress Vegetal E471 in a fluidized bed chamber at 50 °C in a ratio of 80% strain / 20% Biogapress Vegetal E471; tempered at 35 °C for 72 hours, stored at -20 °C. - Sample 6: MCPM-P2 / 20 strain granulated to 180 microns (180 μm, coated + 35 °C, matured for 72 hours); coated with a coating matrix containing lipid (i) Biogapress Vegetal E471 in a fluidized bed chamber at 50 °C in a ratio of 80% strain / 20% Biogapress Vegetal E471; tempered at 35 °C for 72 hours, stored at -20 °C. Samples 0 to 6 are in powder form. Bacterial strain used in samples 0 and 1–6: Lactobacillus rhamnosus GG ATCC 53103. The granulation size was determined by the granulator mesh, and in the examples, 180 μm or 450 μm granules were evaluated.

[0102] Analysis method (A) Flow cytometry analysis method. The flow cytometry analysis method was used according to the method established by the standard ISO 19344:2015(E) and was applied to Samples 1 to 6 at time t0, i.e., after preparation and before subjecting them to scanning electron microscopy (SEM) and X-ray diffraction analysis (see paragraphs (B) and (C)). TFU is total bacterial units or cells; An AFU is a bacterial unit or cell that has a complex (or viable) cell membrane. The samples were stored at −20° C. (stock preparations).

[0103] (B) X-ray diffraction analysis (Figure 1). Analytical methods used: X-ray diffraction to characterize the crystalline structure of lipids. The method was applied to samples 1, 4, 5 and 6. Sample preparation: Samples in powder form were placed in 1 mm diameter glass capillaries.

[0104] diffraction: - Transparency settings - Beamline PROXIMA 2 at SOLEIL syncrotrone (France) - Wavelength: 0.7293Å, - Sample detector width: 400mm - 2D DECTRIS (pixel 75 x 75 μm 2 ) detector - T=22℃, HR 40%

[0105] Analysis: X-ray patterns were analyzed using ESIT FIT2D software and compared with each other. The intensity profiles presented in this report correspond to angular profiles integrated over 180°. The diffraction setup allows for the acquisition of SAXS and WAXS diffraction patterns. SAXS patterns provide information on the stacking structure of lipid lamellae (network width, crystallite size) and The WAXS patterns reveal lateral ordering information within the lipid layers.

[0106] (C) Scanning electron microscopy (SEM) Analytical methods used: SEM FEG (low voltage SEM) to observe the surface of the coating to evaluate the quality of the coating. The method was applied to samples 1, 4, 5 and 6.

[0107] Sample preparation: The powder was deposited on a carbon adhesive fixed to the medium. Care was taken not to break up the powder or overload the deposit. Image acquisition: Observed with a SUPE 55VP di ZEISS electron microscope. A voltage of 1.20 kV was chosen to obtain the best compromise between strength, surface charge and stability of the product under the electron beam. Images at various magnifications (x200, x500, x1500, x3000 and x8000) were acquired to cover a wide field of view and see high resolution details.

[0108] result (A) Flow cytometry analysis method From the bacterial counts in flow cytometry of samples 1 to 6, the following were observed (Table 1): - Samples 1-6 are homogeneous since the TFU data determining the total cell number remain constant for samples 1-3-5 (450 μm granulation) and 2-4-6 (180 μm granulation), which indicates that the observations made in analyses (B) and (C) are consistent; Treatment lethality, assessed by AFU (viable / combined cell number), is inferred to be absent since we started with naked bacterial strains at two different granule sizes (samples 1 and 2) and the AFU data remained essentially unchanged in the methods of coating with lipid matrices (samples 3 and 4) and tempering (samples 5 and 6).

[0109] [Table 1] * 35°C for 72 hours

[0110] (B) X-ray diffraction analysis. (C-I) Sample 1 (Figures 2–5). Sample 1 (non-granular, uncoated freeze-dried bacterial cells) is completely amorphous (no sharp diffraction features).

