Microbial capsule fermentation

Abstract

The disclosure provides methods of obtaining fermentation products and postbiotic products using one or more capsules comprising fermenting microorganisms. The disclosure also provides methods of preparing capsules comprising fermenting microorganisms.

Description

MICROBIAL CAPSULE FERMENTATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of U.S. Provisional Patent Application No. 63 / 700,889, filed September 30, 2024, and U.S. Provisional Patent Application No. 63 / 710,768, filed October 23, 2024, each of which is incorporated by reference in its entirety.FIELD OF THE DISCLOSURE

[0002] The present disclosure pertains to methods for obtaining a fermentation product using capsules including fermenting microorganisms, and methods of making said capsules.BACKGROUND OF THE DISCLOSURE

[0003] In bioprocesses, such as fermentation, one of the most important objectives is to maximize the efficiency and yield of the process. Microbial immobilization through encapsulation is a new technique for building up a higher cell density in the bioreactor. Cell retention techniques, that include cell recycling (Brandberg et al., Enzyme and Microbial Technology, 2007, 40(4), 585-593), cross-linking of cells (Abelyan, Applied Biochemistry and Microbiology, 2000, 36, 310-314), entrapment in a matrix (Taherzadeh et al., Applied Biochemistry and Biotechnology, 2001, 95, 45-57), and encapsulation in a polymer gel membrane (Talebnia and Taherzadeh, Journal of Biotechnology, 2006, 125(3), 377-384), offer some advantages, such as easy product recovery by separation of the cells from the metabolites produced during the fermentation process, the increase in the volumetric productivity and also the reduction of the overall cost (Westman et al., Applied Microbiology and Biotechnology, 96, 1441-1454, 2012). Encapsulation has been widely used in the pharmaceutical field, where it can be used as a drug delivery system (Paulo and Santos, Materials Science and Engineering, 2017, C77: 1327 40). However, encapsulation is increasingly being used in the food, cosmetic, and textile sectors, where it can be applied to preserve the active ingredient during processing and / or storage, to increase nutraceuticals bioavailability, or to ensure a targeted (Reque and Brandelli,Trends in Food Science & Technology, 2021, 114: 1-10) or controlled release of the encapsulated compound (Casanova and Santos, Journal of Microencapsulation, 2016, 33(1): 1-17).

[0004] One of the main applications of encapsulation is linked to probiotic microorganisms: capsules are often used to protect bacteria during gastrointestinal transit and to release them in a target site (Raj am and Subramanian, Beni-Suef University Journal of Basic and Applied Sciences, 2022, 11 : 1 11(1): 1-18). Similarly, recent studies have explored using encapsulation to protect postbiotics during gastrointestinal transit. (See, e.g., Yang, K., et al., Adv. Sci. (Weinh), May 2022, 9(16):e2104089). However, capsules can also be used as reactors, by which the performance of the reaction itself could be improved. For instance, immobilized microorganisms could enable a reaction faster than free microorganisms (Oliveira et al., Food Research International, 2011, 44(7): 2391-2400), or the reaction could be more efficient. Immobilization could allow for faster microbial fermentation due to shortening of the non-productive growth phase or due to higher cell density than free cell fermentation. Other possible effects of immobilization are higher product yields (Ramakrishna and Prakasham, Current Science, 1999, 77(1): 87-100) and that immobilized bacteria can quickly be recovered and reused in fermentation processes (Duarte et al., AMB Express, 2013, 3(1): 1-8).

[0005] A widely used encapsulating material is alginate, a linear heteropolysaccharide of d-mannuronic and 1-guluronic acid extracted from various species of algae (Smidsrod, Haug, & Lian, Acta Chemica Scandinavica, 1972, 26, 79-88). Alginate capsules can be produced by an extrusion technique, for example, obtaining a gel matrix with a pore size of less than 17 nm (Klein et al., European Journal of Applied Microbiology and Biotechnology, 1983, 18, 86-91) good for containing bacteria during the fermentation process (Krasaekoopt et al., International Dairy Journal, 2004, 14(8), 737-743). Alginate- based capsules are carriers capable of releasing or confining bacteria and bioactive compounds in a controlled manner. Furthermore, the rate of the compound release can be controlled, acting on different parameters, such as varying the concentration of crosslinking agent during gel formation (Gerbsch and Buchholz, FEMS Microbiology Reviews, 1995, 16(2-3), 259-269) or reducing pushing forces of solutes and cells (Liu et al., Bioresource Technology, 2008, 99(6), 1904-1910).

[0006] Inoculum preparation involves developing a starter culture to initiate, for example, fermentation processes. It is a multi-step process (depending on the scale) that involvesprogressive microbial propagation to obtain a suitable quantity of viable microorganisms to start a fermentation process. Typically, the microorganism to be used is usually stored in liquid or freeze-dried form, under refrigerated or frozen conditions. The stored microorganism then requires a revitalization phase during which it is nourished with a fresh broth to reactivate its metabolism and growth. This phase can last hours or days, depending on the volume of inoculum with a specific bacterial concentration desired to start the fermentation process.

[0007] At the beginning of a fermentation process, it is typically desirable to have a microbial concentration that is sufficiently high in order to reduce lag time, allowing the fermentation process to start rapidly, prevent the growth of contaminating microorganisms, and to ensure that the microorganism can quickly reach the exponential phase of growth, maximizing the production of the desired product. For these reasons, depending on the concentration desired to start the fermentation and the volume to be fermented (e.g., for industrial scale fermentations), multiple activation steps of the microorganism may be necessary, with a progressive increase in the charge and volumes of inoculum, which require higher inoculum volumes and increased manufacturing time due to the progressive microbial propagations required. Accordingly, improved methods of initiating microbial cultures are needed.

[0008] Provided herein are capsules including fermenting microorganisms that can be readily produced and stored (e.g., stored as frozen capsules) to be used as an inoculum at a future time, directly added to the medium to be fermented. Also provided herein are postbiotic dried capsules to be used as anti-inflammatory products. Specifically, the current approach aims to optimize the fermentation process, reducing the amount of time and volume of inoculum required to obtain fermentation products.SUMMARY OF THE DISCLOSURE

[0009] In one aspect of the present disclosure is provided a method of obtaining a fermentation product comprising the steps of: a) inoculating a culture media with a capsule comprising a fermenting microorganism, wherein optionally prior to the inoculating, the capsule is frozen; and b) fermenting the inoculated culture media under conditions suitable for fermentation, thereby obtaining a fermentation product. In some aspects, prior to step a), the capsule is stored frozen. In some aspects, the capsule is storedfrozen in a glycerol solution. In some aspects, the glycerol solution comprises about 10% to about 50% w / v glycerol. In some aspects, the glycerol solution comprises about 30% w / v glycerol.

[0010] In some aspects, prior to step a), the frozen capsule is incubated at room temperature.

[0011] In some aspects, the capsule comprises a culture broth, a substrate comprising starch and / or fiber, alginate, and / or CaCh. In some aspects, the culture broth is selected from the group consisting of MRS broth, Brain Heart Infusion (BHI) broth, Luria-Bertani (LB) broth, plant-derived broth, functional broth containing plant extracts with antioxidant, antiviral and / or antibacterial activity, culture broth of natural origin, or animal-free broth. In some aspects, the substrate comprising starch and / or fiber is a cereal suspension. In some aspects, the cereal suspension is an oatmeal suspension.

[0012] In some aspects, prior to step a), the capsule is resuspended in a hydrolyzed oatmeal suspension or a culture broth. In some aspects, the hydrolyzed oatmeal suspension comprises about 1% to about 50% w / v oat flour, and about 0.1% to about 5% w / v glucose. In some aspects, the hydrolyzed oatmeal suspension comprises about 15% w / v oat flour and about 1% w / v glucose. In some aspects, the culture broth is selected from the group consisting of MRS broth, Brain Heart Infusion (BHI) broth, Luria-Bertani (LB) broth, plant-derived broth, functional broth containing plant extracts with antioxidant, antiviral and / or antibacterial activity, culture broth of natural origin, or animal-free broth.

[0013] In some aspects, the capsule comprises an alginate layer. In some aspects, the capsule further comprises a chitosan layer. In some aspects, the capsule further comprises a second alginate layer.

[0014] In some aspects, the capsule is produced by extrusion, high shear rate mixing, spray-drying, emulsion, lyophilization, and / or layer-by-layer production.

[0015] In some aspects, the capsule is produced by extrusion using about 0.5% to about 1.5 % w / v alginate and about 0.1M to about 3M CaCh. In some aspects, the capsule is produced by extrusion using 1% w / v alginate and IM CaCh.

[0016] In some aspects, the capsule is of a spherical, filamentous, irregular, or flat geometry. In some aspects, the capsule is of a spherical geometry.

[0017] In some aspects, the method further comprises inoculating the culture media with one or more additional capsules comprising fermenting microorganisms. In some aspects,each of the one or more additional capsules comprises a different fermenting microorganism.

[0018] In another aspect, provided herein is a method of preparing a plurality of capsules comprising a fermenting microorganism, comprising the steps of: (i) preparing a suspension comprising a fermenting microorganism culture and an alginate solution; and (ii) dripping the suspension of step (i) into a calcium chloride solution, thereby forming a plurality of capsules comprising the fermenting microorganism, and incubating the plurality of capsules in the calcium chloride solution.

[0019] In another aspect, provided herein is a method of preparing a plurality of capsules comprising a fermenting microorganism, comprising the steps of: (i) preparing a hydrolyzed oatmeal suspension comprising amylase-treated oatmeal and glucose; (ii) mixing the hydrolyzed oatmeal suspension with a fermenting microorganism culture and alginate solution; and (iii) dripping the mixture of step (ii) into a calcium chloride solution, thereby forming a plurality of capsules comprising the fermenting microorganism, and incubating the plurality of capsules in the calcium chloride solution. In some aspects, the method further comprises rinsing the capsules with distilled water. In some aspects, the method further comprises mixing the capsules with a glycerol solution and freezing the mixture.

[0020] In some aspects, the alginate solution comprises about 0.5% to about 2.0 % w / v alginate. In some aspects, the alginate solution comprises 1% w / v alginate.

[0021] In some aspects, the calcium chloride solution comprises about 0.1M to about 3M CaCh. In some aspects, the calcium chloride solution comprises IM CaCh.

[0022] In some aspects, the capsules are of a spherical, filamentous, irregular, or flat geometry. In some aspects, the capsules are of a spherical geometry.

[0023] In some aspects, the fermenting microorganism culture comprises about 106colony forming units (CFU) / mL to about 1011CFU / mL.

[0024] In some aspects, each capsule comprises about 103CFU / mL to about 109CFU / mL of the fermenting microorganism.

[0025] In some aspects, each capsule comprises about 106CFU / mL of the fermenting microorganism.

[0026] In some aspects, each capsule comprises about 108CFU / mL of the fermenting microorganism.

[0027] In some aspects, step (i) comprises activating an amylase enzyme at about 60 °C to about 80 °C, followed by adding oat flour and glucose, incubating at about 60 °C to about 80 °C, and then incubating at about 80 °C to about 95 °C. In some aspects, step (i) comprises activating an amylase enzyme at about 65 °C, followed by adding oat flour and glucose, incubating at about 65 °C, and then incubating at about 85 °C.

[0028] In some aspects, the hydrolyzed oatmeal suspension comprises about 1% to about 50% w / v oat flour, and about 0.1% to about 5% w / v glucose. In some aspects, the hydrolyzed oatmeal suspension comprises about 15% w / v oat flour and about 1% w / v glucose.

[0029] In some aspects, the fermenting microorganism is selected from the group consisting of a bacterial microorganism and a yeast microorganism. In some aspects, the bacterial microorganism is selected from the group consisting of Lactobacillus species, a Lactococcus species, a Bifidobacterium species, a Lacticaseibacillus species, a Streptococcus species, and an Akkermansia species. In some aspects, the yeast microorganism is selected from a Saccharomyces species.

[0030] In some aspects, the fermenting microorganism is selected from the group consisting of Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Lacticaseibacillus paracasei, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium longum, Streptococcus salivarius, Akkermansia muciniphila, and Saccharomyces boulardii. In some aspects, the Lacticaseibacillus paracasei is Lacticaseibacillus paracasei NPB01.

[0031] In another aspect, provided herein is a kit comprising a capsule comprising a fermenting microorganism as described herein.

[0032] In another aspect, provided herein is an encapsulated fermenting microorganism composition comprising: i) a capsule comprising a fermenting microorganism, wherein the capsule comprises at least one alginate layer; and ii) a glycerol solution. In some aspects, the capsule further comprises a chitosan layer.

[0033] In some aspects, the capsule is of a spherical, filamentous, irregular, or flat geometry. In some aspects, the capsule is of a spherical geometry. In some aspects, the capsule is produced by extrusion, high shear rate mixing mixing, spray-drying, emulsion, lyophilization, and / or layer-by-layer production.

[0034] In some aspects, the capsule comprises a culture broth, a substrate comprising starch and / or fiber, alginate, and / or CaCh. In some aspects, the culture broth is is selected from the group consisting of MRS broth, Brain Heart Infusion (BHI) broth, Luria-Bertani (LB) broth, plant-derived broth, functional broth containing plant extracts with antioxidant, antiviral and / or antibacterial activity, culture broth of natural origin, or animal-free broth. In some aspects, the substrate comprising starch and / or fiber is a cereal suspension. In some aspects, the cereal suspension is an oatmeal suspension.

