Pulicaria incisa infusion for sustaining a healthy microbiome balance

Abstract

The invention concerns Pulicaria incisa (Pi) infusion, compositions and methods comprising Pi infusion for producing or maintaining microbiome balance in a human tissue or organ, and specifically for promoting the growth of probiotic bacteria and / or inhibiting the growth or spread of pathogenic bacteria, e.g., in the gastrointestinal system.

Description

[0001] Pulicaria incisa infusion for sustaining a healthy microbiome balance

[0002] TECHNOLOGICAL FIELD

[0003] The present disclosure generally relates to a novel method and composition for reshaping the microbiome in a human tissue or organ.

[0004] BACKGROUND ART

[0005] References considered to be relevant as background to the presently disclosed subject matter are listed below:

[0006] 1. Hooper, L. V. & Gordon, J. I. Commensal host-bacterial relationships in the gut. Science 292, 1115-1118 (2001).

[0007] 2. Lebeer, S. et al., Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens. Nat Rev Microbiol 8, 171-184 (2010).

[0008] 3. Ilinskaya, O. N. et al., Secretome of Intestinal Bacilli: ANatural Guard against Pathologies. Front Microbiol 8, doi:Unsp 1666 10.3389 / Fmicb.2017.01666 (2017).

[0009] 4. Hall-Stoodley, L. et al., Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2, 95-108 (2004).

[0010] 5. Kolter, R. & Greenberg, E. P. Microbial sciences: the superficial life of microbes. Nature 441, 300-302 (2006).

[0011] 6. Boles, B. R. & Singh, P. K. Endogenous oxidative stress produces diversity and adaptability in biofilm communities. Proceedings of the National Academy of Sciences of the United States of America 105, 12503-12508, (2008).

[0012] 7. Shemesh, M. et al., The biocide chlorine dioxide stimulates biofilm formation in Bacillus subtilis by activation of the histidine kinase KinC. J Bacteriol 192, 6352-6356 (2010).

[0013] 8. Yahav, S. et al., Encapsulation of beneficial probiotic bacteria in extracellular matrix from biofilm-forming Bacillus subtilis. Artificial Cells Nanomedicine and Biotechnology 46, 974-982 (2018). Kimelman, H. & Shemesh, M. Probiotic Bifunctionality of Bacillus subtilis- Rescuing Lactic Acid Bacteria from Desiccation and Antagonizing Pathogenic Staphylococcus aureus. Microorganisms 7, doi:ARTN 407 10.3390 / microorganisms7100407 (2019). Li, Z. H. etal. Biofilm-Inspired Encapsulation of Probiotics for the Treatment of Complex Infections. Adv Mater 30, doi:Artn 1803925 10.1002 / Adma.201803925 (2018). Lopez, D. et al., Biofilms. Cold Spring Harb Perspect Biol 2, a000398, doi: 10.1101 / cshperspect.a000398 (2010). Chu, F. et al., Targets of the master regulator of biofilm formation in Bacillus subtilis . Mol. Microbiol. 59, 1216-1228 (2006). Kearns, D. B. et al., A master regulator for biofilm formation by Bacillus subtilis. Mol Microbiol 55, 739-749 (2005). Vlamakis, H., Chai, Y., Beauregard, P., Losick, R. & Kolter, R. Sticking together: building a biofilm the Bacillus subtilis way. Nat Rev Microbiol 11, 157-168, doi: 10.1038 / nrmicro2960 (2013). Chu, F. et al. A novel regulatory protein governing biofilm formation in Bacillus subtilis. Mol Microbiol 68, 1117-1127 (2008). Burbulys, D. et al., Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay. Cell 64, 545-552 (1991). Shemesh, M. & Chai, Y. A combination of glycerol and manganese promotes biofilm formation in Bacillus subtilis via histidine kinase KinD signaling. J Bacteriol 195, 2747-2754 (2013). McLoon, A. L. et al., Spatial regulation of histidine kinases governing biofilm formation in Bacillus subtilis. J Bacteriol 193, 679-685 (2011). LeDeaux, J. R. et al., Different roles forKinA, KinB, andKinC in the initiation of sporulation in Bacillus subtilis. J Bacteriol 177, 861-863 (1995). Chen, Y. et al. A Bacillus subtilis sensor kinase involved in triggering biofilm formation on the roots of tomato plants. Molecular Microbiology 85, 418-430, (2012). Mhatre, E. et al. From environmental signals to regulators: modulation of biofilm development in Gram -positive bacteria. Journal of basic microbiology 54, 616-632 (2014). Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

[0014] BACKGROUND

[0015] The microbiota of the human gastrointestinal tract (GIT) exists in crosstalk that ranges between mutualism and pathogenicity, fostered by residential and ingested microorganisms. The normal microbiota contributes to food digestion and the development as well as the optimal functioning of the immune system [1], The Gram-positive Bacilli are prominent colonizers of the human GIT and thus have been widely used as probiotic species in clinical applications [2, 3].

[0016] It was recently postulated that biofilms defined as highly structured multicellular communities could allow bacteria to survive in hostile environments, such as oxidative and other environmental stresses [4-7], Accordingly, biofilm-inspired encapsulation could be applied to protect probiotic Bacilli coping with different environmental stresses [7-9], Since beneficial bacteria do not often survive during the transition in GIT, their biofilm-inspired encapsulation could enhance the efficiency and functionality of the probiotic treatment [8, 10],

[0017] Biofilm formation in B. subtilis depends on the synthesis of an extracellular matrix (ECM) that holds the constituent bacteria together

[0011] , The ECM has two main components, an exopolysaccharide synthesized by the products of the epsA-0 operon, and amyloid fibers encoded by the tapA-sipW-tasA operon [12-14], Both operons are under the direct negative control of two biofilm repressors, SinR and AbrB, which synergistically repress the matrix genes [13, 15], De-repression is triggered in part by the action of SinI

[0013] , a small protein antagonist of SinR. Expression of sinl is activated by SpoOA, whose activity is controlled collectively by members of the Kin family, including five histidine kinases (KinA-E) [16-19], These histidine kinases respond to different environmental cues, which contribute to the specificity of the outcome [17, 18, 20],

[0018] Several plant-derived carbohydrates have been linked to the signaling pathway for biofilm formation in B. subtilis and its close relatives [20, 21], Still, their effect in a complex environment, for instance, in combination with other biofilm effectors such as metal ions remained largely uncharacterized. The plant Pulicaria incisa (Lam.) DC. (Pi) is a desert plant grown in the Negev and Arava and belongs to the Asteraceae family. Pi infusion is consumed in place of tea by Bedouin residents in Israel, northern Sinai, Egypt, and Jordan.

[0019] GENERAL DESCRIPTION

[0020] In a first of its aspects, the present invention provides a composition for producing or maintaining microbiome balance in a human tissue or organ, wherein the composition comprises Pulicaria incisa (Pi) infusion and one or more physiologically acceptable carriers.

[0021] In another aspect, the present invention provides a Pulicaria incisa (Pi) infusion for use in producing or maintaining microbiome balance in a human tissue or organ.