[0111] (C.ii) Samples 4, 5, and 6 (Figures 2–5). Samples 4, 5 and 6 are characterized by a series of characteristic diffractions in the SAXS and WAXS regions. It should be noted that the signals for samples 5 and 6 are approximately the same in location and intensity.

[0112] WAXS region (Fig. 4) All samples 4, 5 and 6 show a peak at 4.1 Å (hexagonal packing of lipids). For samples 5 and 6, some WAXS peaks were also observed at about 4.1 Å, which implies the coexistence of other interlamellar reticulum of hexagonal reticulum in the lipid layers.

[0113] SAXS region (Fig. 5) Samples 5 and 6 show a sharp peak at 49.8 Å (and its harmonics) and sample 4 at 52 Å (and its harmonics). The positions of these peaks are related to the network width of the stack of lipid lamellae.

[0114] A comparison of the crystalline characteristics of samples 1, 4, 5 and 6 is shown in Table 2. [Table 2]

[0115] in conclusion - Sample 1 is purely amorphous, without any trace of lamellar order; - All samples 4, 5 and 6 show only one lamellar system. The reticulation width varies slightly from sample to sample; - Systems with hexagonal transverse order are present in all samples 4, 5 and 6; - A second lateral system is also detectable in samples 5 and 6; - Samples 5 and 6 have the same crystallographic structure. Except for sample 1, which is completely amorphous (no sharp diffraction features), all other samples are characterized by the typical lipid diffraction pattern of a "lamellar phase". A "lamellar phase" is a spatial arrangement of lipids that corresponds to a molecular structure of lamellae, in which the lipid chains are more or less perpendicular to the plane of the lamellae, as shown in FIG.

[0116] To sum up - Sample 1: WAXS and SAXS data show no coating; - Sample 4: WAXS and SAXS data confirm the presence of a non-multilayer coating; - Samples 5 and 6: WAXS and SAXS data confirm the presence of a multilayer coating.

[0117] (C) Scanning electron microscopy (SEM) - Sample 1 Sample 1 exhibits large, irregularly shaped structures with typical dimensions of several hundred micrometers (Figure 8). These structures combine to form large, three-dimensional objects (up to millimeters). At low magnification, the surface appears undulating and rough, but overall smooth. At higher magnification (Figure 9), the surface of Sample 1 exhibits striped or geometric patterns and sometimes appears more granular. Rod-shaped bacteria (approximately 0.5 x 2 microns in size) can be seen, often with the formation of very narrow villi. The morphology of bacteria, given by the shape of the cell wall, can be of three basic types: cocci, ellipsoids, and rods; the rod forms have a longer cell axis than the others.

[0118] - Sample 4 (MCPM-P2) Sample 4 is a mixture of small granules, irregularly shaped large particles, and flat particles (Figure 10). At high magnification, no bacteria are clearly visible in Sample 4 (Figure 11).

[0119] - Sample 5 (MCPM-P1 / 20) Sample 5 is a mixture of large, flat particles (tens or hundreds of μm in size) and small, irregularly shaped particles (a few μm to several μm in size). The surface is slightly rough and irregular (Figure 12). At high magnification (Figure 13), bacteria are only slightly visible locally. The surface is fairly smooth with slight irregularities and particles.

[0120] - Sample 6 (MCPM-P2 / 20) Sample 6 is a mixture of small granules, irregularly shaped large particles, and flat particles (Figure 14). At high magnification (Figure 15), the surface is fairly smooth, but some particles are visible, and bacteria are sparsely visible.

[0121] A comparison of the characteristics of samples 1, 4, 5 and 6 under an electron microscope is shown in Figure 3. Table 3 Table 3 is correct and the data remains from the two reports, so there is no need to do it again. [Table 3]

[0122] To sum up, - Samples 1 and 2: the bacterial strain is clearly visible under the electron microscope; - Samples 3, 4, 5 and 6: disappearance / reduction of microorganisms in the optical field. No bare samples were observed in these samples, thus indicating effective microencapsulation.