[0035] In another aspect, provided herein is a method of obtaining a capsule comprising a postbiotic product comprising the steps of preparing a capsule comprising a fermenting microorganism according to a method described herein; fermenting the capsule comprising the fermenting microorganism, thereby obtaining a capsule comprising a fermentation product; and inactivating the capsule comprising the fermentation product, thereby obtaining a capsule comprising a postbiotic product. In some aspects, the inactivating of step c) comprises heat inactivation of the capsule comprising the fermentation product. In some aspects, the heat inactivation comprises conventional heat inactivation of the capsule comprising the fermentation product. In some aspects, the heat inactivation comprises dielectric heat inactivation of the capsule comprising the fermentation product. In some aspects, the dielectric heat inactivation comprises microwave heat inactivation of the capsule comprising the fermentation product. In some aspects, the dielectric heat inactivation comprises radiofrequency heat inactivation of the capsule comprising the fermentation product.

[0036] In some aspects, the method further comprises drying the capsule comprising the postbiotic product. In some aspects, the drying of the capsule comprises lyophilizing the capsule comprising the postbiotic product. In some aspects, the drying of the capsule comprises spray-drying the capsule comprising the postbiotic product.

[0037] In some aspects, the method further comprises mechanical disruption of the capsule comprising the postbiotic product. In some aspects, the mechanical disruption of the capsule comprises comminution of the capsule via high-speed airflow pulverization, ball milling, high-speed rotary strike crushing, vacuum superfine grinding, low- temperature milling, or cryogenic grinding. In some aspects, the mechanical disruption of the capsule comprises lyophilizing the capsule. In some aspects, the mechanical disruption of the capsule comprises spray-drying the capsule.

[0038] In another aspect, provided herein is a postbiotic product described herein for use in the prevention or the therapeutic treatment of a disease in a subject in need thereof selected from the group consisting of infectious and inflammatory diseases, immune- mediated diseases, cancer diseases, skin disorders, gastrointestinal diseases, urogenital tract diseases, neurologic disorders, neuropsychiatric disorders, bone diseases, muscle diseases, malnutrition, metabolic diseases, and any combination thereof. In some aspects, the postbiotic product reduces Myd88 and / or TLR4 expression in the subject.

[0039] In another aspects, provided herein is a use of a postbiotic product described herein in a food, a beverage, a pharmaceutical, a nutraceutical, a cosmetic, or a packaging composition, and wherein the composition further comprises at least one pharmaceutically acceptable vehicle, excipient and / or diluent.BRIEF DESCRIPTION OF THE FIGURES

[0040] FIGs. 1A-1B illustrate experimental overviews of conditions tested with capsules as a culture inoculum during preliminary testing at a IL reactor scale (FIG. 1 A) and at a 30 L reactor scale (FIG. IB). 106colony forming units (CFU) / mL free cell inoculum, and either 106or 108CFU / mL frozen capsules containing Lacticaseibacillus paracasei CBA L74 were compared for bacterial growth kinetics during fermentation.

[0041] FIG. 2 shows bacterial growth curves obtained in freshly prepared capsules (FPC), in frozen capsules (FrC), in capsules prepared with 30% glycerol and then frozen (FrCG), and in the corresponding external suspensions in which they were mixed to ferment (dashed lines). Bars represent standard deviations of three independent tests.

[0042] FIG. 3 shows bacterial growth curves in the fermented media obtained using either standard inoculum free cells (106CFU / mL), frozen capsules as inoculum (106CFU / mL), and frozen capsules as inoculum (108CFU / mL).

[0043] FIG. 4 shows bacterial growth curves of reference alginate-based capsules (Ci), hydrolyzed oatmeal external liquid of Ci (Li), double-coating chitosan-based capules (C2), hydrolyzed oatmeal external liquid of C2 (L2), double-layer alginate-based capsules (C3), and hydrolyzed oatmeal external liquid of C3 (L3) samples determined during 24 hours of fermentation. Each result reported is a mean value of a triplicate analysis and error bars indicate the corresponding standard deviations. Values marked with different letters indicate significant differences (p < 0.05) for minibioreactors.

[0044] FIGs. 5A-5B show lactic acid concentration of all samples after 24 hours of fermentation. FIG. 5A shows the lactic acid content of samples Ci, C2, and C3. FIG. 5B shows the lactic acid content of the external liquid samples Li, L2 and, L3. Each result reported is a mean value of a triplicate analysis and error bars indicate the corresponding standard deviations. Values marked with **, ***, and ****, are significantly different (p < 0.01, p < 0.001 and p < 0.0001, respectively).

[0045] FIG. 6 provides a schema of the intracellular cascade promoted by the binding between lipopolysaccharide (LPS) and toll-like receptor 4 (TLR-4). LPS and TLR4 binding results in the binding of adaptor protein, MyD88, which promotes the activation of transcription factor NF-KB.

[0046] FIGs. 7A-7B show HCT116 cell viability after exposure to different percentages of capsule-related samples, with or withour enzymatic digestion. FIG. 7 A shows HCT116 cell viability (%) by MTT assay, after exposure of cells to different percentages of extracts obtained from cell-free fermented oat (FCO), capsules (Caps), and their external suspensions (ExS). C: negative control (cells without extracts); H2O2 was used as positive cytotoxicity control. Bars represents standard deviations of three independent experimental tests. ****p<0.0001. FIG. 7B shows HCT116 cell viability (%) by MTT assay, after exposure of cells to different percentages of extracts obtained from digested cell-free fermented oat (dFCO), digested capsules (dCaps), and their digested external suspensions (dExS). C: negative control (cell without extracts); H2O2 was used as positive cytotoxicity control. Bars represents standard deviations of three independent experimental tests. **p<0.01; p ****p<0.0001.

[0047] FIGs. 8A-8B show TLR4 (FIG. 8A) and Myd88 (FIG. 8B) expression in LPS- inflammed HCT116 cells after stimulation with digested and undigested (i) 0.1% postbiotic product (CAPS 0.1% and CAPS 0.1%, digested — respectively) and (ii) cell- free, fermented oats (FREE 0.1% and FREE 0.1%, digested — respectively). Values marked with *, **, ***, and ****, are significantly different (p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively).

[0048] FIG. 9 provides a schema of the tandem fermentation. Capsules containing probiotic fermenting microorganisms are used as an inoculum for tandem fermentation in an external suspension. After 24 hours of fermentation, the capsules are collected and suspended in a fresh external suspension to act as an inoculum again.

[0049] FIG. 10 shows bacterial growth (in CFU / mL) over 96 total hours of tandem fermentation. The single, topmost line represents the bacterial load of the capsules. The four, bottomost lines represent the bacterial load of each of the first, second, third, and fourth external suspensions after 24 hours of fermentation, respectively.DETAILED DESCRIPTION OF THE DISCLOSURE

[0050] The present disclosure is directed to methods of preparing a capsule comprising a fermenting microorganism and methods of using said capsule for obtaining a fermentation product.1. Definitions

[0051] In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0052] Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to specific compositions or process steps, as such can vary. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. The terms “a” (or “an”), as well as the terms “one or more,” and “at least one” can be used interchangeably herein.

[0053] Furthermore, “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0055] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0056] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. As used herein, the terms “comprise” and “include” and variations thereof (e.g., “comprises,” “comprising,” “includes,” and “including”) will be understood to indicate the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other component, feature, element, or step or group of components, features, elements, or steps. Any of the terms “comprising,” “consisting essentially of,” and “consisting of’ may be replaced with either of the other two terms, while retaining their ordinary meanings.

[0057] The term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower).

[0058] As used herein, the term “approximately,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain aspects, the term “approximately” refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0059] As used herein, the terms “ug” and “uM” are used interchangeably with “pg” and “pM,” respectively.

[0060] The term “probiotic” as used herein refers to live microorganisms which administered in adequate amounts are able to confer healthy effect on the host. Probiotics are discussed in, e.g., Hill, C., Guarner, F., Reid, G. et al., The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic, Nat Rev Gastroenterol Hepatol 11, 506-514 (2014).

[0061] The term “postbiotic” as used herein refers to a preparation derived from probiotic cells, after their fermentation and inactivation, composed of the same microorganisms or their components and fragments and / or their metabolites, which can confer healthy effect(s) on the host. Postbiotics are also known as “non-viable probiotics”, “inactivate probiotics” or “ghost probiotics” and refer to both non-viable microbial cells and soluble factors secreted by live bacteria or released after their lysis, including various cell surface components, lactic acid, short-chain fatty acids (SCFAs) and bioactive peptides among other metabolites. The bacterial inactivation may occur, for example, by a mild heat treatment.

[0062] The term “fermentation” as used herein refers to a metabolic process by which organic molecules are broken down anaerobically.

[0063] The “fermenting microorganism” as used herein refers to any microorganism, including bacterial and fungal organisms, suitable for use in a desired fermentation process to produce a fermentation product. The fermenting organism can be hexose and / or pentose fermenting organisms, or a combination thereof. Both hexose and pentose fermenting organisms are well known in the art.Suitable fermenting microorganisms are able to ferment, i.e., convert, sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, and / or oligosaccharides, directly or indirectly into the desired fermentation product. Examples of bacterial and fungal fermenting organisms producing ethanol are described, for example, by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

[0064] As used herein, “fermentation product” refers to any substance derived from the fermentation. The fermentation product can be, without limitation, an alcohol (e.g.,_arabinitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1,3- propanediol [propylene glycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane), a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene); an amino acid (e.g., aspartic acid, glutamicacid, glycine, lysine, serine, tryptophan, and threonine); a gas (e.g., methane, hydrogen (H2), carbon dioxide (CO2), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); an organic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-Dgluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3 -hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); 1-3 propane diol, and polyketide. The fermentation product can also be protein.

[0065] As used herein, “capsule” refers to a semi-permeable vehicle that coats or entraps specific materials from an outer environment. Capsules may encapsulate microorganisms, for example, and allow the diffusion of microorganisms, reagents and reaction products across the capsule layer(s). Capsules may have one or more layers, for example, one or more semi-permeable layers. Capsules may be distinguished on the basis of their size, shape, and material composition, for example. With regards to the size, capsules may be distinguished as nanocapsules (with diameters of less than 1 micrometer), microcapsules (if in the order of microns), or macrocapsules (if larger). In terms of morphology, capsules may be distinguished as mononuclear or continuous core / shell capsules, characterized by a spherical geometry and a continuous shell surrounding a continuous core, and multinuclear or polynuclear capsules, which are characterized by irregular shapes and core material droplets dispersed in the shell material. Capsules may be spherical, filamentous, irregular, or flat geometry. Examples of materials used for producing capsules include proteins, such as whey proteins, gelatin, and caseins; lipids, such as phospholipids; and polysaccharides, such as starch, cellulose, and alginate. Capsules may be produced by, for example, processes such as spray-drying, lyophilization, extrusion, high shear rate mixing, exploiting chemical-physical methods such as interfacial polymerization and molecular inclusion complexation coacervation, liposomes, and ionic gelation.

[0066] The term “multiple” as used herein means consisting of, comprising, and / or involving more than one.

[0067] “Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules, or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated, however, that the resulting reaction product can beproduced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture. The term “contacting” may include allowing two species to react, interact, or physically touch, wherein the two species may be a compound as described herein and a protein or enzyme.

[0068] The term “effective amount” refers to an amount of an agent that provides the desired biological, therapeutic, and / or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount can be administered in one or more administrations.

[0069] The term “combination” refers to either a fixed combination in one dosage unit form, or a combined administration where a compound of the present disclosure and a combination partner (e.g. another drug as explained below, also referred to as “therapeutic agent” or “co-agen ’) may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g. synergistic effect. The single components may be packaged in a kit or separately. One or both of the components (e.g., powders or liquids) may be reconstituted or diluted to a desired dose prior to administration. The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g. a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. “Concurrently,” as used herein to, refers to administration of two or more therapeutic agents, where at least part of the administration overlaps in time. Accordingly, concurrent administration includes a dosing regimen when the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s).

[0070] A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g. cancer) and compared to a known normal (non-diseased) individual (e.g. a standard control subject). A standard control can also represent an average measurement or value gathered from a population of similar individuals (e.g.standard control subjects) that do not have a given disease (i.e. standard control population), e.g., healthy individuals with a similar medical background, same age, weight, etc. A standard control value can also be obtained from the same individual, e.g. from an earlier-obtained sample from the patient prior to disease onset. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g. RNA levels, protein levels, specific cell types, specific bodily fluids, specific tissues, synoviocytes, synovial fluid, synovial tissue, fibroblast-like synoviocytes, macrophage-like synoviocytes, etc).

[0071] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0072] The term “inactive” or “inactived,” as used herein to describe, e.g., inactivated microbial biomass, inactivated fermentation products, and inactivated fermenting microorganisms — refers to a state wherein a microorganism is dead, non-reproductive, and / or otherwise metabolically dormant.

[0073] Various aspects of the disclosure are described in further detail in the following subsections.2. Methods of the Disclosure

[0074] As illustrated below, the methods according to the present disclosure are based on the use of encapsulated microorganisms (i.e., one or more capsules including fermenting microorganisms) to perform fermentations. According to one aspect of the disclosure, each fermentation process in the method is carried out using a capsule comprising a fermenting microorganism, wherein the capsule acts as an inoculum for initiation microbial growth and fermentation of a culture media. Additional methods are provided for preparing a plurality of capsules comprising fermenting microorganisms, wherein the capsules comprise one or more layers (e.g., an alginate layer and / or a chitosan layer).

[0075] In one aspect of the present disclosure is provided a method of obtaining a fermentation product comprising the steps of: a) inoculating a culture media with a capsule comprising a fermenting microorganism; and b) fermenting the inoculated culture media under conditions suitable for fermentation, thereby obtaining a fermentation product. In some aspects, prior to the inoculating, the capsule is frozen. In some aspects, prior to the inoculating, the capsule is freshly-prepared. In some aspects, prior to step a), the capsule is stored frozen. In some aspects, the capsule is stored frozen in a glycerol solution.