[0022] In another aspect, the present invention provides a method of producing or maintaining microbiome balance in a human tissue or organ, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of Pulicaria incisa (Pi) infusion or a composition comprising Pulicaria incisa (Pi) infusion.

[0023] In another aspect, the present invention provides a method of treating or preventing gastrointestinal infection by pathogenic bacteria, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of Pulicaria incisa (Pi) infusion or a composition comprising Pulicaria incisa (Pi) infusion.

[0024] In one embodiment, said human tissue or organ is selected from a group consisting of gastrointestinal tract, skin, and respiratory tract.

[0025] In one embodiment, the composition can promote the growth of probiotic bacteria in the gastrointestinal system.

[0026] In one embodiment, the composition can promote biofilm formation by probiotic bacteria in the gastrointestinal system.

[0027] In one embodiment, the composition can inhibit the growth or spread of pathogenic bacteria in the gastrointestinal system.

[0028] In one embodiment, producing or maintaining microbiome balance comprises promoting the growth of probiotic bacteria and / or promoting biofilm formation by probiotic bacteria and / or inhibiting the growth or spread of pathogenic bacteria in the microbiome. In one embodiment the probiotic bacteria comprise probiotic bacilli selected from the group consisting of Bacillus species and Lactobacillus species.

[0029] In some embodiments, said Bacillus species is selected from a group consisting of B. subtilis, B. velezensis, and B. coagulans.

[0030] In one embodiment, said Lactobacillus species is selected from a group consisting of L. rhamnosus, L. plantarum and L. casei.

[0031] In some embodiments, said pathogenic bacteria are selected from the group consisting of Bacillus cercus, Staphylococcus aureus, Salmonella Typhimurium, and Clostridia species.

[0032] In some embodiments, said composition is in the form of a food article, a beverage, a food additive, a food supplement, or botanical drug.

[0033] In some embodiments, said composition is in the form of tablets, capsules, liquid syrups, nasal spray, nasal drops, soft gels, suppositories, patches, and enemas.

[0034] In some embodiments, said composition is a pharmaceutical composition, a nutritional composition, a prebiotic or synbiotic composition.

[0035] In one embodiment, said composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0036] In some embodiments, the pharmaceutical composition is administered by oral, intraperitoneal, intrarectal, subcutaneous, transcutaneous, intramuscular, intravenous, or topical administration.

[0037] In one embodiment, said composition is administered at a concentration of between about Img / kg and lOOmg / kg.

[0038] In one embodiment, said composition is provided orally at a concentration of between about 16mg / kg and about 50mg / kg.

[0039] In one embodiment, said composition is administered or consumed daily.

[0040] In one embodiment, the composition further comprises at least one probiotic commensal gastrointestinal bacterium.

[0041] In one embodiment, the probiotic commensal gastrointestinal bacterium is selected from the group consisting of Bacillus species and Lactobacillus species.

[0042] In some embodiments, the. Bacillus species is selected from a group consisting of B. subtilis, B. velezensis, and B. coagulans.

[0043] In some embodiments, the Lactobacillus species is selected from a group consisting of L. rhamnosus, L. plantarum and L. casei. In one embodiment, said composition is provided in combination with a probiotic or prebiotic product.

[0044] In one embodiment, said composition is enriched with one or more components of the Pi infusion.

[0045] In one embodiment, the one or more components of the E / infusion is myoinositol.

[0046] In another aspect, the present invention provides a probiotic product comprising live probiotic microorganisms and an infusion of the plant Pulicaria incisa (Pi).

[0047] BRIEF DESCRIPTION OF THE DRAWINGS

[0048] To better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0049] Fig- 1 is a schematic representation of an embodiment of the invention exemplifying an antagonistic mode of action triggered by Pi infusion during Bacilli biofilm formation.

[0050] Fig. 2A-B are GC / MS chromatograms of a metabolomic analysis of Pi infusion. Pi infusion was run on an SPE Sep-Pak C18 cartridge and eluted with water to yield the aqueous phase (2A), followed by methanol elution to yield methanolic phase (2B). Extracts were derivatized prior to analysis.

[0051] Fig. 3A-B are graphs showing the percentage (relative abundance) of probiotic bacilli (A) and pathogenic clostridia (B) in fecal samples of mice treated daily for 18 months with Pi infusion (Old Mice + Infusion (200mg / kg)), or (Old Mice + Infusion (600mg / kg)), as compared to untreated old mice (Old Mice + water), and untreated young mice. 16S rRNA library was prepared, sequenced, and microbiome analyses are presented as mean ± SEM (n=14). * p<0.05, ** p<0.01.

[0052] Fig. 4A-D are photographs showing pellicle formation at the air-liquid interface by Bacillus species following incubation of 24 hours in growth medium. B. subtilis, control without Pi infusion (4A); B. subtilis, with Pi infusion (4B); B. cereus, control without Pi infusion (4C); and B. cereus, with Pi infusion (4D).

[0053] Fig. 5A-D are photomicrographs showing microscopic imaging of B. subtilis (A, B) and B. cereus (C, D) following growth in either LB medium (A, C) or LB supplemented with the Pi infusion (B, D). Fig. 6A-D are graphs showing the effect of Pi infusion on growth and sporulation of B. subtilis and B. cereus. The graphs demonstrate the viable counts of B. subtilis (A) and B. cereus (B) growing in the presence of Pi infusion for 6, 8, or 16 hours. The data represent three independent experiments' average log CFU / ml and standard deviation. The graphs also show the sporulation rate (% spores vs. total cell counts) of B. subtilis (C) or B. cereus (D).

[0054] Fig. 7A-B are graphs showing ^-galactosidase activity (in Miler units) of the transcriptional reporters for two major matrix operons (Peps-lacZ and VtapA-lacZ) in response to Pi infusion as compared with control untreated cells.

[0055] Fig. 8A-C are graphs showing antagonistic activity of Bacillus subtilis against pathogenic bacteria. The pathogenic strains S. aureus (8A), Salmonella Typhimurium (8B), and B. cereus (8C) were grown for 24 h at 37°C in the presence of regular media (media) or conditioned media (spent media) obtained from cultures of wild-type (wt) laboratory strain B. subtilis (NCIB3610) that was cultured for 72 hours in LB supplemented with Pi infusion (Pi infusion 1, 3 and 5mg / ml) or with LB alone. The amounts of pathogenic bacteria are shown as log CFU / ml.

[0056] Fig. 9A-B are photographs showing induction of pellicle formation in Bacillus strains 3610, AT- 10 and AT- 16 grown in LB + Pi infusion, as compared with LB alone (A), and robustness of colony biofilm morphology of Bacillus strains 3610, AT- 10 and AT- 16 grown in LB + Pi infusion, as compared with LB alone. Images were taken with a Zeiss Stemi 2000-C microscope with an axiocam ERc 5 s camera (Zeiss). Fig. 9C is a graph showing the amount of Salmonella (log CFU / ml) after growing for 24 h at 37°C in the presence of LB or conditioned media obtained from cultures of wild-type (wt) laboratory strain B. subtilis (3610-CM), AT-10-CM or AT-16-CM that were cultured for 72 hours in LB supplemented with Pi infusion (5mg / ml) or with LB alone.