[0123] Discussion of results 1) The non-granular freeze-dried bacterial strain (comparison blank) is clearly distinguishable by scanning electron microscopy (SEM); moreover, X-ray diffraction analysis does not show any coating. 2) The freeze-dried naked stock was granulated with two different meshes, 180 μm and 450 μm, coated with Biogapress Vegetal E471, and optionally tempered to obtain Sample 4 (not tempered) or Samples 5 and 6 (tempered). Scanning electron microscopy (SEM) shows that the bacterial strain is coated in sample 4 and both samples 5 and 6. X-ray diffraction analysis (WAXS and SAXS) - in samples 5 and 6 - considering the same method, shows the presence of crystalline multilayers that are not present in sample 4 since no tempering step was carried out. Finally, no process mortality was observed for the bacterial strains in samples 5 and 6 subjected to the granulation, coating and tempering process of the present invention, as analyzed by means of flow cytometry. Therefore, by means of the method of the present invention, it is possible to efficiently coat the granular bacterial strains with a lipid matrix and by means of the tempering process to obtain good crystallization (development of lateral multilayers) while maintaining the concentration of viable bacterial strains unchanged or almost unchanged before and after the granulation, coating and tempering method of the present invention.

[0124] Experiment Part 2 The following test (Test D) compares the bacterial viability (bacterial load) of a naked bacterial strain and its corresponding gastroprotective bacterial strain having a coating matrix in crystalline form according to the present invention after heating at controlled humidity.

[0125] Materials and Methods for Study D Analysis sample - Sample 7: naked bacterial strain Lactobacillus rhamnosus GG ATCC 53103 (uncoated and untempered); briefly naked uncoated bacterial strain. - Sample 8: Bacterial strain Lactobacillus rhamnosus GG ATCC 53103 gastroprotected in a coating matrix in crystalline form according to the invention (coating and tempering): coating matrix comprises lipid (i) Biogaress Vegetal E471; for example: coating in a 50°C fluidized bed chamber in a ratio of 80% strain / 20% Biogaress Vegetal E471; tempering at 35°C for 72 hours; briefly coated bacterial strain.

[0126] method Test D was conducted by administering Samples 7 and 8 (naked and coated bacterial strains) to vegetable oil to form a bacterial suspension in oil. The bacterial suspension in vegetable oil was subjected to two different temperatures and controlled humidity (RH: relative humidity), e.g., 30°C-75% RH (Figure 16) and 40°C-75% RH (Figure 17), for times ranging from 0 to 12 months and 0 to 60 days, respectively. The viability of the bacterial strains in the two samples (Samples 7 and 8) was measured during the time ranges.

[0127] Analysis method The viability (bacterial load) of the bacterial strains was assessed by a cytometric method (data are presented in AFU). Flow cytometry analysis was performed according to the method established by the ISO 19344:2015(E) standard. An AFU is a bacterial unit or cell that has a complex (or viable) cell membrane.

[0128] result As reported in Figures 16 and 17, the load of the coated bacterial strain is log-larger than the naked bacterial strain at both 60 days at 40°C and 12 months at 30°C. Thus, it is clear that the gastroprotective bacterial strain with the crystalline morphology coating matrix of the present invention is more resistant than the naked bacterial strain given the same temperature and humidity.

Claims

1. A granular bacterium gastroprotected with a coating matrix having a crystalline structure, wherein the coating matrix comprises or consists of at least one lipid, wherein the at least one lipid has a lamellar arrangement having a crystalline structure, preferably a multilayered crystalline structure-like lamellar arrangement.

