Therapeutic Microbubbles of Probiotics

Abstract

A method of providing therapeutic microbubbles from probiotics includes exposing the bacteria to an inductive treatment during cultivation to induce the bacteria to produce therapeutic microbubbles. The therapeutic microbubbles can be used to treat, for example, colic, gastrointestinal disorders or diseases in infants or children, gastrointestinal pain disorders, bone loss diseases, and / or periodontal diseases.

Description

Technical Field

[0001] The present invention generally relates to therapeutic microbubbles from probiotics and their uses. Background Art

[0002] The Food and Agricultural Organization of the United Nations has defined probiotics as "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host". Probiotics affect the immune function of the host by modulating the microbiota composition, by metabolic activity, or even by directly interacting with the immune system underlying the intestinal mucosa. More than 60% of immune cells are located in the intestinal mucosa, and sampling information on the microbiota structure and composition is converted into local and systemic effects by circulating immune cells. After the interaction of probiotics, immune mechanisms can be activated, as reflected by the release of immune mediators such as cytokines, the production of antibodies, and the activation of lymphocytes and other immune cells. These activated cells, cytokines, and / or compounds released by probiotics exert immunomodulatory functions at different sites within the body via the blood circulation. Probiotics can also prevent or inhibit the proliferation of pathogens and suppress the production of virulence factors of pathogens.

[0003] Several different bacterial strains are currently used as probiotics, including selected strains of lactic acid-producing bacteria such as the genera Lactobacillus and Bifidobacterium. The effectiveness of probiotics is strain-specific, and each strain may promote host health through different mechanisms.

[0004] There are numerous and diverse probiotic supplements, but the beneficial health effects vary between bacterial strains, and little is still known about the ways to control and regulate specific probiotics or biological effects. Each bacterial strain has different mechanisms by which it mediates specific effects to improve health and alleviate symptoms such as gastrointestinal disorders (including diarrhea and constipation), inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and infantile colic. Infantile colic is a condition that can be a great stress for the affected family and severely impair the quality of life. Additionally, infantile colic may potentially have long-term consequences for the baby's later life. The well-studied probiotic strain Lactobacillus reuteri DSM 17938 has been shown to significantly reduce the crying time of colicky infants. However, the onset time of this effect is not immediate and may take one to three weeks for the baby to benefit from the treatment. Therefore, there is a great need for more rapid-acting interventions and even more effective interventions to, for example, reduce the discomfort and crying periods of colicky children. Summary of the Invention

[0005] A general object of the present invention herein is to provide therapeutic microbubbles from probiotics.

[0006] This and other objects are met by embodiments as disclosed herein.

[0007] The invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0008] One aspect of an embodiment relates to a method of generating therapeutic microbubbles. The method includes culturing bacteria of a probiotic strain in a culture medium. The probiotic strain is selected from Lactobacillus strains, Bifidobacterium strains, and combinations thereof. The method further includes exposing the bacteria to an inducible biological treatment during culturing to induce the bacteria to produce therapeutic microbubbles. The inducible biological treatment is selected from co-culturing the bacteria with bacteria of another bacterial strain, culturing the bacteria in the presence of conditioned medium from bacteria of another bacterial strain, and combinations thereof. The other bacterial strain is a Bifidobacterium strain, and the other bacterial strain is different from the probiotic strain.

[0009] Other aspects of the embodiment relate to a probiotic composition comprising bacteria of a probiotic strain and therapeutic microbubbles produced by the probiotic strain or another probiotic strain, for treating colic and / or for treating a disease selected from: gastrointestinal disorders or diseases in infants or children, gastrointestinal pain disorders, bone loss diseases, periodontal diseases, and combinations thereof. The probiotic strain and the other probiotic strain are selected from Lactobacillus strains, Bifidobacterium strains, and combinations thereof.

[0010] A further aspect of the embodiment relates to a probiotic composition for treating colic and / or for treating a disease selected from: gastrointestinal disorders or diseases in infants or children, gastrointestinal pain disorders, bone loss diseases, periodontal diseases, and combinations thereof. The probiotic composition comprises a fast-acting component in the form of therapeutic microbubbles from bacteria of a probiotic strain. The probiotic strain is selected from Lactobacillus strains, Bifidobacterium strains, and combinations thereof. The probiotic composition further comprises a slow-acting component in the form of bacteria of the probiotic strain or another probiotic strain. The other probiotic strain is selected from Lactobacillus strains, Bifidobacterium strains, and combinations thereof. When administered to a subject, the fast-acting component and the slow-acting component together produce an extended therapeutic effect.

[0011] Still other aspects of the embodiment relate to therapeutic microbubbles isolated from bacteria of a probiotic strain for treating colic and / or for treating a disease selected from: gastrointestinal disorders or diseases in infants or children, gastrointestinal pain disorders, bone loss diseases, periodontal diseases, and combinations thereof. The probiotic strain is selected from Lactobacillus strains, Bifidobacterium strains, and combinations thereof.

[0012] Another aspect of the embodiment relates to a bacterial strain, wherein the bacterial strain is Bifidobacterium longum DSM32947 or Bifidobacterium longum DSM 32948.

[0013] A further aspect of the embodiments relates to a probiotic composition comprising a strain of the genus Lactobacillus, preferably a strain of Lactobacillus reuteri, and more preferably a bacterium of a Lactobacillus reuteri strain selected from: Lactobacillus reuteri DSM 17938, Lactobacillus reuteri DSM 32846, and combinations thereof. The composition further comprises a bacterium of a Bifidobacterium longum strain selected from Bifidobacterium longum DSM 32947, Bifidobacterium longum DSM 32948, and combinations thereof, or a conditioned medium from a Bifidobacterium longum strain.

[0014] Therapeutic microbubbles produced by probiotics are able to reproduce the beneficial effects of probiotics, as shown herein. In addition, therapeutic microbubbles are actually more effective than the probiotics that produce them, as demonstrated by their more rapid onset of specific beneficial effects, as shown in the examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The embodiments, together with further objects and advantages thereof, may best be understood by reference to the following description taken in conjunction with the accompanying drawings, in which:

[0016] Figure 1 Measurement of 5'-nucleotidase activity in the conditioned medium after Lactobacillus reuteri DSM 17938 was exposed to different inducible treatments is shown.

[0017] Figure 2 Shown in Figure 2 a is the addition of Lactobacillus reuteri DSM 17938 (DSM), in Figure 2 b is the addition of conditioned medium (CM), in Figure 2 c is the addition of medium (broth), in Figure 2 d is the addition of DSM-derived microbubbles (μV), or in Figure 2 e is the addition of conditioned medium minus microbubbles (CM-μV), on the speed of the propagated contractile complex (PCC) of mouse jejunal segments in vitro. Upper panel: Bar graph showing the mean and standard error. P values obtained from paired t tests are given above the horizontal bars. Lower panel: Single-value difference plots (treatment - control Krebs) with 95% confidence intervals for each matching plot in the upper row. Figures 3 to 7 The same relationship between the upper and lower panels is shown.

[0018] Figure 3 Shown is the addition of DSM ( Figure 3 a), CM ( Figure 3 b), broth ( Figure 3 c), μV( Figure 3d) or CM - μV ( Figure 3 e), on the PCC frequency in the jejunal segment of mice in vitro.

[0019] Figure 4 Shows the effect of adding DSM ( Figure 4 a), CM ( Figure 4 b), broth ( Figure 4 c), μV ( Figure 4 d) or CM - μV ( Figure 4 e), on the PCC peak amplitude in the jejunal segment of mice in vitro.

[0020] Figure 5 Shows the effect of adding DSM ( Figure 5 a), CM ( Figure 5 b), broth ( Figure 5 c), μV ( Figure 5 d) or CM - μV ( Figure 5 e), on the PCC velocity in the colonic segment of mice in vitro.

[0021] Figure 6 Shows the effect of adding DSM ( Figure 6 a), CM ( Figure 6 b), broth ( Figure 6 c), μV ( Figure 6 d) or CM - μV ( Figure 5 e), on the PCC frequency in the colonic segment of mice in vitro.

[0022] Figure 7 Shows the effect of adding DSM ( Figure 7 a), CM ( Figure 7 b), broth ( Figure 7 c), μV ( Figure 7 d) or CM - μV ( Figure 7 e), on the PCC peak amplitude in the colonic segment of mice in vitro.

[0023] Figure 8 Shows Figures 2 to 7 A summary of the jejunal and colonic velocity, frequency, and peak amplitude results presented in

[0024] Figure 9 Shows the effect of microbubbles on TrpV1 signaling. The figure shows the capsaicin - induced responses obtained using microbubbles isolated from Lactobacillus reuteri DSM17938 (DSM - MV), and microbubbles isolated from the Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) JB - 1 bacterial strain (MV - JB - 1).

[0025] Figure 10It is a graph showing the onset timing of responses in a mesenteric nerve discharge model using Lactobacillus reuteri DSM 17938 (DSM) and microvesicles (MV) isolated from Lactobacillus reuteri DSM17938.

[0026] Figure 11 It shows that microvesicles (MV) derived from Lactobacillus reuteri DSM 17938 are immunomodulatory and inhibit IFN-γ and IL-17A responses. Evaluation of the immunomodulatory effects of MV derived from Lactobacillus reuteri DSM 17938 purified in PBMC cultures.( Figure 11 a) PBMC were cultured for 48 hours in the presence of Lactobacillus reuteri (L.r)-MV at ratios of 500:1, 100:1, and 20:1 (MV:cells), followed by quantification of the secretion levels of IL-6, IL-10, IL-17A, and IFN-γ (n = 8).( Figure 11 b) PBMC were stimulated with Staphylococcus aureus( Staphylococcus aureus )(S.a)-CFS (2.5%) in the presence of L.r-MV at ratios of 500:1, 100:1, and 20:1 (MV:cells), followed by quantification of the secretion levels of IFN-γ and IL-17A. Shown are the relative values of Staphylococcus aureus-CFS standardized alone, (n = 8). The box covers the data between the 25th and 75th percentiles, where the median is the center line and the error bars show the minimum to the maximum. The bar graph shows the median with the interquartile range.

[0027] Figure 12 It shows that Lactobacillus reuteri DSM 17938 and microvesicles derived from Lactobacillus reuteri DSM 17938 protect epithelial integrity from the harmful effects of enterotoxigenic Escherichia coli( Escherichia coli )(ETEC).

[0028] Figure 13 It shows the 5'-nucleotidase activity in MV samples obtained from Lactobacillus reuteri DSM 17938 (compared to Lactobacillus reuteri DSM 17938 in the SIM (control) or with Lactobacillus paracasei( L. paracasei) The Lactobacillus reuteri DSM 17938 in the 4% supernatant of LMG-P-17806 (DSM 17938 in SIM or DSM 17938 + 4% supernatant of LMG-P-17806) was cultured in SIM medium with the addition of 4% supernatant from Bifidobacterium longum ATCC BAA-999 or Bifidobacterium longum DSM 32947 (DSM 17938 + 4% supernatant of DSM 32947 or DSM 17938 + 4% supernatant of ATCC BAA-999).

[0029] Figure 14 showed the same results as presented in Figure 13 but the results were normalized with respect to the 5'-nucleotidase activity and optical density of DSM 17938 in SIM.

[0030] Figure 15 The 5'-nucleotidase activity in the control sample (DSM 17938 in SIM) was shown compared to a sample obtained after an inducible biological treatment by co-culturing Lactobacillus reuteri DSM 17938 with 25% cells from Bifidobacterium longum DSM 32947. The results were normalized with respect to the 5'-nucleotidase activity and optical density of DSM 17938 in SIM.

[0031] Figure 16 The 5'-nucleotidase activity in the MV sample was shown after culturing Lactobacillus reuteri DSM 32846 in SIM medium with the addition of 4% supernatant from Bifidobacterium longum DSM 32947 (DSM 32846 + 4% supernatant of DSM 32947) compared to the control Lactobacillus reuteri DSM 32846 in SIM (DSM 32846 in SIM). The results were normalized with respect to the 5'-nucleotidase activity and optical density of DSM 32846 in SIM.

[0032] Figure 17 It was shown that the microvesicles derived from Lactobacillus reuteri DSM 32846 were more effective in inducing IL-6 production compared to the microvesicles isolated from Lactobacillus reuteri DSM 17938.

[0033] Figure 18 and 19 showed the IL-6 induction by the MVs isolated from Lactobacillus reuteri DSM 32846 and Lactobacillus reuteri DSM 17938 after culturing with 4% supernatant from Bifidobacterium longum strains compared to the control samples (DSM 32846 or DSM 17938, respectively).

[0034] Figure 20 Shows the IL-6 production induced by MVs derived from Lactobacillus reuteri DSM17938 after co-culture with 25% of the cells from Bifidobacterium longum DSM32947, compared to the control sample (DSM 17938).

[0035] Figure 21 Shows that MVs derived from Lactobacillus reuteri DSM 32846 are able to partially protect the epithelial monolayer against the ETEC-induced reduction in TEER. The figure also shows the protective effect of MVs derived from Lactobacillus reuteri DSM32846 against ETEC damage to the monolayer in the FITC-dextran flux experiment.

[0036] Figure 22 Shows a comparison between the protective effect of MVs derived from Lactobacillus reuteri DSM 32846 and the effect obtained with MVs derived from Lactobacillus reuteri DSM 17938 in the FITC-dextran flux experiment. Pretreatment of the epithelial cell monolayer with MVs derived from Lactobacillus reuteri DSM 32846 more effectively reduced the leakage of FITC-dextran, especially at lower concentrations of MVs, compared to MVs derived from Lactobacillus reuteri DSM17938. Detailed Description

[0037] The present invention generally relates to therapeutic microvesicles from probiotics and their uses.

[0038] Definition

[0039] Microvesicles (MVs, μVs), also known in the art as, for example, membrane vesicles, outer membrane vesicles, extracellular vesicles, are a proven form of communication used by bacteria and eukaryotic cells. Bioactive MVs released from the cell surface are conserved across microbial life in bacteria, archaea, fungi, and parasites, and MV production has been demonstrated both in vitro and in vivo, suggesting the influence of these surface organelles in microbial physiology and pathogenesis through the delivery of their important signaling molecules, enzymes, and toxins. Bacterial MVs are regularly produced and shed by both Gram-positive and Gram-negative bacteria, and proteomics experiments have shown that the content of such MVs can be different from that of the parental bacteria. MVs may contain lipid molecules, RNA molecules, DNA molecules, and / or proteins. In addition, MVs may also contain surface components of the parental bacteria.

[0040] The MVs produced by probiotics as disclosed herein are referred to herein as therapeutic MVs to indicate that the MVs have therapeutic efficacy. When administered to a subject, this therapeutic efficacy of the MVs can be the same as or at least similar to the probiotic effect of the probiotics that produce the MVs. Accordingly, the MVs will exert therapeutic efficacy in the subject, which will inhibit, treat, or prevent, including delaying the onset of a medical condition, disease, or disorder in the subject, as further described herein.

[0041] A culture medium or growth medium is the starting medium in which bacteria will be cultured.

[0042] A conditioned medium is a culture or growth medium in which bacteria have been cultured. Such conditioned medium thus contains any compounds or reagents released into the medium by the bacteria, including MVs. The bacteria have been removed from the medium and are thus not part of the conditioned medium. For example, a conditioned medium can be obtained by centrifugation, sedimentation, and / or precipitation of a bacterial cell culture to obtain the conditioned medium as the supernatant.

[0043] Cytoplasm is a mixture of cultured bacteria and the medium, including any compounds or reagents released into the medium by the bacteria, such as MVs, i.e., the conditioned medium. Cytoplasm is the end result of fermentation.

[0044] The probiotic strain is preferably a strain of lactic acid-producing probiotics, sometimes also referred to as lactic acid bacteria. Lactic acid-producing bacteria are a group of Gram-positive bacteria with a low %GC content in their genomes, acid-tolerant, generally non-spore-forming, non-respiring, rod-shaped or spherical bacteria that share common metabolic and physiological characteristics. These bacteria produce lactic acid as the main metabolic end product of carbohydrate fermentation. Genera that contain lactic acid-producing bacteria include Lactobacillus, Leuconostoc ( Leuconostoc ), Pediococcus ( Pediococcus ), Lactococcus ( Lactococcus ), and Streptococcus ( Streptococcus ). The genus Bifidobacterium is not included in traditional lactic acid bacteria due to its genetic unrelatedness, but this bacterium has characteristics overlapping with lactic acid bacteria, and it has a metabolism that produces lactic acid as the main end product of fermentation, although it produces much less lactic acid than Lactobacillus. Bifidobacterium is strictly anaerobic and is typically found in high abundance in the large intestine.

[0045] MVs produced by probiotics are important by constituting a means of communication between the probiotics and the surrounding host cells, such as mucosal cells in the human body, such as intestinal mucosal cells of the gastrointestinal system, oral or vaginal mucosa. Thus, MVs can transfer information, such as probiotic information, from bacterial cells to the host. Therefore, there is a need to enhance the production of therapeutic MVs from probiotics, which can be used in probiotic and therapeutic applications. As the experimental data presented herein shows, MVs produced by probiotics can reproduce the probiotic probiotic effects or therapeutic efficacy, as shown by the effects of isolated MVs on pain signaling and gastrointestinal motility. The MVs not only reproduced the effect of probiotics on pain signaling, but were actually more effective, as demonstrated by their ability to act faster than probiotics, resulting in an earlier onset of the observed effect. This finding was very unexpected. As the experimental data presented herein also shows, therapeutic MVs isolated from probiotic strains have immunostimulatory effects and are able to inhibit specific cytokines associated with autoimmune diseases, and they also protect epithelial barrier integrity. As the experimental data presented herein also shows, the production of MVs can be induced by bioprocessing during culture, such as by adding the supernatant from another bacterial strain to the probiotic strain, or by co-culturing the probiotic with bacteria from another bacterial strain.

