Methods of making postbiotic compositions

Abstract

This disclosure provides a method for preparing a postbiotic composition, the method comprising multiple (i.e., two or more) separate microbial fermentations, the fermentations being capable of being carried out in parallel or sequential steps.

Description

Cross-references to related applications

[0001] This application claims priority to the following patents: Italian Patent Application No. 102023000020844, filed October 9, 2023; U.S. Provisional Patent Application No. 63 / 651,254, filed May 23, 2024; U.S. Provisional Patent Application No. 63 / 665,111, filed June 27, 2024; U.S. Provisional Patent Application No. 63 / 667,555, filed July 3, 2024; U.S. Provisional Patent Application No. 63 / 677,259, filed July 30, 2024; and U.S. Provisional Patent Application No. 63 / 688,678, filed August 29, 2024, each of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to a method for preparing postbiotic compositions from various fermentation microbial strains. Background Technology

[0003] Fermentation processes are known to lead to changes in the fermentation substrate, including microbial, chemical, and physical changes, and may result in the acquisition of functionality (Handbook of Fermented Functional Foods, Second Edition - GoogleBooks nd).

[0004] Probiotic products are characterized by the presence of live microorganisms that can confer health benefits to the host (Hill C. et al.; "The International Scientific Association for Probiotics and Prebiotics Consensus Statement on the Scope and Appropriate Use of the Term Probiotic," Nature Reviews Gastroenterology & Hepatology 2014 11(8): 506–14). However, probiotic products are poorly tolerant to heat treatment and are characterized by low stability and short shelf life (Pimentel TC et al.; 2023. "Postbiotics: An Overview of Concepts, Inactivation Technologies, Health Effects, and Driver Trends," Trends in Food Science & Technology 138: 199–214).

[0005] Recently, a new concept based on postbiotic formulations has attracted interest. Currently, postbiotic products are defined as "formulations of non-living microorganisms and / or their components that confer health benefits to the host." In this context, the term "formulation" is intended to refer to intact, inactivated microbial biomass, its components (fimbriae, cell wall components, or other components), substrate, and bacterial metabolites produced during fermentation (Salminen, S., et al. 2021, "The International Scientific Association of Probiotics and Prebiotics (ISAPP) Consensus Statement on the Definition and Scope of Postbiotics," Nature Reviews, Gastroenterology & Hepatology 18(9): 649).

[0006] The activity of postbiotic products is related to the specific microorganisms from which they originate and the fermentation substrate used. In fact, substrate composition affects microbial growth, metabolite production, and thus the bioactivity of the formulation.

[0007] It is known that specific fermentation substrates can significantly improve the production performance of biomass and the biological activity of postbiotics. Furthermore, the growth of microorganisms and the production of functional compounds and / or metabolites are influenced by the composition of the fermentation substrate. Processes using fermentation substrates containing prebiotics are well-known and widely used in the field of biotechnology.

[0008] Postbiotic products are fermented and subsequently inactivated products that have beneficial effects on the health of subjects. These products are characterized by greater stability and extended shelf life, which is particularly attributed to the absence of live microorganisms. In addition, postbiotics are a safer product for debilitated and / or immunocompromised patients (Salminen S. et al., 2021, “The International Scientific Association of Probiotics and Prebiotics (ISAPP) Consensus Statement on the Definition and Scope of Postbiotics,” Nature Reviews, Gastroenterology & Hepatology 18(9): 649).

[0009] Therefore, the object of the present invention is to provide a method for preparing postbiotic compositions, particularly postbiotic compositions having enhanced bioactive properties.

[0010] Another object of the present invention is to provide a method for obtaining a safe metabiotic composition with high stability and long shelf life. Summary of the Invention

[0011] One aspect of the present invention provides a method for preparing a postbiotic composition, the method comprising the steps of: (i) inoculating a variety of fermenting microorganisms into a variety of culture media, wherein different fermenting microorganisms are inoculated into each culture media; (ii) fermenting the inoculated variety of culture media under conditions suitable for fermentation to obtain a variety of fermentation products, each fermentation product containing different fermenting microorganisms; (iii) inactivating the fermenting microorganisms in the variety of fermentation products to obtain a variety of inactivated fermentation products; and (iv) mixing the variety of inactivated fermentation products to obtain a postbiotic composition.

[0012] Another aspect of the present invention provides a method for preparing a metabiotic composition, the method comprising the steps of: (i) inoculating a culture medium with a first fermenting microorganism; (ii) fermenting the culture medium under conditions suitable for the fermentation of the first fermenting microorganism to obtain a first fermentation product containing the first fermenting microorganism; (iii) inactivating the first fermenting microorganism in the first fermentation product to obtain a first fermentation substrate; (iv) inoculating the first fermentation substrate with a second fermenting microorganism; (v) fermenting the first fermentation substrate under conditions suitable for the fermentation of the second fermenting microorganism to obtain a second fermentation product containing the second fermenting microorganism; (vi) inactivating the second fermenting microorganism in the second fermentation product to obtain a second fermentation substrate, thereby obtaining a metabiotic composition; and optionally repeating steps (iv) to (vi) once or more using additional fermenting microorganisms. In some aspects, each fermenting microorganism is a different species from any other fermenting microorganism, thereby obtaining the metabiotic composition.

[0013] In some respects, fermentation microorganisms are selected from the group consisting of bacteria and yeast. In other respects, bacterial microorganisms are selected from the group consisting of *Lactobacillus* (*Lactobacillus*). Lactobacillus ) species, Lactococcus genus ( Lactococcus ) species, Lactobacillus genus ( Lacticaseibacillus ) species, Bifidobacterium genus ( Bifidobacterium ) species, Streptococcus genus ( Streptococcus species, genus Akkermania ( Akkermansia species and Escherichia genus ( Escherichia ) species. In some respects, yeast microorganisms are selected from the genus *Yeast* ( Saccharomyces ) of the species.

[0014] In some respects, fermentation microorganisms were selected from the group consisting of: Lactobacillus paracasei ( Lactobacillus paracasei Lactobacillus rhamnosus ( Lactobacillus rhamnosus Lactobacillus reuteri ( Lactobacillus reuteri) Lactobacillus plantarum (Lactobacillus plantarum Lactobacillus casei ( Lactobacillus casei Lactobacillus paracasei ( Lacticaseibacillus paracasei Bifidobacterium animalis ( Bifidobacterium animalis Bifidobacterium bifidum ( Bifidobacterium bifidum ), Bifidobacterium infantis ( Bifidobacterium infantis) Bifidobacterium breve (Bifidobacterium breve) Bifidobacterium longum (Bifidobacterium longum) Streptococcus salivarius (Streptococcus salivarius) Akkermansia myxophilus ( Akkermansia muciniphila ), Escherichia coli ( Escherichia coli ) and Saccharomyces boulardii ( Saccharomyces boulardii In some respects, *Lactobacillus paracasei* is *Lactobacillus paracasei* NPB01. In some respects, *Bifidobacterium animalis* is a subspecies of *Bifidobacterium animalis*. lactis In some respects, Escherichia coli is Escherichia coli Nissle 1917.

[0015] In some cases, one or more additional components are added to the inactivated fermentation products before inoculation with fermenting microorganisms.

[0016] In some aspects, the method further includes drying the inactivated fermentation product, wherein the dried inactivated fermentation product is optionally rehydrated in water before subsequent inoculation with fermenting microorganisms.

[0017] In some aspects, the method further includes the step of drying the biogenic composition.

[0018] In some respects, the culture media include those selected from the group consisting of: MRS medium, brain and heart infusion (BHI) broth, Luria-Bertani (LB) broth, plant-derived media, functional media containing plant extracts with antioxidant, antiviral, and / or antibacterial activities, naturally derived media, and any combination thereof. In some respects, the brain and heart infusion (BHI) broth is supplemented with porcine gastric mucin (PGM).

[0019] In some respects, the fermentation process is carried out at temperatures ranging from about 25°C to about 45°C.

[0020] In some respects, the inactivation of fermenting microorganisms includes procedures selected from the group consisting of: heat inactivation, preferably at a temperature of 50°C to 100°C for 5 to 120 seconds; chemical treatment; gamma ray or ultraviolet irradiation; high pressure; ultrasonic treatment; and any combination thereof. In some respects, heat inactivation occurs at a temperature of about 50°C to about 100°C for about 5 to about 120 seconds.

[0021] This document also provides a postbiotic composition obtained by the methods described herein, which further comprises lactic acid at a concentration of about 1 g / L to about 30 g / L (based on the total volume of the composition) and / or in an amount of about 0.00015 g / L to about 150 g / L (based on the total volume of the composition) of inactivated fermenting microorganisms. In some aspects, the amount of inactivated fermenting microorganisms includes about 10 5 cells / ml to approximately 10 11 Cells / ml

[0022] This document also provides postbiotic compositions obtained by the methods described herein, which further comprise L-tryptophan or a dipeptide containing L-tryptophan. In some aspects, the composition comprises L-tryptophan at a concentration of at least about 0.01% w / w. In some aspects, the composition comprises L-tryptophan at a concentration of at least about 0.10% w / w. In some aspects, L-tryptophan is present in an amount of at least about 10 mg, at least about 50 mg, or at least about 100 mg.

[0023] This document also provides a metabiotic composition obtained by the method described herein, wherein at least one of the fermenting microorganisms used to obtain the metabiotic composition is *Lactobacillus paracasei* NPB01.

[0024] This document also provides the postbiotic compositions described herein for the prevention or therapeutic treatment of diseases in subjects in need, said diseases being selected from the group consisting of: infectious and inflammatory diseases, immune-mediated diseases, cancerous diseases, skin diseases, gastrointestinal diseases, genitourinary diseases, neurological diseases, neuropsychiatric diseases, skeletal diseases, muscle diseases, malnutrition, metabolic diseases, and any combination thereof.

[0025] This article also provides the metabiotic compositions described herein for promoting healthy aging in mammals. Healthy aging is defined as a continuous process throughout the life course of life that optimizes various opportunities to maintain and improve physical and mental health, independence, and quality of life.

[0026] This article also provides the metabiotic compositions described herein for correcting drug-induced nutritional depletion in mammals.

[0027] This document also provides the postbiotic composition described herein for improving intestinal barrier function in subjects of need by increasing the expression of tight junction proteins and / or mucins, and / or increasing intestinal epithelial cell growth and differentiation. In some aspects, the tight junction proteins include closure proteins and / or ZO-1. In some aspects, the mucins include MUC5AC. In some aspects, treatment with the postbiotic composition resulted in an increase of at least 2-fold, 3-fold, 4-fold, 5-fold, or more in the expression of closure proteins, ZO-1, and / or MUC5AC compared to untreated subjects. In some aspects, treatment with the postbiotic composition resulted in an increase of at least 25%, 50%, 75%, or more in the expression of closure proteins, ZO-1, and / or MUC5AC compared to treatment with a composition containing a single live microorganism.

[0028] This article also provides the postbiotic composition described herein for increasing the expression of β-defensin-2 (HBD-2) in subjects of need. In some aspects, HBD-2 expression was increased by at least 2, 3, 4, 5, 7, 8, or more times after treatment with the postbiotic composition compared to untreated subjects. In some aspects, HBD-2 expression was increased by at least 2, 3, 4, or more times after treatment with the postbiotic composition compared to treatment with a composition containing a single microorganism.

[0029] This article also provides the postbiotic composition described herein for increasing the expression of the antimicrobial peptide LL-37 in subjects in need. In some respects, the expression of the antimicrobial peptide LL-37 was increased by at least about 2-fold, about 3-fold, or more after treatment with the postbiotic composition compared to untreated subjects.

[0030] This document also provides for the use of the metabiotic compositions described herein in food, beverage, pharmaceutical, nutritional supplement, cosmetic, or packaging compositions, wherein said compositions further comprise at least one pharmaceutically acceptable carrier, excipient, and / or diluent. In some aspects, the use is for improving intestinal barrier function in mammals. In some aspects, the use is for improving the innate immune response against infection in mammals. In some aspects, the use is for inducing a tolerance-inducing immune response in mammals. In some aspects, the use is for protecting the skin of mammals from infection.

[0031] This document also provides the use of the post-biotic compositions described herein for increasing the total amount of one or more metabolites of L-tryptophan in mammals. In some aspects, one or more metabolites of L-tryptophan include indole-3-acetic acid, indole-3-lactic acid, and / or L-kynurenine.

[0032] This article also provides the use of the postbiotic compositions described herein for increasing the total amount of L-tryptophan in mammals.

[0033] This article also provides the use of the postbiotic compositions described herein for increasing the ratio of L-tryptophan to large neutral amino acids in mammalian plasma.

[0034] This article also provides the use of the post-biotic compositions described herein for increasing the biosynthesis of serotonin and / or melatonin in mammals.

[0035] This article also provides methods for preventing or treating diseases selected from the group consisting of: infectious and inflammatory diseases, immune-mediated diseases, cancerous diseases, skin diseases, gastrointestinal diseases, genitourinary diseases, neurological diseases, neuropsychiatric diseases, skeletal diseases, muscle diseases, malnutrition, metabolic diseases, and any combination thereof, wherein the methods include administering the postbiotic composition described herein to a subject in need.

[0036] This article also provides a method for improving intestinal barrier function in subjects in need by increasing the expression of tight junction proteins and / or mucins, and / or increasing intestinal epithelial cell growth and differentiation, wherein said method comprises administering the postbiotic composition described herein to the subject. In some aspects, tight junction proteins include closure proteins and / or ZO-1. In some aspects, mucins include MUC5AC. In some aspects, compared with untreated subjects, treatment with the postbiotic composition increased the expression of closure proteins, ZO-1, and / or MUC5AC by at least about 2-fold, about 3-fold, about 4-fold, about 5-fold, or more. In some aspects, compared with treatment with a composition containing a single live microorganism, treatment with the postbiotic composition increased the expression of closure proteins, ZO-1, and / or MUC5AC by at least about 25%, about 50%, about 75%, or more.

[0037] This article also provides a method for increasing the expression of β-defensin-2 (HBD-2) in subjects in need, wherein said method comprises administering the postbiotic composition provided herein to the subject. In some aspects, HBD-2 expression is at least about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 7-fold, about 8-fold, or more after treatment with the postbiotic composition compared to untreated subjects. In some aspects, HBD-2 expression is increased at least about 2-fold, about 3-fold, about 4-fold, or more after treatment with the postbiotic composition compared to treatment with a composition comprising a single microorganism.

[0038] This article also provides a method for increasing the total amount of one or more metabolites of L-tryptophan in a subject in need, wherein said method comprises administering the postbiotic composition provided herein to the subject. In some aspects, one or more metabolites of L-tryptophan include indole-3-acetic acid, indole-3-lactic acid, and / or L-kynurenine.

[0039] This article also provides a method for increasing the total amount of L-tryptophan in the body of a subject in need, wherein the method includes administering the postbiotic composition provided herein to the subject.

[0040] This article also provides a method for increasing the ratio of L-tryptophan to large neutral amino acids in the plasma of subjects in need, wherein the method comprises administering the postbiotic composition provided herein to the subject.

[0041] This article also provides methods for increasing the biosynthesis of serotonin and / or melatonin in subjects in need, wherein the methods include administering the postbiotic composition provided herein to the subject.

[0042] This article also provides methods for promoting healthy aging in subjects in need, wherein the methods include administering the postbiotic composition provided herein to the subjects.

[0043] This article also provides a method for correcting drug-induced nutritional depletion in subjects in need, wherein the method includes administering the postbiotic composition provided herein to the subject.

[0044] This document also provides metabiotic compositions produced by multimicrobial fermentation, wherein the multimicrobial fermentation includes the fermentation of two or more bacterial or yeast species. In some aspects, the metabiotic comprises two or more inactivated fermentation products. In some aspects, the multimicrobial fermentation includes the methods described herein.

[0045] This document also provides a metabiotic composition comprising: (a) a first substrate comprising a first inactivated microorganism and a culture medium in which said microorganism was inactivated; and (b) one or more additional substrates, each additional substrate comprising another inactivated microorganism and a culture medium in which said microorganism was inactivated. In some aspects, the first inactivated microorganism and each additional inactivated microorganism are different species. In some aspects, the metabiotic composition is prepared according to the method described herein.

[0046] This document also provides a kit comprising the metabiotic composition described herein. This document also provides a kit comprising a multimicrobial fermentation substrate and an inoculum containing two or more inactivated microorganisms. In some aspects, the metabiotic composition is prepared according to the methods described herein. In some aspects, the kit also includes documentation containing steps and conditions for use. Attached Figure Description

[0047] Figure 1 A flow chart of the parallel fermentation process is shown.

[0048] Figure 2 A flow chart of the continuous fermentation process is shown.

[0049] Figure 3 A variation of the continuous fermentation process is shown, which allows for optional intermediate storage stages.

[0050] Figures 4A-4B It shows Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus 24-hour growth curve of (LGG) Figure 4A ) and 24-hour growth curves of Lactobacillus paracasei (LP) Figure 4B ).

[0051] Figures 5A-5B The 24-hour lactate accumulation curve of LGG is shown. Figure 5A ) and LP's 24-hour lactate accumulation curve ( Figure 5B ).

[0052] Figure 6 The bacterial growth and lactic acid production of Lacticaseibacillus plantarum (Lpl) after 24 hours of fermentation are shown.

[0053] Figure 7 It shows Lactobacillus casei ( Lacticaseibacillus casei (Lc) Bacterial growth and lactic acid production after 24 hours of fermentation.

[0054] Figures 8A-8B The bacterial growth of different microbial genera (Bifidobacterium bifidum-Bb, Streptococcus salivarius-Ss, and Saccharomyces boulardii-Sb) after 24 hours of fermentation is shown. Figure 8A ) and lactic acid production ( Figure 8B ).

[0055] Figures 9A-9B The growth curves of the continuous fermentation process are shown. Figure 9A ) and lactic acid accumulation curve ( Figure 9B The process includes 24 hours of LGG fermentation and 24 hours of LP fermentation.

[0056] Figures 10A-10B The growth curves of the continuous fermentation process are shown. Figure 10A ) and lactic acid accumulation curve ( Figure 10B The process includes 24 hours of LP fermentation and 24 hours of LGG fermentation.

[0057] Figure 11A-11B The growth curves of the continuous fermentation process are shown. Figure 11A ) and lactic acid accumulation curve ( Figure 11B The process includes 6 hours of LGG fermentation and 24 hours of LP fermentation.