[0057] Fig. 10A-C are graphs showing antagonistic activity against Salmonella Typhimurium (A), Bacillus cereus (B), and Staphylococcus Aureus (C) by plant-isolated Bacillus strains. Bacillus strains (wildtype (wt), AT-10 and AT-16) were grown in the presence of Pi infusion (Img / ml). After 72 hours, their growth medium (conditioned medium - CM) was collected and used to challenge S. Typhimurium, B. cereus, and S. aureus (104CFU / mL). For control samples, pathogens were grown in either LB or LB+Pi infusion under identical conditions. The pathogen’s survival was determined in control and CM using the CFU counting. Fig. 11 is a graph showing the effects of supplementation of either B. subtilis alone or B. subtilis + i infusion (50 pg / mL) on the % survival of C. elegans exposed to B. cereus lawns (B, cereus-treated). *P<0.05, **P<0.01, and *** P<0.001.

[0058] Fig. 12 is a graph showing the amount of Bacillus cereus cells (B. cereus log CFU / ml) in cultures exposed to conditioned media of various B. subtilis biofilm mutant strains (surf, kind, tasA, luxS and eps) or wildtype B. subtilis (wt) which were either grown in LB or in the presence of Img / ml Pi infusion. For control samples, B. cereus was grown in either LB or LB + Pi infusion.

[0059] Fig. 13 is a graph showing the survival rate of B. cereus (log CFU / ml) in the presence of increasing amounts of Myo-inositol (%), with (+ B. sub) or without (- B. sub) supplementation of B. subtilis.

[0060] DETAILED DESCRIPTION OF EMBODIMENTS

[0061] The present invention is based on the surprising finding that an infusion prepared from the plant Pulicaria incisa (also referred to herein as Pi infusion) has a beneficial effect on the gut microbiota of healthy mice. B. subtilis was selected as a model organism for Gram-positive antagonistic (anti-pathogenic bacteria) Bacilli. Thus, it was discovered that Pi infusion triggers a robust biofilm formation by B. subtilis which presumably provides an optimal mode of growth, and that it induces an antagonistic activity of Bacillus strains against pathogenic bacteria, as demonstrated in vivo using a C. elegans model. It is thus postulated that these effects facilitate the establishment of a positive microbiota, as modelled for example in Fig. 1, and as demonstrated in vivo, see for example Fig. 3.

[0062] Accordingly, Pi infusion could be used to enhance and sustain the efficacy of healthy microbiota associated Bacilli either as such or in the context of functional probiotic and / or prebiotic foods.

[0063] The present invention thus provides nutritional and therapeutic methods and compositions comprising Pi infusion for sustaining a healthy microbiome balance in a human tissue or organ, e.g., in the gastrointestinal system, for example by increasing the number of probiotic bacteria and / or decreasing the number of pathogenic bacteria in the microbiome.

[0064] Furthermore, since the effects of the Pi infusion were evident upon oral administration to the mice, the present disclosure further encompasses administration of the Pi infusion as a nutritional supplement or functional food / beverages either alone or in combination with probiotic bacteria.

[0065] Particularly, the Pi infusion may be administered as a beverage (e.g., herbal infusion, tea, nutritional shake, or juice) suitable for daily consumption, or as a food product comprising probiotic bacteria, e.g., dairy products, or prebiotic dietary elements. In addition, the Pi infusion may be dried (e.g., prepared in the form of a powder) and incorporated as such into the food, beverages, probiotic, and / or prebiotic dietary products.

[0066] Therefore, in a first of its aspects, the present invention provides a composition for producing or maintaining microbiome balance in a human tissue or organ, wherein the composition comprises an infusion of the plant Pulicaria incisa (Pi) and one or more physiologically acceptable carriers.

[0067] As used herein the term “microbiome” refers to the community of microorganisms that exist in a particular part of the human body, for example in the gastrointestinal tract, the skin, or the respiratory tract (including the oropharynx and the lungs).

[0068] In various embodiments, producing or maintaining gastrointestinal microbiome balance comprises promoting the growth of probiotic bacteria in the gastrointestinal system, and / or promoting biofilm formation by probiotic bacteria in the gastrointestinal system, and / or inhibiting the growth or spread of pathogenic bacteria in the gastrointestinal system.

[0069] The composition may be a pharmaceutical composition or a nutritional composition.

[0070] As used herein the term “pharmaceutical composition” refers to a composition comprising an infusion of the plant Pulicaria incisa (Pi), wherein said composition is provided as a medicament. As used herein the term “nutritional composition” refers to a composition comprising an infusion of the plant Pulicaria incisa (Pi), wherein said composition is provided as a food supplement, nutrition additive, food, or beverage.

[0071] As used herein the term " Pulicaria incisa (Pi)” refers to a desert plant that belongs to the Asteracea family. The term refers both to wild-growing Pi, namely to uncultivated Pi plants that were not irrigated by human cultivators, as well as to cultivated Pi plants that are grown for agricultural purposes in an enclosed area of land (e.g., a field) and are irrigated by human cultivators. The terms "infusion of the plant Pulicaria incisa (Pi)" or “Pulicaria incisa (Pi) infusion” are used interchangeably herein and refer to an infusion of the aerial parts of Pi plants. The aerial parts of the plant include stems, leaves, and flowers. The infusion can be prepared using any method known in the art, for example by soaking dried aerial Pi parts in boiling water and removing the solid parts, e.g., by centrifugation and filtration. A non-limiting example of a method of preparing the Pi infusion is provided in the Examples below. The infusion can also be prepared from a freshly collected plant. As used herein, the term Pi infusion also encompasses a dried form of the infusion, for example a powder that can be incorporated into food products, beverages, probiotic dietary products, or prebiotic dietary products.

[0072] The terms “probiotic product” and “dietary probiotic product” are used interchangeably herein and refer to a composition or product comprising probiotic (i.e., b enefi ci al / advantageous) bacteri a.

[0073] The terms “prebiotic product” and “dietary prebiotic product” are used interchangeably herein and refer to a composition or product comprising one or more compounds that foster growth or activity of beneficial microorganisms such as bacteria and fungi. Dietary prebiotics are typically nondigestible fiber compounds.

[0074] Profiling of the infusion by gas chromatography-mass spectrometry revealed multiple components and identified chlorogenic acid, fructose, myoinositol sucrose, quercetin, and aucubin as some of its main constituents. A non-limiting example of the Pi infusion content is provided in Example 1, Table 1.

[0075] The term “sustaining a healthy microbiome balance” is used conventionally and refers to the management or care of a subject for the purpose of generating, maintaining, or improving the subject’s microbiome balance between probiotic bacteria and pathogenic bacteria. Sustaining a healthy microbiome balance comprises increasing the number of probiotic bacteria in the microbiome by promoting the growth of probiotic bacteria in the gastrointestinal system, the term also encompasses promoting biofilm formation by probiotic bacteria in the gastrointestinal system. The term further encompasses decreasing the number of pathogenic bacteria in the microbiome by inhibiting the growth or spread of pathogenic bacteria in the gastrointestinal system.