[0046] Accordingly, the present invention describes protocols that can be used to produce therapeutic MVs and / or increase the intrinsic or endogenous production of therapeutic MVs.

[0047] One aspect of the embodiments includes a method for producing therapeutic MVs. The method includes culturing bacteria of a probiotic strain in a culture medium and exposing the bacteria to an inducible bioprocess during the culture to induce the bacteria to produce therapeutic MVs. The probiotic strain is selected from Lactobacillus strains, Bifidobacterium strains, and combinations thereof. In this regard, the inducible bioprocess is selected from co-culturing the bacteria of the probiotic strain with bacteria of another bacterial strain, culturing the bacteria of the probiotic strain in the presence of the conditioned medium of bacteria of another bacterial strain, and combinations thereof. The other bacterial strain is a Bifidobacterium strain, and the other bacterial strain is different from the probiotic strain.

[0048] Thus, in this regard, the production of therapeutic MVs involves culturing bacteria of a probiotic strain and simultaneously stimulating the bacteria to produce therapeutic MVs by exposing the bacteria to an inducible treatment during the culture to induce the production of therapeutic MVs.

[0049] As used herein, the induced production of therapeutic MVs includes stimulating bacteria to produce therapeutic MVs, including promoting, enhancing, or increasing the production of therapeutic MVs by the bacteria. Alternatively or additionally, the induced production of therapeutic MVs includes more efficient release of therapeutic MVs from the bacteria, resulting in a greater number of therapeutic MVs being released by the bacteria compared to when the bacteria are not exposed to the inducing treatment. Alternatively or additionally, the induced production of therapeutic MVs includes the production of more potent or effective therapeutic MVs by the bacteria. In such cases, the therapeutic MVs produced by bacteria exposed to the inducing treatment have enhanced efficacy compared to MVs produced by unstimulated bacteria (i.e., bacteria not exposed to the inducing treatment). Thus, the inducing treatment of the embodiments can be used, for example, to increase the production of therapeutic MVs in bacteria of probiotic strains that already have an inherent or endogenous production of such therapeutic MVs. In such cases, the inducing treatment enhances this inherent or endogenous MV production by the bacteria, resulting in more therapeutic MVs when exposed to the inducing treatment compared to when not exposed. The induced production of therapeutic MVs also includes the induced production of such therapeutic MVs in bacteria of probiotic strains that do not have any significant MV production when not exposed to the inducing treatment. Thus, the induced production of therapeutic MVs by the inducing treatment includes both increasing the inherent or endogenous production of therapeutic MVs in the bacteria and increasing the de novo production of therapeutic MVs in the bacteria.

[0050] As further described herein, the inducing treatment is a bacterial treatment that induces (including increasing) the production of therapeutic MVs by the bacteria. Thus, according to the various embodiments, the bacteria are modified or induced to produce therapeutic MVs by exposing the bacteria to at least one inducing treatment during cultivation.

[0051] The cultivation of the bacteria can be carried out in a suitable culture device, fermenter, or bioreactor according to known cultivation protocols, which include but are not limited to stirred tank bioreactors, airlift bioreactors, hollow fiber bioreactors, and rotating cell culture system (RCCS) bioreactors. The specific cultivation conditions are preferably selected based on the particular probiotic strain.

[0052] In one embodiment, the culture medium containing the therapeutic MVs and the probiotics, i.e., the cell lysate, is preserved, for example, by drying and / or freezing. Common examples of drying include spray drying, freeze drying, spray freeze drying, and vacuum drying.

[0053] The cell lysate can optionally be concentrated before or during preservation to reduce the overall volume of the cell lysate and also to concentrate the bacterial cells, therapeutic MVs, and any other compounds or reagents present therein.

[0054] For example, the cytoplasm can be concentrated to a volume corresponding to about 5% to 95% of the original volume, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% of the original volume.

[0055] Various methods and processes can be employed to concentrate the cytoplasm. For example, the cytoplasm can be concentrated by removing water and optionally other substances such as organic acids, sugars, and salts from the cytoplasm. A filtration device that mainly allows water to pass through a filtration membrane can be used. This process is called osmosis and can be operated in various modes such as reverse osmosis mode, forward osmosis mode. In one embodiment, concentration can also be performed by using chemicals to precipitate the sample. Chemical precipitation can be used to concentrate the cytoplasm by the addition of denaturing solvents or salts. Various types of chromatography setups can also be used for concentration and, for example, separate the input sample according to its chemical properties and size. For example, size exclusion chromatography works by separating samples based on size, trapping smaller molecules inside small pores and excluding larger molecules and particles. Ion chromatography helps to separate molecules with certain charges. For example, anion exchange chromatography can be used to utilize the net negative charge found on bacterial membranes and MVs and bind them to a positively charged chromatography matrix. It can then be eluted by increasing the ionic strength of the surrounding mobile phase. Another concentration technique is ultrafiltration. Ultrafiltration is based on mechanical interactions rather than chemical interactions. The filtration device may have a molecular weight cut-off, meaning that all substances above a certain molecular weight are retained while smaller molecules and salts, etc. pass through. Membranes with highly defined pore sizes are typically employed. These types of ultrafiltration devices and processes are also used in different setups, such as direct flow filtration (DFF) or tangential flow filtration (TFF). Tangential flow filtration (TFF), also known as cross-flow filtration, differs from other filtration systems in that the fluid passes parallel to the filter rather than perpendicular through the membrane. This method is preferred due to its continuous filtration and reproducible performance. The particles passing through the membrane (permeate) are set aside while the remaining portion (retentate) is recycled back to the feed.

[0056] In one embodiment, the bacteria are exposed to an inducing treatment during cultivation to induce (including enhance) the production of therapeutic MVs and the release of therapeutic MVs into the culture medium. This means that after exposure to the inducing treatment, the therapeutic MVs produced by the bacteria of the probiotic strain are released from the bacteria into the culture medium. Additionally or alternatively, at least some of the therapeutic MVs produced by the bacteria can bind to and / or attach to the cell membrane and / or cell wall of the bacteria.

[0057] In one embodiment, the method comprises isolating therapeutic MVs from a culture medium, such as from the cytoplasm or conditioned medium. In one embodiment, isolation of the therapeutic MVs comprises subjecting the culture medium, such as the cytoplasm, to at least one centrifugation at a relative centrifugal force selected within a first interval to obtain a bacteria-depleted supernatant, i.e., conditioned medium, and subjecting the conditioned medium to at least one ultracentrifugation at a relative centrifugal force selected within a second interval to obtain a pellet containing MVs. The second interval is higher than the first interval.

[0058] Perform a first centrifugation or at least one centrifugation at a relative centrifugal force selected within the first interval to remove live bacteria and large debris from the culture medium, thereby forming a discarded pellet and a supernatant containing therapeutic MVs, designated as the conditioned medium above. This first step in the isolation process may comprise a single centrifugation step, but preferably comprises at least two centrifugation steps in order to more effectively remove bacteria and large debris. In the case of at least two centrifugation steps, all steps may be carried out at the same relative centrifugal force. However, it is generally more effective to increase the relative centrifugal force for each successive centrifugation step. The first interval is preferably from 100 × g to 50,000 × g , for example from 200 × g to 25,000 × g , and preferably from 500 × g to 15,000 × g . For example, the first centrifugation step may be 4,000 × g , while the second centrifugation step is 10,000 × g . Alternatively, a single centrifugation step at 600 × g may be used. The supernatant may also or alternatively be run through a micron filter (from 0.20 µm to 0.50 µm, such as 0.45 µm) to remove any debris and / or bacteria remaining from the centrifugation.

[0059] The conditioned medium containing therapeutic MVs is then subjected to at least one ultracentrifugation at a relative centrifugal force selected within the second interval to obtain a pellet containing MVs. This second step may comprise one or more ultracentrifugation steps. In the case of multiple ultracentrifugation steps, all steps may be carried out at the same relative centrifugal force, or the relative centrifugal force may be increased as disclosed above. The second interval is preferably equal to or greater than 75,000 × g ,, for example equal to or greater than 85,000 × g , preferably equal to or greater than 100,000 × g . For example, a relative centrifugal force of 118,000 × g may be used.

[0060] In one embodiment, the separation step further comprises loading the conditioned medium onto a sucrose gradient or sucrose cushion and centrifuging at a relative centrifugal force selected within a second interval.

[0061] Additionally or alternatively, the conditioned medium can be filtered prior to ultracentrifugation. In such a case, a filter with an average pore size of, for example, from 0.20 µm up to 0.50 µm can be used.

[0062] The isolated therapeutic MVs can be preserved, for example, by drying, such as by spray drying, freeze drying, spray freeze drying or vacuum drying, and / or by freezing. In one embodiment, the therapeutic MVs are stable after being preserved for at least 1 month, at least 3 months, at least 5 months or at least 7 months.

[0063] The inducible bioprocessing is selected from co-culturing a probiotic strain with bacteria of another bacterial strain, culturing the probiotic strain in the presence of the conditioned medium from bacteria of another bacterial strain, and combinations thereof. The other bacterial strain is preferably a probiotic strain. For example, the other bacterium can be a Bifidobacterium strain, preferably Bifidobacterium longum strain, and more preferably Bifidobacterium longum DSM 32947 and / or DSM 32948 (deposited at the Leibniz-Institut DSMZ - German Collection of Microorganisms and Cell Cultures (Inhoffenstrasse 7B, D-38124 Braunschweig, Germany) on November 1, 2018 by BioGaia AB under the Budapest Treaty). The Bifidobacterium strains exemplified above are particularly useful for binding to bacteria of the Lactobacillus genus as probiotic strains, and in particular bacteria of the Lactobacillus reuteri strain, such as Lactobacillus reuteri DSM 17938 (deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures (Mascheroder Weg 1b, D-38124 Braunschweig, Germany) on January 30, 2006 by BioGaia AB under the Budapest Treaty), and / or Lactobacillus reuteri DSM 32846 (deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures (Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on July 4, 2018 by BioGaia AB under the Budapest Treaty).

[0064] In a particular embodiment, the bacteria of the probiotic strain can be co-cultured with the bacteria of another bacterial strain. Alternatively or additionally, the conditioned medium from the bacteria of another bacterial strain can be added to the medium containing the bacteria of the probiotic strain.

[0065] In one embodiment, the method further includes exposing the bacteria to an inducible abiotic treatment during cultivation to induce (including increasing) the production of therapeutic MVs by the bacteria. Thus, in this embodiment, the bacteria are exposed to both an inducible biotic treatment and an inducible abiotic treatment.

[0066] Abiotic treatment involves treatment with non-living chemical and physical components that affect living organisms. Biotic treatment involves treatment with biological materials that are either living organisms or derived from living organisms.

[0067] In a specific embodiment, the abiotic treatment is treatment with an abiotic stressor, i.e., an abiotic treatment that induces a stress response in the bacteria of a probiotic strain when exposed to the abiotic treatment during cultivation. In one embodiment, the abiotic stressor is selected from oxidative stress (oxygen treatment), temperature stress, pH stress, ultraviolet (UV) stress, and combinations thereof.

[0068] Oxygen treatment means exposing the bacteria to an increased concentration of oxygen. In one embodiment, the increased concentration of oxygen is a non-toxic concentration of oxygen. In a specific embodiment, exposing the bacteria to oxygen treatment includes exposing relatively oxygen-tolerant anaerobes, microaerophiles, aerobes, and / or facultative anaerobes to an increased oxygen concentration, i.e., an increased non-toxic concentration of oxygen, during cultivation to induce the production of therapeutic MVs by the relatively oxygen-tolerant anaerobes, microaerophiles, aerobes, and / or facultative anaerobes.

[0069] As used herein, oxygen treatment does not involve the addition of any reactive oxygen species (ROS), such as peroxides, including hydrogen peroxide, superoxide, or hydroxyl radicals.

[0070] The increased oxygen concentration implies that the oxygen concentration in the culture medium is higher than the (normal) oxygen concentration, which is, however, non-toxic to the bacteria, and the (normal) oxygen concentration is otherwise selected to be optimal or at least suitable for culturing the bacteria of the probiotic strain. In one embodiment, the non-toxic concentration of oxygen does not result in significant bacterial cell death, which means that the exposed bacteria remain viable at least up to 70%, preferably at least up to 75%, more preferably at least up to 80%, such as at least up to 85% or 90% or even higher compared to when the bacteria are exposed to the normal oxygen concentration. This increase in oxygen concentration can be achieved by adding (injecting) oxygen or air into the culture medium in one or more bursts or pulses or over an extended period of time. Alternatively or additionally, the increase in oxygen concentration can be achieved by agitating or stirring the culture medium containing the bacteria, including increasing the amount or level of agitation or stirring of the culture medium. The oxygen concentration, such as the non-toxic concentration, can vary between different bacterial strains but can generally be set to 0.1 to 10%. In one embodiment, the oxygen concentration is set to 0.5 to 2%. In another embodiment, the oxygen concentration is set to 2 to 5%. In yet another embodiment, the oxygen concentration is set to 5 to 10%.

[0071] A specific aspect of the embodiment includes a method of generating therapeutic MVs. The method includes culturing bacteria of a probiotic strain in a culture medium and exposing the bacteria to an oxidative treatment during the culturing to induce the bacteria to produce therapeutic MVs.

[0072] Thus, in this aspect of the embodiment, (including increasing) MV production is induced in the bacteria by exposing the bacteria to an oxygen treatment (oxidative stress), but not necessarily in combination with exposing the bacteria to any inductive biological treatment.

[0073] In one embodiment, the bacteria of the probiotic strain are selected from relatively oxygen-tolerant anaerobes, microaerophiles, aerobes, and / or facultative anaerobes, preferably selected from aerobes and facultative anaerobes.

[0074] In a specific embodiment, the bacteria are selected from Lactobacillus strains, Bifidobacterium strains, and combinations thereof. Preferred Lactobacillus and Bifidobacterium strains can be selected from the illustrative examples of preferred bacterial strains described below.

[0075] By raising the culture temperature to above the normal temperature used for culturing bacteria in a bioreactor, temperature stress, i.e., so-called heat stress, can be induced. For example, if the normal culture temperature is 37 °C, the temperature can be increased to at least 42 °C, at least 43 °C or at least 44 °C, and more preferably at least 45 °C, such as at least 46 °C, at least 47 °C, at least 48 °C, at least 49 °C or at least 50 °C. Instead of exposing the bacteria to heat stress, the bacteria can be exposed to cold stress, i.e., by lowering the culture temperature to below the normal culture temperature. For example, the culture temperature can be lowered to 10 °C, such as 8 °C or lower, 6 °C or lower, or 4 °C or lower.

[0076] By lowering the pH of the culture medium in which the bacteria are cultured from the normal or baseline pH to an acidic or more acidic pH, pH stress can be induced. Alternatively, the bacteria can be temporarily removed from the culture medium and then exposed to pH stress, and subsequently the bacteria exposed to pH stress are added to the culture medium or fresh culture medium. For example, the pH can be lowered from the normal pH range of 6.5 to 7 to a pH of 2 or lower.

[0077] By exposing the bacteria to UV treatment, e.g., by directing ultraviolet light into the culture medium containing the bacteria, UV stress can be induced.

[0078] In one embodiment, the method further includes exposing the bacteria to a stress inducer during culturing to induce (including increasing) the production of therapeutic MVs by the bacteria. Thus, in this embodiment, the bacteria are exposed to both a stress inducer and an inductive bioprocess and / or an inductive abiotic process.

[0079] In one embodiment, the stress inducer is selected from fructose; sucrose; lysozyme, e.g., from eggs, also known as muramidase or N-acetylmuramide glycanhydrolase; mucin purified, e.g., from porcine intestine; β-lactams such as ampicillin and combinations thereof.

[0080] In a particular embodiment, the stress inducer is sucrose. Sucrose can be added during the culturing of the bacteria to induce the production of MVs by the bacteria. For example, sucrose can be added to the culture medium to obtain a sucrose concentration in the range of 0.3% - 10% in the culture medium, such as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10%.

[0081] Examples of the above inducible treatments can be combined, such as combining multiple, i.e., at least two, abiotic treatments, multiple biotic treatments, treatment with multiple stress inducers, at least one abiotic treatment and at least one biotic treatment, at least one abiotic treatment and treatment with at least one stress inducer, at least one biotic treatment and treatment with at least one stress inducer, or at least one abiotic treatment, at least one biotic treatment, and treatment with at least one stress inducer.

[0082] The duration of exposure to the inducible treatment can be selected based on the specific treatment type, specific probiotic strain, and culture conditions, such as the type of bioreactor. For example, as illustrative but non-limiting examples, bacteria can be exposed to the abiotic treatment for 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.25 hours, 2.5 hours, 2.75 hours, 3 hours, 3.25 hours, 3.5 hours, 3.75 hours, 4 hours, 4.25 hours, 4.5 hours, 4.75 hours, 5 hours, or longer. There can also be longer periods of abiotic stress exposure, such as overnight, 12 hours, 18 hours, 24 hours, or even longer. The addition of the stress inducer can include adding at least one stress inducer one or more times, i.e., at least twice, to the culture medium. The addition of bacteria of another bacterial strain or conditioned medium from such bacteria can also be carried out one or more times.