[0058] Figure 12A-12B The growth curves of the continuous fermentation process are shown. Figure 12A ) and lactic acid accumulation curve ( Figure 12B The process includes 6 hours of LP fermentation and 24 hours of LGG fermentation.

[0059] Figures 13A-13B Bacterial growth during a continuous fermentation process is shown. Figure 13A ) and lactic acid production ( Figure 13B The process includes 6 hours of Lpl fermentation and 24 hours of Lc fermentation.

[0060] Figures 14A-14B Bacterial growth during a continuous fermentation process is shown. Figure 14A ) and lactic acid production ( Figure 14B The process includes 6 hours of Lc fermentation and 24 hours of Lpl fermentation.

[0061] Figures 15A-15B Bacterial growth during a continuous fermentation process is shown. Figure 15A ) and lactic acid production ( Figure 15B The process includes 6 hours of Bb fermentation, 6 hours of Ss fermentation, and 24 hours of Sb fermentation.

[0062] Figure 16 The effects of metagenics obtained from single fermentation of LP and LGG, different combinations of continuous processes, and different compositions of parallel processes on HBD-2 peptide production in Caco-2 cells were shown using ELISA assays compared to controls.

[0063] Figures 17A-17B The effects of metagenic factors obtained from single fermentations of Lpl and Lc, different combinations of continuous processes, and individual components of parallel processes on HBD-2 peptide production in Caco-2 cells were shown using ELISA assays compared to controls. Figure 17A ). ( Figure 17B This paper presents a comparison of metabiotics among all tested species belonging to the Lactobacillus genus.

[0064] Figure 18The effects of metabiotics obtained from single fermentation of Bb, Ss, and Sb, from a single combination of continuous processes, and from a single component of parallel processes on HBD-2 peptide production in Caco-2 cells were shown using ELISA assays, compared to controls.

[0065] Figures 19A-19C This demonstrates the effect of metabiotics obtained from single fermentation of LP and LGG and from parallel processes on the biomarker MUC5AC (intestinal barrier integrity) in human intestinal epithelial cells, compared to controls and commercial metabiotics. Figure 19A ), Closure protein ( Figure 19B ) and ZO-1( Figure 19C The influence of the expression of ).

[0066] Figures 20A-20B The chromatogram of Lactobacillus paracasei NPB01 polysaccharide observed by size exclusion chromatography using Sephacryl HR-300 is shown. Figure 20A ) and the NMR proton spectra of the fraction obtained after purification on a Sephacryl HR-300 (600 MHz, 298 K, D2O) ( Figure 20B ).

[0067] Figures 21A-21B show the HSQC spectra (600 MHz, 298 K, D2O) of the CPS-1 + TA mixture isolated from *Lactobacillus paracasei* NPB01, along with the proton NMR spectrum and repeating unit structure (Figure 21A). Letters indicate the carbohydrate residues reported in the figures and are plotted according to the notational notation for glycans. All monosaccharides are in pyranose form. Arabic numerals indicate the proton / carbon atom ratio of the corresponding residues. Figure 21B The N-acetyl signal (2.05 ppm) of glucosamine (residue A) and the methyl signal (1.35–1.30 ppm) of rhamnose units (C and D residues) are shown.

[0068] Figures 22A-22C The HSQC spectrum (600 MHz, 315 K, D2O) of CPS-1 isolated from *Lactobacillus paracasei* NPB01 is shown, along with its proton spectrum and structure. Figure 22A The letters indicate the carbohydrate residues reported in the diagram and are drawn according to the notational naming conventions for glycans. All monosaccharides are in pyranose form. Arabic numerals indicate the proton / carbon atom of the corresponding residue. Figure 22B The CPS-1 + TA mixture was shown. 1 H NMR spectrum (600 MHz, D2O). Figure 22C Showing pure CPS-1 1 HNMR spectrum (600 MHz, D2O).

[0069] Figure 23 The HSQC spectrum (1200 MHz, 293 K, D2O) of CPS-2 isolated from *Lactobacillus paracasei* NPB01, showing the cyclic proton region and proton NMR spectrum, is presented. Letters indicate the carbohydrate residues reported in the figure and are plotted according to SNFG. Arabic numerals indicate the proton / carbon ratio of the corresponding residue. Specifically, when the substituent "J" is present and the dimer K6→1T is absent, the residue is indicated by a capital letter in parentheses.

[0070] Figures 24A-24B The effects of *Lactobacillus paracasei* NPB01 postbiotic, CPS-1, CPS-2, TA, or control (NT) on LL-37 cells in Caco-2 cells were demonstrated. Figure 24A ) and closing protein ( Figure 24B The effect of [unclear] expression was investigated. The experiment was conducted in triplicate and repeated three times. Data are presented as mean ± SD and analyzed using an unpaired t-test. p <0.05 vs NT; *** p <0.0005CPS-2 vs NT, vs CPS-1 vs Lactobacillus paracasei NPB01 postbiotic; # p <0.05 TA vs. Lactobacillus paracasei NPB01 postbiotic; ## p <0.005 CPS-1 vs Lactobacillus paracasei NPB01 postbiotic, CPS-2 vs Lactobacillus paracasei NPB01 postbiotic.

[0071] Figures 25A-25B The effect of biogenic stimulation on Priestella megaterella MV30 was shown after 6 hours. P. megaterium MV30 ()( Figure 25A ) and Bacillus belesii MV4 ( B. velenzensis MV4 ()( Figure 25B The effect of OD600 absorbance on the light.

[0072] Figures 26A-26B The effect of the direct interaction between metagenes and human intestinal epithelial cells (Caco-2 cells) on cell growth (expressed as a percentage of cell density) was shown, assessed by MTT assay. Figure 26A The effects on cell differentiation were measured using RT-PCR to detect lactase expression. Figure 26B Data represent the mean (± standard deviation, shown as bars) of three independent experiments, each conducted in duplicate. *p<0.05 vs NT, #p<0.05 vs LP, °p<0.05 vs LGG.

[0073] Figures 27A-27B This demonstrates the direct interaction between the metagener and human intestinal epithelial cells (Caco-2 cells) on the tight junction protein closure protein ( ). Figure 27A ) and zonula occludens-1 (ZO-1)( Figure 27B The influence of ) is shown. Data represent the mean (± standard deviation, shown as bars) of three independent experiments, each experiment performed in duplicate. *p<0.05 vs NT, #p<0.05 vs LP, °p<0.05 vs LGG.

[0074] Figure 28 This study demonstrates the effect of the direct interaction between metagenes and human intestinal epithelial cells (Caco-2 cells) on mucus production (MUC2). Data represent the mean (± standard deviation, shown as bars) of three independent experiments, each performed in duplicate. *p<0.05 vs NT, #p<0.05 vs LP, °p<0.05 vs LGG.

[0075] Figure 29 This study demonstrates the effect of the direct interaction between metagenes and human intestinal epithelial cells (Caco-2 cells) on the stimulation of the innate immune peptide antimicrobial peptide LL-37. LL-37 production in cell supernatant was assessed by ELISA. Data represent the mean (± standard deviation, shown as bars) of three independent experiments, each performed in duplicate. *p<0.05 vs NT, #p<0.05 vs LP, °p<0.05 vs LGG.

[0076] Figure 30 The effect of continuous fermentation over time (in hours) on the increase in microbial growth (in CFU / ml) is shown. LP, Bal, Akk, and Ecn correspond to *Lactobacillus paracasei* NPB-01 and *Bifidobacterium animalis* subsp. *animal*. lactis Akkermansia myxophilus and Escherichia coli Nissle 1917.

[0077] Figures 31A-31C The results showed the use of Lactobacillus paracasei NPB-01 and Bifidobacterium animalis subsp. lactis The effect of incubation with *Akermansia myxophilus* and *Escherichia coli* Nissle 1917 on the fold-fold increase in expression of the following intestinal barrier integrity biomarker (in Caco-2 cells): tight junction protein ZO-1 (… Figure 31A ), tight junction protein closure protein ( Figure 31B ) and epithelial mucus protein MUC5AC ( Figure 31C ). Detailed Implementation

[0078] This disclosure relates to a method for preparing postbiotic compositions, the method comprising multiple (i.e., two or more) independent microbial fermentations, which may be carried out in parallel or sequential steps.

[0079] 1. Definition To make this disclosure more readily understandable, certain terms are first defined. As used in this application, each of the following terms shall have the meaning described below unless expressly provided otherwise herein. Further definitions are set forth throughout the application.

[0080] Before describing this disclosure in detail, it should be understood that this disclosure is not limited to specific compositions or process steps, and therefore such compositions or process steps may vary. Unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” as used in this specification and the appended claims include plural indicators. The terms “a / an,” “one or more,” and “at least one” are used interchangeably herein.

[0081] Furthermore, when used herein, “and / or” should be considered as a specific disclosure of each of the two particular features or components of the other, whether or not they possess them. Thus, the term “and / or” as used in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: 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).

[0082] Unless otherwise defined, 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 pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many terms used in this disclosure.

[0083] Units, prefixes, and symbols are represented in their SI-acceptable form. Numerical ranges include the values ​​that define the range. Unless otherwise specified, amino acid sequences are written from left to right with the amino-to-carboxyl orientation. The headings provided herein are not intended to limit the aspects which are derived from the specification as a whole. Therefore, the terms defined below are more fully explained by reference to the specification as a whole.

[0084] It should be understood that wherever the term "comprising" is used to describe an aspect herein, other similar aspects described as "consisting of" and / or "substantially constituted of" are also provided. As used herein, "comprise" and "include" and their variations (e.g., "comprises", "comprising", "includes", and "including") should be understood to mean including the said component, feature, element, or step, or a group of components, features, elements, or steps, but not excluding any other component, feature, element, or step, or a group of components, features, elements, or steps. Any of the terms "comprising", "substantially constituted of", and "consisting of" may be replaced by any of the other two terms while retaining their usual meaning.

[0085] The term “about” is used in this document to mean approximately, roughly, about, or around. When the term “about” is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the indicated value. Typically, the term “about” can modify a value to be higher or lower than the stated value by varying it upwards or downwards (increases or decreases), for example, by 10%.

[0086] As used herein, the term “approximately” when applied to one or more values ​​of interest means a value similar to the stated reference value. In some respects, the term “approximately” means a range of values ​​falling within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in any direction of the stated reference value, unless otherwise stated or otherwise apparent from the context (except where such a number would exceed 100% of the possible value).

[0087] As used in this article, the terms “ug” and “uM” are used interchangeably with “μg” and “μM”, respectively.

[0088] As used herein, the term “probiotic” refers to live microorganisms that, when administered in adequate amounts, are capable of producing health benefits for the host. Probiotics are discussed in, for example, the following literature: 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).

[0089] As used herein, the term "metogenic" refers to preparations derived from fermented and inactivated probiotic cells, composed of the same microorganisms or their components and fragments and / or metabolites, which can confer health benefits to the host. Metabiotics are also known as "inactive probiotics," "phantom probiotics," or "ghost probiotics," and refer to inactive microbial cells and soluble factors secreted or released by live bacteria upon lysis, including various cell surface components, lactic acid, short-chain fatty acids (SCFAs), and bioactive peptides. Bacterial inactivation can be achieved, for example, through mild heat treatment.

[0090] As used in this article, the term "fermentation" refers to the metabolic process in which organic molecules are broken down under anaerobic conditions.

[0091] As used herein, the term “substrate” or “fermentation substrate” refers to a substrate, optionally mixed with nutrients, that is suitable for microbial growth and / or fermentation.

[0092] As used herein, the term "multiple" means consisting of more than one, containing more than one, and / or involving more than one.

[0093] "Intestinal barrier function" refers to the function of the intestinal epithelium in allowing necessary nutrients to pass through the intestinal epithelium to other parts of the body, but preventing potentially harmful substances (such as antigens) from leaving the intestine.

[0094] "Immunotherapy" refers to the treatment of a subject who has a disease or is at risk of infection or disease recurrence by means of methods including inducing, enhancing, suppressing or otherwise altering the immune system or immune response.

[0095] As used herein, the terms “subject,” “individual,” or “patient” refer to any subject, particularly mammalian subjects requiring diagnosis, prognosis, or treatment. Mammal subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, etc.

[0096] The term "contact" is used in its simple and general sense and refers to a process that allows at least two different substances (e.g., chemical compounds including biomolecules or cells) to come close enough to react, interact, or physically touch. However, it should be recognized that the resulting reaction product can be produced directly from the reaction between the added reagents or from intermediates from one or more of the added reagents that can be produced in the reaction mixture. The term "contact" can include allowing two substances to react, interact, or physically touch, wherein the two substances can be the compounds described herein, as well as proteins or enzymes.

[0097] "Administration" means the physical introduction of a composition (including postbiotic compositions) into a subject using any of the various methods and delivery systems known to those skilled in the art. Routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase "parenteral administration" means a method of administration other than enteral and local administration, typically by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as intracorporeal electroporation. In some aspects, the formulation is administered via a non-parenteral route, and in some aspects by oral administration. Other non-parenteral routes include local, epidermal, or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual, or local administration. Application can also be performed, for example, once, multiple times, and / or for one or more extended periods.

[0098] "Treatment" or "therapy" for a subject means any type of intervention or treatment performed on a subject, or administration of an active agent to a subject with the aim of reversing, alleviating, improving, suppressing, slowing down, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, or disorders or biochemical indicators associated with a disease.

[0099] As used herein, “effective treatment” refers to treatment that produces a beneficial effect, such as improving at least one symptom of a disease or condition. A beneficial effect can be manifested as improvement relative to a baseline, that is, improvement relative to measurements or observations made according to the method prior to the start of treatment.

[0100] The term "effective amount" refers to the amount of agent that provides the desired biological, therapeutic, and / or preventive outcome. This outcome may be a reduction, improvement, mitigation, relief, delay, and / or remission of one or more signs, symptoms, or causes of a disease, or any other desired change in a biological system. An effective amount may be administered in a single or multiple doses.

[0101] The term "combination" refers to a fixed combination or combination administration in the form of a single dose unit, wherein the compounds of the present invention and combination partners (e.g., another medicine as explained below, also referred to as "therapeutic agents" or "co-agents") may be administered simultaneously and independently or separately at time intervals, particularly where these time intervals allow the combination partners to exhibit cooperation, such as synergistic effects. Individual components may be packaged in a kit or individually. Before administration, one or both of the components (e.g., powder or liquid) may be reconstituted or diluted to the desired dose. As used herein, the terms "co-administration" or "combination administration," etc., are intended to cover the administration of selected combination partners to a single subject (e.g., a patient) in need, and are intended to include treatment regimens in which the agents are not necessarily administered via the same route of administration or are administered simultaneously. As used herein, "simultaneously" means the administration of two or more therapeutic agents, wherein at least part of the administration overlaps in time. Thus, simultaneous administration includes a dosing regimen in which one or more agents are continued after the administration of one or more other agents has been discontinued.

[0102] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors, which can invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" can include tumors.

[0103] An "immune response" refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, pathogen-infecting cells or tissues, cancer cells or other abnormal cells, or (in cases of autoimmune or pathological inflammation) normal human cells or tissues within the vertebrate body. Immune responses include, for example, T cells (e.g., effector T cells, Th cells, CD4+).+ Cells, CD8 + The activation or suppression of T cells (or Treg cells), or any other cell of the immune system (e.g., NK cells).

[0104] The "innate immune response" refers to the activation of one or more innate white blood cells in the innate immune system (or nonspecific immune system or natural immune system). Activated white blood cells in the innate immune response include natural killer (NK) cells, macrophages, and dendritic cells. The innate immune system differs from the adaptive immune system (or specific immune system), which includes lymphocytes such as CD4+ or CD8+ T cells.

[0105] "Tolerogenic immune response" refers to any immune response that leads to specific immunosuppression against an antigen or cells, tissues, organs, etc., expressing that antigen. Such immune responses include any reduction, delay, or suppression of an adverse immune response specific to the antigen or cells, tissues, organs, etc. They also include any stimulation, generation, induction, promotion, or recruitment of a desired immune response specific to the antigen or cells, tissues, organs, etc. Therefore, a tolerance-inducing immune response includes the absence or reduction of an adverse immune response against an antigen (which can be mediated by antigen-responding cells) and the presence or promotion of suppressive cells. As described herein, a tolerance-inducing immune response includes immune tolerance. "Generating a tolerance-inducing immune response" refers to generating any of the aforementioned immune responses specific to the antigen or cells, tissues, organs, etc., expressing that antigen. A tolerance-inducing immune response can be the result of MHC class I restriction presentation and / or MHC class II restriction presentation and / or B cell presentation and / or CD1d presentation, etc. Tolerogenic immune responses include any reduction, delay, or inhibition of the proliferation and / or activity of CD4+ T cells, CD8+ T cells, or B cells. Tolerogenic immune responses also include a reduction in the production of antigen-specific antibodies. Tolerogenic immune responses may also include any response leading to the stimulation, induction, generation, or recruitment of regulatory cells, such as CD4+ Treg cells, CD8+ Treg cells, Breg cells, etc. In some embodiments, a tolerogenic immune response is a response leading to a conversion to a regulatory phenotype, characterized by the generation, induction, stimulation, or recruitment of regulatory cells. An “adverse immune response” refers to any adverse immune response caused by exposure to an antigen that promotes or exacerbates the disease, condition, or disorder (or its symptoms) described herein, or a symptom of the disease, condition, or disorder described herein. Such an immune response generally has a negative impact on the health of the subject, or is a symptom of a negative impact on the health of the subject. Adverse immune responses include the production of antigen-specific antibodies, the proliferation and / or activity of antigen-specific B cells, or the proliferation and / or activity of antigen-specific CD4+ T cells.

[0106] A “control” or “standard control” refers to a sample, measurement, or value used as a reference, typically a known reference, for comparison with the test sample, measurement, or value. For example, a test sample may be collected from a patient suspected of having a given disease (e.g., cancer) and compared with a known healthy (disease-free) individual (e.g., a standard control subject). A standard control may also represent an average measurement or value collected from a group of similar individuals (e.g., standard control subjects) who do not have a given disease (i.e., a standard control group), such as healthy individuals with similar medical histories, ages, weights, etc. Standard control values ​​may also be obtained from the same individual, for example, from a sample obtained earlier from a patient before the onset of the disease. For example, controls can be designed to compare treatment benefits based on pharmacological data (e.g., half-life) or treatment measures (e.g., comparison of side effects). Controls are also valuable for determining the significance of data. For example, if there is a large difference in the value of a given parameter in the controls, then the change in the test sample will not be considered significant. Technicians will recognize that standard controls can be designed to assess many parameters (such as RNA levels, protein levels, specific cell types, specific body fluids, specific tissues, synovial cells, synovial fluid, synovial tissue, fibroblast-like synovial cells, macrophage-like synovial cells, etc.).