[0076] The compositions of the invention may be administered as such, or may be incorporated into food products, beverages (e.g., juices, herbal infusions, nutritional shakes, or tea), food additives such as corn syrup or probiotic products. The composition may also be provided as a dry powder for mixing in a food product or beverage.

[0077] In one embodiment, the composition of the invention is a nutritional composition administered by feeding.

[0078] In one specific embodiment, the composition of the invention is administered as a beverage.

[0079] In other embodiments, the composition of the invention is a pharmaceutical composition that can be administered and dosed in accordance with good medical practice. For example, the pharmaceutical composition can be introduced to the body by any suitable route including oral, intraperitoneal, intrarectal, subcutaneous, transcutaneous, topical, intramuscular, intraarticular, subconjunctival, or mucosal, e.g., intranasal, or intraocular administration. The compositions may be administered systemically or may be locally administered. Local administration may be facilitated by using an implant that acts to retain the active dose at the site of implantation. The active agent may be formulated for immediate activity, or it may be formulated for sustained release.

[0080] In yet some further embodiments, the composition of the invention may optionally further comprise at least one of pharmaceutically acceptable carrier / s, excipient / s, additive / s diluent / s and adjuvant / s.

[0081] More specifically, pharmaceutical compositions used to treat subjects in need thereof according to the invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, formulations are prepared by uniformly and intimately bringing into association the active ingredients of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0082] The compositions may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, nasal spray, nasal drops, soft gels, suppositories, patches, and enemas.

[0083] The Pi infusion may be diluted to a desired final concentration by mixing with an appropriate vehicle solution, e.g., water, a hydrophilic solution, physiological saline, herbal infusion (other than the Pi infusion), a nutritional beverage, juice, or ethanol. In addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question.

[0084] Still further, the compositions of the invention and any components thereof may be applied as a single daily dose or multiple daily doses.

[0085] In one embodiment, the “daily dose” refers to one administration of Pi infusion per day. In other embodiments, the “multiple daily doses” refers to two or more administrations of Pi infusion per day.

[0086] In another embodiment, the compositions are administered for an unlimited timeperiod (also referred to as chronic administration), for example for one year, two years or more, or throughout the subject’s lifespan. For example, the compositions may be given as a food supplement or a beverage for regular consumption.

[0087] The compositions may be administered once symptoms of microbiome imbalance become evident.

[0088] In another embodiment, the compositions are administered for treating and / or preventing ageing of the microbiome.

[0089] The composition of the invention can be administered alone, or in combination with other active agent(s). In certain embodiments the additional active agents include, but are not limited to, probiotic bacteria, myoinositol, herbs or herbal extracts or infusions (other than the Pi infusion), fruit juice, or an infusion enriched with one of its constituents.

[0090] The compositions of the present invention may be combined with other types of treatments including diet restrictions.

[0091] In another aspect, the present invention provides a method of producing or maintaining gastrointestinal microbiome balance, wherein the method comprises administering to a subj ect in need thereof a therapeutically effective amount of an infusion of the plant Pulicaria incisa (Pi) or a composition comprising an infusion of the plant Pulicaria incisa (Pi).

[0092] By the term “administering” . it is meant that the composition is delivered to a subject by any means or route which is effective to achieve the desired result, including e.g., oral, parenteral, enteral, intraperitoneal, topical, transdermal (e.g., using any standard patch), subcutaneous, intravenous, intra-arterial, intramuscular, buccal, sublingual, ophthalmic, nasal, by aerosol, by inhalation, rectal, vaginal, and intrathecal. In some embodiments the composition of the invention is administered to human subjects in an amount of between about 1 mg / kg to 100 mg / kg per day, depending upon the subject’s physical condition, the severity of disease, mode of administration, the content of the composition (namely, if the composition is enriched with one of the active components of the infusion, e.g., myoinositol, or if the composition is administered together with probiotic bacteria is may be administered at a lower concentration), etc. In specific embodiments the composition of the invention is orally administered to human subjects in an amount of about 16mg / kg (which approximately corresponds to an amount of 200 mg / kg in mice) or about 50 mg / kg (which approximately corresponds to an amount of 600 mg / kg in mice) per day.

[0093] The compositions can be administered at any suitable time, e.g., prior to or after a meal, prior to activity, prior to sleeping and at different times of the day, e.g., in the morning, in the evening etc.

[0094] EXAMPLES

[0095] Materials and Methods

[0096] Growing Pi plants and preparation of Pi infusion

[0097] Pi plant was grown in Arava (Israel). Pi plants were harvested and dried in the oven, at 30°C. The infusions were prepared as described previously (Elmann, A. et al. (2016) Journal of Functional Foods 25, 110-122). The infusions were filtered, and aliquots were stored at -20°C until use. To determine the concentration of each preparation, a sample of the infusion was lyophilized, and the powder was weighed.

[0098] Chemical characterization of Pi infusion

[0099] Sample preparation

[0100] The infusion was lyophilized (Alpha-Christ, Christ, Osterode am Harz, Germany) and stored at -20 °C. Samples (30 mg) and 50 pl of 0.2 mg / mL ribitol (Sigma Aldrich, St. Louis MO, USA) in methanol (Arcos, NJ, USA) as internal standard, were dissolved in 0.2 ml DDW (Synergy® UV Water Purification System, Merck, Darmstadt, Germany). A SEP-PAK Cis cartridge (Waters, Milford, MS, USA) was pre-washed with 1 ml methanol, followed by 1 mL H2O. The sample was loaded on the cartridge and eluted with 1.5 mL DDW. The aqueous phase was collected. Next, the cartridge was washed with 1.5 ml methanol, and the methanolic phase was collected. A 0.5 mL sample of each phase was lyophilized and then derivatized, according to an established protocol (Mahajna, S., et al., Molecules, 2019. 24 (22)).

[0101] Silylation derivatization

[0102] Briefly, 70 pL methoxyamine hydrochloride (Sigma, Aldrich, St. Louis MO, USA) solution (40 mg / mL in pyridine) was added to each lyophilized sample in an Eppendorf tube. Samples were then incubated in a thermoshaker for 90 min, after which, 100 pL N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA; Sigma, Aldrich, St. Louis MO, USA) were added. Samples were mixed in a thermoshaker at 30 °C for 30 min, and 70 pL were used for injection into GC / MS.

[0103] Gas chromatography-mass spectrometry analysis

[0104] GC / MS analysis was performed with an Agilent 6850 GC, equipped with Agilent 5975C single quadrupole MS, CTC-PAL RSI 85 auto-sampler, and HP-5MS capillary column (0.25 pm x 30 m x 0.25 mm). The following conditions were applied: injector temperature 250 °C, initial temperature 50 °C for 5 min, a gradient of 5 °C / min until 180 °C, a gradient of 10 °C / min until 270 °C and a hold time of 10 min, and increasing to 320 °C. The MS parameters were set as follows: source temperature 230 °C, transfer line 325 °C, quadrupole: 150 °C, detector 325, positive ion monitoring and electron ionization (EI)-MS measurement at 70 eV (Mahajna, S. et al. Molecules 2019, 24, 4139). Helium was used as a carrier gas, infused at 0.6 mL / min.