[0083] The probiotic strain is preferably a lactic acid-producing probiotic strain and is particularly selected from the genera Lactobacillus and Bifidobacterium. The genus Lactobacillus includes several species, including Lactobacillus acidophilus ( L. acetotolerans ), Lactobacillus amylolyticus ([[]] L. acidifarinae ), Lactobacillus acidipiscis ([[]] L. acidipiscis ), Lactobacillus acidophilus ([[]] L. acidophilus ), Lactobacillus agilis ([[]] L. agilis ), Lactobacillus alimentarius ([[]] L. algidus ), Lactobacillus amylophilus ([[]] L. alimentarius ), Lactobacillus amylolyticus ([[]] L. amylolyticus ), Lactobacillus amylophilus ([[]] L. amylophilus ), L. amylotrophicus , Lactobacillus amylovorus ([[]] L. amylovorus ), Lactobacillus animalis ([[]] L. animalis ), Lactobacillus antri ([[]] L. antri ), Lactobacillus arvicolae ([[]] L. apodemi ), Lactobacillus aviarius ([[]] L. aviaries ), Lactobacillus bifarmentans ([[]] L. bifermentans ), Lactobacillus brevis ([[]] L. brevis ), Lactobacillus buchneri ([[]] L. buchneri ), Lactobacillus camelliae ([[]]L. camelliae ) Lactobacillus casei L. casei ) Lactobacillus cateniformis L. catenaformis ) L. ceti 、 L. coleohominis ) Lactobacillus collinoides L. collinoides ) Lactobacillus complex L. composti ) Lactobacillus curvatus L. concavus ) Lactobacillus coryniformis L. coryniformis ) Lactobacillus crispatus L. crispatus ) Lactobacillus farciminis L. crustorum ) Lactobacillus flexuosus L. curvatus ) Lactobacillus delbrueckii subsp. bulgaricus L. delbrueckii subsp . bulgaricus ) Lactobacillus delbrueckii subsp. delbrueckii L. delbrueckii subsp . delbrueckii ) Lactobacillus delbrueckii subsp. lactis L. delbrueckii subsp . lactis ) Lactobacillus dextrinicus L. dextrinicus ) L. diolivorans 、 L. equi ) Lactobacillus homohiochii L. equigenerosi ) Lactobacillus farinae L. farraginis ) Lactobacillus farciminis L. farciminis ) Lactobacillus fermentum L. fermentum ) Lactobacillus floricola L. fornicalis ) Lactobacillus fructivorans L. fructivorans ) Lactobacillus graminis L. frumenti ) Lactobacillus fructosus L. fuchuensis ) Lactobacillus gallinarum L. gallinarum ) Lactobacillus gasseri L. gasseri ) Lactobacillus gastricus L. gastricus ) Lactobacillus ghanensis L. ghanensis ) Lactobacillus herbarius L. graminis ) L. hammesii ) Lactobacillus hammesii L. hamster ) Lactobacillus harbinensis L. harbinensis ) L. hayakitensis ) Lactobacillus helveticus L. helveticus ) Lactobacillus hilgardii L. hilgardii ) Lactobacillus homohiochii L. homohiochii ) Lactobacillus iners L. iners ) L. ingluviei ) Lactobacillus intestinalis L. intestinalis ) Lactobacillus jensenii L. jensenii)、Lactobacillus johnsonii L. johnsonii )、Lactobacillus calixensis L. kalixensis )、Lactobacillus kefiranofaciens L. kefiranofaciens )、Lactobacillus kefiri L. kefiri )、 L. kimchi 、Lactobacillus kitasatonis L. kitasatonis )、Lactobacillus kunkeei L. kunkeei )、Lactobacillus leichmannii L. leichmannii )、Lactobacillus lindneri L. lindneri )、Lactobacillus malfermentans L. malefermentans )、Lactobacillus mali L. mali )、Lactobacillus manihotivorans L. manihotivorans )、Lactobacillus mendenhalli L. mindensis )、Lactobacillus mucosae L. mucosae )、Lactobacillus murinus L. murinus )、Lactobacillus nagelii L. nagelii )、 L. namurensis 、Lactobacillus nanensis L. nantensis )、Lactobacillus oligofermentans L. oligofermentans )、Lactobacillus oris L. oris )、Lactobacillus panis L. panis )、Lactobacillus pantheris L. pantheris )、 L. parabrevis 、Lactobacillus parabrevis L. parabuchneri )、Lactobacillus paracasei L. paracasei )、Lactobacillus parabuchneri L. paracollinoides )、Lactobacillus paralimentarius L. parafarraginis )、Lactobacillus parakefiri L. parakefiri )、Lactobacillus paraplantarum L. paralimentarius )、Lactobacillus pentosus L. paraplantarum )、Lactobacillus perolens L. pentosus )、Lactobacillus plantarum L. perolens )、Lactobacillus pontis L. plantarum )、 L. pontis )、 L. protectus 、 L. psittaci 、Lactobacillus rennini L. rennini )、Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus crevioris L. rimae )、Lactobacillus rogosae L. rogosae )、Lactobacillus rossiae L. rossiae )、Lactobacillus ruminis L. ruminis )、 L. saerimneri 、Lactobacillus sakeiL. sakei ) Lactobacillus salivarius L. salivarius ) Lactobacillus sanfranciscensis L. sanfranciscensis ) L. satsumensis , L. secaliphilus , Lactobacillus sakei L. sharpeae ) L. siliginis , L. spicheri , L. suebicus , Lactobacillus thailandensis L. thailandensis ) L. ultunensis , Lactobacillus vaccinostercus L. vaccinostercus ) Lactobacillus vaginalis L. vaginalis ) L. versmoldensis , L. vini , Lactobacillus vitulinus L. vitulinus ) Lactobacillus zeae L. zeae ) and Lactobacillus panis L. zymae ). Preferred examples of such probiotic strains include Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus gasseri and Lactobacillus plantarum. Current preferred examples of such probiotic strains include Lactobacillus reuteri strains such as Lactobacillus reuteri DSM 17938 and Lactobacillus reuteri DSM 32846. Preferred species of the genus Bifidobacterium are Bifidobacterium adolescentis B. adolescentis ), Bifidobacterium breve B. breve ), Bifidobacterium longum, Bifidobacterium animalis B. animalis ), Bifidobacterium infantis B. infantis ), Bifidobacterium thermophilum B. thermophilum ), Bifidobacterium bifidum B. bifidum ) and Bifidobacterium lactis B. lactis ). A further preferred species of the genus Bifidobacterium is Bifidobacterium longum. Current preferred examples of the genus Bifidobacterium are Bifidobacterium longum DSM 32947 and Bifidobacterium longum DSM 32948.

[0084] Lactobacillus reuteri is an oxygen-tolerant (alternatively oxygen-tolerant or relatively oxygen-tolerant) anaerobe, i.e., it can only generate ATP by fermentation.

[0085] A further aspect of the embodiments relates to Bifidobacterium longum DSM 32947, Bifidobacterium longum DSM 32948, and compositions comprising Bifidobacterium longum DSM 32947 and / or Bifidobacterium longum DSM 32948, such as pharmaceutical compositions, nutritional compositions, food supplements and probiotic compositions. In a particular embodiment, the bacteria of the bacterial strain, i.e., Bifidobacterium longum DSM 32947 and / or Bifidobacterium longum DSM 32948, are in a dry or lyophilized form.

[0086] Aspects include a probiotic composition comprising bacteria of a Bifidobacterium strain selected from Bifidobacterium longum subsp. longum DSM 32947, Bifidobacterium longum subsp. longum DSM 32948, and combinations thereof, and another probiotic strain, preferably a Lactobacillus strain, and more preferably a Lactobacillus reuteri strain, and in particular bacteria of a Lactobacillus reuteri strain selected from: Lactobacillus reuteri DSM 17938, Lactobacillus reuteri DSM 32846, and combinations thereof.

[0087] Further aspects include a probiotic composition comprising a probiotic strain, preferably a Lactobacillus strain, and more preferably a Lactobacillus reuteri strain, and in particular a Lactobacillus reuteri strain selected from Lactobacillus reuteri DSM 17938, Lactobacillus reuteri DSM 32846, and combinations thereof, and a conditioned medium from a Bifidobacterium strain selected from: Bifidobacterium longum subsp. longum DSM 32947, Bifidobacterium longum subsp. longum DSM 32948, and combinations thereof.

[0088] The bacteria comprised in the probiotic composition according to the above are preferably comprised in the probiotic composition as dried bacteria, such as freeze-dried or lyophilized, spray-dried or spray-freeze-dried or vacuum-dried bacteria.

[0089] The bacteria of Bifidobacterium longum subsp. longum DSM 32947 or Bifidobacterium longum subsp. longum DSM 32948 present in the above probiotic composition can be provided in dried form, such as in freeze-dried (lyophilized), spray-dried, spray-freeze-dried or vacuum-dried form. If the composition further comprises another probiotic strain, such as bacteria of Lactobacillus reuteri DSM 17938 and / or Lactobacillus reuteri DSM 32846, then these bacteria can also be provided in dried form in the composition, such as in freeze-dried (lyophilized), spray-dried, spray-freeze-dried or vacuum-dried form.

[0090] Bifidobacterium longum subsp. longum DSM 32947 and DSM 32948 have been modified (adapted or evolved) from parental strains through a multi-step selection process to improve growth and reduce problems regarding heterogeneous growth. Thus, Bifidobacterium longum subsp. longum DSM 32947 and DSM 32948 exhibit improved growth. These strains do not exist in nature as they have been forced to evolve, i.e., they are non-natural or non-naturally occurring bacterial strains.

[0091] The multi-step selection of Bifidobacterium from clinical samples involves isolating Bifidobacterium from clinical samples on MRS agar plates. To improve growth and reduce problems regarding heterogeneous growth, the bacteria are subjected to the following procedure:

[0092] 1. Streak plate on MRS agar plates and, after anaerobic incubation at 37 °C for 3 days, select colonies with good growth;

[0093] 2. Inoculate the selected colonies into MRS broth and incubate them at 37 °C during anaerobic conditions;

[0094] 3. Obtain samples and repeat steps 1 and 2 until the desired properties are observed; and

[0095] 4. Suspend the bacteria in 15% glycerol and store at –70 °C.

[0096] The above treatment improves both growth and problems regarding heterogeneous colony morphology.

[0097] By exposing the bacteria to at least one inducible treatment during cultivation according to the invention, the bacteria are modified and / or induced to produce (including increasing) the production of therapeutic MVs, and they preferably release these MVs into the culture medium. The inducible treatment involves a change in the culture conditions which strongly affects the MV production of the bacteria as compared to bacteria not exposed to the inducible treatment during cultivation.

[0098] To evaluate and / or measure the increase in MV production or the change in MV efficiency induced or generated due to different inducible treatments, different methods can be applied. One option is to quantify the MVs by, for example, using a Nanosight instrument or by using flow cytometry or by staining the membranes with a fluorescent dye and thereby quantifying the amount of MVs. For example, fluorescence can be measured (after washing) by using a plate reader (and comparing with a standard curve). A simpler method is to compare the pellet size or the size of the precipitated particles after centrifugation, or to measure the weight of the pellet. A larger particle size or a higher pellet weight means more MVs. Other methods for evaluating MV efficiency are to measure the activity of the MVs in more complex in vitro models or in vivo models, such as those disclosed in the Examples section.

[0099] An important metabolic process in the human body is purine metabolism, in which purines are metabolized and broken down by specific enzymes. Examples of such enzymes are ecto-5'-nucleotidase (CD73), a cell membrane-anchored 5'-nucleotidase that is regarded as a key enzyme in adenosine production. Some probiotics have the 5'-nucleotidase gene and produce active 5'-nucleotidase, and thus are able to produce adenosine. The 5'-nucleotidase activity and thus adenosine production may occur extracellularly, i.e., outside or on the surface of the bacteria, such that it can be present, for example, in the supernatant or other extracellular fluids produced by the bacteria. Thus, active 5'-nucleotidase can be present on the cell surface, for example, in the form of cell wall-anchored 5'-nucleotidase extracellularly in bacterial cells, for example, present in the supernatant and / or as MV membrane-bound 5'-nucleotidase. Thus, in this group of bacteria, the production of adenosine and / or the activity of 5'-nucleotidase (EC 3.1.3.5) can be used as a marker for determining the efficiency of MV production.

[0100] Other possible models include, but are not limited to, models currently used to evaluate the probiotic effects of bacterial strains. For example, this includes preclinical in vitro models in which intestinal motility or pain perception / signal transduction can be measured, which have confirmed typical discomfort associated with, for example, infantile colic and other functional gastrointestinal disorders. Several models are used herein to evaluate the potential effects on infantile colic, for example, the models described in Examples 1, 3, 4, 9, and 10. It also includes cell-based immunostimulation models in which selected cytokines can be evaluated. Another mechanism by which probiotics exert their effects is related to a decrease in mucosal permeability, i.e., protecting epithelial barrier integrity. Thus, the efficiency of MV can be measured in an epithelial permeability ETEC (enterotoxigenic Escherichia coli) challenge in vitro model. Relevant animal models can also be used to investigate the effects of different inductive treatments, and in addition, the effects can be evaluated in human clinical trials.

[0101] Therapeutic MV can optionally be administered to a mammal after preservation, for example, in the form of isolated therapeutic MV, as a probiotic composition further described hereinafter, or as bacteria from a probiotic strain and the processed culture medium, conditioned medium, or cytoplasm of the probiotic strain, such as a dry medium, including a freeze-dried (lyophilized), spray-dried, spray-freeze-dried, or vacuum-dried medium, conditioned medium, or cytoplasm.

[0102] One embodiment relates to a probiotic composition comprising bacteria of a probiotic strain and therapeutic MV produced by the probiotic strain or another probiotic strain. The probiotic strain and another probiotic strain are selected from Lactobacillus strains, Bifidobacterium strains, and combinations thereof.

[0103] In one embodiment, a probiotic strain or another probiotic strain has been exposed to an inducible biological treatment according to the embodiment. Thus, in one embodiment, the therapeutic MVs in the probiotic composition have been produced according to the embodiment by a method for producing therapeutic MVs. Thus, in one embodiment, the probiotic composition comprises bacteria of a probiotic strain and therapeutic microvesicles, wherein the therapeutic microvesicles are produced by the probiotic strain or another probiotic strain as follows: during cultivation, the bacteria of the probiotic strain or another probiotic strain are exposed to an inducible biological treatment to induce the bacteria to produce therapeutic microvesicles. The probiotic strain and the other probiotic strain are selected from Lactobacillus strains, Bifidobacterium strains, and combinations thereof. The inducible biological treatment is selected from co-culturing the bacteria with bacteria of another bacterial strain, culturing the bacteria in the presence of the conditioned medium from bacteria of another bacterial strain, and combinations thereof. The other bacterial strain is a Bifidobacterium strain and the other bacterial strain is different from the probiotic strain and the other probiotic strain.

[0104] In one embodiment, the therapeutic MVs are isolated from bacteria of a probiotic strain such as described above by exposing the bacteria to an inducible treatment during cultivation and then isolating the therapeutic MVs from the culture medium. The isolated therapeutic MVs can then be added to the isolated bacteria of the same probiotic strain used for producing the therapeutic MVs. In this case, the probiotic composition comprises a mixture of the isolated bacteria of the probiotic strain and the therapeutic MVs isolated from the bacteria of the probiotic strain. In another embodiment, the probiotic composition comprises bacteria of a first probiotic strain and therapeutic MVs isolated from bacteria of a second different probiotic strain. In the latter case, it is possible to combine the properties or characteristics of different probiotic strains by mixing the isolated bacteria of a probiotic strain with the therapeutic MVs isolated from the bacteria of another probiotic strain. It is also possible to have a probiotic composition comprising bacteria of a first probiotic strain and therapeutic MVs from the bacteria of the first probiotic strain and therapeutic MVs isolated from bacteria of a second different probiotic strain. In another embodiment, the probiotic composition comprises at least one bacterial strain and a mixture of therapeutic MVs from any of the at least one bacterial strain or therapeutic MVs produced by another bacterial strain.

[0105] The bacteria comprised in the probiotic composition according to the above are preferably comprised in the probiotic composition as dried bacteria such as freeze-dried or lyophilized, spray-dried or spray-freeze-dried or vacuum-dried bacteria.

[0106] As previously described herein, the probiotic strain is preferably a lactic acid-producing probiotic strain (e.g., Lactobacillus), such as Lactobacillus reuteri probiotic strain, and more preferably Lactobacillus reuteri DSM 17938 and / or Lactobacillus reuteri DSM 32846.