[0107] The ranges provided herein are to be understood as abbreviations of all values ​​within the range. For example, the range 1 to 50 should be understood to include any number, combination of numbers, or subrange of numbers selected from the group consisting of 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.

[0108] As used herein, the term "inactivated" or "inactivated" in describing, for example, inactivated microbial biomass, inactivated fermentation products, and inactivated fermenting microorganisms means a state in which the microorganisms are dead, non-reproductive, and / or otherwise metabolically dormant.

[0109] The various aspects of this disclosure are further described in detail in the following sections.

[0110] 2. The method disclosed herein As shown below, the method according to the invention is based on multiple (i.e., two or more) independent microbial fermentations, which can be carried out in parallel or sequential steps. According to the invention, each fermentation process in the method is carried out by fermenting microorganisms on a different fermentation substrate, wherein each fermenting microorganism used is different from any other fermenting microorganism used in the parallel or sequential fermentation steps.

[0111] In the context of this specification, the term "different" means that the fermenting microorganisms used for multiple fermentations according to the present invention are different from each other in terms of their genus, species, or strain. For example, the succession pattern of microorganisms involved in the fermentation steps of the method of the present invention may include species of the genus *Lactobacillus*, species of the genus *Cytobacter*, species of the genus *Bifidobacterium*, and species of the genus *Saccharomyces*; or, as another example, the succession sequence of fermenting microorganisms may consist of different strains of the same microbial species, such as, for example, different strains of a certain species of *Lactobacillus*.

[0112] This invention allows for the advantageous acquisition of postbiotic formulations containing complex mixtures of metabolic byproducts from various microorganisms by utilizing the fermentation capabilities of combinations of individually grown different microorganisms, while avoiding the drawbacks typically associated with simultaneous fermentation processes, such as microbial growth competition and the inhibition of the production of certain metabolites by others.

[0113] It is known in the art that a variety of microorganisms can be used to ferment a substrate. Fermenting microorganisms can be inoculated simultaneously, thus carrying out so-called "mixed" fermentation, or they can be inoculated sequentially, as in winemaking and brewing (Liu W et al.; 2023. "Adjustment of Impact Phenolic Compounds, Antioxidant Activity and Aroma Profile in Cabernet Sauvignon Wine by Mixed Fermentation of Pichia Kudriavzevii and Saccharomyces Cerevisiae"). Food Chemistry: X 18: 100685) and bread making (Fang L., et al.; 2023. “Effects of Mixed Fermentation of Different Lactic Acid Bacteria and Yeast on Phytic Acid Degradation and Flavor Compounds in Sourdough.” LWT The process described in 174: 114438)

[0114] In the method according to the invention, the fermenting microorganisms in the resulting fermentation product are inactivated after each fermentation step. Advantageously, the metabiotic composition contains all and / or components (cellular protein matter, cellular nucleic acid matter, cellular protoplasm matter and / or cell wall components) of various inactivated microorganisms, thereby obtaining different functional properties while reducing the safety risks and poor stability associated with the presence of live bacteria.

[0115] The final or intermediate microbial inactivation in multiple fermentation processes has not yet been described.

[0116] Therefore, a first embodiment of the present invention is a method for preparing a postbiotic composition, comprising the following steps: inoculating at least two culture media each with fermenting microorganisms selected from the group consisting of: Akkermania, Bifidobacterium, Escherichia, Lactobacillus, Lactococcus, Lactobacillus, Yeast, and Streptococcus, wherein the fermenting microorganisms inoculated in each culture medium are different from each other; fermenting the at least two culture media under conditions suitable for the fermentation of the respective inoculated fermenting microorganisms to obtain at least two fermentation products, each fermentation product containing fermenting microorganisms; inactivating the fermenting microorganisms in each fermentation product to obtain at least two inactivated fermentation products; and mixing the at least two inactivated fermentation products to obtain the postbiotic composition.

[0117] Compared to probiotics, the advantages of using postbiotics include, for example, greater stability because they do not contain live bacteria; and a higher level of safety because they reduce the risk of microbial translocation, infection, or enhanced inflammatory responses in individuals with imbalanced or compromised immune systems.

[0118] like Figure 1 As shown, two or more different microorganisms are fermented in parallel, each microorganism fermenting in a medium containing a nutrient source specific to that fermenting microorganism, and the fermentation time allows for optimization of microbial biomass growth and the production of functional and non-functional metabolites in the resulting fermentation products. After fermentation, the fermentation products are subjected to an inactivation treatment suitable for achieving microbial death without altering the bioactive components contained in the products. Subsequently, the inactivated fermentation products are combined by mixing to obtain a postbiotic composition.

[0119] In this embodiment, the method according to the invention envisions the use of at least two culture media, more preferably more than two culture media, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 culture media, each culture media being inoculated with different fermenting microorganisms, thereby achieving multiple parallel fermentation processes.

[0120] According to a second embodiment of the present invention, a method for preparing a postbiotic composition includes the following steps: (i) inoculating a variety of fermenting microorganisms into a variety of culture media, wherein different fermenting microorganisms are inoculated into each culture media; (ii) fermenting the inoculated variety of culture media under suitable fermentation conditions to obtain a variety of fermentation products, each fermentation product containing different fermenting microorganisms; (iii) inactivating the fermenting microorganisms in the variety of fermentation products to obtain a variety of inactivated fermentation products; and (iv) mixing the variety of inactivated fermentation products to obtain a postbiotic composition.

[0121] Figure 2 A schematic diagram of a method for preparing a postbiotic composition according to a second embodiment of the present invention is provided, wherein multiple sequential fermentations are performed, each fermentation being carried out by different microorganisms on a different substrate. In this embodiment, the fermentation product obtained by culturing the fermenting microorganisms is used as the fermentation substrate for the next fermentation step after microbial inactivation, in which the growth of microorganisms different from those previously used is permitted. By sequentially repeating the microbial inoculation, fermentation, and inactivation steps described above, a postbiotic composition comprising all the non-living fermenting microorganisms used and their bioactive components can be obtained.

[0122] Another aspect of the present invention provides a method for preparing a metabiotic composition, the method comprising the steps of: (i) inoculating a culture medium with a first fermenting microorganism; (ii) fermenting the culture medium under conditions suitable for the fermentation of the first fermenting microorganism to obtain a first fermentation product containing the first fermenting microorganism; (iii) inactivating the first fermenting microorganism in the first fermentation product to obtain a first fermentation substrate; (iv) inoculating the first fermentation substrate with a second fermenting microorganism; (v) fermenting the first fermentation substrate under conditions suitable for the fermentation of the second fermenting microorganism to obtain a second fermentation product containing the second fermenting microorganism; (vi) inactivating the second fermenting microorganism in the second fermentation product to obtain a second fermentation substrate, thereby obtaining a metabiotic composition; and optionally repeating steps (iv) to (vi) once or more using additional fermenting microorganisms. In some aspects, each fermenting microorganism is a different species from any other fermenting microorganism, thereby obtaining a metabiotic composition. In some aspects, each fermenting microorganism is the same species.

[0123] According to the invention, steps (iv) to (vi) can be repeated any number of times, such as once, twice, three times, four times, five times, ten times or more.

[0124] Optionally, in the above embodiments, the inactivated fermentation product to be used as a substrate for subsequent fermentation steps may be supplemented with additional components suitable for optimizing microbial growth and the performance of the fermentation process. Typical nutrients include, but are not limited to, carbon, nitrogen, magnesium, and phosphorus sources.

[0125] Suitable microorganisms for parallel or sequential fermentation in the method of the present invention may be selected from genera of the group consisting of: Akkermania, Bifidobacterium, Escherichia, Lactobacillus, Lactococcus, Lactobacillus, Saccharomyces, and Streptococcus.

[0126] In some embodiments, the fermentation microorganisms are selected from the group consisting of bacterial microorganisms and yeast microorganisms. In some aspects, the bacterial microorganisms are selected from the group consisting of species of the genus Akkermansia, species of the genus Bifidobacterium, species of the genus Escherichia, species of the genus Lactobacillus, species of the genus Lactococcus, species of the genus Lactobacillus, and species of the genus Streptococcus. In some aspects, the yeast microorganisms are selected from species of the genus Yeast.

[0127] In some embodiments, the fermenting microorganism is selected from the group consisting of: *Ackermania myxophilus*, *Bifidobacterium animalis*, *Bifidobacterium bifidum*, *Bifidobacterium infantis*, *Bifidobacterium breve*, *Bifidobacterium longum*, *Lactobacillus casei*, *Lactobacillus paracasei*, *Lactobacillus plantarum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, *Lactobacillus paracasei*, *Saccharomyces boulardii*, and *Streptococcus salivarius*. In some embodiments, the fermenting microorganism is *Ackermania myxophilus*. In some embodiments, the fermenting microorganism is *Bifidobacterium animalis*. In some embodiments, the fermenting microorganism is *Bifidobacterium bifidum*. In some embodiments, the fermenting microorganism is *Bifidobacterium infantis*. In some embodiments, the fermenting microorganism is *Bifidobacterium breve*. In some embodiments, the fermenting microorganism is *Bifidobacterium longum*. In some embodiments, the fermenting microorganism is *Lactobacillus casei*. In some embodiments, the fermenting microorganism is *Lactobacillus paracasei*. In some embodiments, the fermenting microorganism is *Lactobacillus plantarum*. In some embodiments, the fermenting microorganism is *Lactobacillus reuteri*. In some embodiments, the fermenting microorganism is *Lactobacillus rhamnosus*. In some embodiments, the fermenting microorganism is *Lactobacillus paracasei*. In some embodiments, the fermenting microorganism is *Lactobacillus plantarum*. In some embodiments, the fermenting microorganism is *Lactobacillus reuteri*. In some embodiments, the fermenting microorganism is *Lactobacillus rhamnosus*. In some embodiments, the fermenting microorganism is *Lactobacillus paracasei*. In some embodiments, the fermenting microorganism is *Saccharomyces boulardii*. In some embodiments, the fermenting microorganism is *Streptococcus salivarius*.

[0128] In one implementation, *Bifidobacterium animalis* is a subspecies of *Bifidobacterium animalis*. lactis .

[0129] In one implementation, the Escherichia coli is Escherichia coli Nissle 1917.

[0130] In some implementations, *Lactobacillus paracasei* is *Lactobacillus paracasei* NPB01.

[0131] In one embodiment, the fermenting microorganism is *Lactobacillus paracasei* NPB-01. In some embodiments, the metabiotic composition comprises teichoic acid. In some embodiments, the composition comprises one or more polysaccharides. In some embodiments, the one or more polysaccharides include one or more capsular polysaccharides. In some embodiments, the teichoic acid is derived from *Lactobacillus paracasei* NPB-01. In some embodiments, one or more polysaccharides are derived from *Lactobacillus paracasei* NPB-01. In some embodiments, one or more capsular polysaccharides are derived from *Lactobacillus paracasei* NPB-01 (e.g., the capsular polysaccharides described in Example 4).

[0132] In one implementation, the *Lactobacillus* microorganisms are selected from the group consisting of: *Lactobacillus paracasei*, *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, *Lactobacillus reuteri*, and *Lactobacillus delbrueckii*. Lactobacillus delbrueckii ).

[0133] Non-limiting examples of Bifidobacterium species suitable for the methods of the present invention include: Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium breve, and Bifidobacterium infantis.

[0134] According to the present invention, the microorganisms of the genus *Lactococcus* are preferably *Lactococcus lactis* (…). Lactococcus lactis ) strains, preferably Streptococcus thermophilus (Streptococcus spp.) Streptococcus thermophilus The strains and / or yeast species are preferably selected from Saccharomyces boulardii and Saccharomyces cerevisiae. Saccharomyces cerevisae ) strains.

[0135] In this invention, any culture medium can be used, which can be selected according to the nutritional requirements of the fermenting microorganisms in order to maximize bacterial growth, the yield and bioactivity of metabiotic metabolites, and to impart specific sensory characteristics to the metabiotic composition.

[0136] Non-limiting examples of suitable culture media include MRS medium, BHI broth (including BHI broth supplemented with PGM), LB broth, plant-derived media, functional media containing plant extracts with antioxidant, antiviral and / or antibacterial activities, naturally derived media, and any combination thereof.

[0137] Those skilled in the art are well aware of the selection of the most suitable culture medium.

[0138] According to the method of the invention, the fermentation step is preferably carried out at a temperature of 25°C to 45°C, more preferably at 37°C, for at least two hours, more preferably for 6 to 24 hours. The pH is typically maintained at a value of 4.0 to 7.0, preferably 6.2. In some aspects, the fermentation step is carried out at a temperature of about 25°C to about 45°C.

[0139] In some respects, there are one or more individual fermentation steps, followed by one or more consecutive fermentation steps. In some respects, a single fermentation step may last up to about 24 hours. In some respects, one or more consecutive fermentation steps may last up to about 6 hours or up to about 12 hours. In some respects, the total consecutive fermentation time may last up to about 36 hours.

[0140] The fermentation process can be carried out in any type of stirred or wave-type bioreactor. Examples of bioreactors that can be used in this invention include, but are not limited to, batch reactors, fed-batch reactors, and CSTR (continuous flow stirred tank reactor) reactors.

[0141] In the method of the present invention, the step of inactivating the fermenting microorganisms in the fermentation product can be carried out using various techniques known in the art. Such techniques include, but are not limited to, heat treatment, chemical treatment (e.g., formalin), gamma ray or ultraviolet irradiation, high pressure, and ultrasonic treatment. Ultrasonic treatment is particularly commonly used to produce cell lysates: in a second embodiment, this method can increase the availability of functional components, thereby enhancing the fermentation performance of the second microorganism.

[0142] Heat inactivation of the fermenting microorganisms in the fermentation product is particularly preferred. Preferably, heat inactivation is carried out at a temperature of about 50°C to about 100°C for about 5 seconds to about 120 seconds. In an exemplary embodiment, heat inactivation is carried out at about 80°C for about 30 seconds.

[0143] In a more preferred embodiment, the fermentation product of the method according to the invention is a cultured broth.

[0144] The method of the present invention may suitably further include the step of drying the biotic composition. The techniques used to perform the drying step of the present invention are known in the prior art and have been described; therefore, their selection and use are within the skill of those skilled in the art.

[0145] The use of freeze drying, granulation and spray drying is mentioned by way of non-limiting examples.

[0146] Alternatively, in embodiments of the invention based on multiple parallel fermentations, the inactivated fermentation product from step (iii) can be processed into a dry form and then mixed together to obtain a dry metabiotic composition, or the wet inactivated fermentation product can be mixed and then processed into a dry form. Figure 1 ).

[0147] In some implementations, one or more additional components are added to the inactivated fermentation product before inoculation with the fermenting microorganism.

[0148] In some embodiments, the method further includes drying the inactivated fermentation product, wherein the dried inactivated fermentation product is optionally rehydrated in water before subsequent inoculation with fermenting microorganisms.

[0149] In some embodiments, the method further includes the step of drying the biogenic composition.

[0150] When the method of the present invention is carried out by means of multiple sequential fermentations, after fermentation and inactivation of the first microorganism, a second microorganism is inoculated into the fermentation substrate, and fermentation of the second microorganism is carried out. Figure 2 According to one embodiment, after inactivation, the fermentation product can be subjected to drying treatment and optionally stored in dry form, and the dried, inactivated fermentation product can be rehydrated, for example, in water, before inoculation with different fermenting microorganisms. Figure 3 ).

[0151] Optionally, in the first and second embodiments described above, the method according to the invention may further include subjecting the fermentation product to a centrifugation step after inactivation to separate the fraction consisting of the inactivated fermenting microorganisms (forming a cell body precipitate) from the remaining portion of the inactivated fermentation product, said remaining portion containing functional components (supernatant) and which can also be used as a fermentation substrate in subsequent fermentation steps or as a fermentation substrate for subsequent fermentation steps. Following the centrifugation step, the resulting cell body precipitate and / or supernatant may optionally undergo a drying stage.

[0152] This document also provides the postbiotic compositions described herein for the prevention or therapeutic treatment of diseases in subjects in need, said diseases being selected from the group consisting of: infectious and inflammatory diseases, immune-mediated diseases, cancerous diseases, skin diseases, gastrointestinal diseases (e.g., celiac disease), genitourinary diseases, neurological diseases, neuropsychiatric diseases, skeletal diseases, muscle diseases, malnutrition, metabolic diseases, and any combination thereof.

[0153] In some respects, administration of the postbiotic compositions described herein results in an increase in the production of one or more of the following in the subject's body: short-chain fatty acids, bile acids, choline metabolites, vitamins, amino acids (e.g., tryptophan), and / or neurotransmitters (e.g., mucosal serotonin release).

[0154] This article also provides the metabiotic compositions described herein for promoting healthy aging in mammals. Healthy aging is defined as a continuous process throughout the life course of life that optimizes various opportunities to maintain and improve physical and mental health, independence, and quality of life.

[0155] This article also provides the metabiotic compositions described herein for correcting drug-induced nutritional depletion (DIND) in mammals. DIND is a side effect of some medications that can cause nutritional deficiencies. These deficiencies may appear months or years after starting medication and can lead to other health problems. Some common medications that can cause DIND include oral contraceptives, cholesterol-lowering statins, antibiotics, and diuretics. In some respects, DIND is caused by glucagon-like peptide-1 (GLP-1) agonists, such as semaglutide.

[0156] This document also provides the postbiotic compositions described herein for improving intestinal barrier function in subjects of need by increasing the expression of tight junction proteins and / or mucins, and / or increasing intestinal epithelial cell growth and differentiation. In some aspects, tight junction proteins include closure proteins and / or ZO-1. In some aspects, mucins include MUC5AC. In some embodiments, treatment with the postbiotic composition increases the expression of closure proteins, ZO-1, and / or MUC5AC by at least about 2-fold, about 3-fold, about 4-fold, about 5-fold, or more compared to untreated subjects. In some embodiments, treatment with the postbiotic composition increases the expression of closure proteins, ZO-1, and / or MUC5AC by at least about 25%, about 50%, about 75%, or more compared to treatment with a composition containing a single live microorganism.