[0105] Data processing and compound annotation

[0106] Data were analyzed by the Unknown Analysis software (Agilent, Santa Clara, CA, USA). The percentage composition of the samples was computed from the GC peak areas. Library searches were conducted using the National Institute of Standards and Technology (NIST) 14 GC / MS Library and mass spectra from the literature. Componentrelative percentages were calculated based on GC peak areas without using correction factors. Commercially available standards were used for 2-hydroxybenzoic acid, 4- hydroxybenzoic acid, genistic acid, protocatechuic acid, quininic acid, gallic acid, ferulic acid, caffeic acid, (2R, -E) catechin, and quercetin (Sigma, Aldrich, St. Louis MO, USA). Processing of fecal samples for microbiome analysis

[0107] DNA Extraction

[0108] DNA was extracted from samples using 400pl Guanidium Thiocyanate (GT) lysis buffer and 20pl proteinase K (from Magcore Genomic DNA Tissue Kit) in beadbeating tubes type C (Geneaid). Beadbeating was performed for 2 minutes using a Biospec machine. Samples were then incubated at 60°Cfor 2 hours and extracted on Magcore machine (RBC Bioscience) using Magcore Genomic DNA Tissue Kit cartridges and protocol.

[0109] PCR1:

[0110] DNA was quantified by nanodrop and 3 pl (~15ng) was used as template for initial PCR. Amplification was performed using PCRbio Hot start ready mix using custom primers covering the V4 region primers from Earth Microbiome Project containing CS1 / CS2 adaptors for 20 cycles in a volume of 15 l.

[0111] SEQ ID NO: ! - CS1 515F (V4_F):

[0112] ACACTGACGACATGGTTCTACAGTGCCAGCMGCCGCGGT

[0113] SEP ID NO:2 - CS2 806R (V4_R):

[0114] TACGGTAGCAGAGACTTGGTCTGGACTACHVGGGTWTCT

[0115] Protocol PCR2:

[0116] A 2pl sample from the PCR1 amplified sample containing CS1 / CS2 adaptors was amplified for 10 cycles in lOpl using Fluidigm Access Array Barcode library according to manufacturer’ s protocol (2 pl barcode per reaction). DNA was purified using Kapa Pure Beads at a ratio of 0.65X and quantified with qubit using Denovix DsDNA high sensitivity assay. DNA size and integrity was quantified by Tapestation using Agilent DNA screen tape and reagents.

[0117] Sequencing

[0118] Samples were run on a dedicated Miseq (Illumina) machine with 30% PhiX using MiSeq Reagent Kit v2 250PE. Demultiplexing was performed using bc!2fastq with default parameters allowing for 1 mismatch. Data was then mapped to PhiX using bowtie2 and unmapped reads were quantified, collected and examined using fastQC.

[0119] Analysis

[0120] Demultiplexed reads were uploaded into CLC genomics workbench (Quigen) and analyzed using their 16S microbiome pipeline. The analysis workflow consisting of quality filtration of the sequence data, and operational taxonomic unit (OTU) clustering was performed with default parameter settings. The adaptor sequence was removed and the reads with a quality score lower than 25 or length <150 were discarded. The maximum number of acceptable ambiguou s nucleotides was set to 2 and the length of the reads was fixed at 200-500bp. Chimeric sequences and singletons were detected and discarded. The remaining unique reads were used for OTU clustering, which was performed by alignment to the SILVA database at 97% sequence similarity.

[0121] Sporulation efficiency assay

[0122] Bacterial strains were revived from -80°C stock, onto LB agar plates, and then one selected colony was grown for 16-18 h at 23 °C under shaking (150 rpm) conditions. Bacterial cultures were diluted (1 : 1000) in LB or LB with Pi (5mg / ml) and subsequently incubated at 37°C under shaking (150 rpm) for 16 h. The total colony forming unit (CFU) was determined at 6, 8, and 16 hours. For spores’ enumeration, Bacillus cultures were heat shocked at 80°C for 20min, while only spores could survive. Spores CFU were determined at 6,8,16 h of bacterial growth.

[0123] Characterizing the effect of Pi infusion on regulation and expression dynamics of the ECM operons in B. subtilis

[0124] The regulation assessment of the two ECM operons, tapA and eps, was conducted using two different types of transcriptional reporters for both operons. The colorimetric promoter-ZacZ reporter was used to quantify the expression levels of the ECM operons

[0017] ; whereas the fluorescently tagged reporters were used to study the expression dynamics of these operons in individual cells during growth and biofilm development using fluorescent microscopy [7],

[0125] Largely, two different experiments have been performed to profile the expression dynamics of the ECM operons by using the above reporter strains. First, through growing the ZacZ reporter harboring strains (fieps-lacZ and VtapA-lacZ) in the growth media supplemented by the Pi infusion prepared from different seasonal batches. Following the collections of cells periodically, the activities of Veps-lacZ and VtapA-lacZ were measured. Second, the Veps-gfp and tapA-cfp fluorescent reporter strains exposed to the Pi infusion prepared from different seasonal batches were applied. The cells were collected periodically from the suspensions and the expression of eps and tapA was examined under fluorescent microscopy.

[0126] Assessing in vitro and in vivo antagonistic activity of Bacillus induced in the presence of Pi infusion

[0127] Antagonistic Bacillus cells were grown in the presence of Pi infusion to collect a conditioned medium (CM). The CM was used to challenge the model enteropathogenic bacteria, such as Bacillus Salmonella and Staphylococcus (104CFU / mL). Their survivability was determined in the control and CM-treated samples using CFU counting. For in vivo experiments, the C. elegans model was used. This model was shown to be an appropriate system for characterizing the health-promoting activities of Bacillus subtilis (Donato et al., (2017) Nature Communications 8, 14332; Smolentseva et al., (2017) Scientific Reports 7, 7137). The C. aenorhabditis elegans N2 strain was continuously maintained at 25°C on nematode-grown media (NGM) with / i. co / z OP 50 as a food source, and synchronization was performed as described previously (Rajasekharan et al., (2018) Sci Rep 8, 11531). Freshly synchronized adult C. elegans were exposed to .A subtilis lawn for 12 or 24 h. After feeding, the nematodes were collected and washed with M9 buffer to remove the B. subtilis cells attached to the outer surfaces of the worms. The M9 buffer containing approximately 30 nematodes (per well) was transferred into 96 well plates. To this, different doses of Pi infusion were supplemented and incubated for 2 days for biofilm formation. The nematodes were subsequently transferred to a lawn containing a pathogen (Bacillus cereus expressing a red-fluorescent protein marker) for infection by incubation for 8 h. Controls were maintained by feeding the C. elegans with E. coli OP50 paired with Pi infusion. The infected nematodes were maintained in M9 buffer for 2 days at 25°C for the establishment of B. cereus biofilms inside C. elegans. The biofilm consortium inside C. elegans and the ability of B. subtilis biofilms to restrict an invading !?, cereus SNQXQ monitored by live-cell imaging and by counting live / dead nematodes by LED-based nematode bioassays. The nematodes were considered dead if they showed no response to blue LED light exposure for 30 seconds. Bacterial accumulation assays inside the nematodes were estimated by sonicating and killing the worms and subsequent plating of the bacterial populations inside the nematode. To achieve this, 400 mg of 1.0 mm silicon carbide particles were added to M9 buffer containing nematodes and vortexed. The process kills and disrupts the nematodes without affecting the bacterial populations. The resulting suspension was serially diluted and plated to estimate the CFU count.