[0076] In one aspect of the present disclosure is provided a method of obtaining a fermentation product comprising the steps of: a) inoculating a culture media with a capsule comprising a fermenting microorganism, wherein optionally prior to the inoculating, the capsule is frozen; and b) fermenting the inoculated culture media under conditions suitable for fermentation, thereby obtaining a fermentation product. In some aspects, prior to step a), the capsule is stored frozen. In some aspects, the capsule is stored frozen in a glycerol solution.

[0077] In some aspects, the glycerol solution comprises about 10% to about 50% w / v glycerol. In some aspects, the glycerol solution comprises about 10% w / v glycerol. In some aspects, the glycerol solution comprises about 20% w / v glycerol. In some aspects, the glycerol solution comprises about 30% w / v glycerol. In some aspects, the glycerol solution comprises about 40% w / v glycerol. In some aspects, the glycerol solution comprises about 50% w / v glycerol.

[0078] In some aspects, prior to step a), the frozen capsule is incubated at room temperature (i.e., the frozen capsule is thawed at room temperature prior to step a).

[0079] In some aspects, the capsule comprises a culture broth, a substrate comprising starch and / or fiber, alginate, and / or CaCh. In some aspects, the culture broth selected from the group consisting of MRS broth, Brain Heart Infusion (BHI) broth, Luria-Bertani (LB) broth, plant-derived borth, functional broth containing plant extracts with antioxidant, antiviral and / or antibacterial activity, culture broth of natural origin, or animal-free broth. In some aspects, the culture broth is MRS broth. In some aspects, the culture broth is animal-free broth. In some aspects, the culture broth is a broth that can support microbial growth, wherein the broth is natural or synthetic in origin. In some aspects, the culture broth is derived from food products, vegetable products, and / or fruitproducts. In some aspects, the substrate comprising starch and / or fiber is a cereal suspension. In some aspects, the cereal suspension is an oatmeal suspension.

[0080] In some aspects, prior to step a), the capsule is resuspended in a hydrolyzed oatmeal suspension or a culture broth. In some aspects, prior to step a), the capsule is resuspended in a hydrolyzed oatmeal suspension. In some aspects, prior to step a), the capsule is resuspended in a culture broth.

[0081] In some aspects, the hydrolyzed oatmeal suspension comprises about 1% to about 50% w / v oat flour, and about 0.1% to about 5% w / v glucose. In some aspects, the hydrolyzed oatmeal suspension comprises about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% w / v oat flour. In some aspects, the hydrolyzed oatmeal suspension comprises about 0.1%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5% w / v glucose.

[0082] In some aspects, the hydrolyzed oatmeal suspension comprises about 15% w / v oat flour and about 1% w / v glucose. In some aspects, the culture broth is MRS broth or animal-free broth. In some aspects, the culture broth is MRS broth. In some aspects, the culture broth is animal-free broth.

[0083] In some aspects, the capsule comprises an alginate layer. In some aspects, the capsule further comprises a chitosan layer. In some aspects, the capsule further comprises a second alginate layer. In some aspects, the capsule comprises a first alginate layer and a second alginate layer. In some aspects, the capsule comprises an alginate layer and a chitosan layer.

[0084] In some aspects, the capsule is produced by extrusion, high shear rate mixing, spray-drying, emulsion, lyophilization, and / or layer-by-layer production. In some aspects, the capsule is produced by extrusion. In some aspects, the capsule is produced by high shear rate mixing. In some aspects, the capsule is produced by spray-drying. In some aspects, the capsule is produced by emulsion. In some aspects, the capsule is produced by lyophilization. In some aspects, the capsule is produced by layer-by-layer production.

[0085] In some aspects, the capsule is mechanically disrupted. In some aspects, the mechanical disruption of the capsule comprises comminution of the capsule via highspeed airflow pulverization, ball milling, high-speed rotary strike crushing, vacuum superfine grinding, low-temperature milling, or cryogenic grinding. In some aspects, themechanical disruption of the capsule comprises lyophilizing the capsule. In some aspects, the mechanical disruption of the capsule comprises spray-drying the capsule.

[0086] In some aspects, the capsule is produced by extrusion using about 0.5% to about 1.5 % w / v alginate and about 0.1M to about 3M CaCh. In some aspects, the capsule is produced by extrusion using about 0.5%, about 1%, or about 1.5 % w / v alginate. In some aspects, the capsule is produced by extrusion using about 0.1M, about 0.5M, about IM, about 1.5M, about 2M, about 2.5M, or about 3M CaCh. In some aspects, the capsule is produced by extrusion using 1% w / v alginate and IM CaCh.

[0087] In some aspects, the capsule is of a spherical, filamentous, irregular, or flat geometry. In some aspects, the capsule is of a spherical geometry. In some aspects, the capsule is of a filamentous geometry. In some aspects, the capsule is of an irregular geometry. In some aspects, the capsule is of a flat geometry.

[0088] In some aspects, the method further comprises inoculating the culture media with one or more additional capsules comprising fermenting microorganisms. In some aspects, each of the one or more additional capsules comprises a different fermenting microorganism.

[0089] In another aspect, provided herein is a method of preparing a plurality of capsules comprising a fermenting microorganism, comprising the steps of: (i) preparing a suspension comprising a fermenting microorganism culture and an alginate solution; and (ii) dripping the suspension of step (i) into a calcium chloride solution, thereby forming a plurality of capsules comprising the fermenting microorganism, and incubating the plurality of capsules in the calcium chloride solution.

[0090] In another aspect, provided herein is a method of preparing a plurality of capsules comprising a fermenting microorganism, comprising the steps of: (i) preparing a hydrolyzed oatmeal suspension comprising amylase-treated oatmeal and glucose; (ii) mixing the hydrolyzed oatmeal suspension with a fermenting microorganism culture and alginate solution; and (iii) dripping the mixture of step (ii) into a calcium chloride solution, thereby forming a plurality of capsules comprising the fermenting microorganism, and incubating the plurality of capsules in the calcium chloride solution. In some aspects, the method further comprises rinsing the capsules with distilled water. In some aspects, the method further comprises mixing the capsules with a glycerol solution and freezing the mixture.

[0091] In some aspects, the alginate solution comprises about 0.5% to about 2.0 % w / v alginate. In some aspects, the alginate solution comprises about 0.5% w / v alginate. In some aspects, the alginate solution comprises about 1% w / v alginate. In some aspects, the alginate solution comprises about 1.5% w / v alginate. In some aspects, the alginate solution comprises about 2% w / v alginate.

[0092] In some aspects, the calcium chloride solution comprises about 0.1M to about 3M CaCh. In some aspects, the calcium chloride solution comprises about 0.1M CaCh. In some aspects, the calcium chloride solution comprises about 0.5M CaCh. In some aspects, the calcium chloride solution comprises about IM CaCh. In some aspects, the calcium chloride solution comprises about 1.5M CaCh. In some aspects, the calcium chloride solution comprises about 2M CaCh. In some aspects, the calcium chloride solution comprises about 2.5M CaCh. In some aspects, the calcium chloride solution comprises about 3M CaCh.

[0093] In some aspects, the capsules are of a spherical, filamentous, irregular, or flat geometry. In some aspects, the capsules are of a spherical geometry. In some aspects, the capsules are of a filamentous geometry. In some aspects, the capsules are of an irregular geometry. In some aspects, the capsules are of a flat geometry.

[0094] In some aspects, the fermenting microorganism culture comprises about 106colony forming units (CFU) / mL to about 1011CFU / mL. In some aspects, the fermenting microorganism culture comprises about 106colony forming units CFU / mL. In some aspects, the fermenting microorganism culture comprises about 107colony forming units CFU / mL. In some aspects, the fermenting microorganism culture comprises about 108colony forming units CFU / mL. In some aspects, the fermenting microorganism culture comprises about 109colony forming units CFU / mL. In some aspects, the fermenting microorganism culture comprises about 1010colony forming units CFU / mL. In some aspects, the fermenting microorganism culture comprises about 1011colony forming units CFU / mL.

[0095] In some aspects, each capsule comprises about 103CFU / mL to about 109CFU / mL of the fermenting microorganism. In some aspects, each capsule comprises about 103CFU / of the fermenting microorganism. In some aspects, each capsule comprises about 104CFU / mL of the fermenting microorganism. In some aspects, each capsule comprises about 105CFU / mL of the fermenting microorganism. In some aspects, each capsule comprises about 106CFU / mL of the fermenting microorganism. In some aspects, eachcapsule comprises about 107CFU / mL of the fermenting microorganism. In some aspects, each capsule comprises about 108CFU / mL of the fermenting microorganism. In some aspects, each capsule comprises about 109CFU / mL of the fermenting microorganism.

[0096] In some aspects, step (i) comprises activating an amylase enzyme at about 60 °C to about 80 °C, followed by adding oat flour and glucose, incubating at about 60 °C to about 80 °C, and then incubating at about 80 °C to about 95 °C. In some aspects, step (i) comprises activating an amylase enzyme at about 65 °C, followed by adding oat flour and glucose, incubating at about 65 °C, and then incubating at about 85 °C.

[0097] In some aspects, the hydrolyzed oatmeal suspension comprises about 1% to about 50% w / v oat flour, and about 0.1% to about 5% w / v glucose. In some aspects, the hydrolyzed oatmeal suspension comprises about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% w / v oat flour. In some aspects, the hydrolyzed oatmeal suspension comprises about 0.1%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5% w / v glucose. In some aspects, the hydrolyzed oatmeal suspension comprises about 15% w / v oat flour and about 1% w / v glucose.

[0098] In some aspects, the fermenting microorganism is selected from the group consisting of a bacterial microorganism and a yeast microorganism. In some aspects, the bacterial microorganism is selected from the group consisting of Lactobacillus species, a Lactococcus species, a Bifidobacterium species, a Lacticaseibacillus species, a Streptococcus species, and an Akkermansia species. In some aspects, the yeast microorganism is selected from a Saccharomyces species.

[0099] In some embodiments, the fermenting microorganism is selected from the group consisting of Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Lacticaseibacillus paracasei, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium longum, Streptococcus salivarius, Akkermansia muciniphila, and Saccharomyces boulardii. In some embodiments, the fermenting microorganism is Lactobacillus paracasei. In some embodiments, the fermenting microorganism is Lactobacillus rhamnosus. In some embodiments, the fermenting microorganism is Lactobacillus plantarum. In some embodiments, the fermenting microorganism is Lactobacillus casei. In some embodiments, the fermenting microorganism is Bifidobacterium bifidum. In some embodiments, the fermenting microorganism isStreptococcus salivarius. In some embodiments, the fermenting microorganism is Saccharomyces boulardii. In some embodiments, the fermenting microorganism is Lacticaseibacillus paracasei. In some embodiments, the Lacticaseibacillus paracasei is Lacticaseibacillus paracasei NPB01.

[0100] In one embodiment, the fermenting microorganism is Lacticaseibacillus paracasei NPB-01. In one embodiment, the microorganisms of the genus Lactobacillus are selected from the group consisting of Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, Limosilactobacillus reuteri, and Lactobacillus delbrueckii.

[0101] Non-limiting examples of microorganisms of the genus Bifidobacterium suitable for use in the methods of the disclosure are the species Bifidobacterium longum, Bifidobacterium animali . Bifidobacterium breve, and Bifidobacterium infantis.

[0102] According to one aspect of the disclosure, the microorganism of the Lactococcus genus is preferably a Lactococcus lactis strain, the microorganism of the Streptococcus genus is preferably a Streptococcus thermophilus strain, and / or the microorganism of the Saccharomyces genus is preferably a strain selected from Saccharomyces boularddi and Saccharomyces cerevisae.

[0103] Any culture medium may be used in the present disclosure, which may be selected based on the nutritional requirements of the fermenting microorganisms in order to maximize bacterial growth, yield of postbiotic metabolites and bioactivity and to confer specific organoleptic characteristics to the postbiotic composition.

[0104] Non-limiting examples of suitable culture media are MRS media, plant-derived media, functional media containing plant extracts with antioxidant, antiviral and / or antibacterial activity, culture media of natural origin, and any combination thereof.

[0105] The selection of the most appropriate culture medium is well known to those skilled in the art.

[0106] According to one aspect of the methods of the disclosure, the fermentation step is carried out at a temperature comprised from about 25°C to about 45°C, preferably at about 37°C, for a time of at least about two hours, preferably for about 6 to about 24 hours. The pH is typically kept at a value comprised from about 4.0 to about 7.0, preferably at about 6.2.

[0107] The fermentation process can be carried out in any type of stirred or wave-type bio-reactor. Examples of useful bioreactors for the present disclosure include, but are notlimited to batch reactors, fed-batch reactors, CSTR (Continuous-flow Stirred-Tank Reactor) reactors.

[0108] In some aspects, the method further comprises inactivating the fermenting microorganism(s) in the fermentation product.

[0109] In one aspect of the methods of the disclosure, the step of inactivating the fermenting microorganism in the fermentation product may be carried out by means of various techniques, as are known in the art. Such techniques include, but are not limited to, heat treatment, chemical treatment (e.g., formalin), gamma or ultraviolet irradiation, high pressure, dielectric heat treatment, radiofrequency, microwave, and sonication. Sonication, in particular is the method commonly used to produce cell lysates: in another embodiment, this method can increase the availability of functional components, enhancing the fermentation performances of the second microorganism.

[0110] Heat inactivation of the fermenting microorganism in the fermentation product can be used. Preferably, heat inactivation is conducted at a temperature comprised from about 50°C to about 100°C for a time of about 5 seconds to about 120 seconds. In an exemplary embodiment, heat inactivation is conducted at about 80°C for about 30 seconds.[OHl] In one embodiment, the fermentation product of the method according to the disclosure is a culture broth.