2. The at least one lipid mono-, di- or triglycerol esterified with saturated or unsaturated fatty acids, preferably saturated fatty acids; - free saturated fatty acids; - free unsaturated fatty acids, preferably monounsaturated; mono-alcohols esterified with saturated or unsaturated fatty acids, preferably saturated fatty acids; - di-alcohols esterified with saturated or unsaturated fatty acids, preferably saturated fatty acids; sucrose fatty acid esters (cane sugar esters), preferably mixtures of mono-, di- and / or tri-sucrose fatty acid esters; wherein said saturated or unsaturated fatty acids esterified with free or esterified glycerol or mono- or di-alcohol or sucrose have a number of carbon atoms ranging from C6 to C32, preferably C14 to C24, more preferably C16, C18 and / or C22, The bacterium of claim 1.

3. said at least one lipid, preferably of plant origin, at least one first lipid, which is a mono-, di- or triglycerol esterified with a saturated or unsaturated fatty acid, preferably a saturated fatty acid; and at least one second lipid, which is a sucrose fatty acid ester (sucrose ester), preferably a mixture of mono-, di- and / or tri-sucrose fatty acid esters; wherein the fatty acids esterified with glycerol or sucrose have a number of carbon atoms ranging from C6 to C32, preferably C14 to C24, more preferably C16, C18, C20 and / or C22; The bacterium of claim 2.

4. The bacterium according to any one of claims 1 to 3, wherein: - the bacteria itself is present in a weight percentage ranging from 60% to 90%; and - a coating matrix comprising or consisting of at least one lipid, in a weight percentage comprised between 10% and 40% relative to the total weight of the gastroprotective granular bacteria; preferably between 65% and 85% of the bacteria itself and between 15% and 35% of the coating matrix; more preferably between 70% and 80% of the bacteria itself and between 20% and 30% of the coating matrix.

5. A composition comprising or consisting of the bacterium of any of claims 1 to 4, optionally containing at least one food-grade or pharmaceutical or cosmetic additive and / or excipient.

6. The bacterium according to any one of claims 1 to 5 is 1 x 10 ６ AFU / g ~ 1 x 10 １４ AFU / g, preferably 1 x 10 ７ AFU / g ~ 1 x 10 １３ AFU / g, more preferably 1 x 10 ８ AFU / g ~ 1 x 10 １２ 6. The composition of claim 5, wherein the composition is present at a concentration in the range of AFU / g, where AFU / g refers to viable cells and complex cell membranes on one gram of the composition.

7. A method for producing the bacterium of any one of claims 1 to 6, comprising: (I) granulating at least one bacterial cell strain through a mesh ranging from 50 microns to 900 microns to obtain granular bacteria; (II) coating the granular bacteria with a coating matrix comprising at least one lipid, preferably of plant origin, according to claim 3 or 4 to obtain the gastroprotective granular bacteria itself; and (III) tempering the gastroprotected granular bacteria itself at a temperature comprised between 25°C and 60°C for a time comprised between 48 hours and 96 hours to obtain gastroprotected granular bacteria with a coating matrix having a crystalline structure; preferably, wherein the at least one lipid adopts a lamellar configuration having a crystalline structure after the tempering step (III).

8. 8. The method of claim 7, further comprising, following step (III), a step (IV) of performing a bacterial count on a sample of granular bacteria gastroprotected with a coating matrix having a crystalline structure obtained in step (III) using an analytical method, wherein the analytical method allows for the detection of the amount of bacterial cells having a complex cell membrane; preferably, the analytical method is flow cytometry.

9. 9. The method of claim 7 or 8, wherein in step (I), the bacteria are granulated through a mesh of 100 microns to 600 microns, preferably 150 to 500 microns, more preferably 180 microns or 450 microns; wherein in step (II), which is preferably carried out in a fluidized bed chamber, the granulated bacteria and the coating matrix are treated in a weight ratio of 6:4 to 9:1, preferably 6.5:3.5 to 8.5:1.5, more preferably 7:3 to 8:2; and wherein in step (III), the gastroprotective granulated bacteria itself is tempered at a temperature of 30°C to 40°C, preferably about 35+1°C, for a time of 60 hours to 84 hours, preferably about 72 hours.

10. Granular bacteria gastroprotected with a coating matrix having a crystalline structure, obtainable by the method of claims 7 to 9.

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