[0128] EXAMPLE 1: Chemical characterization of Pi infusions

[0129] Pi infusions presented a rich metabolomic profile, comprised of a total of 88 compounds, including 13 organic acids (accounting for 6.5% of the total peak area of the methanolic phase), 4 alcohols (0.48%), 11 amino acids (0.97%), 26 sugars and saccharides (40.4%), 3 fatty acids (1.45%), 3 terpenes (8.37%), 35 phenolic compounds (34.8%) and 5 other compounds (6.8%). The main compounds in the 77 infusion and their relative contents in the methanolic and aqueous phases are presented in Table 1, and respective chromatograms are brought in Figure 2. In the methanolic phase saccharides including sucrose (15.35% of total peak area), fructose (11.65%), myo-inositol (6.42%) and talose (4.52) were most abundant, while chi orogenic acid and its derivatives (17.77%), quercetin and its derivatives (8.08%), and aucubin (2.79%) were the most abundant non-saccharides. Saccharides were also the most abundant group of compounds in the aqueous phase, mainly fructose (25.91%), myo-inositol (25.90%), talose (10.31%), and glucose (9.04%), in addition to citric acid (3.81%), galactaric acid (3.57%) and malic acid (1.92%) as the prevalent non-saccharides. The analysis revealed the presence of various chlorogenic acid isomers. Phenolic acids rarely appear in a free form, and typically are esterified with quinic or tartaric acid or a sugar moiety at different locations, giving rise to various structural isomers (Kumar and Goel, BiotechnoL Rep (Amst), 2019. 24: p. e00370). Specifically, chlorogenic acid is a group of caffeic and quinic acid esters, comprising six different isomers (3-O- caffeoylquinic acid (3CQA), 4CQA, 5CQA, 3,4- dicaffeoylquinic acid (3,4-DQA), 3,5-DQA and 4,5-DQA), or 26 compounds when considering cis-trans isomerization (Clifford et al., Food Chemistry, 2008. 106(1): p. 379- 385). These have also been reported to vary in occurrence, bioavailability, and bioactivity (Liang and Kitts, Nutrients, 2015. 8(1)). Similarly, flavonoids, e.g., quercetin, present characteristic versatility due to structural variability, owing to conjugation with various sugar moieties at various positions (Abu-Reidah, et al., Food Research International, 2017. 100: p. 494-500). As GC / MS metabolomic analysis cannot be used for reliable quantification, the current data only provided relative quantification of these compounds.

[0130] Table 1. Main compounds identified in the methanolic and aqueous phases of Pulicaria incisa infusion.

[0131] Compound Rt Total peak Total peak area (%) in the area (%) in methanolic the aqueous phase phase

[0132] Malic acid 21.884 1.925

[0133] 3-Aminophenol 25.702 1.414

[0134] Citric acid 27.333 1.034 3.806

[0135] Quininic acid 28.051 1.754

[0136] Fructose 28.259 11.647 25.913

[0137] Talose 28.634 4.517 10.309

[0138] Glucose 28.646 9.038

[0139] Myo-Inositol 29.433 6.418 25.897

[0140] Quinoline, 5-chloro-8-ethoxy-7- iodo- 29.442 1.825

[0141] Gal actaric acid 30.296 3.575

[0142] Caffeic acid 31.520 1.327

[0143] Sucrose 37.814 15.348 8.261

[0144] Turanose 38.323 1.240

[0145] Aucubin 39.131 2.792

[0146] Catechine (2R-E) 40.487 2.115

[0147] Chlorogenic acid 1 41.573 16.192

[0148] Quercetin 41.974 8.082

[0149] Chlorogenic acid 2 42.115 1.424

[0150] Metabolites were analyzed by GC / MS metabolomics following Cis SPE extraction with methanol (methanolic phase) and DDW (aqueous phase). Presented are metabolites that comprise at least 1% of the total ion count (total peak area) of the respective chromatograms. Quercetin and Quininic acid were identified by commercial standards. Fructose and Quercetin represent the sum of numerous derivative peaks, resulting from N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) derivatization at different locations of the molecules. Chlorogenic acids 1 and 2 may represent different isomers, or different derivatives of the same isomer.

[0151] EXAMPLE 2: Feeding mice with Pi infusion reshapes the microbiota composition.

[0152] To test whether Pi infusion affects the microbiota composition in mice, two months old C57BL / 6 mice were fed daily with Pi infusion (200 or 600 mg / kg, freshly thawed, in the drinking water). After 13 months of treatment, at the age of I S months (considered to be “old mice”), each mouse was transferred to a metabolic cage, and fecal samples were collected and kept at -80°C until DNA extraction. 16s library was prepared, sequenced, and analyzed as described in Materials and Methods above.

[0153] Daily treatment with Pi infusion significantly elevated the abundance of probiotic Bacilli, whereas the abundance of pathogenic Clostridia in the gut microbiome was notably reduced (Fig. 3). The results indicated that the components of the Pi infusion could selectively sustain healthy microbiota balance dominated by probiotic Bacillus species.

[0154] EXAMPLE 3: Pi infusion triggers biofilm formation of B. subtilis by activating a major biofilm-formation pathway.

[0155] The direct effect of Pi infusion on the physiology of several Bacillus species was investigated in vitro. The Pi infusion was supplemented to the growth media to assess a possible effect on bacterial growth, biofilm formation, and spore formation as well as antagonistic activity.

[0156] The bacterial strains were revived from a frozen (-80°C) stock, by placing onto Lysogeny broth (LB) agar plates [BD Difco, US], One selected colony was grown for 16- 18 h at 23°C under shaking (150 rpm) conditions. Bacterial cultures were diluted (1 : 1000) in LB or LB with Pi (l-5mg / ml) and subsequently incubated at 30°C as standing culture for 24 h.

[0157] The phenotypic observations consistently indicated induction of biofilm formation by B. subtilis, but not B. cereus in the presence of Pi infusion (Fig. 4). In addition to macroscopic visualization of the biofilm phenotype, extensive bundling and characteristic chaining were observed in B. subtilis cell cultures, but not in B. cereus cultures, following their exposure to Pi infusion (Figure 5). Bacteria were stained with LIVE / DEAD BacLight Bacterial Viability Kit (Invitrogen). Confocal microscopy was performed using a Leica SP8 laser scanning confocal microscope (Leica, Wetzlar, Germany), equipped with a HC PL APO CS2 63x / l .2 water immersion objective ( Leica, Wetzlar, Germany), solid state lasers with 488 / 552 nm light for excitation of live / dead stained bacteria and Leica Application Suite X software (LASX, Leica, Wetzlar, Germany). Live / Dead emission signals were detected with PMT and HyD (hybrid) detectors in ranges of 495-540 and 565-650 nm, respectively.