[0107] The experimental data presented herein show that therapeutic MVs produced and isolated from probiotic strains are not only able to recapitulate the beneficial effects of the MV-producing probiotics on gastrointestinal motility and pain signaling. Importantly, these therapeutic MVs are actually more effective than the probiotics themselves, as evidenced by the more rapid onset of beneficial effects caused by the therapeutic MVs compared to the bacteria. As the experimental data presented herein also show, therapeutic MVs isolated from probiotic strains have immunostimulatory effects. The MVs are able to reduce specific cytokines associated with autoimmune diseases, and they also show protection of epithelial barrier integrity. The experimental data reveal that the therapeutic MVs of the embodiments can be used to inhibit, treat, or prevent various diseases or conditions previously shown to be inhibited, treated, or prevented by the use of probiotics. In the same way, therapeutic MVs can be expected to produce similar general and / or specific effects when administered to a mammal as compared to probiotics when administered to a subject such as any mammal, and may also produce improved effects. As the experimental data presented herein also show, the production of MVs can be induced by bioprocessing during cultivation, such as by adding the supernatant from another bacterial strain to the probiotic strain, or by adding bacterial cells from a different bacterial strain to the probiotic strain during cultivation (so-called co-cultivation). Such inducible bioprocessing generates more effective or potent MVs in different models compared to MVs from non-induced or non-stimulated bacterial preparations.

[0108] The more rapid onset of beneficial effects visible with therapeutic MVs can be used in the probiotic compositions of the embodiments to achieve extended efficacy when administered to a mammal. Thus, the therapeutic MVs in the probiotic composition induce or produce an early effect in the mammal due to their more rapid onset, while the bacteria of the probiotic strains included in the probiotic composition induce or produce a later but generally extended effect. In addition, therapeutic MVs may also produce enhanced efficacy compared to the efficacy induced by the bacteria of the probiotic strains. This means that the probiotic compositions of the embodiments achieve significantly improved efficacy in mammals compared to administering only probiotics.

[0109] In other words, a composition containing both probiotic cells and therapeutic MVs provides advantages over compositions having either probiotic cells or MVs alone. In a composition containing both probiotic bacterial cells and therapeutic MVs, the more rapid onset of beneficial effects observed with the therapeutic MVs is combined with the extended effect of the probiotic cells to improve the rapid onset and extended efficacy of the probiotic composition when administered to a subject such as a mammal.

[0110] Accordingly, one aspect of the embodiments relates to a probiotic composition comprising a rapid-acting component in the form of therapeutic MVs from bacteria of a probiotic strain. The probiotic strain is selected from Lactobacillus strains, Bifidobacterium strains, and combinations thereof. The probiotic composition further comprises a slow-acting or extended component in the form of bacteria of a probiotic strain that produces MVs or another probiotic strain. The another probiotic strain is selected from Lactobacillus strains, Bifidobacterium strains, and combinations thereof. Thus, when administered to a subject, the rapid-acting component and the slow-acting component together produce improved, early-onset, and extended efficacy.

[0111] In one embodiment, the probiotic strain or another probiotic strain has been exposed to an inducible biological treatment according to the embodiments. Thus, in one embodiment, the therapeutic MVs in the rapid-acting component of the probiotic composition have been produced according to the embodiments by a method for producing therapeutic MVs. Thus, in one embodiment, the probiotic composition comprises a rapid-acting component in the form of therapeutic microbubbles produced by bacteria of a probiotic strain as follows: during cultivation, the bacteria are exposed to an inducible biological treatment to induce the bacteria to produce therapeutic microbubbles. The probiotic strain is selected from Lactobacillus strains, Bifidobacterium strains, and combinations thereof. The inducible biological treatment is selected from co-culturing the bacteria with bacteria of another bacterial strain, culturing the bacteria in the presence of conditioned medium from bacteria of another bacterial strain, and combinations thereof. The another bacterial strain is a Bifidobacterium strain and the another bacterial strain is different from the probiotic strain. The probiotic composition further comprises a slow-acting component in the form of bacteria of a probiotic strain or another probiotic strain. The another probiotic strain is selected from Lactobacillus strains, Bifidobacterium strains, and combinations thereof. The another probiotic strain is different from the another bacterial strain. When administered to a subject, the rapid-acting component and the slow-acting component together produce extended efficacy.

[0112] In one embodiment, the rapid-acting component has an earlier onset of efficacy in a subject compared to the slow-acting component. Thus, the rapid and slow with respect to the rapid-acting component and the slow-acting component define the relative onset of the efficacy induced by these components. In other words, compared to the slow-acting component, the rapid-acting component is the more rapid or more rapid-acting component, and when compared to the rapid-acting component in terms of inducing efficacy in a subject, the slow-acting component can be considered the more slow or more slow-acting component.

[0113] In one embodiment, the rapid-acting component is in the form of isolated therapeutic MVs from bacteria of a probiotic strain.

[0114] The above discussion regarding the use of the same or different probiotic strains for therapeutic MVs and bacteria presented also applies to this embodiment.

[0115] The probiotic composition of the embodiments can be used as a medicament and is particularly useful for treating gastrointestinal disorders.

[0116] One aspect of the embodiment defines a probiotic composition for use as a medicament and in particular for treating colic, which comprises bacteria of a probiotic strain and a therapeutic MV produced by said probiotic strain or another bacterial strain. One aspect of the embodiment also defines a probiotic composition for use as a medicament and in particular for treating colic, which comprises the above-mentioned fast-acting component and slow-acting component.

[0117] In one embodiment, the colic is infantile colic (also known as colic in infancy).

[0118] Other aspects of the embodiment define a probiotic composition comprising bacteria of a probiotic strain and a therapeutic MV produced by said probiotic strain or another bacterial strain, or a probiotic composition comprising a fast-acting component and a slow-acting component, for treating a disease selected from the group consisting of infant or child gastrointestinal disorders or diseases, gastrointestinal pain disorders, bone loss diseases, periodontal diseases, and combinations thereof.

[0119] The therapeutic MV can be produced by bacteria of the same probiotic strain included in the probiotic composition. Alternatively or additionally, the therapeutic MV can be produced by bacteria of another probiotic strain different from the bacteria included in the probiotic composition.

[0120] In one embodiment, the bacteria of the probiotic strain in the probiotic composition have been exposed to an inducible biological treatment according to the embodiment.

[0121] The therapeutic MV isolated from the bacteria of the probiotic strain can be used to treat colic, such as infantile colic. The probiotic strain is selected from the group consisting of Lactobacillus strains, Bifidobacterium strains, and combinations thereof.

[0122] The embodiment also relates to a therapeutic MV isolated from the bacteria of a probiotic strain, which can be used to treat a disease selected from the group consisting of infant or child gastrointestinal disorders or diseases, gastrointestinal pain disorders, bone loss diseases, periodontal diseases, and combinations thereof. The probiotic strain is selected from the group consisting of Lactobacillus strains, Bifidobacterium strains, and combinations thereof.

[0123] The therapeutic MV is preferably from a Lactobacillus reuteri strain, and even more preferably from Lactobacillus reuteri DSM17938 and / or Lactobacillus reuteri DSM 32846.

[0124] The isolated therapeutic MVs in the probiotic composition or the therapeutic MVs are preferably produced by bacteria of an inducible bioprocessed probiotic strain as disclosed herein. One embodiment relates to therapeutic microbubbles isolated from bacteria of a probiotic strain that is exposed to an inducible bioprocess during cultivation to induce the bacteria to produce therapeutic microbubbles. The probiotic strain is selected from Lactobacillus strains, Bifidobacterium strains, and combinations thereof. The inducible bioprocess is selected from co-culturing the bacteria with bacteria of another bacterial strain, culturing the bacteria in the presence of conditioned medium from bacteria of another bacterial strain, and combinations thereof. The other bacterial strain is a Bifidobacterium strain and wherein the other bacterial strain is different from the probiotic strain.

[0125] In one embodiment, the probiotic composition and / or the therapeutic MVs can alternatively be used to treat gastrointestinal disorders. The gastrointestinal disorders are preferably functional gastrointestinal disorders selected from the following: functional esophageal disorders such as functional heartburn, functional chest pain of esophageal origin, functional dysphagia, and globus; functional gastroduodenal disorders such as functional dyspepsia, aerophagia, unexplained excessive belching, chronic idiopathic nausea, functional vomiting, cyclic vomiting syndrome, and rumination syndrome; functional bowel disorders such as irritable bowel syndrome (IBS), functional constipation, functional diarrhea, and unexplained functional bowel disorders; functional abdominal pain syndrome such as functional abdominal pain (FAP), functional gallbladder and sphincter of Oddi disorders such as functional gallbladder disorders, functional biliary sphincter of Oddi disorders, and functional pancreatic sphincter of Oddi disorders; functional anorectal disorders such as fecal incontinence, functional anorectal pain, and functional defecation disorders; and pediatric functional gastrointestinal disorders such as infant reflux, infant rumination syndrome, infant cyclic vomiting syndrome, functional diarrhea, infant dyschezia, and functional constipation.

[0126] In a specific embodiment, the gastrointestinal disorders are selected from gastrointestinal motility disorders, gastrointestinal pain, colic, irritable bowel syndrome, and constipation.

[0127] In one embodiment, the infant or pediatric gastrointestinal disorder or disease is an infant gastrointestinal disorder or disease such as an infant functional gastrointestinal disorder or disease. In a specific embodiment, the infant gastrointestinal disorder or disease is selected from infant gastrointestinal motility disorders, infant gastrointestinal pain, infant colic, infant irritable bowel syndrome, infant food intolerance, infant constipation, infant diarrhea, infant reflux, infant rumination syndrome, infant dyschezia, infant functional constipation, and combinations thereof.

[0128] In another specific embodiment, the infant gastrointestinal disorder or disease is selected from infant colic or infant food intolerance and combinations thereof.

[0129] In another specific embodiment, the infant gastrointestinal disorder or disease is an infant gastrointestinal motility disorder, preferably infant constipation and / or infant diarrhea and combinations thereof.

[0130] In another specific embodiment, the infant gastrointestinal disorder or disease is an infant gastrointestinal motility disorder and / or infant colic.

[0131] In one embodiment, the infant or child gastrointestinal disorder or disease is a child gastrointestinal disorder or disease, such as a child functional gastrointestinal disorder or disease.

[0132] In one specific embodiment, the child gastrointestinal disorder or disease is selected from child reflux, child rumination syndrome, child functional diarrhea, child dyschezia, child functional constipation, and combinations thereof.

[0133] In another specific embodiment, the child gastrointestinal disorder is selected from child reflux, child dyschezia, and combinations thereof.

[0134] In one embodiment, the gastrointestinal pain disorder is selected from functional abdominal pain (FAP), colic, frequent recurrent abdominal pain (FRAP), and combinations thereof.

[0135] In one embodiment, the bone loss disease is selected from osteoporosis, osteopenia, and combinations thereof.

[0136] In one embodiment, the probiotic composition is used for treating osteoporosis or osteopenia.

[0137] In one embodiment, the periodontal disease is selected from periodontitis, gingivitis, and combinations thereof.

[0138] In another embodiment, the probiotic composition is used for treating periodontitis.

[0139] In a further specific embodiment, the gastrointestinal motility disorder is selected from bloating, recurrent obstruction, colic, constipation, gastroesophageal reflux disease, intractable recurrent vomiting, diarrhea, inflammatory bowel disease (IBD), fecal incontinence, frequent recurrent abdominal pain (FRAP), reflux, or food intolerance.

[0140] The embodiments also relate to the use of a probiotic composition or a therapeutic MV isolated from a probiotic strain as a medicament and for manufacturing a medicament for treating gastrointestinal disorders.

[0141] The embodiments further include a method for inhibiting, treating, or preventing a gastrointestinal disorder. The method includes administering to a subject a probiotic composition or a therapeutic MV isolated from a probiotic strain to inhibit, treat, or prevent a gastrointestinal disorder.

[0142] The experimental data presented herein also show that therapeutic MVs isolated from probiotic strains have immunostimulatory effects and are able to inhibit the secretion of IFN-γ and IL-17A. An effect of increasing IL-6 secretion was also observed. Thus, such therapeutic MVs can be used as modulators of human immunity.

[0143] When administered to a subject, the probiotic composition of the present invention and / or the therapeutic MV according to the present invention can be used to inhibit or reduce the amount of the cytokines IFN-γ and / or IL-17A. Accordingly, the probiotic composition and / or the therapeutic MV can be used to inhibit, treat or prevent diseases characterized by abnormal expression of IFN-γ and / or IL-17A, including inflammatory, autoinflammatory and autoimmune diseases. In a particular embodiment, the inflammatory, autoinflammatory and autoimmune diseases are selected from SLE, MCTD, RA, SS, DM, SSc, MS, psoriasis, bone loss, osteoporosis, osteopenia, periodontitis, gingivitis, sarcopenia, cachexia, malnutrition and allergies, such as AD, AR and asthma, as well as food intolerances and allergies.

[0144] When administered to a subject, the probiotic composition of the present invention and / or the therapeutic MV according to the present invention can be used to increase the secretion of the cytokine IL-6. IL-6 is a pleiotropic cytokine that has various functions in the body. Most of such functions and processes are associated with the inflammatory response, yet these functions and responses are important for, for example, protection against pathogens. However, the pro-inflammatory processes in the body must be well balanced, and Lactobacillus reuteri DSM 17938 has also been described as increasing the amount of regulatory T cells (e.g., Liu, Y., Fatheree, N. Y., Dingle, B. M., Tran, D. Q., & Rhoads, J. M. (2013). Lactobacillus reuteri DSM 17938 changes the frequency ofFoxp3+ regulatory T cells in the intestine and mesenteric lymph node inexperimental necrotizing enterocolitis.PloS One, 8 (2), e56547. http: / / doi.org / 10.1371 / journal.pone.0056547 )). The increased expression of IL-6 in combination with a higher frequency of regulatory T cells can be a way to make the immune system more vigilant and improve protection against infection without increasing the risk of inflammation. Accordingly, the probiotic composition and / or the therapeutic MV can be used to balance the anti-inflammatory and pro-inflammatory processes of the immune system.

[0145] Enterotoxigenic Escherichia coli (ETEC) is a type of Escherichia coli and one of the leading bacterial causes of diarrhea in developing countries and the most common cause of traveler's diarrhea. It is estimated that there are approximately 157,000 deaths per year due to ETEC, mostly in children. The main hallmarks of ETEC are the expression of one or more enterotoxins and the presence of pili for attachment to host intestinal cells.

[0146] As the experimental data provided herein show, therapeutic MVs isolated from probiotic strains protect epithelial barrier integrity from the deleterious effects of ETEC, which is a model used to study the effects of epithelial permeability. The main function of the intestinal barrier is to regulate the absorption of nutrients, electrolytes, and water from the lumen into the circulation and, on the other hand, to prevent pathogenic microorganisms and toxic luminal substances from entering the circulation. An intact barrier is thus essential for achieving a healthy state. A disrupted intestinal barrier is associated with several different diseases and conditions such as irritable bowel syndrome (IBS), Crohn's disease, depression, autism spectrum disorder, diverticulosis, periodontitis, osteopenia, and osteoporosis.

[0147] It has also been reported that intestinal barrier alterations may be the main driver of several cachectic features (Bindels et al. (2018)).

[0148] Epithelial barrier integrity is not only important in the gut. An intact barrier is also important, for example, in the oral cavity. The gingival epithelium is the first line of defense in the oral cavity against microbial attack. If disrupted, bacteria can collectively enter the underlying connective tissue, which can lead to inflammation and destruction of the tooth attachment apparatus (DiRienzo (2014)). Accordingly, probiotic compositions and / or therapeutic MVs can be used to inhibit, treat, or prevent diseases or conditions that cause disruption of epithelial barrier integrity, i.e., epithelial barrier dysfunction, including IBS, Crohn's disease, cachexia, osteopenia, osteoporosis, gingivitis, periodontitis, depression, autism spectrum disorder, diverticulosis, and / or to inhibit, treat, or prevent ETEC infection and to inhibit, treat, or prevent diarrhea and / or traveler's diarrhea caused by ETEC or other pathogenic bacteria.

[0149] Compared to probiotics, a faster onset of a medical or probiotic effect as seen with therapeutic MVs can be used to treat a subject suffering from a disease or disorder such as a gastrointestinal disorder, an inflammatory, autoinflammatory or autoimmune disease, an epithelial barrier dysfunction, an ETEC infection, diarrhea and / or traveler's diarrhea. This means that by administering a therapeutic MV or a probiotic composition comprising such therapeutic MVs, then a faster onset of a therapeutic effect such as a medical or probiotic effect can be obtained compared to administering only probiotics. The different timing of the onset of a medical or probiotic effect as seen between therapeutic MVs and probiotics as mentioned previously can also be utilized to achieve a prolonged medical or probiotic effect in a subject. Thus, a therapeutic MV administered to a subject will contribute to a rapid or immediate therapeutic effect in the subject, while a probiotic administered separately or together with the therapeutic MV to the subject will contribute to a slower or delayed therapeutic effect in the subject. Therefore, by administering a therapeutic MV and a probiotic separately or together to a subject, a prolonged medical or probiotic effect can be obtained, i.e., both immediate and delayed.

[0150] The appropriate mode of administration and formulation of a probiotic composition or a therapeutic MV can be selected based on the disease or disorder. The preferred mode of administration is oral. Other modes of administration include nasal, intraocular, topical or some other form of local administration to the skin, rectum, nose, eye, vagina or gums, or intravenous, subcutaneous or intramuscular injection.