[0112] In another aspect, provided herein is a postbiotic product as described herein for use in the prevention or the therapeutic treatment of a disease in a subject in need thereof selected from the group consisting of infectious and inflammatory diseases, immune- mediated diseases, cancer diseases, skin disorders, gastrointestinal diseases, urogenital tract diseases, neurologic disorders, neuropsychiatric disorders, bone diseases, muscle diseases, malnutrition, metabolic diseases, and any combination thereof. In some aspects, use of the postbiotic product reduces Myd88 and / or TLR4 expression in the subject.

[0113] In another aspect, provided herein is a postbiotic product for use in a food, a beverage, a pharmaceutical, a nutraceutical, a cosmetic, or a packaging composition, wherein the composition further comprises at least one pharmaceutically acceptable vehicle, excipient and / or diluent.

[0114] The preferred embodiments described above can be combined with each other as required, and the implementation of these combinations falls within the skills of the person skilled in the art.3. Compositions and Kits of the Disclosure

[0115] Also provided herein are encapsulated fermenting microorganism compositions produced by a method of the disclosure. In another aspect, provided herein is a kit comprising a capsule comprising a fermenting microorganism as described herein.

[0116] In another aspect, provided herein is an encapsulated fermenting microorganism composition comprising: i) a capsule comprising a fermenting microorganism, wherein the capsule comprises at least one alginate layer; and ii) a glycerol solution. In some aspects, the capsule further comprises a chitosan layer.

[0117] In some aspects, the capsule comprises an alginate layer. In some aspects, the capsule further comprises a chitosan layer. In some aspects, the capsule further comprises a second alginate layer. In some aspects, the capsule comprises a first alginate layer and a second alginate layer. In some aspects, the capsule comprises an alginate layer and a chitosan layer.

[0118] In some aspects, the capsule is of a spherical, filamentous, irregular, or flat geometry. In some aspects, the capsule is of a spherical geometry. In some aspects, the capsule is of a filamentous geometry. In some aspects, the capsule is of an irregular geometry. In some aspects, the capsule is of a flat geometry.

[0119] In some aspects, the capsule is produced by extrusion, high shear rate mixing, spray-drying, emulsion, lyophilization, and / or layer-by-layer production. In some aspects, the capsule is produced by high shear rate mixing. In some aspects, the capsule is produced by extrusion. In some aspects, the capsule is produced by spray-drying. In some aspects, the capsule is produced by emulsion. In some aspects, the capsule is produced by lyophilization. In some aspects, the capsule is produced by layer-by-layer production.

[0120] In some aspects, the capsule is produced by extrusion using about 0.5% to about 1.5 % w / v alginate and about 0.1M to about 3M CaC12. In some aspects, the capsule is produced by extrusion using about 0.5%, about 1%, or about 1.5 % w / v alginate. In some aspects, the capsule is produced by extrusion using about 0.1M, about 0.5M, about IM, about 1.5M, about 2M, about 2.5M, or about 3M CaC12. In some aspects, the capsule is produced by extrusion using 1% w / v alginate and IM CaC12.

[0121] In some aspects, the capsule comprises a culture broth, a substrate comprising starch and / or fiber, alginate, and / or CaC12. In some aspects, the culture broth is selected from the group consisting of MRS broth, Brain Heart Infusion (BHI) broth, Luria-Bertani (LB) broth, plant-derived broth, functional broth containing plant extracts withantioxidant, antiviral and / or antibacterial activity, culture broth of natural origin, or animal-free broth. In some aspects, the substrate comprising starch and / or fiber is a cereal suspension. In some aspects, the cereal suspension is an oatmeal suspension. In some aspects, the cereal suspension is an oatmeal suspension.

[0122] In some aspects, the capsule comprises about 103CFU / mL to about 109CFU / mL of the fermenting microorganism. In some aspects, the capsule comprises about 103CFU / mL of the fermenting microorganism. In some aspects, the capsule comprises about 104CFU / mL of the fermenting microorganism. In some aspects, the capsule comprises about 105CFU / mL of the fermenting microorganism. In some aspects, the capsule comprises about 106CFU / mL of the fermenting microorganism. In some aspects, the capsule comprises about 107CFU / mL of the fermenting microorganism. In some aspects, the capsule comprises about 108CFU / mL of the fermenting microorganism. In some aspects, the capsule comprises about 109CFU / mL of the fermenting microorganism.

[0123] A further aspect of the disclosure is a pharmaceutical, nutraceutical, or cosmetic composition, or a food product or food supplement comprising a fermentation product obtained using the methods described herein, in combination with at least one pharmaceutically acceptable vehicle, excipient and / or diluent. Suitable compositions and formulations may be prepared by methods commonly employed using conventional organic and inorganic additives.

[0124] In another aspect, provided herein is a kit comprising a capsule comprising a fermenting microorganism as described herein.

[0125] In various embodiments, kits for use in the laboratory and therapeutic applications described herein are within the scope of the present disclosure. Such kits may comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method disclosed herein, along with a label or insert comprising instructions for use, such as a use described herein.

[0126] Kits may comprise the container described above, and one or more other containers associated therewith that comprise materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carrier, package, container, vial and / or tube labels listing contents and / or instructions for use, and package inserts with instructions for use.

[0127] A label may be present on or with the container to indicate that the composition is used for a specific therapy or non-therapeutic application, such as a prognostic, prophylactic, diagnostic, or laboratory application. A label may also indicate directions for either in vivo or in vitro use, such as those described herein. Directions and or other information may also be included on an insert(s) or label(s), which is included with or on the kit. The label may be on or associated with the container. A label may be on a container when letters, numbers, or other characters forming the label are molded or etched into the container itself. A label may be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. The label may indicate that the composition is used for diagnosing or treating a condition, such as a cancer a described herein.

[0128] The embodiments described above can be combined with each other as required, and the implementation of these combinations falls within the skills of the person skilled in the art.

[0129] The present disclosure is further illustrated by the following examples which should not be construed as further limiting. The contents of all references cited throughout this application are expressly incorporated herein by reference.EXAMPLESExample 1: Encapsulated microorganisms for efficient fermentation

[0130] The composition, size, and the cross-linking of capsule walls can be appropriately chosen and modified, in particular by modulating their permeability, to control capsule diffusion of bacteria. Controlling the permeability of capsules allows the inoculation of media to be performed in a controlled manner.

[0131] Furthermore, the capsules act not only as microorganism release devices but also as small-scale fermenters, “minibioreactors”, in which it is possible to obtain high performance and process yields due to an active and continuous growth of microorganisms and production of metabolites, which diffuse during the fermentation process towards the external environment.

[0132] Various techniques, such as emulsion, spray-drying, lyophilization, interfacial polymerization, molecular inclusion complexation, coacervation, liposomes, and ionic gelation can be used to produce capsules for specific purposes. Capsules ranging in sizefrom a few micrometers to a few millimeters can be used as a ready -to-use inoculum (e.g., 2-4 mm).

[0133] Capsules made of different materials (such as culture broth, for example, MRS or animal-free broth; cereal suspension, for example, oat suspension; alginate and CaC12) can be utilized as a ready -to-use inoculum. Capsules infused with different bacterial concentrations (ranging from 103 CFU / mL to 1010 CFU / mL, preferably 106-108 CFU / mL) can be used as a ready-to-use inoculum.

[0134] In the current study, 4mm spherical capsules produced by ionic gelation using an oatmeal suspension as material along with alginate and CaCh, were tested.Materials and MethodsStrain

[0135] Lacticaseibacillus paracasei CBA L74 (Heinz Italia S.p.A), a gram-positive and homofermentative strain, was used as the fermenting microorganism. L. paracasei CBA L74 has been shown to have protective effects against rotavirus infection in human enterocytes (Bruno C et al. Sci. Rep. 2022; 12(1):6268). It was stored at -80°C in an animal-free broth (20 g / L Bacto Yeast Extract (BD Biosciences, Milan, Italy), 0.5 g / L MgSCU (Sigma-Aldrich, Milan, Italy), 50 g / L glucose (Sigma-Aldrich), 0.5 g / L citric acid (Sigma-Aldrich)) with 20% glycerol. To revitalize it, 9 mL of the same animal-free broth was used, and an incubation of 24 h at 37°C was carried out. After the revitalization, a bacterial concentration of about 108CFU / mL was obtained.Oatmeal Suspension

[0136] Whole oat flour (Le Farine magiche, Lo Conte Group) was purchased in a local market. A suspension made with 15% w / v of oat flour in water, with the addition of 1% w / v glucose, was prepared.

[0137] The oatmeal suspension was pretreated according to Lentini et al., Fermentation, 2022, 8(11), 632. Briefly, an enzymatic pretreatment using 0.036% w / v amylase (E- BLAAM, Megazyme) was carried out to prevent starch gelatinization during the following sterilization phase. In particular, water and amylase were heated at 75°C for enzyme activation. The temperature was maintained for 20 min. After that, oat flour (15% w / v) and glucose (1% w / v) were added, and the temperature was maintained at 75°C foranother 25 min. The suspension was then further heated at 90°C with a holding of 20 min and, finally, sterilized in an autoclave at 134°C for 40 min.Encapsulation

[0138] Ionic gelation was carried out by direct extrusion, carried out using a peristaltic pump (Watson Marlow, 401U / D1), to which a silicone tube was mounted, with the possibility of regulating the flow rate. The hydrolyzed oatmeal suspension was mixed with the revitalized bacterial culture, and with 1% w / v Na-alginate (Alginic acid sodium salt from brown algae, Sigma Aldrich). The mixture was dripped into a 1 M calcium chloride solution through a peristaltic pump (nozzle size 2 mm). The flow rate was set at 5 ml / min. The capsules were left to soak for 20 minutes, then collected and rinsed in distilled water for a few minutes. To understand whether the capsules could be used as a ready -to-use inoculum, once produced, a portion of them were directly used as fresh inoculum, and a portion were stored at -20°C, before being used for a fermentation process. Among the frozen capsules, two different conditions were tested: capsules stored without any cryoprotectant and capsules prepared with 30% v / v glycerol. Frozen capsules were kept for 10 min. at room temperature and were then used as an inoculum.Experimental ApparatusIL reactor

[0139] A I L reactor was used to carry out preliminary inoculum and fermentation tests, carried out to compare freshly-prepared capsules or frozen capsules (FIG. 1 A). The system consisted of a batch reactor vessel with an external jacket circling service fluid from a thermostatically controlled water bath. The mixing system, with its versatile connection to a motor, allowed for easy adjustments of the stirring speed, adapting to various process requirements. A silicone gasket placed on the edge of the vessel and a metal ring around the head plate ensured the reactor’s hermetic sealing. The head plate was equipped with an input for the insertion of the In Pro 3100 probe (Mettler Toledo, Milan, Italy), connected to the M300 transmitter (Mettler Toledo, Milan, Italy), which was helpful for inline temperature / pH measurements, and an input connected to a tank containing a 4M NaOH solution, fed by a peristaltic pump used for controlling pH.30 L reactor

[0140] A 30 L reactor was used to perform inoculation and fermentation tests, which were carried out to study the fermentation process with capsules prepared by varying the encapsulated bacterial concentration (FIG. IB). The 30L vessel was made on AISI 316. On the top an AISI cover was fixed using fast lock. On this cover were located several vent ports (vent valves, safety valves and measuring instruments such thermocouple and pHmeter). The vessel was of jacketed type to supply a suitable amount of a hot / cold process fluid (water) to control the process condition.

[0141] The mixing system was composed of a shaft on which two impellers were mounted. The first one was 6-plane bladed and positioned at 10 cm from liquid surface; the second one was a two bladed impeller located at the bottom of the vessel. The rotation of the shaft was assured by a 220 V motor. The thermal system, able to control the temperature of service fluid (water), supplied to the reactor jacket. Temperatures of the process fluid and of the product were monitored and controlled by a thermocouple (K type) interfaced to an on-line acquisition data system. The temperature of the process fluid supplied to external jacket was controlled by a thermal resistance and by cold water suitably mixed.Fermentation Protocols

[0142] To understand whether frozen capsules showed the same inoculum ability of freshly prepared capsules, preliminary fermentation tests were carried out in a 1-L rector. Freshly prepared capsules or frozen capsules were used as an inoculum, by suspending them in an external liquid, consisting of the same hydrolyzed oatmeal suspension used to produce capsules. A fixed ratio 1 :3 between the volume used to produce the capsule (333 mL) and the volume of the external suspension (1000 mL) was used in the preliminary fermentation tests carried out in a 1 L reactor.

[0143] To understand whether capsules prepared with different bacterial concentrations (106 / 108 CFU / mL) could further improve the process, fermentation tests were carried out in a 30 L reactor. A fixed ratio of 1 : 1000 between the volume used to produce the capsule (30 mL) and the volume of the external suspension (30000 mL) was used. The temperature was controlled for the entire process time (24h) at 37°C, while the pH was left free.Analyses

[0144] Capsules obtained by ionic gelation and the external hydrolyzed oatmeal suspensions in which they were put to carry out preliminary fermentation tests were characterized. Capsules were preliminarily broken in sterile sodium citrate solution (1% (w / w), pH 6.0, Sigma-Aldrich) in a 1 : 10 proportion (capsule weight / sodium citrate volume).

[0145] Aseptically sampling was carried out at specific times: after 10 min from the bacterial inoculation (tO) and after 2, 4, 6, 16, 18, 20, 22, and 24 h from the beginning. Sampling at t28 and t46 (i.e., 28 and 46 hours, respectively, from the beginning) were also carried out to compare long-term fermentation using frozen ready-to-use capsules versus standard inoculum-free cells. Samples were subjected to a microbiological characterization.