[0158] Furthermore, growth curve analysis shows that A. subtilis cells grow readily in the medium supplemented by the infusion (Figure 6A). However, the growth of B. cereus was apparently inhibited by the Pi infusion (Figure 6B).

[0159] The sporulation efficiency was unaffected in A. subtilis cultures, but it was notably induced in A. cereus even during early stages of growth (Fig. 6D).

[0160] A confocal laser scanning microscope (CLSM) was used to visualize B. subtilis morphology following exposure to 5 mg / mL of Pi infusion. Cyan fluorescent protein (CFP) expression of YC189 strain under the control of promoter for tapA was visualized using excitation wavelength of 543nm. The microscopic imaging of B. subtilis cells demonstrated that expression of the tapA operon was notably induced, and substantial biofilm formation was observed following exposure of B. subtilis to the infusion. Moreover, the activity of the eps and tapA promoters was quantified using the f>- galactosidase assay. Cells were harvested and re-suspended in phosphate-buffered saline (PBS) solution. The optical densities (OD) of the cell samples were normalized to an ODeoo of 1.0. One-milliliter aliquots of bacterial cell suspensions were collected and assayed as described by Chu et al., 2008

[0015] , A significant upregulation in the expression of both operons responsible for ECM production and biofilm formation was detected (Fig. 7 A, B).

[0161] EXAMPLE 4: Pi infusion triggers a robust antibacterial activity by B. subtilis

[0162] In addition to inducing biofilm formation, the Pi infusion also triggered in B. subtilis cells a robust antagonistic antibacterial activity (Fig. 8A-C). The antagonistic activity was assessed by growing the pathogenic strains S. aureus, Salmonella Typhi murium, and B. cereus for 24 h at 37°C in the presence of conditioned media obtained from cultures of wild-type (wt) laboratory strain B. subtilis (NCIB3610) that was cultured for 72 hours in LB supplemented with Pi infusion (Pi infusion 1, 3 and 5mg / ml). As can be seen in Fig. 8, the triggered antagonistic activity was found to be most significant against Gram positive bacteria rather than Grain negative Salmonella. Next, several Bacillus isolates derived from the Pi plant tissue were examined. Colonytype biofilm and pellicle formation were characterized for selected Pi plant tissue derived Bacillus strains (i.e., AT-10 and AT-16) grown in either LB (medium alone) or LB + Pi for 48h as described previously

[0017] , As can be seen in Fig. 9, supplementation of the Pi infusion triggered colony-type biofilm and pellicle formation in Bacillus strains and facilitated a potent antagonistic activity against Salmonella, as well as against other pathogenic bacteria, i.e., Bacillus cereus and Staphylococcus Aureus (Fig. 10).

[0163] EXAMPLE 5: Pi infusion enhances the survival of C. elegans exposed to pathogenic bacteria

[0164] To further substantiate the probiotic potential of B. subtilis cells, the effect of Pi infusion on their survivability and antagonistic activity were tested using the C. elegans (nematode) model.

[0165] Preparation of B. subtilis and B. cereus lawns

[0166] B. subtilis were first treated with 50 pg / mL of Pi infusion and grown in LB for 5 h. After incubation, the cells were taken, pelleted, resuspended in M9 buffer, and used to prepare lawns on LB agar.

[0167] Overnight cultures of B. cereus were incubated on LB plates for 48 h at 37 °C to produce lawns.

[0168] The nematode assay

[0169] Adult nematodes were placed in the B. subtilis lawns and were allowed to feed on these lawns for an overnight incubation. Next, the nematodes were washed and transferred to B. cereus lawns. The synchronized adult nematodes were placed in the plates and were allowed to feed on the B. cereus lawn for 6 hours at 22 °C, after which time the nematodes were collected and washed with M9 buffer. Approximately <30 nematodes were placed in each well in 96-well microtiter plates containing 200 mL of M9 media. Controls were maintained in the M9 medium alone. The survival rates of the nematodes were monitored for 7 days, and the results were expressed as percentages of live nematodes following three independent measurements. Non-treated groups (namely nematodes that were not placed on B. cereus lawns) have an almost 90% survival rate.

[0170] As can be seen in Fig. 11, pre-feeding on B. subtilis cells supplemented with the Pi infusion rescued the nematodes from the detrimental effect of later feeding on the pathogenic B. cereus lawns. Namely, B. subtilis cells supplemented with the Pi infusion significantly enhanced the survival rate of C. elegans.

[0171] EXAMPLE 6: B. Subtilis cells with the biofilm mutation AtasA are not antagonistic to pathogenic bacteria

[0172] Next, various B. subtilis strains which are mutated in genes related to biofilm formation were tested. These biofilm mutant cells included mutations in surf, kind, tasA, luxS and eps genes. The mutant cells were inoculated into either LB or LB supplemented with Pi infusion (1 mg / ml) for 72 h in 25C°. Conditioned media were collected and used to challenge Bacillus cereus cells. For control samples, B. cereus was grown in either LB or LB + Pi infusion under identical conditions. The bacterial survival was determined in control and CM-treated cultures using CFU counting. As can be seen in Figure 12, CM of various mutant bacillus subtilis strains supplemented with Pi showed an antagonistic activity against B. cereus. However, the AtasA mutant cells could not produce antagonistic substance in response toPi infusion. This result points to an involvement of the TasA protein in the observed antagonistic activity of B. subtilis against pathogenic bacteria.

[0173] TasA is a functional amyloid protein that forms fibers resistant to adverse physicochemical conditions that confer biofilms with structural stability [Romero et al., 2010 PNAS 107:2230-2234],

[0174] TapA appears to favor the transition of TasA into the fiber state, and the signal peptidase SipW processes both proteins into their mature forms [Camara-Almiron et al., 2020 Nature Communications 11 : 1859], The ability of amyloids to perform a transition from monomers into fibers represents a structural, biochemical, and functional versatility that microbes exploit in different contexts and for different purposes. One of the possible roles of TasA protein could be associated with antimicrobial activity during developmental stages of B. subtilis. it was indeed shown that the recombinant TasA has a broad-spectrum antibacterial activity [Stover and Driks, 1999 J Bacteriol 181 : 1664-1672], Without wishing to be bound by theory, it is therefore suggested that TasA protein inhibits other organisms in the environment, thus conferring a competitive advantage to B. subtilis cells during developmental stages.

[0175] EXAMPLE 7: Myo-inositol significantly enhanced the antagonistic activity of B. subtilis towards B. cereus

[0176] As shown in Example 1, GC / MS metabolomic analysis of the Pi infusion presented a rich metabolomic profile, comprised of a total of 88 compounds, including organic acids, alcohols, amino acids, sugars and saccharides, fatty acids, terpenes, phenolic compounds, and other compounds. The relative contents of myo-inositol in the methanolic and aqueous phases following column extraction with methanol (methanolic phase) and DDW (aqueous phase) are 6.42% of total peak area and 25.90%, respectively (Barak et al 2023, Antioxidants. 12: 32. https: / / doi.org / 10.3390 / antioxl2010032). As can be seen in Figure 13, treatment of B. subtilis with myo-inositol significantly enhanced its antagonistic activity towards B. cereus.