[0151] Based on the disease or disorder to be treated, the mode of administration and the relevant formulation, the appropriate dose of a probiotic composition or a therapeutic MV as defined herein can be readily selected. For example, the dose and the dosing regimen are selected to ensure that a therapeutic MV or a probiotic composition administered to a subject according to the invention can result in the desired therapeutic effect, prophylactic effect or health benefit. Thus, preferably the dose is a therapeutically or prophylactically effective dose that is suitable for the subject to be treated and the type of disease or disorder. For example, a daily dose of bacteria of 10 4 to 10 10 , for example 10 5 to 10 9 , or 10 6 to 10 8 , or 10 8 to 10 10 total CFU can be used. The preferred daily dose is about 10 8 total CFU, for example 10 7 to 10 9 or 10 8 to 10 9 CFU of bacteria. For example, a daily dose of 10 4 to 10 14 , for example 10 5 to 10 13 can be used.6 to 10 12 or 10 8 to 10 12 or 10 10 to 10 12 or 10 10 to 10 14 the total daily dose of the number of therapeutic MVs. A preferred daily dose is about 10 10 therapeutic MVs, such as 10 9 to 10 11 or 10 10 to 10 11 the total number of therapeutic MVs. Another preferred daily dose is about 10 9 therapeutic MVs, such as 10 8 to 10 10 the total number of therapeutic MVs. Another preferred daily dose is about 10 8 therapeutic MVs, such as 10 7 to 10 9 the total number of therapeutic MVs. Another example would be to use MVs produced by a fixed number of bacteria, such as 10 8 or 10 9 CFU of bacteria.

[0152] The present invention also relates to a method for inhibiting, treating or preventing colic in a subject, particularly infantile colic and / or diseases selected from the following: gastrointestinal disorders or diseases in infants or children, gastrointestinal pain disorders, bone loss diseases, periodontal diseases, and combinations thereof. The method comprises administering to the subject a probiotic composition and / or therapeutic MVs according to the present invention.

[0153] As used herein, inhibiting a disease or disorder includes delaying the onset of the disease or disorder or symptoms associated with the disease or disorder.

[0154] The subject is preferably a mammalian subject, and more preferably a human subject.

[0155] As described in the embodiments disclosed herein, a protocol for the production and isolation of therapeutic MVs from the well-studied Lactobacillus reuteri DSM 17938 bacterial strain was described, with the aim of investigating and exploiting the probiotic effects of the probiotic strain and its therapeutic MVs. The probiotic effects of the isolated MVs were compared to those of the whole bacterium. It was found that the MVs were able to reproduce the beneficial effects of the whole bacterium in in vitro models to study gastrointestinal motility and the beneficial effects of the whole bacterium in in vitro models to study pain signaling. Surprisingly, not only did the MVs indeed reproduce the probiotic effects of the bacterium, but they were even more effective compared to the whole bacterium, as evidenced by their ability to act faster in the nerve signaling model, which led to an earlier onset of the observed effects. To further strengthen the results, another Lactobacillus reuteri strain (Lactobacillus reuteri DSM 32846) was investigated, which also showed similar effects. Additionally, when the bacteria were subjected to inductive treatments, including co-culture experiments with certain other bacterial strains or with conditioned media from other bacterial strains, which enhanced the ability of the bacteria to produce and release MVs, the effects of Lactobacillus reuteri DSM 17938 and Lactobacillus reuteri DSM 32846 could be further improved.

[0156] To study the therapeutic MVs from probiotic strains and their effects in different physiological models, the isolated MV fraction was isolated by culturing the bacteria followed by isolation of the released MVs. The present inventors have identified improved methods of culturing probiotics for increasing the production of MVs that retain and also display enhanced bioactivity. Examples

[0157] Example 1 – Alterations in Enzymatic Activity Associated with MV Production

[0158] Lactobacillus reuteri DSM 17938 was cultured and subjected to different inductive treatments at specific time points. The responses to these inductive treatments were determined using enzymatic assays and compared to the responses obtained with control treatments. The inductive treatments involved inductive stress on the bacteria during their growth phase, and then the effect on enzymatic activity was measured in the bacterial conditioned media.

[0159] Materials and Methods

[0160] Culture / sample collection

[0161] Lactobacillus reuteri DSM 17938 was inoculated from a frozen stock into 25 mL of de Man-Rogosa-Sharpe (MRS) medium under normal culture conditions, i.e., anaerobically cultured overnight at 37 °C. Then, the bacteria (20 mL) were re-inoculated into 200 mL of MRS, and different inducing treatments were applied, as described in more detail below. Bacterial samples were centrifuged at 5000x g for 10 minutes, the supernatant was transferred to a new tube, and then centrifuged at 10,000 x g for 10 minutes. The supernatant was filtered through a 0.45 μm filter and kept on ice, and then further centrifuged at 32 000 rpm for 3 hours at 4 °C using an ultracentrifuge (Beckman SW 32 Ti Rotor, swinging bucket, 30 mL tubes). The supernatant was discarded (poured out gently with the help of a pipette). The pellet was carefully resuspended in a resuspension medium (phosphate buffered saline (PBS)). Depending on the particle size, the resuspension volume varied between 100 – 300 μL. The samples were aliquoted and stored at -70 °C.

[0162] Oxygen treatment

[0163] Increased oxygen concentration (oxidative stress) was simulated by strong shaking of the bacterial culture. The oxidative stress simulation was continuously maintained under shaking for 24 hours.

[0164] Heat treatment

[0165] Temperature-induced stress was induced by raising the temperature from T = 37 °C to T = 50 °C at the time point when the optical density (OD) reached approximately 1.6 absorbance, and maintaining at T = 50 °C for 20 minutes. After this high-temperature treatment, the bacteria were cultured under normal conditions at T = 37 °C. The total culture time was 24 hours.

[0166] pH treatment shift

[0167] pH-induced stress was induced by lowering the pH from pH 6.5 to pH 2 at the time point when the optical density (OD) reached approximately 1.6 absorbance. The pH shift was obtained by centrifuging the bacterial cells and adding simulated gastric juice to the bacterial pellet to reach pH 2. The lowered pH was maintained for 10 minutes, and then the supernatant was added back to the bacterial cells to normalize the pH to 6.5. Since two different samples were obtained, the culture times were different. They were named differently as shown in Figure 1 pH: samples were obtained after 24 hours of culture time; pH + gastric juice: samples were obtained directly after 10 minutes of gastric juice induction.

[0168] Co-culture treatment

[0169] In this treatment, Lactobacillus reuteri DSM 17938 was co-cultured with Bifidobacterium longum DSM 32947 and Bifidobacterium longum DSM 32948 in SIM (synthetic intestinal medium, with the formulation described below) by adding the supernatant, i.e., the conditioned medium, from Bifidobacterium longum DSM 32947 or Bifidobacterium longum DSM 32948. As a control, Lactobacillus reuteri DSM 17938 was grown in SIM.

[0170] Table 1 - Formulation of SIM (synthetic intestinal medium)

[0171]

[0172] Adjust the pH to 6.8; autoclave at 121 °C for 15 minutes. Add the sterile-filtered sugar and electron acceptor solutions before inoculation. Final concentration: 15 mM each. Sugar: galactooligosaccharide (GOS) or glucose. Electron acceptor: citrate, 1,2-propanediol, or fructose.

[0173] Enzymatic activity

[0174] The samples obtained from the above-mentioned inducible treatment were thawed and then tested in a 5'-nucleotidase activity assay using the Crystal Chem 5’-Nucleotidase Assay Kit (Crystal Chem, Elk Grove Village, IL, USA). Briefly, the procedure was performed in two steps. First, reagent 1 (CC1) containing AMP was added to the supernatant samples to convert AMP to adenosine by any 5'-nucleotidase present in the supernatant samples. Adenosine was further hydrolyzed to inosine and hypoxanthine by the components in reagent 1. In the second step, reagent 2 (CC2) was added to convert hypoxanthine to uric acid and hydrogen peroxide, which were used to generate a quinone dye that was kinetically measured at 550 nm in a spectrophotometer. The 5'-nucleotidase activity in the samples was determined by calculating the absorbance change between 3 and 5 minutes and comparing it with the values from the calibration samples.

[0175] Results

[0176] As can be seen from Figure 1As can be seen from the results presented, 5'-nucleotidase activity was increased by oxygen treatment (DSM 17938 + O2), as well as by co-culture of Lactobacillus reuteri DSM 17938 with Bifidobacterium longum subsp. longum DSM 32947 or Bifidobacterium longum subsp. longum DSM in SIM medium (DSM17938 + DSM32947 in SIM or DSM17938 + DSM32948 in SIM).

[0177] Compared to Lactobacillus reuteri DSM 17938 grown as a control (DSM17938 in MRS medium or DSM17938 in SIM medium), none of the other inductive treatments induced any increase in 5'-nucleotidase activity.

[0178] Example 2 - Specific culture conditions are associated with increased MV production

[0179] Lactobacillus reuteri DSM 17938 was cultured and subjected to different inductive treatments. Nanoparticle tracking analysis (NTA) was used to determine the amount of MV produced as a result of these inductive treatments, and the results were compared to the responses obtained with control treatments. The inductive treatments involved oxygen treatment and sucrose treatment, and the effect on MV production was then measured in the bacterial conditioned medium.

[0180] Materials and methods

[0181] Oxygen treatment

[0182] Lactobacillus reuteri DSM 17938 was cultured under normal culture conditions, i.e., anaerobically in bottles / flasks at 37 °C in de Man-Rogosa-Sharpe (MRS) medium. Oxygen treatment was induced by strong shaking of the bacterial culture for 24 hours. As a control, Lactobacillus reuteri DSM 17938 was cultured under normal culture conditions, i.e., anaerobically in bottles / flasks at 37 °C in MRS medium without increasing the oxygen concentration (anaerobic treatment).

[0183] Sucrose treatment

[0184] Lactobacillus reuteri DSM 17938 was cultured under normal culture conditions, i.e., anaerobically in bottles / flasks at 37 °C in Lactobacillus Carrying Medium (LCM) medium. Stress was induced by adding sucrose (2% final concentration in LCM) to the bacterial culture at the start of fermentation. The total culture time was 24 hours. As a control, Lactobacillus reuteri DSM 17938 was grown under normal culture conditions with the addition of 2% glucose instead of 2% sucrose.

[0185] Nanoparticle tracking analysis (NTA)

[0186] The physicochemical characterization of MVs was investigated using NTA. The MVs were appropriately diluted with particle-free PBS (filtered through 0.02 µm) to obtain a concentration within the recommended measurement range (1 - 10×10 8 particles / ml), and directly tracked using a NanoSight NS300 system (NanoSightTM technology, Malvern, UK). The analysis was performed according to the following instrument settings:

[0187] 1. Sample loading: The sample was loaded into the O-ring top plate fixed to the laser module (laser beam of 488 nm) using an injection pump.

[0188] 2. Sample measurement: After optimizing the image, videos were acquired at 25 °C using a high-sensitivity sCMOS camera, and after capture at an injection pump speed of 50 in script control mode (3 videos / measurement for 90 seconds), analysis was performed using NTA software (version 3.2).

[0189] 3. Sample analysis: The sample was captured and analyzed by applying instrument optimization settings, which were the optimal visualization of particles adjusted by the application software (camera level, focus, and detection threshold) to optimize the analysis results for different samples. Further settings such as blur, minimum track length, and minimum expected size were set to "auto", and the viscosity was set to 0.890 cP. The NTA software was optimized, and then each particle was tracked on a frame-by-frame basis, and its Brownian motion was tracked and measured frame-by-frame by capturing the video file. The software individually tracked a number of particles and calculated their hydrodynamic diameter using the Stokes-Einstein equation. Multiple videos with a duration of 90 seconds were recorded, generating a repeated histogram averaged for each sample.

[0190] Results

[0191] Both oxygen treatment and the addition of sucrose resulted in an increase in MV production compared to the corresponding control, as shown in Table 2 below.

[0192] Table 2 - MV production

[0193]

[0194] Example 3 - Isolated Bacterial Microvesicles Replicated the Effect of Bacteria on Intestinal Motility

[0195] Materials and Methods

[0196] Animals

[0197] Adult male Swiss Webster mice (6 - 8 weeks old) were obtained from Charles River Laboratories (Wilmington, MA, USA). The animals were housed at 4 - 5 per cage under a 12 - hour light / dark cycle and had free access to food and water. Following cervical dislocation, subsequent procedures were performed in vitro according to the McMaster Animal Ethics Research Board (AREB) (permit 16 - 08 - 30).

[0198] Tissue flotation bath recordings

[0199] Tissue flotation bath recordings were performed as described by Wu et al. (2013). Jejunum and colon segments at least 4 cm in length were excised and fixed in a 20 - mL tissue flotation bath filled with oxygenated Krebs at 34 °C. The oral end of the segment was cannulated, and the contents were flushed from the lumen by gravity perfusion of Krebs, aerated with carbogen, using a Mariotte bottle. Once cleared, the anal end of the segment was cannulated to a silicon outflow tube. The luminal compartment was perfused with room - temperature Krebs at 5 mL / min. The serosal compartment was perfused with Krebs, aerated with carbogen and heated to 34 °C, at a rate of 2 mL / min. Oxygenated Krebs consisted of (mmol L -1 ) the following: 118 NaCl, 4.8 KCl, 25 NaHCO3, 1.0 NaH2PO4, 1.2 MgSO4, 11.1 glucose, and 2.5 CaCl2, bubbled with carbogen (95% O2 and 5% CO2). Prior to recording, the luminal pressure was adjusted to 2 - 3 hPa by increasing and decreasing the height of the inflow and outflow tubes. Treatments were applied by opening and closing separate cocks to stop the luminal flow of Krebs and initiate the flow of bacteria. Lactobacillus reuteri DSM 17938 was applied at a concentration of 8 - log colony - forming units (CFU) / mL. Conditioned medium from Lactobacillus reuteri DSM 17938, microbubbles produced by Lactobacillus reuteri DSM 17938, and conditioned medium with microbubbles removed were applied at a concentration equal to that of the whole bacteria.

[0200] Video recordings

[0201] A JVC video webcam placed 7 cm above the tissue section was used to record videos. Using NCHDebut Video Capture, videos were recorded and stored in MOV file format at a frame rate of 10 fps and an aspect ratio of 4:3. The recording duration varied from 20 minutes to 40 minutes. Using VideoPad Video Editor, with a forced aspect ratio of 4:3, the video was magnified to 4 cm. The video was converted to black and white by adjusting the color curve and applying a two-color filter. The black and white video was output at a resolution of 400 x 300 pixels at 10 fps.

[0202] Analysis

[0203] All generation, manipulation, and analysis of the spatio-temporal diameter maps were performed as described in Wu et al. (2013). The StMap plugin of NIHImage J software was used to analyze the video recordings. Using an edge detection routine, the diameter at each position across the intestine was represented as a hue value from 0 to 255. Intestinal contractions with smaller diameters were close to a hue value of 0 and were represented as darker black regions. Expanded or relaxed regions were close to a hue value of 255 and were white. The software generated spatio-temporal maps throughout the duration of the video. The map showed alternating dark and light hues based on the position, time, and diameter along the intestine. The spatio-temporal map ran from the oral cavity to the anus on the vertical axis and across time on the horizontal axis. The propagation contraction complex (PCC) velocity was determined by measuring the slope of large dark contractions. The PPC frequency was determined by measuring the number of contractions between intervals. The amplitude was measured as the height of the peak contraction (intestinal diameter).

[0204] Bacteria

[0205] Lactobacillus reuteri DSM 17938 from the original strain was grown in de Man-Rogosa-Sharpe (MRS) medium, harvested after 48 to 72 hours, washed in phosphate buffered saline (PBS), and aliquoted at 1.1 ml at 1 × 10 10 CFU / mL and stored at -20°C, and its microbubbles were separated as described below.

[0206] MV was isolated from Lactobacillus reuteri DSM 17938 broth culture (48 - 72 hours). After centrifugation at 600 × g for 30 minutes, the supernatant was filtered through a 0.22 µm filter and centrifuged at 100,000 × g in PBS twice, resuspended in sterile PBS with a volume corresponding to the initial Lactobacillus reuteri DSM 17938 culture, and representing 1 × 1012 A 0.5 ml aliquot of CFU / ml was stored at −80 °C. MV was quantified by the number of live bacteria in the reference culture and was also normalized by the protein content measured by NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA) (always 5–8 mg / ml protein, 25–60 ng / ml DNA, and 18–30 ng / ml RNA; n = 10). Unless otherwise stated, the MV preparation was used at an equivalent of 10 10 CFU / ml throughout the experiment.

[0207] Bacteria were diluted to a concentration of 8-log CFU / mL for later use.

[0208] Results

[0209] Lactobacillus reuteri DSM 17938 and its culture products were applied luminal to an in vitro preparation of the murine jejunum and colon to determine whether microvesicles produced by Lactobacillus reuteri DSM 17938 could recapitulate the effects of the parental bacterium on intestinal motility. Conditioned medium (CM) was defined as the growth medium (broth) in which Lactobacillus reuteri DSM 17938 bacteria had been cultured. The bacteria were separated from the CM by centrifugation, and the remaining CM was applied luminal to the in vitro intestinal preparation as described above. Microvesicles (MV) were isolated from Lactobacillus reuteri DSM 17938 cultured for 72 h by centrifugation and then resuspended in Krebs buffer. The conditioned medium remaining after removal of microvesicles and bacteria (CM-MV) was then applied luminal to the tissue. As a negative control, the growth medium (broth) used to culture the bacteria was applied separately. The effects of these treatments were compared to a Krebs buffer control, and three parameters of propagating contraction complexes (PCC) in the intestinal segments were measured: velocity, frequency, and amplitude.