[0146] For microbiological characterization, samples were serially diluted in 0.9 % NaCl solution and spread plate method on different agar plates was carried out: MRS agar (Oxoid, Basingstoke, UK) was used for the enumeration of Lactobacilli. Plates were then incubated at 37°C for 48h.ResultsPreliminary Experimental Phase - IL Scale

[0147] Fermentation tests were conducted to compare the ability of freshly prepared and frozen capsules with and without glycerol to act as inoculum. Tests were carried out on a IL reactor, using a fixed ratio of 1 :3 between the volume used to produce the capsule (333 mL) and the volume of the external suspension (1000 mL). Three different types of capsules were tested: freshly prepared capsules (FPC), as a control, frozen capsules (FrC), and capsules prepared with glycerol and then frozen (FrCG). The bacterial growth results for the capsules and external suspensions are shown in FIG. 2.

[0148] The frozen capsules, with or without glycerol, initially had a lower bacterial concentration compared to freshly prepared capsules, although the difference was not statistically significant. This decrease is likely due to the freezing and rapid thawing process the capsules underwent. However, starting from tl 6, the bacterial growth curves of the capsules followed a similar trend, and this trend continued until the end of the process. For instance, at t24, the bacterial concentrations were as follows: for freshly prepared capsules, it was 1.44*109± 2.92* 109CFU / mL; for frozen capsules, it was6.24*108± 3.49*109CFU / mL; and for frozen capsules with glycerol, it was 1.05*109± 5.06* 109CFU / mL. This demonstrates that fermentation occurred in the capsules in the same way, regardless of whether they were fresh or frozen. Moreover, the growth trend of external suspensions was also comparable, with bacterial concentrations of 7.40* 105± 1.31 * 105CFU / mL for the suspension inoculated from fresh capsules, 1.10* 106± 1.15 * 106CFU / mL for the suspension inoculated from frozen capsules, and 2.30*106± 4.23 *106CFU / mL for the suspension inoculated from frozen capsules prepared with glycerol at t24.

[0149] These findings indicate that the capsules can serve as a ready -to-use inoculum, being particularly promising in the use of frozen capsules to have in stock, enabling the production of a probiotic product with a bacterial concentration of 2.30* 106CFU / mL within 24 hours, from the fermented external liquid. Moreover, no significant differences were found between the frozen capsules, with or without glycerol. These results also suggest that capsules can protect bacteria on their own.Experimental Phase - 30L Scale

[0150] Fermentation tests were conducted to compare the fermentation performances using either i) standard inoculum-free cells (106CFU / mL in the fermenting medium at the beginning of the process); ii) frozen ready -to-use capsules as inoculum loaded with 106CFU / mL (hypothetical bacterial concentration in the reactor at tO equal to 103CFU / mL); or iii) frozen ready -to-use capsules as inoculum loaded with 108CFU / mL (hypothetical bacterial concentration in the reactor at tO equal to 105CFU / mL).

[0151] The results of the study are shown in FIG. 3. With standard inoculum-free cells (106CFU / mL at the beginning of the fermentation), the maximum bacterial load (7.5xl08CFU / mL) is obtained after 20 hours of fermentation, while in the frozen ready-to-use capsules - 106CFU / mL, and frozen ready-to-use capsules - 108CFU / mL, the maximum bacterial load is achieved after 24h, obtaining 2xl08CFU / mL and, L lxlO9CFU / mL, respectively. Therefore, by reducing the revitalization time for the frozen ready-to-use capsules as inoculum (108CFU / mL), a value corresponding to the standard inoculum -free cell (106CFU / mL) is reached starting from 18 hours of fermentation and slightly longer times (24 h) are required for the frozen ready-to-use capsules as inoculum (106CFU / mL), which offer advantages such as reduction of the inoculation volume required.

[0152] Moreover, the capsules act as a generator of bacteria that diffuse and gradually inoculate the fermented media by exploiting their semi-permeability. As shown in FIG. 3, when frozen capsules are used, at the beginning of the fermentation (tO) the bacterial concentration in the fermented medium is zero and gradually increases by diffusion after two hours, reaching a bacterial load lower than 102CFU / mL (an initial bacterial load much lower than that commonly used in fermentation processes).

[0153] The results obtained herein indicate that the capsule inoculum has several advantages compared to the standard free cell method. The capsules save time because there is no need to repeat the revitalization phase of the strain before starting a fermentation process each time. By creating a frozen stock of capsules, it is also possible to save hours or even days, depending on the scale of the process; using frozen ready -to- use capsules reduced the fermentation time, since the entire process lasts 24 hours compared to 44 hours of standard procedure (24 hours for inoculum preparation + 20 hours of fermentation) to ferment a volume of 30 L. The capsule inoculum also limits the lag phase, normally observed with the standard inoculation, leading to a more rapid start of the fermentation process. Additionally, the capsules act as a mini-bioreactor, capable of increasing the microbial population inside. The microbes are released into the medium while being protected from contamination and inhibition by the substrate / product. Importantly, the capsules allow the use of lower microbial loads than standard free-cell inoculum, due to the protection offered, with volume (costs) saving. In the standard free cell method, a starting microbial load of 106CFU / mL is conventionally used. With the standard free cell method, it is usually not recommended to reduce the starting load in order to allow the process to start quickly and for the selected starter strain to prevail over possible contaminating microorganisms.

[0154] The saving of inoculum volume can be understood through numerical examples. Considering a volume of 10000 L to be fermented, for a standard inoculation procedure (bacteria concentration at tO in reactor 106CFU / mL), it will be necessary to use 100 L of inoculum with a bacterial concentration of 108CFU / mL. Using ready -to-use capsules as inoculum (in a ratio 1 ready -to-use capsules: 1000 fermenting medium), 10 L of volume of Capsules will be needed.

[0155] For capsules with a bacterial concentration of 108CFU / mL (hypothetical bacterial concentration in the reactor at tO equal to 105CFU / mL), 10 L of inoculum with a bacterial concentration of 108CFU / mL will be needed. For capsules with a bacterial concentrationof 106CFU / mL (hypothetical bacterial concentration in the reactor at tO equal to 103CFU / mL), 0.10 L of inoculum with a bacterial concentration of 108CFU / mL will be needed.Table 1: Numerical examples of the volume savings offered by the capsule inoculum

[0156] From the results obtained, it is possible to optimize process conditions in which the volumes of frozen ready -to-use capsules are further reduced by at least one order of magnitude. (eSt. ratio lready-to-use capsules: 1000 fermenting medium is investigated, ratio Iready-to-use capsules: 10000 fermenting medium).

[0157] An additional study was conducted to characterize the diffusion process, and the diffusion coefficient (D) of bacteria flowing from the capsules into the liquid in the first two hours of the process was estimated.

[0158] Based on the simplifying hypothesis that the capsules are spherical and the cells might be considered as macro-molecules (Liu et al., Bioresource Technology, 2008, 99(6), 1904-1910), the diffusion coefficient of the lactic acid bacteria flowing from the capsule towards the external liquid was calculated using Fick’s second law. If the capsules are approximate to spheres the Fick’s law for a spherical system is: dC d2C 2 dC— D (- — + - — ) ot o r or

[0159] To solve the problem and obtain a first estimate of the diffusion coefficient D, the following simplifying hypotheses can be made. To simplify, it can be assumed that the concentration changes mainly near the sphere’s surface and that the concentration gradient is constant. The concentration in the external liquid is much lower than that in the sphere.

[0160] Considering that the capsule has a radius equal to 2 mm, D « 1011m2 / s; an acceptable value and comparable with the the work of Liu et al., equal to D « 2.7 x 10-10m2 / s.Example 2: Influence of additional coating materials on capsule fermentation

[0161] Several authors have investigated the possibility of using a double coating technique to enhance the mechanical and chemical stability of alginate gel, control or / and reduce cell release, and improve the viability of encapsulated cells (Ching et al., Critical Reviews in Food Science and Nutrition, 2017, 57(6): 1133-1152; Loh et al., Colloid and Polymer Science, 2012, 290(7), 619-629; Simo et al., Carbohydrate Polymers, 2017, 170, 1-14). In the present study, probiotic hydrolyzed oatmeal capsules were produced by extrusion using alginate (1% w / v) and CaCh (IM) and suspended in the same hydrolyzed oatmeal suspension, carrying out the fermentation process at 37 °C for 24h. Three different capsule formulations were studied: one used as a reference and prepared without using additional coating materials, and the others formulated by (i) an additional chitosan layer (4.42 g / L) and (ii) an additional alginate (0.01 g / mL) double layer to understand the possibility of confining bacteria in the capsules without inhibiting the fermentation process through the presence of a double coating. During the fermentation period, samples of capsules and outer liquids were taken to monitor the process, and the bacterial growth and lactic acid production achieved using different capsule formulations were compared. Therefore, the biokinetic parameters were estimated for each minibioreactor system to evaluate the formulation capable of also providing a kinetic improvement of the process.Materials and MethodsStrain and Feedstock

[0162] Lacticaseibacillus paracasei CBA L74 (Heinz Italia S.p.A.) was used as a probiotic starter for the fermentation trials. The microorganism was stored at -80 °C in cryovials with glycerol (20%) and before the inoculation phase was reactivated through incubation at 37°C for 24 h in 9 mL of an animal-free broth (20 g / L Bacto Yeast Extract, BD Biosciences; 0.5 g / L MgSCU, Sigma-Aldrich; 50 g / L Glucose, Sigma-Aldrich; 0.5 g / L citric acid, Sigma- Aldrich). The bacterial load in the inoculum broth was 108CFU / mL.Whole oatmeal (Le Farine Magiche, Lo Conte Group) was purchased in a local store in Naples, Italy.Substrate preparation

[0163] A hydrolyzed oatmeal suspension was prepared to produce both the minibioreactors and the external liquids in which capsules were suspended to perform the fermentation process. The oatmeal suspension (15% w / v of oat flour with 1% w / v of added glucose) was pre-treated by enzymatic hydrolysis using amylase (0.054% w / v). This step had a dual objective: to avoid starch gelling during the successive sterilization process of the medium and minimize any mixing problems inside the bioreactor during the fermentation process (Gallo et al., Journal of Food Processing and Preservation, 2021, 45(4), el5299). The sterilization phase was carried out in an autoclave at 134°C for 40 min (Lentini et al., Fermentation, 2022, 8(11), 632).Encapsulation Process

[0164] The capsule production involved direct extrusion techniques according to the modified protocol of Saez-Orviz et al. (Applied Sciences, 2021, 11(23), 11404). Three different capsule formulations were obtained:Reference alginate-based capsules:

[0165] Mixing phase: hydrolyzed oatmeal suspension was mixed with Na-alginate (0.01 g / mL) and the strain with an initial bacterial concentration of 6 log [CFU / mL],

[0166] Extrusion process: the mixture was dropped, through a peristaltic pump into a calcium chloride solution (IM) for 20 minutes at room temperature. The nozzle sizes and the flow rate were 2 mm and 5 mL / min, respectively.

[0167] Rinsing phase: all capsules were rinsed in distilled water for a few minutes.

[0168] Collecting phase: all capsules were collected for the next fermentation step.

[0169] Double-coating chitosan-based capsules', the encapsulation steps were the same as those listed for the reference alginate-based capsules.

[0170] Addition of chitosan coating: capsules were suspended in a solution of 4.42 g / L chitosan, and 0.44% glacial acetic acid, with a pH of 5.7-6.0 adjusted by NaOH 1 M; and kept at room temperature under gentle agitation for 40 min to obtain an additional coating. This further step should allow the formation of a strong composite hydrogel inthe pH range of 4-6 with alginate (Efentakis and Buckton, Pharmaceutical development and technology, 2002, 7(1), 69-77; and Liu et al., Biomaterials, 2005, 26(15), 2705- 2711).

[0171] Rinsing phase: all capsules were rinsed in distilled water for a few minutes.

[0172] Collecting phase: all capsules were collected for the next fermentation step.

[0173] Double-layer alginate -based capsules: the encapsulation steps were the same as those listed for the reference alginate-based capsules.

[0174] Addition of alginate coating: capsules were resuspended in a Na-alginate bath (0.01 g / mL) for another 20 minutes to obtain an additional layer (Jin et al., Journal of Micro-and Nano-Manufacturing, 2017, 5(4), 041007).

[0175] Rinsing phase: all capsules were rinsed in distilled water for a few minutes.

[0176] Collecting phase: all capsules were collected for the next fermentation step.Fermentation protocol

[0177] Fermentation tests were carried out by suspending the capsules in a hydrolyzed oatmeal suspension, the same used to produce capsules, as external liquid with a ratio of 1 :3. The fermentation process was conducted by placing falcon filled with a total volume of 20 mL in agitation through a wheel incubating at 37°C, without pH control, for 24 h.Bacterial count, lactic acid, and secondary metabolite determination

[0178] To allow sample homogeneity, before analyses, all capsules were broken and dissolved in 1% (w / w) sodium citrate solution at pH 6.

[0179] To evaluate the bacterial load in both capsules and external liquid, sampling at time 0 and after 2, 4, 6, 16, 18, 20, 22, and 24 h from the beginning of the fermentation was performed. Samples were then serially diluted and spread-plated on Petri plates filled with De Man, Rogosa, and Sharpe (MRS) agar (Oxoid, Basingstoke, UK) to monitor the Lactobacillus growth. MacConkey agar (Oxoid, Basingstoke, UK) and Gelatin Peptone Bios Agar (Biolife, Milan, Italy) were used to control the presence of microbial contaminants in the samples. All plates were incubated at 37 °C for 48 h before reading. During the incubation period anaerobic kits (Anaerogen Compact, Oxoid, Basingstoke, UK) were used to guarantee anaerobic growth conditions for MRS plates. The bacterial count was expressed in CFU / mL.

[0180] After 24 hours of fermentation, the lactic acid concentration of both capsules and external liquid samples was determined by high-performance liquid chromatography (HPLC), using Agilent Technologies 1100, equipped with an Agilent Synergi Hydro-RP C18 column (250 mm x 4.6 mm and a pore size of 4 pm) with a visible / UV detector. The mobile phase consisted of 0.27% (w / v) KH2PO4 aqueous solution at a pH=1.5 modified with H3PO4 with a column temperature of 60 °C and a flow rate of 1 mL / min. The detection was set at 210 nm.