Claims

CLAIMS:

1. A composition for producing or maintaining microbiome balance in a human tissue or organ, wherein the composition comprises Pulicaria incisa (Pi) infusion and one or more physiologically acceptable carriers.

2. The composition of claim 1, wherein said human tissue or organ is selected from a group consisting of gastrointestinal tract, skin, and respiratory tract.

3. The composition of claim 1 or 2, capable of promoting the growth of probiotic bacteria in the gastrointestinal system.

4. The composition of any one of claims 1 to 3, capable of promoting biofilm formation by probiotic bacteria in the gastrointestinal system.

5. The composition of any one of claims 1 to 4, capable of inhibiting the growth or spread of pathogenic bacteria in the gastrointestinal system.

6. The composition of claim 3 or claim 4 wherein said probiotic bacteria comprise probiotic bacilli selected from the group consisting of Bacillus species and Lactobacillus species.

7. The composition of claim 6 wherein said Bacillus species is selected from a group consisting of B. subtilis, B. velezensis, and B. coagulans.

8. The composition of claim 6 wherein said Lactobacillus species is selected from a group consisting of L. rhamnosus. L. plantarum and L. casei.

9. The composition of claim 5 wherein said pathogenic bacteria are selected from the group consisting of Bacillus cercus, Staphylococcus aureus, Salmonella Typhimurium, and Clostridia species.

10. The composition according to any one of the preceding claims wherein said composition is in the form of a food article, a beverage, a food additive, a food supplement, or botanical drug.

11. The composition according to any one of the preceding claims wherein said composition is in the form of tablets, capsules, liquid syrups, nasal spray, nasal drops, soft gels, suppositories, patches, and enemas.

12. The composition according to any one of the preceding claims wherein said composition is a pharmaceutical composition, a nutritional composition, a prebiotic or synbiotic composition.

13. The composition according to claim 12 wherein said composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

14. The composition according to claim 13 wherein said pharmaceutical composition is administered by oral, intraperitoneal, intrarectal, subcutaneous, transcutaneous, intramuscular, intravenous, or topical administration.

15. The composition according to any one of the preceding claims wherein said composition is administered at a concentration of between about Img / kg and lOOmg / kg.

16. The composition according to claim 15 wherein said composition is provided orally at a concentration of between about 16mg / kg and about 50mg / kg.

17. The composition according to any one of the preceding claims wherein said composition is administered or consumed daily.

18. The composition according to any one of the preceding claims, further comprising at least one probiotic commensal gastrointestinal bacterium.

19. The composition according to claim 18 wherein the probiotic commensal gastrointestinal bacterium is selected from the group consisting of Bacillus species and Lactobacillus species.

20. The composition according to claim 19 wherein the Bacillus species is selected from a group consisting of B. subtilis, B. velezensis, and B. coagulans.

21. The composition according to claim 19 wherein the Lactobacillus species is selected from a group consisting of L. rhamnosus. L. plantarum and L. casei.

22. The composition according to any one of the preceding claims wherein said composition is provided in combination with a probiotic or prebiotic product.

23. The composition according to any one of the preceding claims wherein said composition is enriched with one or more components of the Pi infusion.

24. The composition according to claim 23 wherein said one or more components of the Pi infusion is myoinositol.

25. A Pulicaria incisa (Pi) infusion for use in producing or maintaining microbiome balance in a human tissue or organ.

26. The infusion of claim 25, wherein said human tissue or organ is selected from a group consisting of gastrointestinal tract, skin, and respiratory tract.

27. The infusion of claim 25 or 26, capable of promoting the growth of probiotic bacteria in the gastrointestinal system.

28. The infusion of any one of claims 25 to 27, capable of promoting biofilm formation by probiotic bacteria in the gastrointestinal system.

29. The infusion of any one of claims 25 to 28, capable of inhibiting the growth or spread of pathogenic bacteria in the gastrointestinal system.

30. A method of producing or maintaining microbiome balance in a human tissue or organ, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of Pulicaria incisa (Pi) infusion or a composition comprising Pulicaria incisa (Pi) infusion.

31. The method of claim 30 wherein producing or maintaining microbiome balance comprises promoting the growth of probiotic bacteria and / or promoting biofilm formation by probiotic bacteria and / or inhibiting the growth or spread of pathogenic bacteria in the microbiome.

32. The method of claim 30 or 31, wherein said human tissue or organ is selected from a group consisting of gastrointestinal tract, skin, and respiratory tract.

33. A method of treating or preventing gastrointestinal infection by pathogenic bacteria, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of Pulicaria incisa (Pi) infusion or a composition comprising Pulicaria incisa (Pi) infusion.

34. The method of claim 31 wherein said probiotic bacteria comprise probiotic bacilli selected from the group consisting of Bacillus species, and Lactobacillus species.

35. The method of claim 34 wherein said Bacillus species are selected from a group consisting of B. subtilis, B. velezensis, and B. coagulans.

36. The method of claim 34 wherein said Lactobacillus species are selected from a group consisting of L. rhamnosus. L. plantarum and L. casei.

37. The method of any one of claims 31 to 33 wherein said pathogenic bacteria are selected from the group consisting of Bacillus cercus, Staphylococcus aureus, Salmonella Typhimurium, and Clostridia species.

38. The method according to any one of claims 30 to 37 wherein said composition is in the form of a food article, a beverage, a food additive, a food supplement, or herbal drug.

39. The method according to any one of claims 30 to 38 wherein said composition is in the form of tablets, capsules, liquid syrups, nasal spray, nasal drops, soft gels, suppositories, patches, and enemas.

40. The method according to any one of claims 30 to 39 wherein said composition is a pharmaceutical composition, a nutritional composition, or a prebiotic composition.

41. The method according to claim 40 wherein said composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

42. The method according to claim 41 wherein said pharmaceutical composition is administered by oral, intraperitoneal, subcutaneous, transcutaneous, intramuscular, intravenous, or topical administration.

43. The method according to any one of claims 30 to 42 wherein said composition is administered at a concentration of between about Img / kg and lOOmg / kg.

44. The method according to claim 43 wherein said composition is provided orally at a concentration of between about 16mg / kg and about 50mg / kg.

45. The method according to any one of claims 30 to 44 wherein said composition is administered or consumed daily.

46. The method according to any one of claims 30 to 45, wherein said composition further comprises at least one probiotic commensal gastrointestinal bacterium.

47. The method according to claim 46 wherein the probiotic commensal gastrointestinal bacteria is selected from the group consisting of Bacillus species, and Lactobacillus species.

48. The method according to claim 47 wherein the Bacillus species are selected from a group consisting of B. subtilis, B. velezensis, and B. coagulans.

49. The method according to claim 47 wherein the Lactobacillus species are selected from a group consisting of L. rhamnosus. L. plantarum and L. casei.

50. The method according to any one of claims 30 to 49 wherein said composition is provided in combination with a probiotic product.

51. The method according to any one of claims 30 to 50 wherein said composition is enriched with one or more components of the Pi infusion.

52. The composition according to claim 51 wherein said one or more components of the Pi infusion is myoinositol.

53. A probiotic product comprising live probiotic microorganisms and an infusion of the plant Pulicaria incisa (Pi).

Images