[0157] This document also provides the postbiotic compositions described herein for increasing the expression of β-defensin-2 (HBD-2) in subjects of need. In some embodiments, HBD-2 expression is increased by at least about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 7-fold, about 8-fold, or more after treatment with the postbiotic composition compared to untreated subjects. In some embodiments, HBD-2 expression is at least about 2-fold, about 3-fold, about 4-fold, or more after treatment with the postbiotic composition compared to treatment with a composition comprising a single microorganism.

[0158] This article also provides the postbiotic composition described herein for increasing the expression of the antimicrobial peptide LL-37 in subjects of need. In some respects, the expression of the antimicrobial peptide LL-37 increased by at least about 2-fold, about 3-fold, or more after treatment with the postbiotic composition compared to untreated subjects.

[0159] This document also provides the use of the metabiotic compositions described herein in food, beverage, pharmaceutical, nutritional supplement, cosmetic, or packaging compositions, wherein said compositions further comprise at least one pharmaceutically acceptable carrier, excipient, and / or diluent. In some embodiments, the use is for improving intestinal barrier function in mammals. In some embodiments, the use is for improving the innate immune response of mammals against infection. In some embodiments, the use is for inducing a tolerance-inducing immune response in mammals. In some embodiments, the use is for protecting the skin of mammals from infection.

[0160] This article also provides the use of the post-biotic compositions described herein for anti-aging effects, including improving gut microbiota composition, lowering cholesterol, and promoting SCFA production by modulating the gut microbiota.

[0161] This article also provides the use of the post-genetic compositions described herein for anticancer effects, including reducing the growth rate of human colon cancer cells in vitro.

[0162] This document also provides the use of the postbiotic compositions described herein for antibacterial activity, including antibacterial activity against Escherichia coli and inhibition of Staphylococcus aureus. S. aureus ).

[0163] This article also provides the use of the postbiotic compositions described herein for anti-biofilm effects, including having anti-biofilm activity against Escherichia coli biofilms.

[0164] This article also provides the use of the post-biotic compositions described herein for anti-inflammatory effects, including protecting human colonic muscle from pathogenic Escherichia coli, exhibiting anti-inflammatory activity against the porcine intestinal epithelial cell line IPEC-J2, promoting dendritic cell maturation, and inducing the secretion of the anti-inflammatory cytokine IL-10 in vivo.

[0165] This article also provides the use of the post-biotic compositions described herein for antioxidant effects, including 2,2′-adiazono-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, 2,2-diphenyl-1-picrylhydrazine (DPPH) radical scavenging activity, and iron reducing ability.

[0166] This article also provides the use of the postbiotic compositions described herein for immunomodulatory effects, including balancing IL-8 mRNA expression induced by surface molecules such as lipoteichoic acid, stimulating pro-inflammatory IL-12 and TNF-α cytokines, improving the adhesion of probiotics to the epithelium, enhancing the expression of tight junction proteins, and improving intestinal mucosal barrier function.

[0167] This article also provides the use of the post-genetic compositions described herein for anti-tumor effects, including inhibiting the proliferation of cancer cells and tumor cells.

[0168] This article also provides the use of the post-genetic compositions described herein for the effects of inflammatory bowel disease (IBD), including reducing neutrophils in crypt and surface epithelial cells, and reprogramming intraepithelial CD4+ T cells to CD4+. + CD8aa + Immune regulatory T cells.

[0169] This article also provides methods for preventing or treating diseases selected from the group consisting of: infectious and inflammatory diseases, immune-mediated diseases, cancerous diseases, skin diseases, gastrointestinal diseases, genitourinary diseases, neurological diseases, neuropsychiatric diseases, skeletal diseases, muscle diseases, malnutrition, metabolic diseases, and any combination thereof, wherein the methods include administering the postbiotic composition described herein to a subject in need.

[0170] This article also provides methods for improving intestinal barrier function in subjects in need by increasing the expression of tight junction proteins and / or mucins, and / or increasing intestinal epithelial cell growth and differentiation. In some aspects, tight junction proteins include closure proteins and / or ZO-1. In some aspects, mucins include MUC5AC. In some embodiments, treatment with the postbiotic composition increases the expression of closure proteins, ZO-1, and / or MUC5AC by at least about 2-fold, about 3-fold, about 4-fold, about 5-fold, or more compared to untreated subjects. In some embodiments, treatment with the postbiotic composition increases the expression of closure proteins, ZO-1, and / or MUC5AC by at least about 25%, about 50%, about 75%, or more compared to treatment with a composition containing a single live microorganism.

[0171] This document also provides a method for increasing the expression of β-defensin-2 (HBD-2) in subjects in need, wherein the method comprises administering the postbiotic composition provided herein to the subject. In some embodiments, HBD-2 expression is at least about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 7-fold, about 8-fold, or more after treatment with the postbiotic composition compared to untreated subjects. In some embodiments, HBD-2 expression is increased at least about 2-fold, about 3-fold, about 4-fold, or more after treatment with the postbiotic composition compared to treatment with a composition comprising a single microorganism.

[0172] This article also provides a method for increasing the total amount of one or more metabolites of L-tryptophan in a subject in need, wherein said method comprises administering the postbiotic composition provided herein to the subject. In some aspects, one or more metabolites of L-tryptophan include indole-3-acetic acid, indole-3-lactic acid, and / or L-kynurenine.

[0173] This article also provides a method for increasing the total amount of L-tryptophan in the body of a subject in need, wherein the method includes administering the postbiotic composition provided herein to the subject.

[0174] This article also provides a method for increasing the ratio of L-tryptophan to large neutral amino acids in the plasma of subjects in need, wherein the method comprises administering the postbiotic composition provided herein to the subject.

[0175] This article also provides methods for increasing the biosynthesis of serotonin and / or melatonin in subjects in need, wherein the methods include administering the postbiotic composition provided herein to the subject.

[0176] This article also provides methods for promoting healthy aging in subjects in need, wherein the methods include administering the postbiotic composition provided herein to the subjects.

[0177] This article also provides a method for correcting drug-induced nutritional depletion in subjects in need, wherein the method includes administering the postbiotic composition provided herein to the subject.

[0178] This document also provides metabiotic compositions produced by multimicrobial fermentation, wherein the multimicrobial fermentation includes the fermentation of two or more bacterial or yeast strains. In some embodiments, the metabiotic comprises two or more inactivated fermentation products. In some embodiments, the multimicrobial fermentation includes the methods described herein.

[0179] This document also provides a metabiotic composition comprising: (a) a first matrix comprising a first inactivated microorganism and a culture medium in which said microorganism was inactivated; and (b) one or more additional matrices, each additional matrix comprising another inactivated microorganism and a culture medium in which said microorganism was inactivated. In some aspects, the first inactivated microorganism and each additional inactivated microorganism are different species. In some embodiments, the first inactivated microorganism and each additional inactivated microorganism are the same species. In some embodiments, the metabiotic composition is prepared according to the methods described herein.

[0180] The above preferred embodiments can be combined with each other as needed, and the implementation of these combinations is within the skill level of those skilled in the art.

[0181] As mentioned above, the present invention advantageously provides a method for producing metabiotic compositions containing unique metabolic activity, since each individual fermentation and inactivation process affects the quality and quantity of the final metabiotic produced and may result in different metabiotics with different effects and properties.

[0182] As will be described in the experimental section below, it has been surprisingly found that the method according to the invention is able to obtain synergistically active postbiotic compositions with beneficial properties, including, for example, immune-enhancing effects in human colon cells.

[0183] Therefore, the present invention also relates to a metabiotic composition obtainable by means of the method of the present invention as defined above, characterized in that it contains lactic acid in a concentration ranging from 1 to 30 g / L (based on the total volume of the composition) and / or inactivated fermenting microorganisms in a concentration ranging from 0.00015 g / L to 150 g / L (based on the total volume of the composition).

[0184] The preferred lactic acid concentration in the postbiotic composition according to the invention is in the range of 5 to 25 g / L, more preferably in the range of 10 to 20 g / L.

[0185] The preferred amount of inactivated fermenting microorganisms is in the range of 0.0005 g / L to 100 g / L, or in the range of 0.05 g / L to 50 g / L, or in the range of 0.2 g / L to 5 g / L.

[0186] 3. The compositions and kits disclosed herein This document also provides compositions comprising postbiotics produced by the methods of this disclosure. In a preferred embodiment of the postbiotic composition according to the invention, the postbiotic composition is suitable for use as a human and animal food supplement or food ingredient, for example, for food biopreservation.

[0187] This document also provides metabiotic compositions produced by multimicrobial fermentation, wherein the multimicrobial fermentation includes the fermentation of two or more bacterial or yeast species. In some aspects, the metabiotic comprises two or more inactivated fermentation products. In some aspects, the multimicrobial fermentation includes the methods described herein.

[0188] This document also provides a metabiotic composition comprising: (a) a first substrate comprising a first inactivated microorganism and a culture medium in which said microorganism was inactivated; and (b) one or more additional substrates, each additional substrate comprising another inactivated microorganism and a culture medium in which said microorganism was inactivated. In some aspects, the first inactivated microorganism and each additional inactivated microorganism are different species. In some aspects, the metabiotic composition is prepared according to the method described herein.

[0189] In some aspects, the metabiotic composition comprises cellular metabiotics. In some aspects, the metabiotic composition comprises cell-free metabiotics. In some aspects, the metabiotic composition comprises: inactivated microorganisms, primary microbial metabolites, secondary microbial metabolites, cell-free supernatant (CSF), cell-free waste culture medium (CFSM), cell lysates, short-chain fatty acids (SCFA), vitamins, enzymes, proteins, peptides, organic acids, flavonoid-derived metabiotics, terpene-derived metabiotics, extracellular polysaccharides (EPS), peptidoglycans, lipoteichoic acid (LTA), phenol-derived metabiotics, cell wall fragments, and lipopolysaccharides (LPS).

[0190] This document also provides a postbiotic composition obtained by the methods described herein, which further comprises lactic acid at a concentration of about 1 g / L to about 30 g / L (based on the total volume of the composition) and / or in an amount of about 0.00015 g / L to about 150 g / L (based on the total volume of the composition) of inactivated fermenting microorganisms. In some aspects, the amount of inactivated fermenting microorganisms includes about 10 5 cells / ml to approximately 10 11 Cells / ml

[0191] This document also provides postbiotic compositions obtained by the methods described herein, which further comprise L-tryptophan or a dipeptide containing L-tryptophan. In some aspects, the composition comprises L-tryptophan at a concentration of at least 0.01% w / w. In some aspects, the composition comprises L-tryptophan at a concentration of at least 0.10% w / w. In some aspects, L-tryptophan is present in an amount of at least 10 mg, at least 50 mg, or at least 100 mg.

[0192] This document also provides a metabiotic composition obtained by the method described herein, wherein at least one of the fermenting microorganisms used to obtain the metabiotic composition is *Lactobacillus paracasei* NPB01.

[0193] This document also provides metagenic compositions obtained by the methods described herein, wherein the metagenic compositions further comprise one or more polysaccharides derived from *Lactobacillus paracasei* NPB01. In some aspects, the one or more polysaccharides derived from *Lactobacillus paracasei* NPB01 include teichoic acid and / or one or more capsular polysaccharides (e.g., the capsular polysaccharides described in Example 4).

[0194] In another embodiment, the postbiotic composition according to the invention is used as a medicine, particularly as a beneficial modulator of: gut microbiota structure and function, immune system, cell growth and differentiation, intestinal barrier, brain function, and skin health. More preferably, the postbiotic composition according to the invention can be used to treat and / or prevent infectious and inflammatory diseases, immune-mediated diseases, cancer, skin diseases, gastrointestinal diseases, genitourinary diseases, and brain diseases.

[0195] According to the present invention, the postbiotic composition can be administered via any suitable route of administration. For example, the composition can be administered orally to animals (including humans). In the case of food compositions or nutritional supplements, the postbiotic composition can be simply incorporated into conventional foods or food supplements. Exemplary pharmaceutical formulations include capsules, microcapsules, tablets, granules, powders, lozenges, pills, suspensions, and syrups. In another embodiment, the composition is in a form intended for rectal administration to animals (including humans), for example, as a rectal suppository or enema. For topical application, the pharmaceutical composition can be formulated as a gel, ointment, cream, or ointment.

[0196] Suitable formulations can be prepared using common methods and conventional organic and inorganic additives. The amount of the active ingredient in the pharmaceutical composition may be at a level that will exert the desired therapeutic effect.

[0197] A further aspect of the invention is a pharmaceutical, nutritional supplement, or cosmetic composition, or a food or food supplement comprising a combination of the postbiotic composition as described above with at least one pharmaceutically acceptable carrier, excipient, and / or diluent.

[0198] For the preparation of food according to the present invention, the post-biotic composition can be incorporated into edible materials using standard techniques well known to those skilled in the art. For example, the aforementioned post-biotic composition can be added directly to edible materials, or it can be used to prepare intermediate compositions (e.g., food additives or premixes) suitable for subsequent addition to edible materials.

[0199] According to the present invention, the food may be in the form of fermented food, processed food, health food or dietary supplement.

[0200] This article also provides a kit comprising the postbiotic composition described herein.

[0201] This document also provides a kit comprising a multimicrobial fermentation substrate and an inoculum containing two or more inactivated microorganisms. In some embodiments, a metabiotic composition is prepared according to the methods described herein. In some embodiments, the kit also includes documentation containing usage steps and conditions.

[0202] In various embodiments, the kits described herein for laboratory and therapeutic applications are all within the scope of this disclosure. Such kits may include a carrier, package, or container that is partitioned to contain one or more containers, such as vials, test tubes, etc., each containing a separate element to be used in the methods disclosed herein, and a label or insert containing instructions for use (such as those described herein).

[0203] The kit may include the containers described above, as well as one or more other associated containers containing the materials required from a commercial and user perspective, including buffers, diluents, filters, needles, syringes; carriers, packaging, containers, vials and / or test tubes listing the contents and / or instructions for use, and packaging inserts with instructions for use.

[0204] Labels may be present on or affixed to the container to indicate that the composition is intended for specific therapeutic or non-therapeutic applications, such as prognostic, preventative, diagnostic, or laboratory applications. Labels may also indicate instructions for in vivo or in vitro use, as described herein. Instructions and / or other information may also be included in inserts or labels accompanying the kit or on the kit. Labels may be on or affixed to the container. Labels may be on the container when the letters, numbers, or other characters forming the label are molded or etched into the container itself. Labels may be affixed to the container when the label is present within a receiver or carrier that also houses the container, for example, as a packaging insert. Labels may indicate that the composition is intended for the diagnosis or treatment of a condition, such as cancer as described herein.

[0205] The above implementation schemes can be combined with each other as needed, and the implementation of these combinations is within the skill level of those skilled in the art.

[0206] The invention is further illustrated by the following embodiments, which should not be construed as further limitations. All references cited throughout this application are expressly incorporated herein by reference.

[0207] Example Example 1: Materials and Methods The bacterial species selected in the following examples are most commonly used in fermented foods and food supplements, and they are considered probiotic microorganisms that have beneficial effects on human health.

[0208] Regarding testing of eukaryotic genera, *Saccharomyces boulardii* is most commonly used as a dietary supplement or in combination with other probiotics to maximize gut health benefits; therefore, we hypothesize that we will test it, as well as in combination with other genera.

[0209] The bacteria used in this experiment belong to the family Lactobacillus, specifically the genus Lactobacillus, including *Lactobacillus paracasei*, *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, and *Lactobacillus casei*. All Lactobacilli are Gram-positive, facultative anaerobic, and homofermentative microorganisms.

[0210] Microbial Lactobacillus paracasei provided by Science Power Srl, Lactobacillus rhamnosus GG (LGG) was isolated from a commercial food supplement (Dicoflor capsules), Lactobacillus casei was isolated from dairy products, and Lactobacillus plantarum was isolated from a commercial food supplement (Femelle capsules).

[0211] Other genera of microorganisms were also used; they belong to the genera Bifidobacterium, Streptococcus, and Saccharomycetes. Specifically, Bifidobacterium bifidum was isolated from a commercial food supplement (Bifidobacterium L1fe nutritional capsules), Streptococcus salivarius was isolated from a commercial food supplement (Bactolactis tablets), and Saccharomyces boulardii was isolated from a commercial food supplement (Fair & Pure capsules).

[0212] Table 1. Microbial Abbreviations

[0213] Specifically, the fermentation process was carried out under the following conditions: Microorganisms were inoculated into the broth and fermented separately for 24 hours, followed by heat inactivation. In this experiment, powders from the two processes obtained by spray drying were mixed (50:50 and 80:20) (i.e., LP 50% + LGG 50% and LP 80% + LGG 20%).

[0214] The first type of microorganism is inoculated into the broth and allowed to ferment for 6 or 24 hours. After reduction, the second type of microorganism is added to the waste broth for a second fermentation of 24 hours, followed by a second reduction.

[0215] Specifically, the following conditions were tested: fermentation of the first microorganism (LP or LGG) for 24 hours + fermentation of the second microorganism (LGG or LP) for 24 hours; fermentation of the first microorganism (LP or LGG) for 6 hours + fermentation of the second microorganism (LGG or LP) for 24 hours.

[0216] Then all fermentation products are dried using a spray drying process.

[0217] In all test cases, the process was characterized based on microbial parameters, which included defining microbial growth curves and corresponding chemical parameters, and quantifying lactic acid produced as the main metabolite of microbial metabolism.

[0218] Experimental groups using other Lactobacillus species (Lpl and Lc) and other genera (Bb, Ss and Sb) Although not all combinations of the first experimental group were tested, the combinations that are most indicative for the stated purpose have been studied based on the results obtained therefrom.

[0219] Microorganisms (Lpl, Lc, Bb, Ss, and Sb) were inoculated into broth and fermented separately for 24 hours, followed by heat inactivation. The inactivated broth was then dried using a spray drying process. The resulting powders were also mixed to obtain a metabiotic composition (Lpl 50% + Lc 50% and Bb 34% + Ss 33% + Sb 33%).

[0220] For other Lactobacillus species: The first microorganism (Lpl) was inoculated into the broth and fermented for 6 hours. After reduction, the second microorganism (Lc) was added to the inactivated broth for a second fermentation of 24 hours, followed by a second reduction. Similarly, the fermentation process was carried out continuously by reversing the order of the microorganisms. The fermented and inactivated broth was then dried by a spray drying process.