[0181] To check for the possible presence of secondary metabolites, such as acetic, propionic, and butyric acids, gas chromatography, (GC), was used through an Agilent technologies 6890 instrument, employing a capillary Poraplot Q column (25 mm x 0.32 mm). The mobile phase was helium gas with a flow rate of 200 mL / min.Fermentation parameters

[0182] To check the fermentation process in the minibioreactors (capsules) some kinetic parameters were evaluated.

[0183] Doubling time (td), was the time needed for the microorganism to double in number during the exponential growth phase. It was calculated using Equation (1) (Colucci Cante et al., Applied Sciences, 2021, 77(7), 2921):where texpis the time necessary to reach the exponential phase and n is the number of generations, calculated by Equation (2) (Colucci Cante et al., 2021):Nf= Nox 2n(2) where Noand N are the numbers of cells at the beginning and the end of the exponential growth phase texp, respectively.

[0184] The constant growth rate, k, is defined in Equation (3):Statistical analysis

[0185] Statistical analysis was performed using Prism 9®. Fermentation tests and analyses of lactic acid and secondary metabolite were carried out in triplicate and, for each experimental data, mean values, and standard deviations (n = 3) were calculated. Theirstatistical significance was evaluated by one-way ANOVA followed by Tukey’s multiple comparisons test, accepting as significant only results with p < 0.05.Results

[0186] The fermentation process starts with minibioreactors (i.e., capsules comprising the fermenting microorganism) suspended in the external liquid (1 :3) and was carried out for 24 hours. FIG. 4 shows bacterial growth curves of reference alginate-based capsules (Ci), hydrolyzed oatmeal external liquid of Ci (Li), double-coating chitosan-based capules (C2), hydrolyzed oatmeal external liquid of C2 (L2), double-layer alginate-based capsules (C3), and hydrolyzed oatmeal external liquid of C3 (L3) samples determined during 24 hours of fermentation. The test was conducted under a controlled temperature (37 °C). The bacterial growth curve for both capsules (Ci, C2, and C3) and external liquids (Li, L2, and L3) are shown.

[0187] FIG. 4 indicates the efficiency of the minibioreactors; Ci, C2, and C3 showed the same growth trend from time zero to 16 hr of fermentation in which stationary is reached; all minibioreactors can be defined as optimal fermenters as a maximum bacterial load of 8 logs for C2, and C3 and, 9 logs for Ci was obtained conferring probiotic properties (Minelli and Benini, Microbial Ecology in Health and Disease, 2008, 20(4), 180-183). From 16 hr to the end of the process (excluding 20 hr) the bacterial load values of Cl were slightly higher than those achieved for C2 and C3, with statistically significant differences; this was probably due to the double coating of capsules C2 and C3, which may limit access and therefore the exchange of the macro and micronutrients required by Lactobacilli during the fermentation process. According to De Prisco and Mauriello (Trends in Food Science & Technology, 2016, 48, 27-39), the migration of nutrients through the increasingly dense molecular structures of capsules is limited, therefore suggesting that this is a key point in the development and growth of probiotics. Rodrigues et al. (Food and Bioprocess Technology, 2012, 5, 2748-2757) indicated that a double coating on a capsule could be more protective for the encapsulated strain, but could also act as a barrier to the exchange of nutrients. As previously stated by Voo et al. (Journal of Bioscience and Bioengineering, 2011, 111(3), 294-299), the double coating of chitosan may cause additional restrictions on nutrient mass transfer, which potentially reduces cell growth; furthermore, a concentration of approximately 8 logs was obtained after 24 hours of fermentation in the alginate capsules; where MRS broth was used both as an internalsubstrate and as an external liquid in which the capsules are suspended (the external liquid was replaced by fresh medium after 12 h of fermentation).

[0188] To understand the potential confinement capacity of the bacteria inside the capsules, the microbial concentration in the external liquids (Li, L2 and L3) was monitored (FIG. 4). Already at time zero the presence of bacteria was found in all external liquids Li, L2 and L3 whose values were not significantly different and equal to: 1.0 x 10 ± 2 CFU / mL, 5.3 x 10 ± 1 .5 x 10 CFU / mL, and 9.0 x 102± 3.9 x 102CFU / mL, respectively. Therefore, despite the addition of double coatings at C2 and C3, there was a release of bacteria through the capsule membrane into L2 and L3, probably due to the presence of microbial cells placed on the external surface of the gel matrix, following the contraction of the capsules during their formation (Zohar-Perez et al., Biotechnol Bioeng, 2004, 88:671-674). Covarrubias et al. (Applied microbiology and biotechnology, 2012, 93(6), 2669-2680) observed a greater tendency of microorganisms to settle on the surface of the capsules, so their number at time zero resulted to be greater than inside the capsule, thus causing their release into the external liquid.

[0189] From time zero to 24 hr in all external liquids it is possible to observe a bacterial growth trend influenced by two indistinguishable mechanisms, the release of the bacteria and the local growth kinetics; the same growth delta was achieved, reaching a final bacterial growth of 7.4 x 105± 4.3 x 104CFU / mL, 2.4 x 105± 1.3 x 105CFU / mL, and 9.8 x 106± 1.3 x 106CFU / mL in Li, L2, an L3, respectively. It has been reported by Zohar- Perez et al. (Biotechnol Bioeng, 2004, 88:671-674) that immediately after an immobilization procedure, Escherichia coli and Trichoderma asperellumdis distribution in alginate-gel beads was nonhomogeneous with a greater presence of cells on the surface of the capsules. Therefore, adding more layers to the capsule structure does not allow an effective confinement, since the cell release from the surface beads is the main source of free cells in the fermentation medium, with subsequent growth in the external liquid (Champagne et al., Appl. Environ. Microbiol., 1992, 58, 1429-1434; Voo et al., 2011). In the study reported by Yuan et al. (Food Hydrocolloids, 2023, 145, 109094) the survival of probiotics via in situ re-culture in calcium alginate gel beads was studied: a bacterial load after 24 hours of fermentation of approximately 8 logs was reached in the external MRS- based liquid, 2 orders of magnitude higher than that found in the liquids in this study, this is probably because MRS is an elective culture broth for Lactobacilli.

[0190] Furthermore, the absence of contaminants during the process was noted as significant quantities of acetic, propionic, and butyric acids were not detected (data not shown) and no bacterial growth was noted on MacConckey agar and Gelatine Peptone Bios gar.

[0191] Lactic acid concentrations for Ci, C2, and C3, and Li, L2, and L3 samples after 24 hours of fermentation are shown in FIGs 5A and 5B, respectively. In all capsules Ci, C2, and C3 and external liquids Li, L2, and L3, lactic acid content was present after 24 hours of fermentation. The production of lactic acid inside the capsules reflected the growth trend, obtaining the maximum concentration in Ci (15.9 ±1.1 g / L). Bahry et al. (Journal of Biotechnology, 2019, 306, 81-88) reached a lactic acid content of 22 g / L inside alginate beads during a fermentation process carried out to valorize a solid carob waste using immobilized Lacticaseibacillus rhamnosus.

[0192] Contrary to what happens with bacterial growth, in the external liquids the highest content of lactic acid was obtained in Li, as it is both produced by the growing bacteria and partly derived from the diffusion through the single-layer capsules into the liquid during the fermentation process. The diffusion phenomenon of metabolites such as lactic acid in L2 and L3 is probably limited by the presence of multiple-layer structures, as also reported by De Prisco and Mauriello (Trends in Food Science & Technology, 2016, 48, 27-39), and as hypothesized for the limited entrance of nutrients. Idris and Suzana (Process Biochemistry, 2006, 41(5), 1117-1123) performed a lactic fermentation by suspending capsules based on alginate (1% w / v) and the probiotic bacterium Lacticaseibacillus delbrueckii in a pineapple waste, obtaining after 24 hours of processing a lactic acid content in the external liquid of 7.5 g / L comparable with what was obtained in L2 and L3.

[0193] The doubling time of the microorganism td, the number of generations n, and the constant growth rate k, evaluated for the minibioreactors Ci, C2, and C3 during the exponential growth phase of fermentation, are reported in Table 2.

[0194] Table 2. Fermentation parameters of minibioreactors (Ci, C2, and C3) evaluated during the exponential growth phase: doubling time (td); generation number (n); constant growth rate (k).

[0195] As shown in Table 2, Ci pointed out almost double differences with C2 and C3 with a lower doubling time td(0.98 h) and higher values of both n and k (10.17 and 1.02 h’1, respectively). These results indicate a better duplication capacity of the microorganism during the exponential growth phase when an additional coating does not cover the capsule, as also demonstrated by Raymond et al. (Artificial Cells, Blood Substitutes, and Biotechnology, 2004, 32(2), 275-291), who studied the encapsulation of yeasts in uncoated k-carrageenan microspheres and coated with chitosan, and estimated doubling times of 1.8 and 2.7 hours during the exponential phase, respectively. Moreover, Galazzo and Bailey (Biotechnology and Bioengineering, 1990, 36(4), 417-426) obtained a doubling time in calcium alginate spheres of Saccharomyces cerevisiae higher than the values found in Ci, C2, and C3 and equal to 2.8 hours.

[0196] This study highlights that all the capsules worked well as a fermenter since a bacterial load considered a probiotic is achieved even if the exchange of nutrients and metabolites in the double-layer capsules (C2 and C3) is limited; only Ci manages to reach the maximum final bacterial load of approximately 9 logs at the end of the fermentation process. The minibioreactor system consisting of Ci and Li was also found to be the most efficient for the production of lactic acid, reaching a final value of 15.9 ± 1.1 g / L, and 10 ± 0.8 g / L, respectively. All the kinetic parameters analyzed in this study, namely doubling time (td), generation number (n), and constant growth rate (k), also confirm a better duplication capacity of the microorganism during the exponential growth phase when a double coating does not cover the capsule. Based on the results obtained in this study, the minibioreactor system made up of Ci and Li was the most efficient in terms of bacterial growth.Example 3: Use of Postbiotic Capsules as Anti-Inflammatory Products

[0197] Capsules allow for obtaining a dried postbiotic, similar to those obtained by the spray-drying process. Typically, after fermentation, the microorganism is killed and then subjected to spray drying, which is an expensive method requiring costly equipment. As described above, capsules enable a fermentation process. Encapsulated microorganisms (i.e., encapsulated probiotics) can subsequently be inactivated, and the capsules can be ground to obtain a dried postbiotic product through a more cost-effective process than, e.g., spray drying.

[0198] Postbiotic oatmeal alginate capsules were tested to assess their use as antiinflammatory products. To assess how digestion affects the anti-inflammatory properties of the postbiotic capsules, digested and undigested postbiotic capsules were tested, along with digested and undigested cell-free, fermented oatmeal.Background

[0199] Lipopolysaccharides (LPS), a major component of the outermembrane of Gramnegative bacteria, can potently activate the innate immune response through toll-like receptors (TLRs), including Toll-like receptor 4 (TLR4). Park, B. & Lee, JO. Recognition of lipopolysaccharide pattern by TLR4 complexes. Exp Mol Med 45, e66 (2013). TLRs are transmembrane proteins, which are present on the surface of immune cells (macrophages and dendritic cells) and non-immune cells (epithelial and endothelial). Duan, T., et al., Toll-Like Receptor Signaling and Its Role in Cell-Mediated Immunity. Front Immunol. 13:812774 (2022); Khakpour S., et al., Vascular endothelial cell Toll-like receptor pathways in sepsis. Innate Immun. 21(8):827-46 (2015). FIG. 6 provides a schema of the LPS and TLR-4 intracellular cascade. LPS binds to TLR4 via the adapter protein, MD-2, and the dimerization of the resultant LPS / TLR4 / MD-2 complex results in the binding of adaptor proteins TIRAP and MyD88 — which promotes the activation of the transcription factor NF-KB, and the subsequent production of pro-inflammatory cytokines, such as TNF-oc, IL-1, and IL-6. Maeshima, N. & Fernandez, R.C., Recognition of Lipid A Variants by the TLR4-MD-2 Receptor Complex. Front. Cell. Infect. Microbiol. 3:3 (2013).Materials and Methods

[0200] Postbiotic Production'. Postbiotic capsules were produced and fermented in an oat suspension as described, e.g., in Examples 1 and 2. After fermentation, the dried postbiotic capsules and dried postbiotic external suspensions were produced via bacterial inactivation and drying. Select dried postbiotic capsules and external suspensions were subsequently subject to in vitro digestion before sample preparation.

[0201] Bacterial Inactivation'. Microwave inactivation was performed using a microwave oven (Whirlpool Extraspace, MWF 427 SL). The sample (capsules immersed in the external suspension in which they were fermented) was sterilely placed in a Pyrex glass bottle (100 mL), which was subsequently placed in the microwave. The sample was microwaved at 800 W for 40 seconds followed by 10 “hits,” wherein each “hit” comprised microwaving the sample at 800 W for 5 seconds with a subsequent 10 second pause. The microwave was operated at 800 W, but only 238 W was transferred to the samples, as calculated in previous studies. See M. Di Domenico, “Dielectric heating of food and polymeric materials,” PhD Thesis, University of Naples Federico II, 2021. [Online], Available: www.fedoa.unina.it / 13637 / . A STM-5 probe (Luxtron Fluoroptic Temperature Probe) connected to a reader (Luxtron FOT Lab kit) was introduced into the capsule to monitor the temperature in real-time until the internal temperature of the capsule reached 85°C for a sufficient duration to achieve microbial inactivation, confirmed through microbiological counting on a plate.