[0210] The results were reproducibly confirmed with 24 h preparations.

[0211] Lactobacillus reuteri DSM 17938 and its products reduce small intestine motility

[0212] Jejunum PCC velocity

[0213] Lactobacillus reuteri DSM 17938, CM, and MV all reduced the PCC velocity in the jejunum to a similar extent. When applied luminal, Lactobacillus reuteri DSM 17938 significantly reduced the jejunal PCC velocity by 34% (p = 0.0067, n = 20) ( Figure 2a). CM reproduced the effect of the parental bacteria and reduced the jejunal PCC velocity by 29% (p = 0.0107, n = 28)( Figure 2 b). As a negative control, the broth used as the culture medium for the bacteria was tested independently and had a negligible effect on jejunal PCC velocity (5% reduction) compared to the Krebs control (p = 0.0877, n = 20)( Figure 2 c). Microvesicles isolated from 72-hour cultures significantly reduced the jejunal PCC velocity by 19% (p = 0.0002, n = 20)( Figure 2 d). When applied to the lumen, CM-MV did not alter the jejunal PCC velocity (p = 0.5203, n =20)( Figure 2 e).

[0214] Jejunum PCC frequency

[0215] A reduction in the PCC frequency in the jejunum was also produced by Lactobacillus reuteri DSM 17938, CM, and MV. Lactobacillus reuteri DSM 17938 significantly reduced the PCC frequency in the jejunum by 26% (p = 0.0482, n = 20)( Figure 3 a). Similarly, CM reduced the PCC frequency by 21% (p = 0.0139, n = 28)( Figure 3 b). When applied to the lumen, the broth did not significantly alter the jejunal PCC frequency (6% reduction) (p = 0.2424, n = 20)( Figure 3 c). Comparable to the bacteria, microvesicles significantly reduced the jejunal PCC frequency by 26% (p = 0.0004, n = 20)( Figure 3 d). The jejunal PCC frequency was not significantly affected by the luminal addition of CM-MV (p = 0.3408, n =20)( Figure 3 e).

[0216] Jejunum PCC amplitude

[0217] Except for microvesicles, the PCC amplitude in the jejunum was not significantly altered in any treatment group. Microvesicles reduced the jejunal PCC amplitude by 17% (p = 0.0453, n = 20)( Figure 4 d), although this effect was not present in the Lactobacillus reuteri DSM 17938 or CM trials (p = 0.3917, n = 20 and p = 0.1989, n = 28, respectively, Figure 4a and 4b). Broth and CM-MV did not alter the jejunal PCC amplitude (p = 0.8472 and p = 0.5627, n = 20) ( Figure 4 c and 4e).

[0218] Lactobacillus reuteri DSM-17938 and its products increase colonic motility parameters

[0219] Colonic PCC velocity

[0220] Colonic contractility was stimulated by the addition of Lactobacillus reuteri DSM 17938, CM or MV. Lactobacillus reuteri DSM 17938 significantly increased the contraction rate of PCC in the colon by 65% (p = 0.0004, n = 20) ( Figure 5 a). This was reproduced by CM, which significantly increased the PCC rate in the colon by 72% (p = 0.0021, n = 28) ( Figure 5 b). Broth continued to have a minor effect on gut motility, increasing the colonic PCC rate by 8%, but not within the 0.05 significance range (p = 0.1861, n = 20) ( Figure 5 c). Microbubbles significantly increased the PCC rate in the colon by 24% (p = 0.0051, n = 20), but to a lesser extent than that produced by Lactobacillus reuteri DSM 17938 and CM ( Figure 5 d). Luminal application of CM-MV failed to significantly alter the colonic PCC rate (p = 0.6475, n = 20) ( Figure 5 e).

[0221] Colonic PCC frequency

[0222] Lactobacillus reuteri DSM 17938, CM and MV all stimulated colonic motility by increasing the frequency of PCC contractions. Lactobacillus reuteri DSM 17938 significantly increased the colonic PCC frequency by 30% (p = 0.0231, n = 20) ( Figure 6 a). At the same volume, CM significantly increased the PCC frequency in the colon by 31% (p = 0.0073, n = 28) ( Figure 6 b). Broth increased the colonic PCC frequency by as little as 4%, but not significantly (p = 0.7219, n = 20) ( Figure 6 c). Similar to that visible for PCC rate, microbubbles increased the frequency of colonic PCC by 18% (p = 0.0424, n = 20); ( Figure 6 d). CM-MV did not significantly affect the PCC frequency in the colon (p = 0.3298, n = 20) (Figure 6 e).

[0223] Colonic PCC amplitude

[0224] The amplitude of PCC in the colon was not significantly affected by Lactobacillus reuteri DSM 17938 or any of the other treatment groups ( Figure 7 a - e).

[0225] Conclusion

[0226] Lactobacillus reuteri DSM 17938 has a region - specific effect on intestinal motility; it decreases jejunal and increases colonic PCC velocity and contraction frequency. This study confirmed that both microvesicles and conditioned medium reproduced the effect of Lactobacillus reuteri DSM 17938 on intestinal motility in both the small intestine and the colon. Additionally, these results were not seen when conditioned medium (CM - MV) was applied after microvesicle removal. All results have been summarized in Figure 8 .

[0227] These results confirm the role of microvesicles in probiotic signaling between Lactobacillus species and the host organism and its mechanism of action within the microbiome - gut - brain axis. This shows that microvesicles produced or shed by bacteria are responsible for the changes in intestinal motility induced by Lactobacillus reuteri DSM 17938.

[0228] Example 4 - Isolated Bacterial Microvesicles Reproduce the Bacterial Effect on Pain Signaling

[0229] In the presence of 10 µM capsaicin, the effect of microvesicles isolated from Lactobacillus reuteri DSM 17938 culture medium on pain signaling was tested using Jurkat cells expressing TrpV1.

[0230] Materials and Methods

[0231] Cell culture

[0232] Jurkat cells (clone E6 - 1 (ATCC® TIB - 152 6 , ATCC) were suspended at a concentration of approximately 5 × 10 TM cells / mL in 20 mL total volume of 2% fetal bovine serum (FBS) Roswell Park Memorial Institute (RPMI) medium.

[0233] Lactobacillus reuteri DSM 17938 was cultured, harvested, and stored as described in Example 3 above. Isolation and preparation of microvesicles from Lactobacillus reuteri DSM 17938 were also completed as described in Example 3 above (48 hours). The bacteria were diluted to 10 10Final concentration of CFU / ml and stored frozen at -80 °C until used in experiments.

[0234] Reference calcium flux measured by flow cytometry

[0235] Dissolve 50 μg each of two dyes, Fluo-3 AM (F1242; Sigma) and Fura Red AM (F3021; Sigma), in 100 μL of 0.1% pluronic acid (PLURONIC® F127 dissolved in dimethyl sulfoxide (DMSO)). Then add 50 μL of the Fluo-3 and 100 μL of the Fura Red solutions to 20 mL of Jurkat cells, resulting in a Fluo-3 / Fura Red ratio of 1:2.5. Then incubate the cells at 37 °C for 1 hour and wash with PBS (centrifuge at 300 × g g for 10 minutes). Then resuspend the cells in Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium containing 2% FBS.

[0236] On the day of the experiment, thaw the bacteria, wash three times in PBS, and then add to the cell culture. Alternatively, thaw, wash, and add microbubbles isolated from the bacteria (as described in Example 3) to the cell culture. Incubation continues for an additional hour at 37 °C. Then centrifuge the cell suspension as above and resuspend in PBS containing 1.25 mM Ca 2+ 2+.

[0237] All experiments were performed on a BD FACSCelesta (BD Bioscience, Mississauga, Canada) equipped with the following lasers: a blue laser emitting at 488 nm, a red laser emitting at 640 nm, and a violet laser emitting at 406 nm. Calibration was performed using BD PMT Beads (BD, Mississauga, Canada). Compensation was run with monochromatic BD CompBeads.

[0238] For FACS experiments, transfer 1 mL of the cell suspension to a 5 mL tube (Falcon 352235) with a cell strainer cap and centrifuge for 1 minute before analysis. Capsaicin was prepared from a 100 mM stock solution and diluted to 100 μM in PBS containing calcium and magnesium.

[0239] Record the background for 30 seconds corresponding to non-specific calcium fluxes. All samples are collected at a constant flow rate (number of cells / second) for a fixed time (30 or 60 seconds). Immediately before recording by FACSCelesta, 100 μl of capsaicin solution (resulting in a final concentration of 10 μM) is added to the cell suspension.

[0240] Recording continues at a rate of 400 - 600 events / second for a total of 30 to 60 seconds.

[0241] Both Fluo-3 and Fura Red are excited at 488 nm, where Fluo-3 emission is detected at 575 nm and Fura Red emission is detected at 610 nm. Data are collected in histograms showing the ratio of purple / blue Fluo-3 fluorescence relative to time and the ratio of purple / Fura Red fluorescence relative to time.

[0242] The reference analysis of Fluo-3 / Fura Red is measured by excitation via the blue laser (488 nm). Emission is detected through two different filter sets: the increase in emission is monitored by the purple laser (610 / 20 nm), and the decrease in emission is detected by the blue laser (575 / 25 nm). Using the Kinetics tool in FlowJo software (Tree Star Inc., OR, USA), the reference 'Fluo-3 / Fura Red Ratio' is calculated as the increase in signal stimulated by the purple laser divided by the decrease in signal stimulated by the blue laser (406 nm / 488 nm).

[0243] Results

[0244] Capsaicin itself or MV isolated from another bacterial strain JB-1 (MV-JB-1) is used as a control. Regarding the capsaicin-activated readout ( Figure 9 ) is calcium entry into Jurkat cells, which increases the ratio of the FLuo-3 / Fura Red ratio. Calcium entry into Jurkat cells is induced by capsaicin itself at 10 μM, and this response is significantly blocked by using MV isolated from Lactobacillus reuteri DSM 17938 (DSM-MV) at a high concentration corresponding to 10 11 CFU / mL of Lactobacillus reuteri DSM 17938. Corresponding to 10 9 - 10 10 CFU / mL of Lactobacillus reuteri DSM 17938 at a lower concentration of Lactobacillus reuteri DSM 17938MV has a similar but slightly reduced effect, while corresponding to 10 11High concentrations of MVs from Lactobacillus rhamnosus JB-1 at CFU / mL had no significant effect on the response to capsaicin treatment.

[0245] Example 5 - The onset of the effect of isolated bacterial microvesicles on spinal nerve discharge was faster compared to that obtained with whole bacteria.

[0246] The inventors have surprisingly observed herein that microvesicles from Lactobacillus reuteri DSM 17938 not only indeed reproduce the effect of whole bacteria on mesenteric nerve discharge, but also that the MVs are capable of generating an enhanced effect compared to whole bacteria. This finding was obtained by analyzing the amount of time taken from treatment initiation to peak response (onset of full effect) when using isolated Lactobacillus reuteri DSM 17938 microvesicles compared to using whole bacteria.

[0247] Materials and Methods

[0248] Lactobacillus reuteri DSM 17938 was grown, harvested, and stored as described in Example 3 above. Isolation and preparation of microvesicles from Lactobacillus reuteri DSM 17938 were completed according to Example 3 above.

[0249] Jejunal mesenteric nerve recordings were performed as previously described (Perez - Burgos et al. (2013); Perez - Burgos et al. (2015)). Briefly, a 3 cm segment of mouse jejunum was excised and fixed to an agar - coated Petri dish filled with oxygenated Krebs buffer and the L - type calcium channel blocker nifedipine (3 µM). The luminal contents of the jejunum were rinsed with Krebs, and the oral and anal ends were cannulated with silicone tubing to allow treatment to flow through the jejunal segment. Mesenteric nerve bundles were carefully dissected from the jejunal segment by gently scraping away the attached mesentery with fine forceps. The Petri dish containing the nerve preparation was then fixed to a microscope stage and continuously perfused with warm oxygenated Krebs using a pump.

[0250] Exposed nerve bundles were aspirated onto a glass micropipette attached to a patch - clamp electrode holder. Multi - unit electrical activity from the nerve bundles was recorded using a Multi - Clamp 700B amplifier and a Digidata 1440A signal converter. A control period of 15 - 30 minutes was recorded during luminal Krebs perfusion. Luminal Lactobacillus reuteri DSM 17938 or microvesicles were applied for a duration of 20 - 30 minutes immediately after the control.

[0251] The single-unit activity of multi-unit electrical activity was analyzed using principal component analysis (PCA) and spike waveform analysis in the Dataview program (Heitler (2007)). The time to reach the peak response was measured from the time of treatment initiation to the time of visible response (change in firing rate).

[0252] Results

[0253] As Figure 10 shown, compared to whole bacteria (DSM), Lactobacillus reuteri MV (MV) was able to initiate a neural firing response more rapidly, resulting in an earlier onset of effect.

[0254] Example 6 – Cultivation and Isolation Protocol

[0255] The general workflow for MV preparation includes the following steps: cultivation, removal of intact bacteria, and isolation of MV from the culture filtrate, and optionally preconcentration and purification. The choice of a particular method depends on many factors, such as the amount of material to be processed and the purity required for subsequent applications.

[0256] Culture conditions

[0257] The present inventors identified cultivation parameters for the bacterial strain that result in the production of therapeutic MVs and have improved effects in preferred models of gastrointestinal motility and gastrointestinal pain. Lactobacillus reuteri DSM 17938 was cultivated under normal cultivation conditions, i.e., anaerobically in a bottle / flask in de Man-Rogosa-Sharpe (MRS) medium at 37 °C for 24 hours with the addition of 2% sucrose as an inducer.

[0258] Isolation conditions

[0259] The preparation of bacterial MVs includes the steps of cultivating the bacteria as defined above followed by MV isolation. To obtain a high number, high purity, and MV fraction with retained biological effects, the bacterial supernatant was centrifuged at 4000 rpm for 20 minutes (Beckman high-speed centrifuge with JA-18 fixed-angle rotor), followed by a second centrifugation at 10,000 × g for 20 minutes, and then filtered through a 0.45 µm filter. These first steps removed live bacteria and large debris. The pellet was then discarded, and the supernatant containing MVs was loaded onto a sucrose cushion and ultracentrifuged at 118,000 × at 4 °C for 20.5 hours. Finally, the pellet was washed and centrifuged at 118,000 × at 4 °C g g ​Centrifuge overnight to obtain the therapeutic MV as a pellet.

[0260] Example 7 – Immunostimulation by Microvesicles (MVs) Derived from Lactobacillus reuteri DSM 17938

[0261] In this example, it has been shown that MVs from cell-free supernatants (CFS) derived from Lactobacillus reuteri DSM 17938 have immunostimulatory and interferon-γ (IFN-γ) inhibitory activities, showing that gut bacteria-derived extracellular MVs can be important regulators of human immunity.

[0262] Materials and Methods

[0263] Ethical statement and isolation of peripheral blood mononuclear cells

[0264] Healthy, anonymous adult volunteers (18 - 65 years old) were included in this study, and the study has been approved by the Regional Ethic’s Committee at Karolinska Institute, Stockholm, Sweden {Dnr 04 - 106 / 1 and 2014 / 2052 - 32}. All study subjects gave their written informed consent. Venous blood was collected in heparinized vacuum tubes (BD Biosciences Pharmingen) and diluted with RPMI-1640 cell culture medium supplemented with 20 mM HEPES (HyClone Laboratories, Inc.). Peripheral blood mononuclear cells (PBMCs) were then isolated by Ficoll-Hypaque (GE Healthcare Bio-Sciences AB) gradient separation. PBMCs were washed in RPMI-1640, resuspended in a freezing medium containing 40% RPMI-1640, 50% fetal bovine serum, and 10% DMSO, gradually frozen in a freezing container (Mr Frosty, Nalgene Cryo 1°C; Nalge Co.), and stored in liquid nitrogen.

[0265] In vitro stimulation of PBMC

[0266] PBMCs were thawed, washed, and viability was evaluated by trypan blue staining followed by counting with a 40× optical microscope. At 1×10 6At a final concentration of cells / ml, the cells were resuspended in cell culture medium (RPMI-1640 supplemented with HEPES (20 mM), penicillin (100 U / ml), streptomycin (100 μg / ml), L-glutamine (2 mM) (all from HyClone Laboratories, Inc.) and 10% fetal bovine serum (Gibco of Life Technologies)). The cells were seeded into flat-bottom cell culture plates and incubated at 37 °C in a 5% CO2 atmosphere. Staphylococcus aureus cell-free supernatant (CFS) was used as a stimulant at 2.5% (v / v), and isolated MVs from Lactobacillus reuteri DSM 17938 were added to PBMCs at MV / cell ratios of 500:1, 100:1, and 20:1.

[0267] Isolation of microvesicles

[0268] Lactobacillus reuteri DSM 17938 bacterial cells were grown in de Man Rogosa Sharpe medium (Oxoid) at 37 °C for 24 h. The bacterial cells were removed from the culture broth by centrifugation at 5,000 × g for 10 min at 4 °C, and then by a second centrifugation at 10,000 × g for 10 min at 4 °C. The supernatant was then filtered using a 0.45 µm pore filter (Millipore). The cell-free supernatant was concentrated using an Amicon Ultra filter unit with a MwCO of 100 kDa, which removed proteins and other molecules with a molecular weight below 100 kDa. The supernatant was loaded onto a 12% sucrose cushion with 50 mM Tris buffer pH 7.2 at a volume ratio of 5:1 and centrifuged at 118,000 × g for 3 h at 4 °C using a Beckman coulter Optima L – 80XP ultracentrifuge (Beckman coulter, USA). The supernatant was discarded, the pellet was resuspended in PBS buffer, and ultracentrifuged a second time (118,000 × g for a total of 3 h at 4 °C). The pellet was then dissolved in PBS, aliquoted, and stored at -70 °C.