[0221] Other genera: The first microorganism (Bb) was inoculated into the broth and fermented for 6 hours. After reduction, the second microorganism (Ss) was added to the inactivated broth, and a second fermentation was carried out for 6 hours, followed by a second reduction. The third microorganism (Sb) was inoculated, and a third fermentation was carried out for 24 hours, followed by further reduction. The fermented and inactivated broth was then dried using a spray drying process.

[0222] In all test cases, the process was characterized based on microbial parameters, which defined the growth of the microorganisms and the corresponding chemical parameters, and quantified the lactic acid produced.

[0223] strains and resuscitation For all microorganisms belonging to the Lactobacillus family and Streptococcus salivarius, the microorganisms were stored in cryovials containing 20% ​​glycerol at -80°C and revived by incubation in MRS broth (Sigma-Aldrich) at 37°C for 24 hours; Bifidobacterium bifidum was revived by incubation in BSM broth (Millipore) at 37°C for 24 hours; and Saccharomyces boulardii was revived by incubation in yeast and mold broth (Difco) at 37°C for 24 hours.

[0224] The cell density in the inoculated broth was 10-1 8 CFU / mL.

[0225] Fermentation Fermentation was carried out using a batch reactor (1L) equipped with an external jacket for circulating working fluid (water) from a temperature-controlled water bath. MRS medium was inoculated (1% v / v), and fermentation was carried out for specific hours by setting a stirring speed of 81 rpm, according to each specific implementation in the two experimental groups.

[0226] The fermentation process was carried out under controlled temperature and pH conditions (37°C and 6.2, respectively). Fermentation samples were aseptically removed from the reactor at specific times.

[0227] Analytical methods Microbiological analysis For Lactobacillus and Streptococcus, Bifidobacterium and Yeast counts, serial dilutions and platings were performed on Petri dishes filled with De Man, Rogosa, and Sharpe (MRS) agar (Oxoid, Basingstoke, UK), BSM agar (Millipore) and yeast agar (Difco).

[0228] MacConkey agar (Oxoid, Basingstoke, UK) and gelatin peptone bios agar (Biolife, Milan, Italy) were used to control for microbial contamination. All plates were incubated at 37°C for 48 hours before reading the results.

[0229] lactic acid production Lactic acid production was monitored by high-performance liquid chromatography (HPLC) using an Agilent Technologies 1100 equipped with a Phenomemenx Synergi Hydro-RP C18 column (250 mm × 4.6 mm, 4 μm pore size) and a visible / ultraviolet detector. The mobile phase consisted of a 0.27% KH2PO4 aqueous solution modified with H3PO4 at pH 1.5, and the flow rate was 1 mL / min. The column temperature was set at 60 °C, and the wavelength was 210 nm.

[0230] Biological tests Cell model All experiments used the human intestinal epithelial cell line Caco-2 (American Center for Type Culture Collection, Teddington, Middlesex TW11 0LY, United Kingdom). These cells reached the morphological and functional characteristics of human small intestinal epithelial cells 14 days after confluence. Cells were grown in Dulbecco's Modified Eagles's Medium (DMEM-Gibco, Berlin, Germany) supplemented with 20% fetal bovine serum (Lonza, Visp, Switzerland), 1% L-glutamine (Lonza), 1% essential amino acids, and 1% penicillin / streptomycin (Lonza). Cells were plated and cultured at 37°C in 5% CO2. All experiments were performed when cells reached full functional differentiation (14 days after confluence).

[0231] Cell stimulation protocols Prior to cell stimulation, the inventors determined the protein content of the metabiotic product using the Bradford method and normalized it to protein concentration. A dose-response experiment determined the appropriate dose to be 11.5 mg / mL. Human intestinal epithelial cells were stimulated with the studied metabiotic product for 48 hours. Cells stimulated only with culture medium served as a control. All experiments were performed in triplicate and repeated twice.

[0232] Analysis of the production of the innate immune peptide HBD-2 Following cell stimulation, the supernatant was collected and stored at -80°C. The concentration of HBD-2 was determined using a specific ELISA kit (Hycult Biotech, Uden, The Netherlands) with a sensitivity limit of 0.1 ng / ml.

[0233] Real-time PCR analysis of intestinal barrier integrity markers After collecting the supernatant, cells were also collected and centrifuged at 1200 rpm for 10 minutes. Total RNA was then extracted using TRIZOL (Gibco BRL, Paisley, Scotland) according to the manufacturer's instructions. Real-time PCR was performed using TaqMan probes for the following genes: closure protein (Hs00170162_m1), closure band small protein-1 (ZO-1, Hs01551871_m1), and mucin 5AC (MUC5AC, Hs01365616_m1). The TaqMan probes for these genes were counted and tested by the manufacturer's quality control (QC) department (Applied Biosystems, Waltham, Massachusetts, United States). The amplification conditions used were as follows: 40 cycles of 50°C for 2 minutes, 95°C for 10 minutes, and 95°C for 25 seconds and 60°C for 1 minute. The expression of each gene was normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs02786624_g1) to obtain the relative quantification of the transcripts of interest. The relative expression of each gene was calculated using the 2-ΔΔCT method: (ΔΔCT = ΔCT sample - ΔCT control). Each sample was analyzed in triplicate.

[0234] The characterization of the fermentation process for all test microorganisms and conditions is reported below in Example 2.

[0235] Example 2: Results LGG and LP microorganisms were fermented, inactivated, and dried separately, then mixed together. Growth curves for 24-hour culture of LGG and LP were obtained separately, as shown in the figure. Figure 4A As shown in (LGG) and 4B(LP).

[0236] During the 24-hour bacterial growth process, the inventors also characterized the fermentation of the two microorganisms in terms of lactic acid production, and the results were as follows: Figure 5A As shown in (LGG) and 5B(LP).

[0237] The inventors' results demonstrate that both test microorganisms can ferment the broth to a final concentration of approximately 10. 9 The fermentation product yielded approximately 20 g / L of CFU / ml and final lactic acid production. The fermentation product was inactivated to kill the bacterial population, thus obtaining metabiotics, which were then dried via a spray-drying process.

[0238] The inactivated fermentation products, obtained from the dehydration process, were then mixed in powder form to form a postbiotic composition (LP / LGG ratios of 50:50 and 80:20), which was used to stimulate CaCo-2 cells to examine the biological effects of the composition.

[0239] For testing other Lactobacillus species, *Lactobacillus plantarum* (Lpl) and *Lactobacillus casei* (Lc) were fermented separately for 24 hours. Figure 6 and Figure 7 The report presents the bacterial growth curves and lactic acid production curves obtained after 24 hours of fermentation.

[0240] The results demonstrate that both tested microorganisms can effectively ferment the broth, achieving a final concentration of 10. 9 The final lactate concentrations at CFU / ml, Lpl, and Lc were 18.46 g / L and 11.95 g / L, respectively.

[0241] The fermented broth was inactivated to kill the bacterial population, thereby obtaining metagenes, which were then dried via a spray drying process for subsequent biological assays.

[0242] The inactivated fermentation powder was then mixed to form a postbiotic composition (Lpl 50% + Lc 50%), which was then tested on Caco-2 cells.

[0243] For testing of other microbial genera, *Bifidobacterium bifidum* (Bb), *Streptococcus salivarius* (Ss), and *Saccharomyces boulardii* (Sb) were fermented individually for 24 hours. Figure 8A and Figure 8B The report presents the bacterial growth curves and lactic acid production curves obtained after 24 hours of fermentation.

[0244] The results showed that all three different microorganisms could effectively ferment the broth, with the final concentration of Bb reaching 10. 8 CFU / ml, the final concentrations of Ss and Sb reached 10. 9 CFU / ml; after 24 hours of fermentation, lactic acid production was detected in Bb, Ss and Sb at 14 g / L, 8 g / L and 3 g / L respectively.

[0245] The three fermented broths were then inactivated to kill the bacterial population, thereby obtaining metagenes, which were then dried by a spray drying process for bioassays. The inactivated fermented powders were then mixed to form a metagene composition (Bb 34% + Ss 33% + Sb 33%), and tested on Caco-2 cells.

[0246] The results obtained from multiple sequential fermentations (the second embodiment of the method of the present invention) are as follows: A 48-hour fermentation process was conducted using *Lactobacillus rhamnosus* (LGG) as the primary microorganism and *Lactobacillus paracasei* (LP) as the secondary microorganism. In short, after the initial 24-hour fermentation with LGG, heat treatment was performed to kill the bacterial biomass, and then LP was inoculated into the inactivated fermentation product without adding any fresh nutrients to the broth. At the end of the second 24-hour fermentation with LP, a second heat inactivation step was performed.

[0247] like Figure 9A As shown, during the first 24 hours of fermentation, LGG showed a microbial growth (Δlog) of 2 log, with a final bacterial concentration of 1.9 * 10^9 CFU / mL. After the first microorganism was inactivated, the second microorganism (LP) was inoculated, which showed an increase in growth (Δlog) of approximately 1 log, with a final bacterial concentration of 3.4 * 10^7 CFU / mL.

[0248] The entire process produced 18 g / L of lactic acid, almost all of which was generated during the first 24 hours of fermentation (LGG); in the second stage of the process, the lactic acid concentration remained roughly constant. Figure 9B ).

[0249] As a further experiment, the method of the present invention was carried out by reversing the inoculation order of the fermenting microorganisms. The first inoculated microorganism, LP, was fermented in a culture broth for 24 hours, followed by mild heat treatment to inactivate the bacterial biomass. Without adding any nutrients to the fermentation broth, a second 24-hour fermentation was carried out by inoculating with a second microorganism (LGG). At the end of the second 24-hour fermentation with LGG, heat inactivation was performed again. During the first 24-hour fermentation, the microbial growth of LP was 2 log (Δlog), with a final bacterial concentration of 5.7 * 10^8 CFU / mL. After microbial inactivation, LGG was inoculated with an initial bacterial concentration of 6 log. Over the next 24 hours, an increase in growth (Δlog) close to 2 log was observed, with a final bacterial concentration of 1.8 * 10^8 CFU / mL. Figure 10A ).like Figure 10B As shown, approximately 20.5 g / L of total lactic acid was observed during this process.

[0250] The inventors conducted further experiments on multiple sequential fermentations according to the present invention by evaluating bacterial growth and lactic acid production after fermentation with LGG for 6 hours, followed by fermentation with LP for 24 hours.

[0251] like Figure 11AAs shown, at the end of 6 hours of fermentation, LGG reached a bacterial load of 1.58 * 10^8 CFU / mL, an increase (Δlog) of approximately 2 log compared to time 0. Furthermore, in this experiment, a second microorganism (LP) was added after the first fermentation microorganism was inactivated, and no nutrients of any type were added to the fermentation broth. After 24 hours of fermentation, LP reached a bacterial growth of 2.6 * 10^8 CFU / mL, with a Δlog of 2 log.

[0252] At the end of the above-described sequential fermentation, the final concentration of lactic acid was observed to be 20 g / L, with a maximum production of 22 g / L at 28 hours. Figure 11B ).

[0253] The inventors conducted a sequential fermentation experiment again by reversing the microbial inoculation order. At the end of 6 hours of fermentation, LP reached a bacterial load of 3.6 * 10^7 CFU / mL. After 24 hours of treatment, LGG growth increased by 2 log, with a final bacterial concentration of 2.8 * 10^9 CFU / mL. Figure 12A ).

[0254] In this experiment, lactic acid production was at the same level observed in the LP (24h) + LGG (24h) experiment, with a concentration of 20.7 g / L. Figure 12B ).

[0255] Under the testing conditions described above for the methods of the present invention, a microbial concentration sufficient to guarantee functional efficacy (above 10^7 CFU / mL) was obtained. Lactic acid production also averaged approximately 20 g / L. For other Lactobacillus species, *Lactobacillus plantarum* (Lpl) was used as the first microorganism and *Lactobacillus casei* (Lc) as the second microorganism, and fermentation was carried out for 30 hours. In short, after the initial 6 hours of fermentation with Lpl, heat treatment was performed to kill the bacterial biomass, and then Lc was inoculated into the inactivated fermentation broth without the addition of any fresh nutrients. At the end of 24 hours of fermentation with Lc, a second heat inactivation step was performed.

[0256] like Figure 13A and 13B As shown, after the first 6 hours of fermentation, Lpl showed an increase in microbial growth (Δlog) of 2log, with a bacterial load reaching 3.6 * 10^8 CFU / mL and a lactic acid concentration of 2 g / L. Figure 13BThe first microorganism was then subjected to a mild heat treatment to inactivate it, and a second microorganism (Lc) was inoculated into the fermentation broth. After 24 hours of fermentation, Lc showed a growth increase (Δlog) of 2 log, with a bacterial load of 1.34*10^8 CFU / mL. Throughout the process, 14 g / L of lactic acid was produced.

[0257] Further experiments were conducted by reversing the inoculation order of the fermenting microorganisms. The first inoculated microorganism, Lc, fermented in the culture medium for 6 hours, followed by mild heat treatment to inactivate the bacterial biomass. A second microorganism, Lpl, was then inoculated and fermented for an additional 24 hours without adding any nutrients to the fermentation broth. At the end of the second fermentation, heat inactivation was performed again. Figure 14A and 14B As shown, in the first 6 hours of fermentation, Lc showed an increase in microbial growth (Δlog) of approximately 2 log, with a final bacterial load of 9.5 * 10^7 CFU / mL and lactic acid production of 0.9 g / L; subsequently, a mild heat treatment was performed to inactivate the first microorganism. The second microorganism (Lpl) was inoculated into the fermentation broth, and after 24 hours of fermentation, it showed an increase in growth (Δlog) of 2 log and a bacterial load of 5.6 * 10^8 CFU / mL. Throughout the process, 18.5 g / L of lactic acid was produced.

[0258] For testing of other genera, *Bifidobacterium bifidum* (Bb) was used as the first microorganism for 6 hours of fermentation, *Streptococcus salivarius* (Ss) as the second microorganism for 6 hours of fermentation, and *Saccharomyces boulardii* (Sb) as the third microorganism for 24 hours of fermentation, for a total fermentation process of 36 hours. In short, after the initial 6-hour fermentation with Bb, heat treatment was performed to kill the bacterial biomass. Then, *Ss* was inoculated into the inactivated fermentation broth without adding any fresh nutrients; the broth fermented for another 6 hours with *Ss*. At the end of this period, a heat inactivation step was performed again. *Sb* was then inoculated without adding any fresh nutrients to the fermentation broth, and a third fermentation of 24 hours was performed. After this period, heat treatment was performed to inactivate the bacterial biomass.

[0259] like Figure 15A and 15BAs shown, in the first 6 hours of fermentation, Bb showed a 2log increase in microbial growth (Δlog), reaching a bacterial load of 2.0 * 10^8 CFU / mL and a lactic acid concentration of 1.2 g / L. A mild heat treatment was performed to inactivate the first microorganism, followed by inoculation with a second microorganism (Ss). Ss showed a 2log increase in growth (Δlog) within 6 hours, reaching a bacterial load of 1.0 * 10^8 CFU / mL and a total lactic acid concentration of 2.1 g / L. An additional heat treatment was performed to kill the biomass of Ss, followed by inoculation with Sb. After 12 hours of fermentation, bacterial growth reached 1.0 * 10^8 CFU / mL, which remained essentially constant (7.0 * 10^7 CFU / mL) for the remaining 12 hours of the process. Over the entire process (36 hours), 7.8 g / L of lactic acid was produced.

[0260] Fermentation results demonstrated that all tested bacteria (LP, LGG, Lpl, Lc, Bb, Ss, and Sb) exhibited good microbial growth (reaching 8 and 9 log CFU / ml) for both single fermentation and continuous processes, thus indicating that they possess probiotic properties (the minimum concentration for probiotic products is generally considered to be 10). 6 CFU / mL or g, and should be consumed at 10 per day. 8 Up to 10 9 Only a certain total amount of probiotic microorganisms can have a probiotic effect, and good lactic acid production is also required.

[0261] Surprisingly, in the sequential fermentation method according to the present invention, the first microorganism consumes the nutrients in the broth, while the second and finally the third inoculated microorganisms can ferment effectively, producing large amounts of biomass and lactic acid. Therefore, the above experiments demonstrate that the "post-biotic broth" can be used as a fermentation medium for other microorganisms, thereby ensuring sufficient metabolic activity.

[0262] Biological results Evaluation of the immunobiological marker β-defensin-2 on Caco-2 cells The method according to the present invention can obtain postbiotic compositions with significant biological activity and high added value.

[0263] In order to evaluate the biological effects of the postbiotic composition according to the present invention, the inventors investigated the ability of the postbiotic product obtained by double fermentation, reduction and subsequent drying of MRS broth to modulate innate immunity.

[0264] The metagenic product obtained by parallel or continuous fermentation of LP and LGG was used as a stimulant for Caco-2 cells (48 hours) at a dose of 11.5 mg / ml. At the end of stimulation, the concentration of HBD-2, a marker of innate immunity, in the supernatant was measured. The unstimulated (NT) supernatant was used as a control. Figure 16 The results show the HBD-2 concentration in the supernatant of Caco-2 cells after stimulation with a metagenic composition prepared according to the following methods: control (NT); MRS fermented with LP alone for 24 h (LP (24h)); MRS fermented with LGG alone for 24 h (LGG (24h)); MRS fermented with LP for 6 h followed by LGG for 24 h (LP (6h) + LGG (24h) sequentially); MRS fermented with LP for 24 h followed by LGG for 24 h (LP (24h) + LGG (24h) sequentially); MRS fermented with LGG for 6 h followed by LP for 24 h (LGG (6h) + LP (24h) sequentially); MRS fermented with LGG for 24 h followed by LP for 24 h (LGG (24h) + LP (24h) sequentially); MRS fermented with LGG for 24 h followed by LP for 24 h (LGG (24h) + LP (24h) sequentially). (24h) sequence); MRS was fermented separately with LP and LGG, inactivated, dried and mixed at 50% (LP(50%) + LGG(50%) in parallel); MRS was fermented separately with LP and LGG, inactivated, dried and mixed at 80%:20% (LP(80%) + LGG(20%) in parallel).

[0265] Similarly, metagenics from other Lactobacillus species were tested. Lpl (Lpl(24h)) fermented alone for 24 hours, Lc (Lc(24h)), Lpl(24h), Lc(24h) fermented alone for 24 hours, Lpl(6h) + Lc(24h) produced from a continuous process, Lc(6h) + Lpl(24h) produced from a continuous process, and Lc(50%) + Lpl(50%) produced from a mixture of single fermentations (parallel processes) were tested on Caco-2 cells to evaluate HBD-2 production.