[0202] Drying After bacterial inactivation, the capsules were separated from the external suspension and dried for 4 hours at 37°C, with an airflow of 0.1-0.3 m / s, in a dryer (CL252, Trevi). See S. Saez-Orviz et al.. “Lactic acid bacteria co-encapsulated with lactobionic acid: Probiotic viability during in vitro digestion,” Applied Sciences (Switzerland), vol. 11, no. 23, 2021, doi: 10.3390 / appl 12311404. The external suspensions were diluted at a 1 :4 ratio with maltodextrin added at 1 : 1 ratio (relative to the initial solid content in the oat-based liquid) and dried by Spray Drying (Xiamen Ollital Technology, OLT-SD8000B) with the following operating parameters: Qfeed (volumetric flow rate of the feed solution) = 5 mL / min; Qair-iN (volumetric flow rate of the inlet air) = 50 L / min; Patomization (atomization pressure) = 3.2 bar; Tair-iN (temperature of the inlet air) = 200 °C.

[0203] Two-Step in vitro Digestion: Select samples were subject to in vitro digestion according to the INFOGEST protocol, with slight modifications. See A. Brodkorb etal.,“INFOGEST static in vitro simulation of gastrointestinal food digestion,” Nature Protocols 2019 14:4, vol. 14, no. 4, pp. 991-1014, Mar. 2019, doi: 10.1038 / s41596-018- 0119-1. The protocol involved two phases: (1) preparation of the digestive solutions and (2) digestion.

[0204] Step 1 — Preparation of the Pise stive Solutions'. Three different digestive solutions were prepared: Simulated Salivary Fluid (SSF), Simulated Gastric Fluid (SGF), and Simulated Intestinal Fluid (SIF). All reagents were purchased by Sigma Aldrich. Fluids were stored at 8°C until used for the digestion test. Enzymes were added to the fluids at the time of the digestion test. Human salivary amylase (Sigma-Aldrich, 1031) was added to SSF to obtain a final concentration of 75 U / mL; porcine pepsin (Sigma-Aldrich, P6887) was added to SGF to obtain a final concentration of 2000 U / mL; porcine pancreatin (Sigma-Aldrich, P7545) was added to SIF to achieve a trypsin activity of 100 U / mL; and bile salts (Sigma-Aldrich, B8631) were added to SIF to reach a concentration of 10 mM.

[0205] Step 2 — Digestion'. The digestion tests comprised three phases: (i) the oral phase,(ii) gastric phase, and (iii) intestinal phases. First, the oral phase comprised (i) mixing the sample with the SSF in a ratio of 1 :3 and inclubating for 2 minutes. Second, the gastric phase comprised (ii) mixing the bolus derived from the oral phase with the SGF in a ratio of 1 : 1, subsequently adding IM HC1 to decrease the pH of the mixture to 3, and incubating the sample for 2 hours at 37 °C. Third, the intestinal phase comprised (iii) mixing the resultant chyme with the SIF, subsequently adding 1 M NaOH to increase the pH of the mixture to 7, and incubating the sample for a final 2 hours at 37 °C.

[0206] Sample Preparation'. Digested samples were treated with an Amicon 3KDa ultracentrifuged tube (Millipore), according to the manufacturer's instructions, to remove mineral salts added during digestion that are potentially toxic to cells. All samples were dissolved in PBS, obtaining a final solid concentration of 5mg / mL. Samples were then centrifuged at 3000 rpm for 10 minutes, and the supernatants — hereinafter referred to as extracts throughout Example 3) — were used for the subsequent tests.

[0207] Mammalian Cell Culture. HCT116, a colorectal adenocarcinoma cell line, was used as a cell model. HCT116 cells were cultured in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin / streptomycin. All reagents were purchased from EuroClone SpA. HCT116 cellswere cultured at 37°C under 5% CO2 conditions. The culture medium was changed every 1 / 2 days.

[0208] Preliminary Cytotoxicity Assay. A MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay was implemented to determine any toxicity of the postbiotic products. The MTT assay was carried out using HCT116 cells (i) without extract (negative control), (ii) with 200 pM H2O2 (positive control), and (iii) with different percentages of extracts. Briefly, 1.5 x 104HCT116 cells were seeded in 96-well plates and incubated in a complete medium for 20 hours at 37°C. After incubation, the complete medium was removed, and the cells were washed with RPMI. They were then incubated for 24 hours at 37°C in RPMI containing 1% penicillin / streptomycin and various concentrations of extracts (0.03%, 0.06%, 0.1%, 0.3%, 1%, and 2%). After the incubation with the extracts, the culture medium was removed, and the cells were washed with 100 pL RPMI without phenol red. Cell viability was assessed using the MTT assay. 100 pL of MTT solution (0.5 mg / ml in RPMI without phenol red) was added to each well, and the cells were incubated for 3 hours at 37°C. After incubation with MTT, 100 pL of 0.1 M HC1 in isopropanol were added to each well to solubilize and extract the formazan from the cells. The absorbance at 595 nm was then measured using a microplate reader (Bio-Rad 3350). The data obtained were expressed as a percentage of viability, assuming the absorbance value of cells grown in the absence of extracts as 100%. Each experiment was performed in triplicate and the cells were maintained in a thermostat at 37°C in an atmosphere of 5% CO2. A 0.1% concentration of the postbiotic product was selected for subsequent analysis because it was not cytotoxic to HCT116 cells.

[0209] Anti-Inflammatory Activity Assay : HCT116 cells were exposed to LPS (to promote inflammation) and subsequently stimulated with 0.1% postbiotic product (postbiotic dried capsules). Specifically, 2.5* 105cells were plated in multiwell plates and incubated at 37°C in 5% CO2 conditions for 24 hours in RPMI medium supplemented with 10% heat- inactivated FBS, 1% L-glutamine, and 1% penicillin / streptomycin. After incubation, the culture medium was replaced with RPMI medium containing 0.1% v / v extracts of the undigested samples and 0.1% v / v of the digested samples and Ipg / ml of LPS for the treatment group, while the control group received regular RPMI medium. After 24 hours incubation, the supernatants were collected, supplemented with a cocktail of protease inhibitors, and centrifuged to remove cellular debris. The cells were washed with RPMI,detached with trypsin, and lysed in RIPA buffer containing protease and phosphatase inhibitors. The lysates were then centrifuged, and the protein concentration was assayed.

[0210] Protein Concentration Assay. The protein concentration of the cell lysates was determined using a colorimetric microassay, according to the Bradford method. A calibration curve was prepared using known concentration solutions (1.12; 2.24; 4.48; 6.72; 8.96; 14; 18; 24 ng / pL) of BSA in TBS (130 mM NaCl, 20 mM Tris-HCl pH 7.4). 2 pL of cell lysate were mixed with 400 pL of TBS and each aliquot was analyzed in duplicate. 100 pL of Bradford reagent (Biorad) were added to both the standard solutions and the samples. The colour change of this reagent from red to blue is proportional to the protein concentration of each sample, which was detected by measuring the absorbance at 595 nm using a spectrophotometer. The protein concentration of the samples in pg / pL was calculated from the absorbance values by extrapolating from the calibration curve constructed using the absorbance of standard albumin and the known concentrations.

[0211] Western Blotting-. Western blot analysis was performed to monitor expression of TLR-4 and Myd88. Cell lysates treated with postbiotics were separated by SDS-PAGE, which consists of two gels. The upper part called the stacking gel, concentrates the protein sample in the wells for consistent migration. The lower part, the running gel, separates the proteins based on their molecular weight. A stock solution of acrylamide and bis-acrylamide at 30% and 0.8%, respectively, was used to prepare the gel. The separation gel consisted of a 10% polyacrylamide matrix. Additionally, a 6% polyacrylamide matrix containing 137 mM Tris-HCl at pH 6.8 and 0.1% SDS was layered on the running gel. Polymerization was catalyzed using ammonium persulfate (APS) and TEMED. The running buffer (pH 8.3) was prepared by dissolving glycine and Tris in water and adding SDS. 20 pg aliquots of each cell lysate were prepared in a denaturing and reducing buffer and incubated at 100°C for 5 minutes before fractionation by electrophoresis. The electrophoresis run was performed at 30 mA and stopped when the dye flowed off the slides. The analysis was done using a Mini-Protean® Tetra Cell vertical chamber by Biorad. Protein transfer from the gel to the membrane, the Western Blotting, was carried out. Briefly, after the electrophoresis, the gel was soaked in transfer buffer (25 mM Tris, 192 mM glycine, 15% methanol, pH 8.3) for 20 minutes at 4 °C. Nitrocellulose membranes were hydrated in distilled water for 30 minutes and then equilibrated in the transfer buffer. Migration of proteins onto nitrocellulose membranes was accomplished using an electric field (Semi-dry; Bio-Rad). The membrane wassandwiched between cellulose sheets soaked in transfer buffer, with the gel placed on top of the membrane and additional cellulose sheets placed on top of the gel. The applied current intensity was 223 mA for 30 minutes.

[0212] The membrane was then subjected to the blocking to saturate any non-specific binding sites with antibodies, for one hour at 37 °C with 5% milk or 2% BSA in T-TBS (130 mM NaCl, 20 mM Tris-HCl, 0.05% Tween 20, pH 7.5). After the blocking the membrane was washed three times with T-TBS for 15 minutes and then incubated with a primary antibody overnight at 4 °C. The following primary antibodies were used: (i) for TLR4: anti-TLR4 IgG from rabbit (1 : 1000) - Sigma Aldrich; (ii) for pNFkB: anti-pNFkB IgG from rabbit (1 : 1000) - Cell Signalling Technology; (iii) for NFkB: anti-NFkB IgG from rabbit (1 : 1000) - Cell Signalling Technology; (iv) for MyD88: anti-MyD88 IgG from rabbit (1 : 1000) - Cell Signalling Technology; and (v) for p-actin: Anti- p-actin from mouse (1 : 1000) - Sigma Aldrich. The following secondary antibodies were used: (i) for TLR4, pNFkB, and MyD88: GAR-HRP IgG from goat (1 :200,000) - Immunoreagents; and (ii) for NFkB and p-actin: GAR-HRP IgG from goat (1 :60,000) - Immunoreagents.

[0213] To detect TLR4, MyD88, pNFkB, and NFkB — the membrane was incubated overnight at 4 °C with a primary antibody in 3% BSA. The membrane was then washed three times for 15 minutes, and incubated at 37 °C for one hour with GAR-HRP secondary antibody in 1% milk in T-TBS. Subsequently, the membrane was washed four times for 30 minutes with T-TBS. The membrane was incubated in the dark with a chemiluminescence solution for 5 minutes. After removing the excess solution, a photographic plate was placed on the membrane and closed in an exposure chamber. The optimal exposure time was 15 minutes. Following this, the plate was immersed in a developer and fixative solution. The markers were normalized with respect to actin, a structural protein of the cytoskeleton. To do this, each membrane was washed with T- TBS and "stripped" by incubating it in the stripping solution (25 mM Glycine, 1% SDS, pH 2) for 30 minutes at room temperature. The membrane was then treated for actin detection by incubating it with a primary anti- actin antibody produced in mouse, diluted 1 : 1000 in 3% BSA in T-TBS overnight at 4°C. Afterward, the membrane was washed and incubated with a secondary antibody GAM-HRP, diluted 1 :30,000 in 1% milk in T-TBS for 1 hour at 37°C. Protein detection was performed, and the optimal exposure time was found to be 7 minutes. Quantitative densitometry of the various markers was performed by analyzing the digital images of the X-ray photographic plates using UN-SCAN-ITsoftware. The band intensities were calculated as Integrated Optical Densities (IOD). The concentration of the different markers was then quantified following normalization with P-actin, and the results were expressed as arbitrary units.Results

[0214] The results of the MTT assays on non-digested samples are shown in FIG. 7A. MTT assay was carried out using cells without extract as a negative control, and 200 pM H2O2, as a positive cytotoxicity control. Different percentages of extracts (0.03, 0.06, 0.1, 0.3, 1, and 2% of a suspension of 5pg / pL of solid content) were tested. The negative control had 100% cell survival, while the positive cytotoxicity control observed a reduction of about 75% of the cell population. The undigested extracts, on the other hand, provided excellent results, since cell survival did not appear to be altered at any of the concentrations used, suggesting excellent tolerability by the cells. Based on the results, subsequent tests were carried out by setting a concentration of 0.1% v / v.

[0215] The results of the MTT assays on digested samples are shown in FIG. 7B. The negative control achieved 100% cell survival, while the positive cytotoxicity control showed a reduction in cell population, as expected. The digested extracts, however, did not provide the same results as those obtained with the undigested samples, as cell survival appeared altered starting from about 1% for all digested samples. Specifically, the digested fermented oat extracts had a cell viability reduction of about 25%, the digested capsules had a cell viability reduction of about 35%, and the digested external suspensions had a cell viability reduction of about 60%. These results are likely attributed to the effect of the enzymes used during the digestion tests. The salts, however, removed through the ultracentrifugation phase cannot be held responsible for cytotoxicity. Based on the results, subsequent tests were carried out using concentrations lower than in the undigested samples: 0.1 % v / v.

[0216] As shown in FIGs. 8A and 8B respectively — TLR4 and Myd88 expression was reduced by postbiotic capsule stimulation, regardless of whether the sample was digested or undigested. Myd88 expression was not affected by the undigested, capsule-free sample, but Myd88 expression was reduced in the digested, capsule-free sample. TLR4 expression was reduced in both the digested and undigested, capsule-free sample. This data suggests that the postbiotic capsules have anti-inflammatory activity, regardless of how they are produced.Example 4: Tandem Fermentation

[0217] Tandem fermentation methods were evaluated to assess the suitability of using one capsule as a mimumum inoculum, wherein the capsule can be preserved for further, subsequent fermentation.Background

[0218] Capsules containing probiotic fermenting microorganisms can be used as an inoculum for "tandem" fermentation. These capsules can be suspended in an external suspension, such as a cereal suspension or culture broth, with the same composition used to produce the capsules. Due to their permeability, the capsules allow the diffusion of bacteria, which can then ferment the external suspension. After a chosen fermentation time, e.g., 24 hours, the capsules can be collected and suspended in a fresh external suspension to act as an inoculum again. This tandem fermentation process can be repeated multiple times.Materials and Methods

[0219] Postbiotic capsules were prepared as described in Examples 1 and 2.Tandem Fermentation

[0220] All fermentation steps were carried out at 37°C in a Falcon tube using a wheel as agitation system. Each cell-free external suspension comprised cereal suspension or culture broth prior to the addition of the capsule.