[0269] Experimental procedures

[0270] Isolated MVs from Lactobacillus reuteri DSM 17938 were added to PBMCs at MV / cell ratios of 500:1, 100:1, and 20:1 and incubated for 48 h. Cell culture supernatants were collected and cytokine induction was analyzed using ELISA.

[0271] ELISA

[0272] Using a sandwich ELISA kit according to the manufacturer's instructions, the secretion levels of cytokines IL-1ra (R&D Systems - BioTechne), IL-1Β, IL-6, IL-10, IL-17A, and IFN-γ (MabTech AB) were measured in the cell culture supernatants. Absorbance was measured at a wavelength of 405 nm using a microplate reader (Molecular Devices Corp.), and the results were analyzed using SoftMax Pro 5.2 rev C (Molecular Devices Corp.).

[0273] Statistics

[0274] All statistical tests were performed using GraphPad Prism (GraphPad Software). All data were considered non-parametric, and thus Dunn's multiple comparison or Mann-Whitney t-test was employed. Differences were considered significant when p < 0.05, and the following significance levels were used: *p < 0.05; **p < 0.01.

[0275] Results

[0276] MVs from Lactobacillus reuteri DSM 17938 clearly induced the production of both IL-6 and IL-10 in a concentration-dependent manner, while no induction of IFN-γ or IL-17A was detected ( Figure 11 a). Furthermore, addition of the isolated MVs to Staphylococcus aureus-stimulated PBMCs significantly inhibited the secretion of IFN-γ and IL-17A to a similar extent as the high MV fraction ( Figure 11 b).

[0277] Example 8 – Lactobacillus reuteri-derived microvesicles (MVs) protect epithelial barrier integrity against the deleterious effects of enterotoxigenic Escherichia coli

[0278] Materials and methods

[0279] Isolation of extracellular microvesicles (MV)

[0280] Lactobacillus reuteri DSM 17938 bacterial cells were grown in Man-Rogosa-Sharpe medium, harvested after 24 hours, and centrifuged at 5,000 × g for 10 minutes at 4°C, and then centrifuged at 10,000 × g for 10 minutes at 4°C to remove them from the culture broth. Thereafter, any remaining cells were removed from the supernatant by filtration through a 0.45 µm pore filter. The supernatant was concentrated using an Amicon Ultra filter (100 kDa), which removed proteins and other molecules below 100 kDa. The supernatant was centrifuged at 118,000 × g for 3 hours at 4°C using a Beckman coulter Optima L – 80XP ultracentrifuge (Beckman coulter, USA). The supernatant was discarded, the pellet was resuspended in PBS buffer, and a second ultracentrifugation was performed (118,000 × g for a total of 3 hours at 4°C). Then the pellet was dissolved in PBS, aliquoted, and stored at -70°C.

[0281] In vitro intestinal permeability (Caco-2 / HT29 cell co-culture)

[0282] Epithelial cell culture (Caco-2 / HT29)

[0283] Caco-2 and HT29 cells were grown separately in Dulbecco's Modified Eagle Medium (DMEM) in tissue culture flasks at 37°C in an atmosphere of 5% CO2 and 90% relative humidity, the medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin and streptomycin. Caco-2 and HT29 cells were grown in 25 cm 2 tissue culture flasks and split at 80 - 90% confluence using a 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) solution. Cells were seeded at a density of 6×10 4 cells / 25-cm 2 flask.

[0284] Cell co-culture

[0285] Caco-2 and HT29 cells were seeded onto the apical chamber of Transwell inserts (Transwell-COL collagen-coated membrane filters) at a ratio of 9:1 and grown in 12-well Transwell plates, with 1×10 5 cells / cm 2Final density. Cells were maintained under the same conditions and allowed to grow for 21 days, with medium replacement (0.5 ml in the apical side and 1.5 ml in the basolateral side) every other day to allow the cells to become differentiated.

[0286] Cell layer integrity

[0287] Two methods were used to determine the integrity of the cell layer: determination of trans-epithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran (FITC-dextran) permeability.

[0288] The Millicell electrical resistance system (Millipore, Darmstadt, Germany) was used to determine the integrity of the cell monolayer during the experiment by measuring trans-epithelial electrical resistance (TEER). Three different areas were selected to detect the TEER value in each well, and the average value was the final result. A TEER value higher than 250 Ω cm 2 was used for permeability studies.

[0289] The inoculated Caco-2 / HT29 cells were pretreated with live Lactobacillus reuteri DSM 17938 cells at a multiplicity of bacteria (MOB) of 100, or extracellular microvesicles (MV) from Lactobacillus reuteri DSM 17938 cells at a multiplicity of MV of 200 for 6 hours, and then challenged with ETEC (pathogenic enterotoxigenic Escherichia coli, known for its destructive effect on epithelial integrity) at a multiplicity of infection (MOI) of 100 for an additional 6 hours. TEER was measured before pretreatment and challenge with ETEC, and subsequently at hourly intervals throughout the challenge. To quantify the paracellular permeability of the monolayer, 1 mg / mL of 4 kDa fluorescein isothiocyanate-dextran (FITC-dextran; Sigma) was added to the apical side of the insert at the onset of challenge with ETEC. After incubation for 6 hours, samples were taken from the basolateral compartment. Then, using a FLUOstar Omega Microplate Reader (BMGLabtech, Ortenberg, Germany), the diffused fluorescent tracer was analyzed in triplicate by fluorometry (excitation, 485 nm; emission, 520 nm).

[0290] Results

[0291] Challenge with ETEC induced a decrease in TEER. Both Lactobacillus reuteri-derived MV and bacterial cells were able to partially protect the epithelial monolayer from this challenge ( Figure 12 ). At 6 hours after ETEC challenge, the TEER decline reached 35% for the ETEC group, and pretreatment with both Lactobacillus reuteri bacterial cells and MV showed significantly higher TEER compared to the ETEC-treated group.

[0292] In the FITC-dextran flux experiment, the protective effects of Lactobacillus reuteri-derived MVs and bacterial cells against ETEC-induced monolayer damage were also evident ( Figure 12 ). Pretreatment of monolayers with Lactobacillus reuteri-derived MVs and bacterial cells reduced the leakage of FITC-dextran compared to the ETEC group.

[0293] Thus, both Lactobacillus reuteri-derived MVs and bacterial cells showed protective effects against ETEC-induced damage of Caco-2 / HT29 co-culture monolayers.

[0294] Example 9 - Alterations in Enzymatic Activities Associated with MV Production

[0295] Lactobacillus reuteri DSM 17938 and Lactobacillus reuteri DSM 32846 were cultured and subjected to a number of inductive biological treatments. With respect to alterations in MV production, the responses to these inductive treatments were determined using enzymatic assays and compared to the responses obtained for a control (i.e., bacterial cultures without inductive treatment). Enzymatic activities were then measured in the bacterial conditioned media.

[0296] Materials and Methods

[0297] Culture and biological treatment

[0298] Lactobacillus reuteri DSM 17938 or Lactobacillus reuteri DSM 32846 was inoculated from frozen stocks into 25 mL of de Man-Rogosa-Sharpe (MRS) medium under normal culture conditions, i.e., anaerobically cultured overnight at 37°C. Then, the bacteria (40 mL) were re-inoculated into 400 mL of SIM together with the supernatant (4%) from other bacterial cultures or by adding other bacterial cells (25%, washed and suspended in PBS), and then cultured for an additional 48 hours. The bacterial samples investigated are summarized in the table below.

[0299] Table 3 - Overview of Bacterial Samples and Treatments

[0300]

[0301] Bifidobacterium longum ATCC BAA-999 and Lactobacillus paracasei LMG-P-17806 are commercially available bacterial strains and have been deposited at the ATCC (American Type Culture Collection) and the Belgian Coordinated Collections of Microorganisms, Microbiology Laboratory, respectively.

[0302] The experimental setup involves probiotic strains and different biological treatments and has been designed to mimic the real-scale-up situation in a production setup or the situation in the human gastrointestinal tract. A 4% supernatant of the biological treatment during cultivation was chosen as the relevant concentration, which is on the one hand high enough to potentially have an effect, but on the other hand not too high due to the risk of having components from the inducible live bacteria as part of the final product. If two (or more) bacterial strains are administered together as a combined composition, a 25% cell biological treatment at a higher concentration was chosen to mimic the effect that would occur locally in the human gastrointestinal tract.

[0303] Sampling

[0304] The bacterial sample was first centrifuged at 5000 x g for 10 minutes. After that, the supernatant was transferred to a new tube and then centrifuged a second time at 10,000 x g for 10 minutes to remove bacterial cells and bacterial cell debris. The supernatant now containing the MV was filtered through a 0.45 μm filter and kept on ice, and then further centrifuged at 32,000 rpm for 3 hours at 4°C using an ultracentrifuge (Beckman SW 32 Ti Rotor, swinging bucket, 30 mL tubes). The supernatant was discarded (poured out gently with the help of a pipette). The pellet containing the MV was carefully resuspended in a resuspension medium (phosphate buffered saline (PBS)) and centrifuged again at 32,000 rpm at 4°C to wash away the remnants of the medium. Depending on the particle size, the resuspension volume varied between 100 – 300 μL. The samples were aliquoted and stored at -70°C.

[0305] Enzymatic activity

[0306] 5'-Nucleotidase activity was used as a measure of the change in the number and / or potency of MVs produced by different inducible biological treatments. Samples obtained from the above biological inducible treatments were thawed and then tested in a 5'-nucleotidase activity assay using the Crystal Chem 5’-Nucleotidase Assay Kit (Crystal Chem, Elk Grove Village, IL, USA). Briefly, the procedure was carried out in two steps. First, reagent 1 (CC1) containing AMP was added to the supernatant sample to convert AMP to adenosine by any 5'-nucleotidase present in the supernatant sample. Adenosine was further hydrolyzed to inosine and hypoxanthine by components in reagent 1. In the second step, reagent 2 (CC2) was added to convert hypoxanthine to uric acid and hydrogen peroxide, which was used to generate a quinone dye that was kinetically measured at 550 nm in a spectrophotometer. The 5'-nucleotidase activity in the sample was determined by calculating the change in absorbance between 3 and 5 minutes and comparing it with values from calibration samples.

[0307] Results

[0308] Figure 13 The 5'-nucleotidase activity (for control and treated samples) in MV samples obtained from Lactobacillus reuteri DSM 17938 is shown. As can be seen from the figure, compared with Lactobacillus reuteri DSM 17938 in SIM (control) or Lactobacillus reuteri DSM 17938 with 4% supernatant from Lactobacillus paracasei LMG-P-17806 (DSM 17938 in SIM or DSM 17938 + 4% LMG-P-17806 supernatant), the 5'-nucleotidase activity was increased in samples obtained from culturing Lactobacillus reuteri DSM 17938 in SIM medium with the addition of 4% supernatant from Bifidobacterium longum ATCC BAA-999 or Bifidobacterium longum DSM32947 (DSM 17938 + 4% DSM32947 supernatant or DSM 17938 + 4% ATCC BAA-999 supernatant). When Lactobacillus reuteri DSM 17938 was cultured with the supernatant from Bifidobacterium longum DSM32947, the effect of the inducible biological treatment on 5'-nucleotidase activity was the most significant, but an effect was also obtained with the supernatant from Bifidobacterium longum ATCC BAA-999. The optical density (OD) scores from each sample showed that the relative cell counts did not change significantly between treatments.

[0309] Figure 14 The results presented in Figure 13Same as presented in, but normalized with respect to the 5'-nucleotidase activity and optical density of DSM 17938 in SIM, making it easy to compare the fold change in 5'-nucleotidase activity between different experiments.

[0310] Figure 15 The results (normalized) in further show that by co-culturing Lactobacillus reuteri DSM17938 with 25% cells from Bifidobacterium longum DSM 32947 in SIM medium, the 5'-nucleotidase activity was increased after the inducible biological treatment compared to the control sample (i.e., DSM 17938 in SIM). The OD scores from these samples show the relative differences in the cell numbers obtained by these different biological treatments (i.e., the scores increased in the inducible biological treatment samples due to the higher total number of bacterial cells).

[0311] Figure 16 Shows the normalized values of 5'-nucleotidase activity obtained in the control and treated samples using Lactobacillus reuteri DSM 32846. As can be seen from the figure, the enzymatic activity was increased by culturing Lactobacillus reuteri DSM 32846 in SIM medium with the addition of 4% supernatant from Bifidobacterium longum DSM 32947 (DSM 32846 + 4% DSM 32947 supernatant) compared to the control Lactobacillus reuteri DSM 32846 in SIM (DSM 32846 in SIM). The OD scores from these samples show the relative differences in the cell numbers obtained by these different biological treatments.

[0312] Example 10 – Improvement in the antagonistic effect of Lactobacillus reuteri DSM 17938 on TrpV1-mediated pain signaling after co-culture with Bifidobacterium longum DSM 32947 or Bifidobacterium longum ATCC BAA-999

[0313] Using neurons expressing TrpV1 obtained from rat dorsal root ganglia (DRG) and the Cellectricon Cellaxess Elektra platform, the effect of a biological treatment on the production of MVs by Lactobacillus reuteri DSM 17938 was tested in an in vitro electric field stimulation (EFS) model. Lactobacillus reuteri DSM 17938 was cultured as a control in a simulated intestinal medium for 48 hours (DSM 17938 control SIM 48 h), or cultured in SIM for 48 hours using an inductive biological treatment consisting of co-culture with 25% bacterial cells of either of two Bifidobacterium longum strains, DSM 32947 or ATCC BAA-999. (The formulation of the SIM medium can be found in Example 1). MVs were isolated from different bacterial preparations according to Example 6. Primary rat DRG neuron cultures were cultured in 384-well plates together with nerve growth factor (NGF) for 48 hours to simulate peripheral sensitization. Then, the antagonistic effect of the obtained MV preparations on capsaicin-induced TrpV1 activation was evaluated. On the day of the experiment, DRG cultures were stained with a Ca 2+ indicator (Ca5; no-wash screening kit) to enable imaging of calcium transients triggered by capsaicin (a specific TrpV1 agonist). First, the EC 50 of capsaicin was determined in separate EFS experiments, and then this concentration was added to all DRG cultures to induce TrpV1 activation. Then, the effect of the MV preparations on TrpV1 activation was evaluated in a dose-response format (six concentrations were tested in triplicate in each plate), with the starting concentration being 1:10 of the original MV stock solution concentration and using a 1:3 dilution step. Before running the EFS experiment, DRG cultures were incubated with the MV preparations for 1 hour. The plates were placed on the Cellaxess platform, and a series of EFS protocols were applied. These EFS protocols included trains of pulses to capture excitability changes occurring in the DRG cultures due to incubation with the MV preparations. Then, the effect of the MV preparations on capsaicin-induced TrpV1 activation was analyzed, and the EC 50 value of each preparation was determined (EC 50 is generally described as the half-maximal effective concentration and refers to the concentration of a substance that induces a response halfway between the baseline and the maximum after a specified exposure time). Here, EC 50 represents the stock solution concentration (%) of MVs extracted from 400 ml of liquid bacterial culture). The experiment was performed twice and the mean EC 50 values were calculated. The EC 50 value of the control experiment was 7.4, while the EC 50Much lower values (1.8 for DSM 17938 + DSM 32947; 2.7 for DSM 17938 + ATCC BAA - 999). These results show that the inducible bioprocessing increases the antagonistic effect of the MV preparation on TrpV1 signaling, i.e., a smaller amount of the inducible MV preparation is required to inhibit capsaicin - induced TrpV1 signaling compared to the control MV preparation. An important aspect mentioned here is that the control (DSM 17938 SIM 48 hours) has previously shown an inhibitory effect on TrpV1 signaling (see Example 4) and can thus be considered a positive control. In summary, by exposing the bacteria to the inducible bioprocessing during cultivation according to the present invention, therapeutic MVs are induced in the bacteria. This in turn improves the inhibitory / blocking effect on capsaicin - induced TrpV1 activation.

[0314] Table 4 - Bacterial EC regarding TrpV1 inhibition 50 Value

[0315]

[0316] EC 50 The values are presented as the stock concentration % of the MV extracted from 400 ml of liquid bacterial culture and show an increased antagonistic effect of the MV preparation from DSM 17938 on TrpV1 in response to the inducible bioprocessing.

[0317] Example 11 - Immunomodulatory alterations due to bioprocessing during cultivation

[0318] In this example, it has been shown that the MV from the cell - free supernatant (CFS) of Lactobacillus reuteri DSM 32846 has a stronger immunomodulatory effect (increased IL - 6) compared to the MV from the cell - free supernatant (CFS) of Lactobacillus reuteri DSM 17938. It has also been shown that the immunomodulatory effect of the MV from the cell - free supernatant (CFS) of Lactobacillus reuteri DSM32846 or Lactobacillus reuteri DSM 17938 is improved after bioprocessing during cultivation.