[0266] Caco-2 cells were stimulated for 48 hours with the five metagenic agents described above (at a dose of 11.5 mg / ml). Cells stimulated with culture medium alone were used as a control.

[0267] Biological results of CaCo-2 cells ( Figure 17A and 17BThe results showed that postbiotics obtained from continuous and parallel processes resulted in higher levels of innate immune stimulation than those promoted by single fermentation. Continuous processes ensured more significant HBD-2 production compared to parallel processes.

[0268] As for the testing using other microbial genera, the metagenerics of Bb (Bb(24h)) fermented alone for 24 hours, Ss (Ss(24h)) fermented alone for 24 hours, Sb (Sb(24h)) fermented alone for 24 hours, Bb(24h), Ss(24h), Sb(24h), Bb(6h) + Ss(6h) + Sb(24h) produced by continuous processes, and Bb(34%) + Ss(33%) + Sb(33%) produced by mixing (parallel fermentation) from single fermentation were tested on Caco-2 cells to evaluate the production of HBD-2.

[0269] Caco-2 cells were stimulated for 48 hours with the aforementioned metagenic agent (at a dose of 11.5 mg / ml). Cells stimulated with culture medium alone were used as a control.

[0270] Biological results of CaCo-2 cells ( Figure 18 The results showed that postbiotics obtained by sequential and parallel fermentation resulted in higher levels of innate immune stimulation than those promoted by single fermentation. The sequential process ensured more significant HBD-2 production compared to the parallel process.

[0271] Biological results from CaCo-2 cells demonstrate that the method of the present invention, whether in a parallel fermentation embodiment or a sequential fermentation embodiment using and combining different microbial genera, leads to the production of a metabiotic composition that promotes synergistic effects, thereby providing a higher level of innate immune stimulation than that promoted by a single microorganism. Notably, a difference was observed between sequential and parallel fermentation according to the present invention: the parallel process ensures twice the production of HBD-2 compared to a single microorganism, while sequential fermentation results in three times the production. Therefore, in the sequential fermentation embodiment, the method of the present invention leads to combined metabolism of the two or three involved microorganisms, thereby amplifying the biological reaction and potentially allowing the production of new metabolites, which could promote greater production of functional markers.

[0272] As for the parallel fermentation of LP and LGG, both formulations (LP50% + LGG50% and LP80% + LGG20%) (50:50; 80:20 LP / LGG) showed synergistic effects on HBD-2, with no significant difference.

[0273] Expression of intestinal barrier integrity biomarkers on Caco-2 cells The integrity of the intestinal barrier plays a vital role in protecting against infection and many chronic non-communicable diseases, such as obesity, allergies, autoimmune diseases, cancer, and central nervous system degenerative diseases.

[0274] The effects of metabiotics obtained from single fermentation of LP (LP(24h)) and LGG (LGG(24h)) and metabiotics obtained from a mixture of single fermentations (parallel process) (LP 80% + LGG 20%) on the intestinal barrier system were evaluated and compared with commercial metabiotics. Cells stimulated with cell culture medium alone (without metabiotics) were used as controls.

[0275] As shown in Figure 19, incubation with commercial metabiotics did not establish any effect on the three biomarkers (tight junction proteins, closure proteins and ZO-1, and epithelial mucosa protein MUC5AC); conversely, LP (24h) and LGG (24h) were able to positively modulate all analyzed biomarkers. Similarly, in this case, the metabiotic composition LP 80% + LGG 20% obtained through parallel processes was found to have significantly greater stimulatory effects on all analyzed biomarkers than other metabiotics.

[0276] In summary, the above results demonstrate that, compared to fermentation methods utilizing single or multiple bacteria simultaneously reproducing in the same substrate, the method of the present invention yields metabiotic compositions exhibiting unique biological characteristics. These results can depend on various factors, including the absence of growth competition between bacteria and the absence of inhibition of certain metabolite production by other metabolites. Individual bacteria cannot achieve significant levels of certain biological activities; therefore, these activities can be combined to obtain complementary or synergistic effects.

[0277] The method according to the invention can also achieve bioactivity at significantly lower doses than conventional processes, thereby limiting quantitative use and / or achieving more pronounced effects at the same dose, with undeniable economic and / or functional advantages.

[0278] Example 3: Composition of post-biotic capsules containing L-tryptophan source L-Tryptophan is a protein-forming amino acid and is therefore used in protein biosynthesis. Furthermore, L-Tryptophan is metabolized into compounds such as nicotinic acid mononucleotide, nicotinamide dinucleotide, and serotonin, each with specific biochemical functions that influence physiology. L-Tryptophan is normally ingested through dietary intake of L-Tryptophan-containing proteins. Additionally, L-Tryptophan can also be obtained as an amino acid through dietary supplements.

[0279] Non-protein L-tryptophan metabolism in mammals occurs in mammalian cells and selected microbial cells; gut microbial L-tryptophan metabolism thus contributes to the collection of L-tryptophan metabolites in mammalian feces and circulation (Dodd D. et al.). Nature 2017, 551(7682):648-652). Notably, some L-tryptophan metabolites, including indole-3-lactic acid (ILA) and indole-3-acetic acid (IAA), appear to be produced entirely by gut microbes rather than by host cells (Lamas B. et al., 2017, 551(7682):648-652). Nat. Med. (2016, 22(6):598-605). Consistent with this, fecal levels of IAA and L-kynurenine were significantly reduced in germ-free mice compared to conventional mice. Therefore, gut microbiota composition and L-tryptophan availability are the main determinants of the bioavailability of these compounds.

[0280] The health effects of L-tryptophan metabolites have been inferred through mechanistic studies, animal studies, and human association studies. Kynurenine and IAA are associated with psychological functions such as mood, appetite, and anxiety, presumably through their effects on neuroinflammation and the metabolism of L-tryptophan and Trp to serotonin across the blood-brain barrier (Ogyu K. et al.). Neurosci. Biobehav. Rev. 2018, 90:16-25 and Osadchiy V. et al., PLOS One 2018, 13(8):e0201772). Kynurenine promotes the expansion of RORγt (+)IL-22 (+) ILC3 cells in the intestinal mucosa, thereby stimulating the proliferation of mucus-producing goblet cells, thus maintaining the integrity of the intestinal barrier (Qi H et al., 2018, 13(8):e0201772). Commun. Biol. 2019, 2:171). It has been described that ILA can protect against inflammatory bowel disease by regulating mucosal CD4+ T cell differentiation (Cervantes-Barragan L. et al., 2019, 2:171). Lactobacillus reuteri ( Limosilactobacillus reuteri ) induces gutintraepithelial CD4(+)CD8alphaalpha(+) T cells. Science 2017, 357(6353):806-810). Some L-tryptophan metabolites are agonists of the aryl hydrocarbon receptor (AhR) (Krishnan S. et al., 2017, 357(6353):806-810). Cell Rep.AhR is a transcription factor that regulates the expression of genes involved in xenobiotic metabolism, immunity, and interleukin-22 expression in various organs, including the liver, intestine, lungs, and brain. Therefore, AhR influences various health conditions, such as chronic inflammatory diseases of the intestine (colitis), lungs (e.g., bronchial asthma), and brain (e.g., major depressive disorder). Other L-tryptophan metabolites, such as indole-3-propionic acid, have been reported to bind to pregnane-X receptors, thereby modulating intestinal barrier function (Venkatesh M. et al., 2018, 23(4): 1099-1111). Immunity 2014, 41(2):296-310). Therefore, combining L-tryptophan with the postbiotic composition described herein can provide beneficial metabolism of L-tryptophan in the subjects.

[0281] The post-biotic composition prepared according to the method of the present invention can be prepared together with L-tryptophan for filling into hydroxypropyl methylcellulose (HPMC) capsules (size 00 or other sizes). L-tryptophan can be added in the form of a free amino acid or a modification thereof, or in the form of a dipeptide, or added to a protein. The post-biotic composition is prepared with 50 mg, 250 mg, or 800 mg of L-tryptophan and filled into HPMC capsules. The capsules may further contain amino acids selected from L-ornithine, L-aspartic acid, L-lysine, and L-arginine. Alternatively, the capsules can be formulated using pH-dependent polymers such as cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HPMCP) 50 and 55, copolymers of methacrylic acid and methyl methacrylate (e.g., Eudragit® S 100, Eudragit® L, Eudragit® FS, and Eudragit® P4135 F).

[0282] The capsules may further contain other carbohydrate components selected from arabinoyl xylan, barley fiber, oat fiber, rye fiber, wheat bran fiber, inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS), resistant starch, beta-glucan, glucomannan, galactoglucomannan, guar gum, and xylooligosaccharides.

[0283] The capsules may further contain one or more plant extracts selected from ginger, cinnamon, grapefruit, parsley, turmeric, olive fruit, ginseng, horseradish, garlic, broccoli, spirulina, pomegranate, cauliflower, kale, coriander, green tea, onion, and milk thistle.

[0284] The capsules may further contain astaxanthin, charcoal, chitosan, glutathione, monacolin K, phytosterols, phytosterols, sulforaphane, collagen, hyaluronic acid and / or phosphatidylcholine.

[0285] The capsule may contain other vitamins selected from biotin, vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B9 (folic acid), vitamin C (ascorbic acid), vitamin D (calciferol), vitamin E (tocopherol and tocotrienols), and vitamin K (quinones), or minerals selected from sulfur, iron, chlorine, calcium, chromium, cobalt, copper, magnesium, manganese, molybdenum, iodine, selenium, and zinc.

[0286] Example 4: Effects of Lactobacillus paracasei NPB01 cell wall polysaccharides on cell viability, immune response, and epithelial barrier of human intestinal epithelial cells. Lactobacillus paracasei is a Gram-positive, homofermentative lactic acid bacterium commonly used in fermented foods and postbiotic products. Kiousi DE (E. et al., Front Microbiol. 2022;13:922689). *Lactobacillus paracasei* strains have been isolated from various environments, including dairy products, plants, and the human gastrointestinal tract. Consuming foods fermented with *Lactobacillus paracasei* strains may confer beneficial health effects on humans. Bengoa AA (E. et al., Foods. 2021; 10(10):2239). This study investigated the structure of cell wall polysaccharides from a novel *Lactobacillus paracasei* strain isolated from the human gut microbiota (named *Lactobacillus paracasei* NPB01) and tested their effects on cell viability, immune response, and epithelial barrier of human intestinal epithelial cells. The results of this study further reveal the safety characteristics of metabiotic compositions containing *Lactobacillus paracasei* NPB01.

[0287] method Bacterial culture *Lactobacillus paracasei* NPB01 (DSM 34367) is a Gram-positive homofermenting bacterium stored at -80°C in MRS broth (Oxoid, Basingstoke, UK) containing 20% ​​glycerol. Before each fermentation, it was revived for 24 hours at 37°C using fresh MRS broth (1:10). At the end of the revival, the bacterial concentration was found to be 10-1. 8 CFU / mL.

[0288] The revived bacteria were then used as an inoculum (1% v / v) for fermenting 1 L of fresh MRS broth. Fermentation was carried out at 37°C for 24 hours, and a concentration of 10 was observed at the end of the process. 8 CFU / mL. Metabiotics were obtained by inactivating bacteria at 85°C for 20 seconds, and the product was stored as a lyophilized composition.

[0289] Bacterial cell wall polysaccharide extraction Polysaccharides derived from *Lactobacillus paracasei* metageners were extracted as previously described (Vinogradov, EV et al., *Carbohydr. Res.* 2015, 413: 93-99). The resulting precipitate was suspended in 20 mL of water and extracted with 50% n-butanol while stirring at room temperature for 1 hour. The organic phase was discarded, and the aqueous phase was deproteinized by adding 5% TCA while stirring at 4°C for 48 hours. The suspension was then centrifuged (9000 rpm, 15 min, 4°C), and 50% HF aqueous solution was added to the precipitate while stirring at 4°C for 48 hours. Finally, the suspension was centrifuged (7500 rpm, 30 min, 4°C), and the supernatant was freeze-dried to obtain a final yield of 0.79 mg / g stem cells.

[0290] In the second step, bacterial polysaccharides were extracted. The polysaccharides (starting with 2 g of precipitate) were suspended in 30 mL of water and autoclaved (120°C, cyclic sterilization for 20 minutes). The supernatant obtained after centrifugation (8000 rpm x 10 minutes, 4°C) was filtered through a 0.45 µm filter membrane and then freeze-dried. The crude product was obtained, with a final yield of 333 mg / g bacterial cells.

[0291] Bacterial polysaccharide purification The crude metagenic product was purified using a combination of chromatographic techniques. First, size exclusion chromatography was performed on a Sephacryl HR-300 resin (1.5 x 45 cm, Cytiva 17-0599-01), with column packing and elution with 50 mM ammonium bicarbonate, and the eluent was monitored using a refractive index detector. Next, anion exchange chromatography was performed on a Q-sepharose fast-flow (1 x 1.5 ml, Cytiva 17-0510-01) to separate the mixture of teichoic acid and capsular polysaccharide 1 (CPS-1), generally yielding pure polysaccharides free of nucleic acid contaminants. Elution was performed using a stepwise NaCl gradient (10, 100, 200, 400, 700, and 1000 mM); teichoic acid was eluted using a NaCl gradient from 200 mM to 1 M. Each solution was concentrated by freeze-drying and desalted on a Biogel P10 (1.5 x 10.5 ml), allowed in water, and the eluent was monitored with a refractive index detector.

[0292] GLC-MS analysis By converting polysaccharides derived from Lactobacillus paracasei metagener to acetylated polysaccharides... O 2-methyl glycosides were subjected to compositional analysis. Subsequently, the acetylated 2-methyl glycosides obtained by the reaction of the monosaccharide with an optically pure alcohol (R-(-)-octanol) were analyzed. O-Octanyl derivatives were used to determine the absolute configuration of the monosaccharides. The derivatives were identified by comparing the retention times of the peaks in the samples with those of internally constructed standards (acetylated monosaccharides functionalized with pure octanol and its racemic mixture) using GLC-MS analysis. The sugar linkage pattern of capsular polysaccharide 2 (CPS-2) was determined by partial methylation and acetylated aldose methods (DeCastro, C. et al., Methods Enzymol. 2010; 480:89-115). Finally, all these derivatives were analyzed using GLC-MS on an Agilent Technologies 7820A (Santa Clara, CA, USA) equipped with a 5977B mass spectrometer, a 7693A autosampler, and an HP-5ms capillary column (Agilent, 30 m × 0.25 mm inner diameter, 0.25 mm film thickness, flow rate 1.2 ml / min, He as carrier gas). Electron collision mass spectra were recorded using an ionization energy of 70 eV. The temperature program used was as follows: hold at 150°C for 3 minutes, increase the temperature from 150°C to 280°C at a rate of 3°C / minute, and hold at 300°C for 5 minutes.

[0293] acetylation O α-Methylglycoside The polysaccharide sample (0.2 mg) was treated with 1 ml HCl / MeOH (1.25 M, 80 °C, 16 h), followed by acetylation in 100 ml pyridine with 50 ml acetic anhydride (80 °C, 30 min). The phosphodiester bonds of teichoic acid were first hydrolyzed with 50 ml 50% HF at 25 °C for 5 h.

[0294] Acetylated octyl glycoside (OAG) Polysaccharides derived from *Lactobacillus paracasei* metagenic precursors were treated overnight at 60°C with 100 mL of R-(-)-octanol and 15 mL of acetyl chloride, and excess octanol was removed under an air stream. Finally, octylglycosides were acetylated at 80°C by adding 100 mL of pyridine and 50 mL of acetic anhydride for 30 minutes.

[0295] Partially methylated alditol acetate (AAPM) The polysaccharide sample (0.5 mg) was dissolved in anhydrous DMSO (1 mL), and then powdered NaOH was added with a stirring spatula. The suspension was stirred at room temperature for 4 hours, with frequent sonication to promote the dissolution of NaOH in DMSO. Subsequently, the deprotonated alcohol group was methylated with 200 mL of CH3I and stirred at 25°C for 17 hours. The solution was extracted five times with H2O / CHCl3 (3:1 v / v), centrifuged at 2500 rpm for 3 minutes each time, and the upper layer was replaced with H2O. The sample was then hydrolyzed at 120°C with 200 mL of 2 M trifluoroacetic acid for 2 hours. Afterwards, the anomeric functional groups were labeled and reduced by adding a small amount of NaBD4 and 200 mL of EtOH. The sample was kept sealed at 25°C for 1 hour. Excess NaBD4 was destroyed with a few drops of ice-cold AcOH, followed by neutralization with MeOH. Finally, the sample was acetylated with acetic anhydride (50 ml) and pyridine (100 ml).

[0296] NMR spectroscopy Recordings were made using Bruker 600 MHz and Bruker 1.2 GHz spectrometers equipped with a z-axis gradient reverse cryoprobe. 1 ¹H and 2D NMR spectra. The sample was dissolved in 550 mL of D₂O, and the spectra were calibrated using internal standard acetone (d). H = 2.225ppm; d C = 31.45 ppm). Totally correlated spectroscopy (TOCSY) and nuclear Overhauser enhanced spectroscopy (NOESY) experiments were performed using a 2048 × 512 point dataset (t1 × t2). 13 In the C domain, using a dataset of 2048 × 512 points, single-quantum coherence with proton decoupling is achieved. 1 Heteronuclear single quantum coherence (HSQC) and heteronuclear multibond correlation (HMBC) experiments were conducted in H-detection mode. HSQC was performed using sensitivity enhancement and phase-sensitive modes via echo / reverse echo gradient selection, with multiplicity editing performed in the selection step. HMBC was optimized for long-range coupling constants, using a low-pass J filter to suppress single-bond correlations, gradient pulse selection, and a 60 ms delay to evolve long-range correlations. The HMBC spectrum was optimized for coupling constants of 6–15 Hz. For transformations, the data matrices from both homonuclear and heteronuclear experiments were extended to 4096 × 2048 points, and transformations were performed using qsine or sine window functions (Speciale I. et al., Carbohydr. Polym. 2022; 277:118885).