[0221] Tandem fermentation was performed as follows: (1) a first, cell-free external suspension was inoculated with a capsule containing probiotic fermenting microorganisms and fermented for 24 hours; (2) the capsule was removed from the first external suspension and added to a second, cell-free external suspension; (3) the first and second external suspensions were fermented in tandem (i.e., simultaneously fermented) for 24 hours; (4) the capsule was removed from the second external suspension and added to a third, cell-free external suspension; (5) the first, second, and third external suspensions were fermented in tandem for 24 hours; (6) the capsule was removed from the third external suspension and added to a fourth, cell-free external suspension; (7) the first, second, third, and fourth external suspensions were fermented in tandem for 24hours. FIG. 9 provides a schema of the tandem fermentation. The bacterial load of each external suspension, and the capsule, was measured every 24 hours.Results

[0222] The bacterial load of the capsule increased over the first 48 hours of fermentation and remained relatively constant over the second 48 hours of fermentation. See FIG. 10. The bacterial load of the first, second, third, and fourth external suspensions increased by a similar amount after 24 hours of fermentation in the respective, cell-free external suspensions. See FIG. 10. These results suggest that tandem fermentation is a suitable means of using one capsule as a minimum inoculum.

[0223] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary embodiments of the present disclosure as contemplated by the inventor(s), and thus, are not intended to limit the present disclosure and the appended claims in any way.

[0224] The foregoing description of the specific embodiments will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0225] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0226] The contents of all cited references (including literature references, U.S. or foreign patents or patent applications, and websites) that are cited throughout this application are hereby expressly incorporated by reference as if written herein in their entireties for any purpose, as are the references cited therein. Where any inconsistencies arise, material literally disclosed herein controls.

[0227] While various specific aspects have been illustrated and described, the above specification is not restrictive. It will be appreciated that various changes can be made without departing from the spirit and scope of the disclosure(s). Many variations will become apparent to those skilled in the art upon review of this specification.

Claims

WHAT IS CLAIMED IS:

1. A method of obtaining a fermentation product comprising the steps of: a) inoculating a culture media with a capsule comprising a fermenting microorganism, wherein optionally prior to the inoculating, the capsule is frozen; and b) fermenting the inoculated culture media under conditions suitable for fermentation, thereby obtaining a fermentation product.

2. The method of claim 1, wherein prior to step a), the capsule is stored frozen.

3. The method of claim 2, wherein the capsule is stored frozen in a glycerol solution.

4. The method of claim 3, wherein the glycerol solution comprises about 10% to about 50% w / v glycerol.

5. The method of claim 4, wherein the glycerol solution comprises about 30% w / v glycerol.

6. The method of any one of claims 1 to 5, wherein prior to step a), the frozen capsule is incubated at room temperature.

7. The method of any one of claims 1 to 6, wherein the capsule comprises a culture broth, a substrate comprising starch and / or fiber, alginate, and / or CaCh.

8. The method of claim 7, wherein the culture broth is is selected from the group consisting of MRS broth, Brain Heart Infusion (BHI) broth, Luria-Bertani (LB) broth, plant-derived broth, functional broth containing plant extracts with antioxidant, antiviral and / or antibacterial activity, culture broth of natural origin, or animal-free broth.

9. The method of claim 7, wherein the substrate comprising starch and / or fiber is a cereal suspension.

10. The method of claim 9, wherein the cereal suspension is an oatmeal suspension.

11. The method of any one of claims 1 to 10, wherein prior to step a), the capsule is resuspended in a hydrolyzed oatmeal suspension or a culture broth.

12. The method of claim 11, wherein the hydrolyzed oatmeal suspension comprises about 1% to about 50% w / v oat flour, and about 0.1% to about 5% w / v glucose.

13. The method of claim 12, wherein the hydrolyzed oatmeal suspension comprises about 15% w / v oat flour and about 1% w / v glucose.

14. The method of claim 11, wherein the culture broth is is selected from the group consisting of MRS broth, Brain Heart Infusion (BHI) broth, Luria-Bertani (LB) broth, plant-derived broth, functional broth containing plant extracts with antioxidant, antiviral and / or antibacterial activity, culture broth of natural origin, or animal-free broth.

15. The method of any one of claims 1 to 14, wherein the capsule comprises an alginate layer.

16. The method of claim 15, where the capsule further comprises a chitosan layer.

17. The method of claim 15 or 16, wherein the capsule further comprises a second alginate layer.

18. The method of any one of claims 1 to 17, wherein the capsule is produced by extrusion, high shear rate mixing, spray-drying, emulsion, lyophilization, and / or layer-by-layer production.

19. The method of any one of claims 1 to 18, wherein the capsule is produced by extrusion using about 0.5% to about 1.5 % w / v alginate and about 0. IM to about 3M CaCh.

20. The method of claim 19, wherein the capsule is produced by extrusion using 1% w / v alginate and IM CaCh.

21. The method of any one of claims 1 to 20, wherein the capsule is of a spherical, filamentous, irregular, or flat geometry.

22. The method of claim 21, wherein the capsule is of a spherical geometry.

23. The method of any one of claims 1 to 22, further comprising inoculating the culture media with one or more additional capsules comprising fermenting microorganisms.

24. The method of claim 23, wherein each of the one or more additional capsules comprises a different fermenting microorganism.

25. A method of preparing a plurality of capsules comprising a fermenting microorganism, comprising the steps of:(i) preparing a suspension comprising a fermenting microorganism culture and an alginate solution; and(ii) dripping the suspension of step (i) into a calcium chloride solution, thereby forming a plurality of capsules comprising the fermenting microorganism, and incubating the plurality of capsules in the calcium chloride solution.

26. A method of preparing a plurality of capsules comprising a fermenting microorganism, comprising the steps of: preparing a hydrolyzed oatmeal suspension comprising amylase-treated oatmeal and glucose; mixing the hydrolyzed oatmeal suspension with a fermenting microorganism culture and alginate solution; and dripping the mixture of step (ii) into a calcium chloride solution, thereby forming a plurality of capsules comprising the fermenting microorganism, and incubating the plurality of capsules in the calcium chloride solution.

27. The method of claim 25 or 26, further comprising rinsing the capsules with distilled water.

28. The method of claim any one of claims 25 to 27, further comprising mixing the capsules with a glycerol solution and freezing the mixture.

29. The method of any one of claims 25 to 28, wherein the alginate solution comprises about 0.5% to about 2.0 % w / v alginate.

30. The method of any one of claims 25 to 29, wherein the calcium chloride solution comprises about 0. IM to about 3M CaCh.

31. The method of claim 29, wherein the alginate solution comprises 1% w / v alginate.

32. The method of claim 30, wherein the calcium chloride solution comprises IM CaCh.

33. The method of any one of claims 25 to 32, wherein the capsules are of a spherical, filamentous, irregular, or flat geometry.

34. The method of claim 33, wherein the capsules are of a spherical geometry.

35. The method of any one of claims 25 to 34, wherein the fermenting microorganism culture comprises about 106colony forming units (CFU) / mL to about 1011CFU / mL.

36. The method of any one of claims 1 to 35, wherein each capsule comprises about 103CFU / mL to about 109CFU / mL of the fermenting microorganism.

37. The method of any one of claims 1 to 36, wherein each capsule comprises about 106CFU / mL of the fermenting microorganism.

38. The method of any one of claims 1 to 36, wherein each capsule comprises about 108CFU / mL of the fermenting microorganism.

39. The method of any one of claims 26 to 38, wherein step (i) comprises activating an amylase enzyme at about 60 °C to about 80 °C, followed by adding oat flour and glucose, incubating at about 60 °C to about 80 °C, and then incubating at about 80 °C to about 95 °C.

40. The method of claim 39, wherein step (i) comprises activating an amylase enzyme at about 65 °C, followed by adding oat flour and glucose, incubating at about 65 °C, and then incubating at about 85 °C.

41. The method of any one of claims 26 to 40, wherein the hydrolyzed oatmeal suspension comprises about 1% to about 50% w / v oat flour, and about 0.1% to about 5% w / v glucose.

42. The method of claim 41, wherein the hydrolyzed oatmeal suspension comprises about 15% w / v oat flour and about 1% w / v glucose.

43. The method of any one of claims 1 to 42, wherein the fermenting microorganism is selected from the group consisting of a bacterial microorganism and a yeast microorganism.

44. The method of claim 43, wherein the bacterial microorganism is selected from the group consisting of Lactobacillus species, a Lactococcus species, Bifidobacterium species, a Lacticaseibacillus species, a Streptococcus species, and an Akkermansia species.

45. The method of claim 43, wherein the yeast microorganism is selected from a Saccharomyces species.

46. The method of any one of claims 1 to 42, wherein the fermenting microorganism is selected from the group consisting of Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Lacticaseibacillus paracasei, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium breve,Bifidobacterium longum, Streptococcus salivarius, Akkermansia muciniphila, and Saccharomyces boulardii.

47. The method according to claim 46, wherein the Lacticaseibacillus paracasei is Lacticaseibacillus paracasei NPB01.

48. A kit comprising the capsules comprising the fermenting microorganism of any one of claims 25 to 47.

49. An encapsulated fermenting microorganism composition comprising: i) a capsule comprising a fermenting microorganism, wherein the capsule comprises at least one alginate layer; and ii) a glycerol solution.

50. The encapsulated fermenting microorganism composition of claim 49, wherein the capsule further comprises a chitosan layer.

51. The encapsuled fermenting microorganism of claim 49 or 50, wherein the capsule is of a spherical, filamentous, irregular, or flat geometry.

52. The encapsulated fermenting microorganism of claim 51, wherein the capsule is of a spherical geometry.

53. The encapsulated fermenting microorganism of any one of claims 49 to 52, wherein the capsule is produced by extrusion, high shear rate mixing, spray-drying, emulsion, lyophilization, and / or layer-by-layer production.

54. The encapsulated fermenting microorganism of any one of claims 49 to 53, wherein the capsule comprises a culture broth, a substrate comprising starch and / or fiber, alginate, and / or CaCh.

55. The encapsulated fermenting microorganism of claim 54, wherein the culture broth is selected from the group consisting of MRS broth, Brain Heart Infusion (BHI) broth, Luria-Bertani (LB) broth, plant-derived broth, functional broth containing plant extracts with antioxidant, antiviral and / or antibacterial activity, culture broth of natural origin, or animal-free broth.

56. The encapsulated fermenting microorganism of claim 54, wherein the substrate comprising starch and / or fiber is a cereal suspension.

57. The encapsulated fermenting microorganism of claim 56, wherein the cereal suspension is an oatmeal suspension.

58. A method of obtaining a capsule comprising a postbiotic product comprising the steps of: a) preparing a capsule comprising a fermenting microorganism according to the method of any one of claims 25 to 47; b) fermenting the capsule comprising the fermenting microorganism, thereby obtaining a capsule comprising a fermentation product; and c) inactivating the capsule comprising the fermentation product, thereby obtaining a capsule comprising a postbiotic product.

59. The method of claim 58, wherein the inactivating of step c) comprises heat inactivation of the capsule comprising the fermentation product.

60. The method of claim 59, wherein the heat inactivation comprises conventional heat inactivation of the capsule comprising the fermentation product.

61. The method of claim 59, wherein the heat inactivation comprises dielectric heat inactivation of the capsule comprising the fermentation product.

62. The method of claim 61, wherein the dielectric heat inactivation comprises microwave heat inactivation of the capsule comprising the fermentation product.

63. The method of claim 61, wherein the dielectric heat inactivation comprises radiofrequency heat inactivation of the capsule comprising the fermentation product.

64. The method of any one of claims 58-63, further comprising drying the capsule comprising the postbiotic product.

65. The method of claim 64, wherein the drying of the capsule comprises lyophilizing the capsule comprising the postbiotic product.

66. The method of claim 64, wherein the drying of the capsule comprises spray-drying the capsule comprising the postbiotic product.

67. The method of any one of claims 58-66, further comprising mechanical disruption of the capsule comprising the postbiotic product.

68. The method of claim 67, wherein the mechanical disruption of the capsule comprises comminution of the capsule via high-speed airflow pulverization, ball milling, high-speed rotary strike crushing, vacuum superfine grinding, low-temperature milling, or cryogenic grinding.

69. The method of claim 67, wherein the mechanical disruption of the capsule comprises lyophilizing the capsule.

70. The method of claim 67, wherein the mechanical disruption of the capsule comprises spray-drying the capsule.

71. A postbiotic product according to any one of claims 58-70 for use in the prevention or the therapeutic treatment of a disease in a subject in need thereof selected from the group consisting of infectious and inflammatory diseases, immune-mediated diseases, cancer diseases, skin disorders, gastrointestinal diseases, urogenital tract diseases, neurologic disorders, neuropsychiatric disorders, bone diseases, muscle diseases, malnutrition, metabolic diseases, and any combination thereof.

72. The use of claim 71, wherein the postbiotic product reduces Myd88 and / or TLR4 expression in the subject.

73. Use of the postbiotic product according to claims 71 or 72 in a food, a beverage, a pharmaceutical, a nutraceutical, a cosmetic, or a packaging composition, and wherein the composition further comprises at least one pharmaceutically acceptable vehicle, excipient and / or diluent.

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