[0319] Materials and methods

[0320] Ethical statement and isolation of peripheral blood mononuclear cells

[0321] The present study included healthy, anonymous adult volunteers (18 - 65 years old), and the study has been approved by the Regional Ethic’s Committee at the Karolinska Institute, Stockholm, Sweden {Dnr 04 - 106 / 1 and 2014 / 2052 - 32}. All study subjects gave their written informed consent. Venous blood was collected in heparinized vacuum tubes (BD Biosciences Pharmingen) and diluted with RPMI - 1640 cell culture medium supplemented with 20 mM HEPES (HyClone Laboratories, Inc.). Peripheral blood mononuclear cells (PBMC) were then isolated by Ficoll - Hypaque (GE Healthcare Bio - Sciences AB) gradient separation. PBMC were washed in RPMI - 1640, resuspended in a freezing medium containing 40% RPMI - 1640, 50% fetal bovine serum, and 10% DMSO, gradually frozen in a freezing container (Mr Frosty, Nalgene Cryo 1°C; Nalge Co.), and stored in liquid nitrogen.

[0322] In vitro stimulation of PBMC

[0323] PBMC were thawed, washed and the viability was evaluated by trypan blue staining followed by counting with a 40× optical microscope. At a final concentration of 1×10 6 cells / ml, the cells were resuspended in cell culture medium (RPMI - 1640 supplemented with HEPES (20 mM), penicillin (100 U / ml), streptomycin (100 µg / ml), L - glutamine (2 mM) (all from HyClone Laboratories, Inc.) and 10% fetal bovine serum (Gibco by Life Technologies)). The cells were seeded into flat - bottom cell culture plates and incubated at 37°C in a 5% CO2 atmosphere. Isolated MVs from different bacterial preparations, described in more detail below, were added to the PBMC at an MV / cell ratio of 500:1.

[0324] Isolation of microvesicles

[0325] Lactobacillus reuteri DSM 17938 or DSM 32846 bacterial cells were grown in SIM (simulated intestinal medium, the formulation described in Example 1) at 37 °C for 48 hours. For experiments in which different biological treatments were investigated, Lactobacillus reuteri DSM 17938 or DSM 32846 bacterial cells were grown in SIM at 37 °C for 48 hours in the presence of supernatant from separately grown Bifidobacterium longum DSM 32947 or Bifidobacterium longum ATCC BAA - 999. Additionally, Lactobacillus reuteri DSM17938 bacterial cells were grown in the presence of 25% bacterial cells from Bifidobacterium longum DSM 32947.

[0326] Bacterial cells were removed from the culture broth by centrifugation at 5,000 × g for 10 minutes at 4 °C, and then by a further centrifugation at 10,000 × g for 10 minutes at 4 °C. The supernatant was then filtered using a 0.45 µm pore filter (Millipore). The cell - free supernatant was concentrated using an Amicon Ultra filter unit with a MwCO of 100 kDa, which removes proteins and other molecules with a molecular weight below 100 kDa. The supernatant was centrifuged at 118,000 × g for 3 hours at 4 °C using a Beckman coulter Optima L – 80XP ultracentrifuge (Beckman coulter, USA). The supernatant was discarded, the pellet was resuspended in PBS buffer, and ultracentrifuged a second time (118,000 × g for a total of 3 hours at 4 °C). The pellet was then dissolved in Neurobasal A + supplement B27 + Glutamax, aliquoted, and stored at - 70 °C

[0327] Experimental procedures

[0328] Isolated MVs from the different bacterial preparations above were added to PBMCs at an MV / cell ratio of 500:1 and incubated for 48 hours. Cell culture supernatants were collected and cytokine induction was analyzed using ELISA.

[0329] ELISA

[0330] According to the manufacturer's instructions (MabTech AB), a sandwich ELISA kit was used to measure the secretion level of the cytokine IL-6 in cell culture supernatants. Absorbance was measured at a wavelength of 405 nm using a microplate reader (Molecular Devices Corp.), and the results were analyzed using SoftMax Pro 5.2 rev C (Molecular Devices Corp.).

[0331] Results

[0332] Compared to the microvesicles isolated from Lactobacillus reuteri DSM 17938, the microvesicles from Lactobacillus reuteri DSM 32846 were more effective in inducing IL-6 production ( Figure 17 ). In addition, compared to the control, the MVs isolated from both Lactobacillus reuteri strains increased the induced production of IL-6 after co-culture with Bifidobacterium longum strains (both 4% supernatant and 25% cells) ( Figure 18 , 19 and 20). These results showed that the isolated MVs were more effective after bioprocessing during cultivation.

[0333] Example 12 – Lactobacillus reuteri-derived microvesicles (MVs) protect epithelial barrier integrity against the harmful effects of enterotoxigenic Escherichia coli

[0334] Materials and methods

[0335] Isolation of extracellular microvesicles (MV)

[0336] Lactobacillus reuteri DSM 17938 or Lactobacillus reuteri DSM 32846 bacterial cells were grown in Man-Rogosa-Sharpe medium, harvested after 24 hours, and removed from the culture broth by centrifugation at 5,000 × g for 10 minutes at 4°C, and then at 10,000 × g for 10 minutes at 4°C. Any remaining cells were removed from the supernatant by filtration using a 0.45 µm pore filter. The supernatant was concentrated using an Amicon Ultra filter (100 kDa), which removed proteins and other molecules with a molecular weight below 100 kDa. The supernatant was centrifuged at 118,000 × g for 3 hours at 4°C using a Beckmancoulter Optima L – 80XP ultracentrifuge (Beckman coulter, USA). The supernatant was discarded, the pellet was resuspended in PBS buffer, and a second ultracentrifugation was performed (118,000 × at 4°C)g (Total of 3 hours). Then the precipitate was dissolved in PBS, aliquoted, and stored at -70 °C.

[0337] In vitro intestinal permeability (Caco-2 / HT29 cell co-culture)

[0338] Epithelial cell culture (Caco-2 / HT29)

[0339] Caco-2 and HT29 epithelial cells were grown in Dulbecco's Modified Eagle Medium (DMEM) in tissue culture flasks at 37 °C in an atmosphere of 5% CO2 and 90% relative humidity. The medium was supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin and streptomycin. Caco-2 and HT29 cells were grown in 25 cm 2 tissue culture flasks and split at 80 - 90% confluence using a 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) solution. Cells were seeded at a density of 6×10 4 cells / 25-cm 2 flask.

[0340] Cell co-culture

[0341] Caco-2 and HT29 cells were seeded onto the apical chamber of Transwell inserts (Transwell-COL collagen-coated membrane filters) at a ratio of 9:1 and grown in 12-well Transwell plates, with a final density of 1×10 5 cells / cm 2 in each insert. Cells were maintained under the same conditions and allowed to grow for 21 days, with the medium replaced every other day (0.5 ml in the apical side and 1.5 ml in the basolateral side) to allow the cells to become differentiated.

[0342] Cell layer integrity

[0343] The integrity of the cell layer was determined using two methods: measurement of transepithelial electrical resistance (TEER) and determination of fluorescein isothiocyanate-dextran (FITC-dextran) permeability.

[0344] The integrity of the cell monolayer during the experiment was determined by measuring transepithelial electrical resistance (TEER) using a Millicell resistance system (Millipore, Darmstadt, Germany). Three different areas were selected to detect the TEER value in each well, and the average value was the final result. A TEER value higher than 250 Ω cm 2 was used for permeability studies.

[0345] Inoculated Caco-2 / HT29 cells were pretreated with live Lactobacillus reuteri DSM 17938 or Lactobacillus reuteri DSM32846 cells at a multiplicity of 100 bacteria (MOB), or with extracellular microvesicles (MV) from Lactobacillus reuteri DSM 17938 or Lactobacillus reuteri DSM32846 cells at a multiplicity of 200 MV (MOM) for 6 hours, and then challenged with ETEC (pathogenic enterotoxigenic Escherichia coli, known for its destructive effects on epithelial integrity) at a multiplicity of infection (MOI) of 100 for an additional 6 hours. TEER was measured before pretreatment and challenge with ETEC, and subsequently every hour throughout the challenge period. To quantify the paracellular permeability of the monolayer, 1 mg / mL of 4 kDa fluorescein isothiocyanate-dextran (FITC-dextran; Sigma) was added to the apical side of the insert at the onset of challenge with ETEC. After incubation for 6 hours, samples were taken from the basolateral compartment. The diffused fluorescent tracer was then analyzed in triplicate by fluorometry (excitation, 485 nm; emission, 520 nm) using a FLUOstar Omega Microplate Reader (BMG Labtech, Ortenberg, Germany).

[0346] Results

[0347] Challenge with ETEC induced a decrease in TEER, showing that MV derived from Lactobacillus reuteri DSM 32846 were able to partially protect the epithelial monolayer from this challenge ( Figure 21 ). At 6 hours after ETEC challenge, the TEER decline in the ETEC group reached 27%, while pretreatment with MV derived from Lactobacillus reuteri DSM 32846 at 10, 50, 100, and 200 MOM showed significantly higher TEER compared to the ETEC-treated group. Untreated cells remained at approximately 90%.

[0348] In the FITC-dextran flux experiment, this protective effect of MV derived from Lactobacillus reuteri DSM 32846 against ETEC-induced damage to the monolayer was also evident ( Figure 21 ). Pretreatment of the monolayer with MV derived from Lactobacillus reuteri DSM 32846 at 10, 50, 100, and 200 MOM reduced the leakage of FITC-dextran compared to the ETEC group.

[0349] Therefore, MV derived from Lactobacillus reuteri DSM 32846 showed a protective effect against ETEC-induced damage to the Caco-2 / HT29 co-culture monolayer.

[0350] At Figure 22In this study, as shown in the FITC-dextran flux experiment, the protective effect of Lactobacillus reuteri DSM 32846-derived MVs was compared with that obtained with Lactobacillus reuteri DSM 17938-derived MVs. Pre-treatment of epithelial cell monolayers with Lactobacillus reuteri DSM 32846-derived MVs more effectively reduced the leakage of FITC-dextran, especially at lower concentrations of MVs, compared to Lactobacillus reuteri DSM 17938-derived MVs. This shows that Lactobacillus reuteri DSM 32846-derived MVs are more effective than Lactobacillus reuteri DSM 17938-derived MVs in protecting epithelial barrier integrity.

[0351] The above-described embodiments should be understood as several illustrative examples of the present invention. Those skilled in the art will understand that various modifications, combinations, and variations can be made to the embodiments without departing from the scope of the present invention. In particular, when technically possible, the different partial solutions in different embodiments can be combined in other configurations. However, the scope of the present invention is defined by the appended claims.

[0352] References

[0353] Abusleme, L. et al. IL-17; overview and role in oral immunity and microbiome. Oral Dis. 23(7): 854-865 (2017).

[0354] Bindels, L. et al. Increased gut permeability in cancer cachexia: mechanisms and clinical relevance. Oncoraget . Volume 9, (No.26), pp: 18224-18238, (2018).

[0355] DiRienzo, J. Breaking the Gingival Epithelial Barrier: Role of the Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin in Oral Infectious Disease. Cells. 3, 476-499, (2014).

[0356] Funauchi, M. et al. Serum level of interferon-gamma in autoimmune diseases, Tohoku J Exp Med. 164(4): 259-267 (1991).

[0357] Gu, Z. W. et al. Neutralization of interleukion-17 suppresses allergic rhinitis symptoms by downregulating Th2 and Th17 responses and upregulating the Treg response. Oncoraget . Volume 8, (No.14), pp: 22361-22369, (2017).

[0358] Heitler, W. J. DataView: A Tutorial Tool for Data Analysis. Template-based Spike Sorting and Frequency Analysis. J. Undergrad. Neurosci. Educ. 6,A1-7 (2007).

[0359] Klonowska, J. et al. New Cytokines in the Pathogenesis of Atopic Dermatitis – New Therapeutic Targets. Int. J. Mol Sci . 19, 3086 (2018).

[0360] Koga, C. et al. Possible Pathogenic Role of Th17 Cells for Atopic Dermatitis. Journal of Investigative Dermatology . 128, 2625-2630, (2008).

[0361] Lee, Youngkyun. The role of interleukin-17 in bone metabolism and inflammatory skeletal diseases. BMB Reports . 46 (1): 479-483 (2013).

[0362] Perez-Burgos, A. et al. Psychoactive bacteria Lactobacillus rhamnosus (JB-1) elicits rapid frequency facilitation in vagal afferents. Am. J. Physiol. Gastrointest. Liver Physiol. 304, G211–G220 (2013).

[0363] Perez-Burgos, A. et al. Transient receptor potential vanilloid 1 channel in rodents is a major target for antinociceptive effect of the probiotic L. reuteri DSM 17938. J. Physiol. 17, n / a-n / a (2015).

[0364] Pollard, K. M et al. Interferon-γ and systemic autoimmunity, Discov. Med. 16 (87), 123-131 (2013).

[0365] Tachibana, K. et al. IL-17 and VEGF are increased and correlated to systemic inflammation, immune suppression, and malnutrition in patients with breast cancer. International Journal of Immunopathology and Pharmacology . Volume 15(3) 219-228, (2017).

[0366] Wang, Y-H. et al. The cytokine family and their role in allergic inflammation. Curr Opin Immunol. 20(6): 697-702, (2008).

[0367] Wu, R. Y. et al. Spatiotemporal maps reveal regional differences in the effects on gut motility for Lactobacillus reuteri and rhamnosus strains. Neurogastroenterol. Motil . 25, e205–e214 (2013).

[0368] Zbikowska-Gotz, M. et al. Expression of IL17A concentration and effector functions of peripheral blood neutrophils in food allergy hypersensitivity patients. International Journal of Immunopathology and Pharmacology. Vol. 29(1), 90 - 98, (2015).

[0369] Zhang, J. et al. Changes of serum cytokines-related Th1 / Th2 / Th17 concentration in patients with postmenopausal osteoporosis. Gynecological Endrocrinology , 31:3, 183 - 190 (2015).

Claims

1. A method for producing therapeutic microbubbles, the method comprising: culturing bacteria of a probiotic strain in a culture medium, wherein the probiotic strain is selected from the group consisting of Lactobacillus strains, Bifidobacterium strains, and combinations thereof; and exposing the bacteria to an inducible biological treatment during the culturing to induce the bacteria to produce therapeutic microbubbles, wherein the inducible biological treatment is selected from co-culturing the bacteria with bacteria of another bacterial strain, culturing the bacteria in the presence of conditioned medium from bacteria of another bacterial strain, and combinations thereof, wherein the other bacterial strain is a Bifidobacterium strain, and wherein the other bacterial strain is different from the probiotic strain, wherein the probiotic strain is selected from Lactobacillus reuteri DSM 17938, Lactobacillus reuteri DSM 32846, and combinations thereof, and the other bacterial strain is selected from Bifidobacterium longum DSM 32947, Bifidobacterium longum DSM 32948, and combinations thereof.

2. The method according to claim 1, wherein exposing the bacteria comprises exposing the bacteria to an inducible biological treatment during the culturing to induce the production of therapeutic microbubbles and the release of the therapeutic microbubbles into the culture medium.

3. The method according to claim 2, further comprising separating the therapeutic microbubbles from the culture medium.

4. The method according to any one of claims 1 to 3, further comprising exposing the bacteria to an inducible abiotic treatment during the culturing to induce the bacteria to produce therapeutic microbubbles, wherein the inducible abiotic treatment is an oxygen treatment, wherein the oxygen treatment refers to exposing the bacteria to an increased concentration of oxygen, and the increased concentration of oxygen is a non-toxic concentration of oxygen.

5. The method according to claim 4, wherein the bacteria are agitated during culturing and / or bubbling.

6. Use of therapeutic microbubbles isolated from bacteria of a probiotic strain in the preparation of a medicament for treating infantile colic, wherein the probiotic strain is Lactobacillus reuteri DSM 17938, and wherein the therapeutic microbubbles are produced according to the method of any one of claims 1 - 5.

7. Use of therapeutic microbubbles isolated from bacteria of a probiotic strain in the preparation of a medicament for treating gastrointestinal disorders or diseases in infants or children, wherein the probiotic strain is Lactobacillus reuteri DSM 17938, and wherein the therapeutic microbubbles are produced according to the method of any one of claims 1 - 5.

8. The use according to claim 6 or 7, wherein the therapeutic microbubbles are in the form of freeze-dried microbubbles.

9. A bacterial strain, wherein the bacterial strain is Bifidobacterium longum DSM 32947 or Bifidobacterium longum DSM 32948.

10. The bacterial strain according to claim 9, wherein the bacterial strain is in a dried or freeze-dried form.

11. A probiotic composition comprising: bacteria of a Lactobacillus reuteri strain selected from: Lactobacillus reuteri DSM 17938, Lactobacillus reuteri DSM 32846, and combinations thereof; and Bacteria of a Bifidobacterium longum strain selected from Bifidobacterium longum DSM 32947, Bifidobacterium longum DSM 32948, and combinations thereof, or a conditioned medium from a Bifidobacterium longum strain.

12. Use of the probiotic composition according to claim 11 for the preparation of a medicament for treating infantile colic.

13. Use of the probiotic composition according to claim 11 for the preparation of a medicament for treating gastrointestinal disorders or diseases in infants or children.

14. Use of the probiotic composition according to claim 11 for the preparation of a medicament for treating gastrointestinal pain disorders.

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