[0297] Spectra of the TA+CPS-1 mixture were acquired at 298 K. TOCSY and NOESY mixing times were set to 100 ms and 200 ms, respectively, and recorded along with the COSY experiment, with 24 scans. 100 scans were recorded for HMBC and HSQC-TOCSY, and 60 scans for the HSQC experiment. Spectra of the pure CPS-1 isolate were recorded at 315 K, with 32 scans for the TOCSY experiment and 70 scans for the HSQC experiment. The TOCSY mixing time was set to 100 ms. CPS-2 spectra were recorded at 293 K. TOCSY and NOESY mixing times were set to 100 ms and 200 ms, respectively, with 16 scans recorded for each spectrum and the COSY spectrum. HSQC, HMBC, and HSQC-TOCSY spectra were acquired with 128, 512, and 192 scans, respectively. All spectra were converted and analyzed using Bruker Topspin NMR data analysis software.

[0298] Human intestinal epithelial cell line and cell assay Caco-2 cells (ATCC, Middlesex, United Kingdom; accession number HTB-37) were grown in Duchenne modified Eagle medium supplemented with 10% FBS, 1% non-essential amino acids, 1% sodium pyruvate, and 1% penicillin / streptomycin (Thermo Fisher Scientific), containing a high glucose concentration (4.5 g / L) and L-glutamine. Cells were incubated at 37°C in a humidified environment containing 5% carbon dioxide. The medium was changed every 2 days.

[0299] Fifteen days after confluence, Caco-2 cells were stimulated for 48 hours with four different concentrations (1, 10, 100, and 1000 µg / ml) of precipitated Lactobacillus paracasei NPB-01 post-generant, or with pure CPS-1, CPS-2, and TA. Cells exposed only to the culture medium were used as negative controls (NT). Cells were then harvested and stored at -20°C for subsequent use.

[0300] The toxicity of *Lactobacillus paracasei* NPB-01 post-biotic, CPS-1, CPS-2, and TA cells was examined by trypan blue exclusion assay, as previously reported (W. Strober. Curr. Protoc. Immunol. 111 (1)(2015), p. A3-B24), by counting the number of live Caco-2 cells treated and untreated at known concentrations (1 to 1000 mg / ml). Aliquots of cell suspension were gently mixed with an equal volume of 0.2% trypan blue dye (0.5% dissolved in PBS) and incubated at room temperature for 3 minutes. Live and dead cells were counted using a hemocytometer under an inverted microscope.

[0301] The antimicrobial peptide LL-37, an innate immune compound, exerts antimicrobial effects against fungal, bacterial, and viral pathogens (Ridvard KE and Overhage J. Antibiotics. 2021; 10(6):650) and was selected as a biomarker for immune responses. Closure proteins, key peptides involved in the regulation of tight junction networks (Brunner J et al., Adv. Drug Deliv. Rev. 2021: 171:266-288), were selected as biomarkers for epithelial barrier regulation.

[0302] The expression of LL-37 and the closing protein was determined by quantitative real-time PCR. In short, total RNA was extracted from stimulated Caco-2 cells using TRIzol reagent (GibcoBRL, Paisley, UK). RNA samples were analyzed using a NanoDrop 2000c spectrophotometer (Thermo Scientific), and purity was verified by A260 / 280 and A260 / 230 absorbance ratios. RNA was reverse transcribed into cDNA using a high-capacity RNA-to-cDNA™ kit (Life Technologies, Waltham, MA, USA) according to the manufacturer's instructions. Complementary DNA (cDNA) was stored at -80°C until use. Quantitative real-time PCR (qRT-PCR) analysis was performed using Taqman GeneExpression Master Mix (Applied Biosystems, Vilnius, Lithuania) to evaluate the expression of LL-37 (Hs00189038_m1) and the closing protein (Hs05465837_g1). TaqMan probes used for these genes were inventoried and tested at the Applied Biosystems manufacturing facility (QC). Data were analyzed using a comparative threshold cycling method. mRNA expression levels were normalized using the glucuronidase β (GUS-B) gene (TaqMan probe: Hs00939627_m1).

[0303] Statistical analysis The Kolmogorov-Smirnov test was used to determine whether the variable was normally distributed. For normally distributed continuous variables, descriptive statistics were reported as mean and standard deviation. Differences between groups were compared using a one-way ANOVA test. Differences between continuous variables were compared using an independent samples t-test. A difference was considered statistically significant when p < 0.05. All data were collected into a dedicated database and analyzed using GraphPad Prism 7.

[0304] result Isolation and identification of polysaccharides from Lactobacillus paracasei Polysaccharides were isolated from the metagenics produced by fermentation of *Lactobacillus paracasei* MRS. The precipitate was suspended in water and extracted by heat treatment (120°C, 20 min). After centrifugation, the crude metagenic product remained in solution.

[0305] Subsequently, the crude polysaccharide underwent multiple purification steps using size exclusion chromatography and anion exchange chromatography. Four main fractions were separated using size exclusion chromatography on a Sephacryl HR-300. Figure 20A), and through 1 1H NMR analysis of its spectrum ( Figure 20B Two important polysaccharide fractions, RBC / 16 / A0 and RBC / 16 / A, were identified through this purification. RBC / 16 / A0 eluted from a flat line in the size exclusion chromatography chromatogram and represented Lactobacillus paracasei CPS (CPS-1), which had a higher molecular weight (MW) than the RBC / 16 / A fraction (CPS-2).

[0306] Both RBC / 16 / A0 and RBC / 16 / A fractions were further purified by anion exchange chromatography on a Q-sepharose fast flow surface, eluted with an ascending NaCl gradient, and then desalted on Biogel P-10 resin and eluted in pure water. Finally, two different polymers, teichoic acid (TA) and capsular polysaccharide (CPS-1), were isolated from fraction RBC / 16 / A0, while a second capsular polysaccharide (CPS-2) was obtained from fraction RBC / 16 / A. Specifically, teichoic acid was purified in a final yield of 0.24 mg / g. 干细胞 It was eluted, while CPS-1 and CPS-2 were eluted at a final yield of 8 mg / g. 干细胞 and 56 mg / g 干细胞 It was washed away. Therefore, CPS-2 was the most abundant of the three glycans.

[0307] By analyzing a complete set of isonuclear and heteronuclear 2D NMR experiments (COSY, TOCSY, NOESY, ... 1 H- 13 C HSQC, 1 H- 13 The structure of *Lactobacillus paracasei* glycans was determined by CHMBC. The spin system of each residue was specified using COSY and TOCSY spectroscopy, while using... 1 H, 13 C HSQC spectroscopy specifies the carbon chemical shift value.

[0308] For CPS-1 and TA, the complete 2D NMR spectrum was acquired in their mixed state. Purification was then performed by anion exchange chromatography, and the mixture was... 1 H, 13 C HSQC (Figure 21A) and proton ( Figure 22B ) Spectrum and spectrum of pure polysaccharides ( Figure 22A and 22C The comparison confirmed their purity.

[0309] For CPS-2, combined 13 C HSQC and 1 H NMR spectroscopy ( Figure 23 The information from the sample allowed for the differentiation and identification of 15 major anomeric signals between 5.3 and 4.6 ppm, a crowded cyclic proton region (4.5–3.2 ppm), a group of acetyl signals between 2.1 and 1.9 ppm, and several methyl signals at approximately 1.3 ppm, all attributed to different rhamnose units. Anomeric signals are labeled with capital letters. Furthermore, CPS-2 of *Lactobacillus paracasei* NPB01 is a small-sized polymer that was actually eluted as the most retained by size exclusion chromatography. Figure 20A Finally, methylation analysis further confirmed the CPS-2 monosaccharide linkage (De Castro, C. et al., Methods Enzymol. 2010; 480:89-115), which identified the presence of terminal rhamnose, 2-substituted rhamnose, 3-substituted rhamnose and 2,3-substituted rhamnose, terminal glucose, 6-substituted glucosamine and 3,6-substituted glucosamine, and 3-substituted galactosamine. Other unlabeled (and smaller) peaks were impurities.

[0310] Cell viability, immune response regulation, and epithelial barrier in human intestinal epithelial cells To determine whether *Lactobacillus paracasei* NPB01 postbiotics, CPS-1, CPS-2, or TA were toxic to human intestinal epithelial cells, cell viability was assessed using a trypan blue exclusion assay for 2 hours, during which cells were incubated with CPS-1, CPS-2, or TA at concentrations up to 1000 μg / ml for 48 hours. None of these compounds were found to be cytotoxic to Caco-2 cells. Since no cytotoxic effects were recorded at the highest dose (1000 μg / ml), this concentration was considered safe for studying the biological effects of these compounds in human intestinal epithelial cells.

[0311] Lactobacillus paracasei NPB01 postbiotic stimulated the expression of LL-37 and closure protein in human intestinal epithelial cells. Similarly, purified CPS-2 upregulated the expression of both biomarkers in human cells. Purified CPS-1 and TA both only regulated closure protein expression. Dose-response experiments revealed that 1 µg / ml Lactobacillus paracasei NPB01 postbiotic and 10 µg / ml CPS-2 were effective doses for regulating LL-37 expression. For regulating closure protein expression, 10 µg / ml Lactobacillus paracasei NPB01 postbiotic, 100 µg / ml CPS-1, 1 µg / ml CPS-2, and 10 µg / ml TA were found to be effective doses.

[0312] Figures 24A-24BThe effects of higher effective doses of *Lactobacillus paracasei* NPB01 on postbiotics, CPS-1, CPS-2, and TA were demonstrated. Stimulation with CPS-2 resulted in stronger effects on LL-37 and closure protein. Stimulation with CPS-1 resulted in stronger expression of closure protein compared to TA stimulation.

[0313] These results indicate that the postbiotic of *Lactobacillus paracasei* NPB01, as well as the polysaccharides TA, CPS-1, and CPS-2 derived from *Lactobacillus paracasei* NPB01, can exert beneficial effects on immune responses and intestinal barrier biomarkers in human intestinal epithelial cells. These effects are similar to those reported for another *Lactobacillus paracasei* strain (i.e., CBAL74) (Paparo L. et al., Benef. Microbes. 2018; 9(1):165-172), suggesting that different *Lactobacillus paracasei* strains can exert similar beneficial effects on human cell function.

[0314] Example 5: Postbiotic Regulation of Human Gut Microbiota The direct interactions between metabiotics and the microbes of the human gut microbiota remain largely unexplored. (Bacillus belye) MV4 and Priestella megaterium MV30 These are two strains of Bacillus belonging to the Firmicutes phylum, recently isolated from healthy gut microbiota (Vittoria M. et al., Microorganisms. 2023; 11(8):1978). These two microbes play a role in protecting the host from infection, oxidative stress, and inflammation. Furthermore, these microbes contribute to the optimal production of short-chain fatty acids in the intestinal lumen. Short-chain fatty acids, particularly butyrate, are responsible for regulating many beneficial effects on human health at both the intestinal and parenteral levels (De Filippis F. et al., NatureComm. 2021; 12(1):1-11). Specifically, at the intestinal level, butyrate can improve ion absorption, cell proliferation, cell differentiation, intestinal barrier function, immune regulation, oxidative stress, intestinal motility, visceral sensation, and rectal compliance; and at the extraintestinal level, butyrate can improve insulin sensitivity, cholesterol synthesis, energy expenditure, ammonia clearance, stimulate β-oxidation of very long chain fatty acids, stimulate peroxisome proliferation, cystic fibrosis transmembrane transport regulator (CFTR) function, neurogenesis, and fetal hemoglobin (HbF) production (Canani, RB et al., World J Gastroenterol 2011; 17(12):1519-1528).

[0315] In this study, two different metabiotic products were evaluated to regulate Bacillus belysinus. MV4and Priestella megaterium MV30 The ability to grow.

[0316] method strains and pre-culture *Priestella megaterium* MV30 and Bacillus vesiculosus MV4 The samples were incubated overnight at 37°C in minimal microbial growth medium M9 (Sigma Aldrich St. Louis, MA, USA). After pre-incubation, absorbance at 600 nm was measured using a spectrophotometer. The samples were then diluted with M9 broth to achieve an absorbance of 0.1 in each well of the multi-well plate, which was the absorbance value selected before stimulation with the postbiotic product.

[0317] Post-natal products Two different epigenetic products were evaluated. The first product (LP6h: LGG24h) was obtained through two sequential fermentations in MRS broth (Sigma-Aldrich). In the first fermentation, *Lactobacillus paracasei* NPB01 (LP) was inoculated into the MRS broth and fermented at 37°C for 6 hours. After heat inactivation, *Lactobacillus rhamnosus* GG (LGG) was inoculated into the same broth and fermented at 37°C for 24 hours. After the second fermentation, heat inactivation and spray drying were performed to obtain the dried epigenetic product (LP6h: LGG24h).

[0318] The second postbiotic product was obtained through two separate fermentation processes: first, MRS broth was fermented with *Lactobacillus paracasei* NPB01, while a second fermentation was carried out with *Lactobacillus rhamnosus* GG. In both cases, fermentation lasted 24 hours at 37°C. At the end of fermentation, heat inactivation and spray drying resulted in two separate dried postbiotic products. These powders were then mixed at a ratio of LP:LGG 80:20.

[0319] Stimulate with post-biotic products result *Priestella megaterium* MV30 and Bacillus vesiculosus MV4 Stimulate with each epigenetic product for 6 hours and read the absorbance at 600 nm. For example... Figures 25A-25B As shown, compared to the control, the presence of each metabiotic product promoted the growth of *Priscilla megaterium*. MV30 The OD600 increased by nearly 300% ( Figure 25A ), prompting Bacillus belesii MV4 The OD600 increased by nearly 150% ( Figure 25B These results indicate that the postbiotic products produced using the methods described herein resulted in a significant increase in two species of the gut microbiota.

[0320] Example 6: Effects of postbiotic products on human intestinal epithelial cells method After-generation products tested The metabiotics tested in this study were produced by the following microorganisms: 1) *Lactobacillus paracasei* NPB01 (LP); 2) *Lactobacillus rhamnosus* GG (LGG); 3) LP:LGG (50:50), obtained by parallel fermentation; 4) LP:LGG (80:20), obtained by parallel fermentation; 5) LP 6h + LGG 24h, obtained by continuous fermentation; and 6) LGG 6h + LP 24h, obtained by continuous fermentation. Each product was derived from MRS fermentation and subsequently inactivated at 85°C for 20 seconds.

[0321] Cell model The effects of the postbiotic product produced using the methods described herein on monolayer human intestinal epithelial Caco-2 cells (American Center for Type Culture Collection, Middlesex, UK; accession number: HTB-37) were tested.

[0322] Cells were grown in Duchenne Eagle Medium (DMEM; Gibco, Berlin, Germany) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% non-essential amino acids (Gibco), 1% sodium pyruvate (Gibco), 1% penicillin / streptomycin (Gibco), and containing high glucose concentration (4.5 g / L) and L-glutamine. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. The medium was changed every 2 days.

[0323] Human intestinal epithelial cell stimulation protocol Caco-2 cells were stimulated 15 days after confluence.

[0324] Cells exposed only to the culture medium were used as a negative control (NT). The supernatant was then harvested and stored at -20°C. Total RNA was extracted from both stimulated and untreated cells using TRIzol reagent (Gibco BRL, Paisley, UK) and stored at -20°C for further use.

[0325] Cell proliferation assay Human intestinal epithelial cell proliferation assay was performed using MTT (bromine salt of 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazol) (Sigma-Aldrich, Milan, Italy).

[0326] Cells (10) 4Cells per well were seeded in 24-well plates (Corning, Inc., New York, NY, USA) with or without different doses (0.115-575 mg / ml) of post-genetic agents and cultured at 37°C in a 5% CO2 incubator for 48 hours. NFM was used as a control.

[0327] Cell viability was monitored by adding 5 mg / ml MTT solution and incubating for 1 hour. The culture medium was then removed, and the transformed dye was dissolved in acidic isopropanol (0.04–0.1 N HCl in anhydrous isopropanol). Absorbance was read at 570 nm using an Epoch microplate spectrophotometer (Bioteck, Winooski, VT, USA).

[0328] Quantitative Real-Time PCR Total cellular RNA was extracted from cells using TRIzol reagent (Gibco BRL, Paisley, UK). RNA (1 μg) was reverse transcribed into cDNA using a high-capacity RNA-to-cDNA™ kit (Life Technologies, Waltham, MA, USA) according to the manufacturer's instructions at 37°C. Complementary DNA (cDNA) was stored at -20°C until use. Quantitative real-time PCR (qRT-PCR) was performed to determine gene expression. After hot start, the amplification protocol was as follows: 40 cycles were performed on a Light Cycler 7900HT (Applied Biosystems, Grand Island, NY, USA), each cycle consisting of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute. Gene expression was quantified using the comparative Ct method and normalized to Ct values ​​of glucuronidase (GUS) messengers.

[0329] ELISA assessment of innate immune peptides and antimicrobial peptide LL-37 The concentration of LL-37 in cell supernatant was measured using a specific human ELISA assay kit (Elabscience Biotechnology Inc. Wuhan, Hubei), with a detection limit of 1.56 ng / ml. ELISA was performed according to the manufacturer's recommendations.

[0330] Statistical analysis The Kolmogorov-Smirnov test was used to determine whether the variables were normally distributed. Unpaired t-tests were used to analyze the data. All statistical tests were two-tailed, with p < 0.05. All data were collected into a dedicated database and analyzed by a statistician using GraphPad Prism 9.3.0 (La Jolla, CA USA).

[0331] result The direct interaction between metabiotics and human intestinal epithelial cells has produced a variety of beneficial effects on human health, as evidenced by the observed effects on human intestinal epithelial cells. The effects of the metabiotic composition were found to be dose-dependent. Under all experimental conditions, the optimal effective dose of metabiotics was 11.5 mg / ml.

[0332] Figures 26A-26B The study describes the effects of direct interactions between metagenes and human intestinal epithelial cells on cell growth and differentiation (measured by results of lactase expression).

[0333] Figures 27A-27B The study reported its effect on the permeability of human intestinal epithelial cell monolayers. Upregulation of tight junction (TJ) proteins, namely closure protein and atresia 1 (ZO-1), observed after postbiotic stimulation confirmed a positive regulation of intestinal permeability. No effect was observed in unstimulated cells (NT).

[0334] Postbiotics can also upregulate MUC2 expression ( Figure 28 ).

[0335] like Figure 29 As shown, metabiotics significantly increase the production of the antimicrobial peptide LL-37 in human intestinal epithelial cells.

[0336] Example 7: Further effects of postbiotic products on human intestinal epithelial cells Basic principles The following four microorganisms were combined and propagated using continuous fermentation: *Lactobacillus paracasei* NPB-01 (LP), *Bifidobacterium animalis* subsp. lactis (Bal), Akkermansia myxophilus (Akk), and Escherichia coli Nissle 1917 (Ecn) are members of the following four major phyla of the gut microbiota: Firmicutes, Actinobacteria, Verruciformis, and Proteobacteria.

[0337] Materials and methods Material LP is provided by Science Power Srl; Bal is isolated from the commercial food supplement OptiBac Bifido & Fibre; Akk is isolated from the feces of healthy children; and Ecn is isolated from the commercial food supplement EcN Integratore Alimentare.

[0338] Single fermentation conditions Culture medium and fermentation Each microorganism underwent a single 24-hour fermentation: LP and Bal were grown in MRS broth for 24 hours and then inactivated by mild heat treatment; Akk was grown in BHI broth supplemented with porcine mucoprotein for 24 hours and then inactivated by mild heat treatment; Ecn was grown in LB broth for 24 hours and then inactivated by mild heat treatment.

[0339] Bacterial growth Bacterial growth was measured after a single fermentation, expressed as CFU / mL.

[0340] dry The obtained single-strain postbiotic composition was dried by spray drying for subsequent biological assays.

[0341] Continuous fermentation conditions culture medium To promote the suitable growth of all microorganisms, the fermentation medium contains: 34% MRS, 33% BHI supplemented with porcine mucoprotein, and 33% LB broth.

[0342] Fermentation First, LP was fermented in the fermentation medium for 6 hours, followed by a mild heat treatment to inactivate the fermentation broth. Then, Bal was added to the fermentation broth and fermented for 6 hours, followed by a mild heat treatment to inactivate the fermentation broth. Next, Akk was added to the fermentation broth and fermented for 12 hours, followed by a mild heat treatment to inactivate the fermentation broth. Finally, Ecn was added to the fermentation broth and fermented for 12 hours, followed by a mild heat treatment to inactivate the fermentation broth.

[0343] Bacterial growth After each consecutive fermentation step, the bacterial growth was measured in CFU / mL.

[0344] dry The obtained multi-strain postbiotic composition was dried by spray drying for subsequent biological assays.

[0345] Biological evaluationAs described in Example 4 above, Caco-2 cells were cultured and then stimulated with a metagenic composition obtained from the following routes: (i) single fermentation (LP, Bal, Akk, and Ecn) and (ii) continuous fermentation (LP+Bal+Akk+Ecn). The expression of ZO-1, closure protein, and MUC-5 was measured.

[0346] result After a single fermentation, the bacterial growth of LP reached 5 × 10⁻⁶. 8 CFU / mL; After a single fermentation, the bacterial growth of Bal reached 7 × 10⁻⁶. 8 CFU / mL; After a single fermentation, the bacterial growth of Akk reached 3.1 × 10⁻⁶. 8 CFU / mL; and the bacterial growth of Ecn after a single fermentation reached 1.2 × 10⁻⁶. 9 CFU / mL.

[0347] For continuous fermentation processes ( Figure 30 ): LP showed a 2 log increase in microbial growth (Δlog), reaching a bacterial concentration of 4.8 x 10⁻⁶ after the first 6 hours of fermentation. 8 CFU / mL; Bal showed a growth increase of (Δlog)2 log, reaching a bacterial load of 2.9 x 10⁻⁶ after 6 hours of fermentation. 8 CFU / mL; Akk showed a growth increase (Δlog) of 2 log, reaching a bacterial concentration of 4.4 x 10⁻⁶ after the subsequent 12 hours of fermentation. 8 CFU / mL; and Ecn showed a growth increase (Δlog) of 1 log, reaching a bacterial load of 7.0 x 10⁻⁶ after the final 12 hours of fermentation. 7 CFU / mL.

[0348] For biological evaluation ( Figures 31A-31C The LP single-strain postbiotic composition showed the highest increase in closure protein, ZO-1, and MUC5AC; however, the multi-strain postbiotic composition (LP+Bal+Akk+Ecn) showed a significantly greater stimulating effect than any single-strain (LP, Bal, Akk, or Ecn) postbiotic composition.

[0349] It should be understood that the Detailed Description section, rather than the Summary and Abstract section, is intended to interpret the claims. The Summary and Abstract section may set forth one or more, but not all, exemplary embodiments of this disclosure as conceived by the inventors, and is therefore not intended to limit this disclosure and the appended claims in any way.

[0350] The foregoing description of specific embodiments so fully reveals the general nature of this disclosure that, without departing from the general conception of this disclosure, others can readily modify and / or adapt various applications of such specific embodiments by applying knowledge in the art, without much experimentation. Therefore, based on the teachings and guidance presented herein, such modifications and adaptations are intended to be within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the phrases or terms used herein are for descriptive rather than limiting purposes, and that the terminology or phrases in this specification should be interpreted by those skilled in the art based on the teachings and guidance.

[0351] The breadth and scope of this disclosure should not be limited by any of the above exemplary embodiments, but should be defined only by the appended claims and their equivalents.

[0352] All references cited throughout this application (including bibliographic references, U.S. or foreign patents or patent applications, and websites) are expressly incorporated herein by reference as if their entirety were included for any purpose. In the event of any inconsistency, the material disclosed herein shall prevail.

[0353] While various specific aspects have been shown and described, the foregoing description is not restrictive. It should be understood that various changes can be made without departing from the spirit and scope of the invention. Many changes will become apparent to those skilled in the art upon reading this description.

Claims

1. A method for preparing a postbiotic composition, comprising the following steps: (i) Inoculating multiple fermenting microorganisms into multiple culture media, wherein different fermenting microorganisms are inoculated into each culture medium; (ii) Fermenting the inoculated multiple culture media under suitable fermentation conditions to obtain multiple fermentation products, each containing different fermentation microorganisms; (iii) Inactivate the fermentation microorganisms in the multiple fermentation products to obtain multiple inactivated fermentation products; as well as (iv) Mix the various inactivated fermentation products to obtain a metabiotic composition.

2. A method for preparing a postbiotic composition, comprising the following steps: (i) Inoculate the culture medium with the primary fermentation microorganism; (ii) Fermenting the culture medium under conditions suitable for the fermentation of the first fermenting microorganism, thereby obtaining a first fermentation product containing the first fermenting microorganism; (iii) Inactivate the first fermentation microorganism in the first fermentation product to obtain the first fermentation substrate; (iv) Inoculate the first fermentation substrate with the second fermentation microorganism; (v) Ferment the first fermentation substrate under conditions suitable for the fermentation of the second fermentation microorganism, thereby obtaining a second fermentation product containing the second fermentation microorganism; (vi) Inactivate the second fermentation microorganism in the second fermentation product to obtain a second fermentation substrate, thereby obtaining a post-biotic composition; as well as Optionally, steps (iv) to (vi) may be repeated once or multiple times using additional fermenting microorganisms.

3. The method of claim 2, wherein each fermenting microorganism is a species different from any other fermenting microorganism.

4. The method according to any one of claims 1 to 3, wherein the fermenting microorganism is selected from the group consisting of bacterial microorganisms and yeast microorganisms.

5. The method according to claim 4, wherein the bacterial microorganism is selected from the group consisting of: species of the genus *Lactobacillus*, species of the genus *Lactococcus*, species of the genus *Bifidobacterium*, species of the genus *Cytobacter*, species of the genus *Streptococcus*, species of the genus *Ackermania*, and species of the genus *Escherichia*.

6. The method according to claim 4, wherein the yeast microorganism is selected from species of the genus *Saccharomyces*.

7. The method according to any one of claims 1-4, wherein the fermenting microorganism is selected from the group consisting of: Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium longum, Streptococcus salivarius, Akkermansia myxotroph, Escherichia coli, and Saccharomyces boulardii.

8. The method according to claim 7, wherein the *Lactobacillus paracasei* is *Lactobacillus paracasei* NPB01.

9. The method according to claim 7, wherein *Bifidobacterium animalis* is a subspecies of *Bifidobacterium animalis*. lactis .

10. The method of claim 7, wherein the Escherichia coli is Escherichia coli Nissle 1917.

11. The method according to any one of claims 1 to 10, wherein one or more additional components are added to the inactivated fermentation product prior to inoculation with the fermenting microorganism.

12. The method according to any one of claims 1 to 11, further comprising drying the inactivated fermentation product, wherein the dried inactivated fermentation product is optionally rehydrated in water prior to subsequent inoculation with fermenting microorganisms.

13. The method according to any one of claims 1 to 12, further comprising the step of drying the post-genetic composition.

14. The method according to any one of claims 1 to 13, wherein the culture medium comprises a culture medium selected from the group consisting of: MRS medium, brain heart infusion (BHI) broth, Luria-Bertani (LB) broth, plant-derived culture medium, functional culture medium containing plant extracts having antioxidant, antiviral and / or antibacterial activity, naturally derived culture medium, and any combination thereof.

15. The method according to any one of claims 1 to 14, wherein the fermentation step is carried out at a temperature of about 25°C to about 45°C.

16. The method according to any one of claims 1 to 15, wherein the inactivation of the fermenting microorganism comprises a procedure selected from the group consisting of: heat inactivation, preferably at a temperature of 50°C to 100°C for 5 to 120 seconds; chemical treatment; gamma ray or ultraviolet irradiation; high pressure; ultrasonic treatment; and any combination thereof.

17. The method of claim 16, wherein the thermal inactivation occurs at a temperature of about 50°C to about 100°C for about 5 to about 120 seconds.

18. A metabiotic composition obtained by the method according to any one of claims 1 to 17, further comprising lactic acid at a concentration of about 1 g / L to about 30 g / L based on the total volume of the composition and / or inactivated fermenting microorganisms at a concentration of about 0.00015 g / L to about 150 g / L based on the total volume of the composition.

19. The metabiotic composition according to claim 18, wherein the amount of inactivated fermenting microorganisms comprises about 10 5 cells / ml to approximately 10 11 Cells / ml 20. A metabiotic composition obtained by the method according to any one of claims 1 to 17, further comprising L-tryptophan or a dipeptide containing L-tryptophan.

21. The metagenic composition of claim 20, wherein the composition comprises L-tryptophan at a concentration of at least 0.01% w / w.

22. The metagenic composition according to claim 20 or 21, wherein the composition comprises L-tryptophan at a concentration of at least 0.10% w / w.

23. The epigenetic composition according to any one of claims 20 to 22, wherein the L-tryptophan is present in an amount of at least 10 mg, at least 50 mg, or at least 100 mg.

24. A metabiotic composition obtained by the method according to any one of claims 1 to 17, wherein at least one of the fermenting microorganisms used to obtain the metabiotic composition is *Lactobacillus paracasei* NPB01.

25. An epigenetic composition according to any one of claims 18 to 24, for the prevention or therapeutic treatment of a disease in a subject in need, said disease being selected from the group consisting of: infectious and inflammatory diseases, immune-mediated diseases, cancerous diseases, skin diseases, gastrointestinal diseases, genitourinary diseases, neurological diseases, neuropsychiatric diseases, skeletal diseases, muscle diseases, malnutrition, metabolic diseases, and any combination thereof.

26. A metabiotic composition according to any one of claims 18 to 24, used to improve intestinal barrier function in subjects of need by increasing the expression of tight junction proteins and / or mucins, and / or increasing intestinal epithelial cell growth and differentiation.

27. The use according to claim 26, wherein the tight junction protein comprises a closure protein and / or ZO-1.

28. The use according to claim 26, wherein the mucin comprises MUC5AC.

29. The use according to claim 27 or 28, wherein, compared with untreated subjects, the expression of closure protein, ZO-1 and / or MUC5AC increased by at least 2, 3, 4, 5 or more after treatment with the postbiotic composition.

30. The use according to any one of claims 27 to 29, wherein, after treatment with the postbiotic composition, the expression of closure protein, ZO-1 and / or MUC5AC is increased by at least 25%, 50%, 75% or more compared with treatment with a composition comprising a single live microorganism.

31. A postbiotic composition according to any one of claims 18 to 24, for increasing the expression of β-defensin-2 (HBD-2) in a subject in need.

32. The use according to claim 31, wherein, compared with untreated subjects, HBD-2 expression increased by at least 2, 3, 4, 5, 7, 8 or more times after treatment with the post-biotic composition.

33. The use according to claim 31 or 32, wherein HBD-2 expression is at least 2, 3, 4 or more times after treatment with the postbiotic composition compared to treatment with a composition comprising a single microorganism.

34. A postbiotic composition according to any one of claims 18 to 24, for increasing the expression of the antimicrobial peptide LL-37 in a subject in need.

35. The use according to claim 34, wherein, compared with untreated subjects, the expression of the antimicrobial peptide LL-37 increased by at least about 2-fold, about 3-fold, or more after treatment with the postbiotic composition.

36. Use of the epigenetic composition according to any one of claims 18 to 24 in a food, beverage, pharmaceutical, nutritional supplement, cosmetic, or packaging composition, wherein the composition further comprises at least one pharmaceutically acceptable medium, excipient, and / or diluent.

37. Use of the postbiotic composition according to claim 36 for improving intestinal barrier function in mammals.

38. Use of the postbiotic composition according to claim 36 for improving the innate immune response of mammals against infection.

39. Use of the postbiotic composition according to claim 36 for inducing a tolerance-inducing immune response in mammals.

40. Use of the post-biotic composition according to claim 36 for protecting the skin of mammals from infection.

41. Use of the postbiotic composition according to any one of claims 18 to 24 for increasing the total amount of one or more metabolites of L-tryptophan in mammals.

42. The use according to claim 41, wherein the one or more metabolites of L-tryptophan include indole-3-acetic acid, indole-3-lactic acid and / or L-kynurenine.

43. Use of the postbiotic composition according to any one of claims 18 to 24 for increasing the total amount of L-tryptophan in mammals.

44. Use of the postbiotic composition according to any one of claims 18 to 24 for increasing the ratio of L-tryptophan to large neutral amino acids in mammalian plasma.

45. Use of the post-biotic composition according to any one of claims 18 to 24 for increasing the biosynthesis of serotonin and / or melatonin in mammals.

46. ​​Use of the postbiotic composition according to any one of claims 18 to 24 for promoting healthy aging in mammals.

47. Use of the postbiotic composition according to any one of claims 18 to 24 for correcting drug-induced nutritional depletion in mammals.

48. A method for preventing or treating a disease selected from the group consisting of: infectious and inflammatory diseases, immune-mediated diseases, cancerous diseases, skin diseases, gastrointestinal diseases, genitourinary diseases, neurological diseases, neuropsychiatric diseases, skeletal diseases, muscle diseases, malnutrition, metabolic diseases, and any combination thereof, wherein the method comprises administering the postbiotic composition according to any one of claims 18 to 24 to a subject in need.

49. A method for improving intestinal barrier function in a subject in need by increasing the expression of tight junction proteins and / or mucins, and / or increasing intestinal epithelial cell growth and differentiation, wherein the method comprises administering the subject a metabiotic composition according to any one of claims 18 to 24.

50. The method according to claim 49, wherein the tight junction protein comprises a closure protein and / or ZO-1.

51. The method of claim 49, wherein the mucin comprises MUC5AC.

52. The method according to claim 50 or 51, wherein, compared with untreated subjects, the expression of closure protein, ZO-1 and / or MUC5AC increased by at least 2, 3, 4, 5 or more after treatment with the postbiotic composition.

53. The method according to any one of claims 50 to 52, wherein, compared with treatment with a composition comprising a single microorganism, the expression of closure protein, ZO-1 and / or MUC5AC is increased by at least 25%, 50%, 75% or more after treatment with the postbiotic composition.

54. A method for increasing the expression of β-defensin-2 (HBD-2) in a subject in need, wherein the method comprises administering the subject the postbiotic composition according to any one of claims 18 to 24.

55. The method of claim 54, wherein, compared with untreated subjects, HBD-2 expression is at least 2, 3, 4, 5, 7, 8 or more times after treatment with the postbiotic composition.

56. The method according to claim 54 or 55, wherein HBD-2 expression increases by at least 2, 3, 4 or more times after treatment with the postbiotic composition compared to treatment with a composition comprising a single microorganism.

57. A method for increasing the total amount of one or more metabolites of L-tryptophan in a subject in need, wherein the method comprises administering the subject an afterbiotic composition according to any one of claims 18 to 24.

58. The method of claim 57, wherein one or more metabolites of L-tryptophan include indole-3-acetic acid, indole-3-lactic acid, and / or L-kynurenine.

59. A method for increasing the total amount of L-tryptophan in a subject in need, wherein the method comprises administering the subject the postbiotic composition according to any one of claims 18 to 24.

60. A method for increasing the ratio of L-tryptophan to large neutral amino acids in the plasma of a subject in need, wherein the method comprises administering the subject an epigenetic composition according to any one of claims 18 to 24.

61. A method for increasing the biosynthesis of serotonin and / or melatonin in a subject in need, wherein the method comprises administering the subject an afterbiotic composition according to any one of claims 18 to 24.

62. A method for promoting healthy aging in a subject in need, wherein the method comprises administering to the subject an epigenetic composition according to any one of claims 18 to 24.

63. A method for correcting drug-induced nutritional depletion in a subject in need, wherein the method comprises administering the subject the postbiotic composition according to any one of claims 18 to 24.

64. An epigenetic composition produced by multimicrobial fermentation, wherein the multimicrobial fermentation comprises the fermentation of two or more bacterial or yeast strains.

65. The epigenetic composition of claim 64, wherein the epigenetic comprises two or more inactivated fermentation products.

66. The metabiotic composition according to claim 64 or 65, wherein the multimicrobial fermentation comprises the method according to claim 1 or 2.

67. An epigenetic composition comprising: a. A first substrate comprising a first inactivated microorganism and a culture medium in which said microorganism was inactivated; and b. One or more additional substrates, each additional substrate containing additional inactivated microorganisms and a culture medium in which said microorganisms are inactivated.

68. The metabiotic composition according to claim 67, wherein the first inactivated microorganism and each of the other inactivated microorganisms are different species.

69. The epigenetic composition according to claim 67 or 68, wherein the epigenetic composition is prepared by the method according to any one of claims 1-17.

70. A kit comprising the post-genetic composition according to any one of claims 18-26 or 64-69.

71. A kit comprising a multimicrobial fermentation substrate and an inoculum containing two or more inactivated microorganisms.

72. The kit according to claim 70 or 71, wherein the post-biotic composition is prepared by the method according to any one of claims 1-17.

73. The kit according to any one of claims 70-72, further comprising documentation containing usage steps and conditions.

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