Pro-biotic compositions and methods for treatment of stress

Abstract

A method of reducing stress in a subject includes consumption by or administration to the subject of a probiotic composition comprising Lactobacillus delbrueckii, Bifidobacterium bifidum, or Lactobacillus delbrueckii and Bifidobacterium bifidum. Other components such as non-pathogenic bacteria, fungi, and an enzyme may also be included.

Description

Attorney Docket No. BHMP106WO Pro-Biotic Compositions and Methods for Treatment of Stress CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. provisional application number 63 / 582,892, filed on September 15, 2023. This prior application is incorporated herein by reference in its entirety. FIELD

[0002] This disclosure relates generally to a method and composition of improving or balancing a microbiome of a human gut. More particularly, the disclosure relates to a method of treating stress, and / or balancing the level of Lactobacillus delbrueckii subsp. Bulgaricus and Bifidobacterium bifidum in the gut of a subject in need thereof. BACKGROUND

[0003] Lactobacillus delbrueckii subsp. Bulgaricus (“L. delbrueckii”) is a gram-positive, non- spore forming bacteria that plays a vital role in the fermentation of dairy products, particularly in the production of yogurts and other fermented milk products. L. delbrueckii, has been used as a probiotic to support gut health and gastrointestinal microbiome balance.

[0004] Bifidobacterium Bifidum (“B. bifidum”) is a gram-positive, anaerobic bacteria that is commonly found in the human gastrointestinal tract, particularly in infants and breast-fed babies. B bifidum plays a role in the fermentation of carbohydrates and fibers to produce beneficial short-chain fatty acids which are associated with overall colon and gut health.

[0005] Biofilms are formed when unicellular microorganisms live together and form a community that is protected by an exopolysaccharide (EPS) matrix. This EPS matrix typically is a conglomeration of proteins, polysaccharides and extracellular DNA. Biofilm-associated microorganisms differ from their planktonic (freely suspended) counterparts. It is believed that biofilm-forming cells co-aggregate with each other to form coordinated groups attached to a biotic or abiotic surface, the cells are surrounded by a protective EPS matrix, communicate effectively through quorum sensing, and have low metabolic activity that limits the impact of conventional antimicrobials (both antifungal and anti-bacterial agents) acting against actively metabolizing cells or cells in stationary phase.

[0006] Microorganisms, including bacteria, fungi, and archaea form biofilms. For a given subject (e.g., a human) many microorganisms (termed microbiota) exist on or within various regions of said subject, e.g., the gut, the skin, the vagina, the respiratory tract, and the mouth.Attorney Docket No. BHMP106WO There is an inextricable link between a host subject’s gastrointestinal microbiota and digestion, immunity and metabolism. Gastrointestinal microbiota are important for vitamin and metabolites biosynthesis and digestion of complex macromolecules such as polysaccharides. Gastrointestinal microbiota, under normal conditions, aid in prevention of colonization of pathogenic microorganisms and in maintaining the integrity and function of the intestinal barrier, and supporting the immune system.

[0007] However, undesirable or unbalanced biofilms may be involved in a significant percentage of human microbial infections (Potera, C. (1999) SCIENCE283:1837-8). It has been proposed that four criteria define biofilm etiology during an infection include whether the pathogenic bacteria or fungi are surface associated or adherent to a substratum; whether the bacteria are in clusters, encased in a matrix of bacterial, fungal or host constituents; whether the infection is localized; and whether the infection is resistant to antibiotic and anti-fungal therapies despite the antimicrobial sensitivity of the constituent planktonic organisms (Parsek, M. R. & Singh, P. K. (2003) ANN REV. MICROBIOL.57:677-701; Ghannoum, M., et al. (2015) MICROBIALBIOFILMS. Second Ed., Am. Soc. for Microbiology).

[0008] With respect to biofilms in the gut, it is now believed that bacteria and fungi can exist for example as biofilms on the colonic epithelium, within the mucus layer covering it, and on food particles in the lumen (MacFarlane, S. & MacFarlane, G. T. (2006) APPL. ENVIRON. MICROBIOL.72:6204-11; Probert, H. M. & Gibson, G. R. (2002) CURR. ISSUES INTEST. MICROBIOL.3:23-7). The biofilms in the GI tract populated at least with pathogenic bacteria form a dense filamentous film encased within an exopolysaccharide (EPS) matrix. Such biofilms are impenetrable to antimicrobials and host immune cells, and are difficult to treat.

[0009] Various associations between microbiome gut flora and systemic or localized effects on other parts of the body have been theorized. However, little in the way of specific associations has been established with reasonable specificity and reproducibility. SUMMARY

[0010] The inventors have assembled a gut microbiome database. A bioinformatics analysis of this gut microbiome database was performed focusing on the stress cohort, i.e., the group of people that reported experiencing various levels of stress. A differential abundance of several probiotic organisms of interest that are associated with lower stress cohorts when compared to higher stress cohorts. Despite the advances made to date, there is need for improved compositions and methods for treating stress through the gut microbiome.Attorney Docket No. BHMP106WO

[0011] As discussed herein, microbiome analysis was conducted on healthy subjects that were distributed into high, moderate, and low stress groups based on their stress frequency answer. Differential abundance analysis using the ANCOMBC2 method and correlation analysis using the SECOM method were performed on the abundance of bacterial and fungal organisms between the cohorts. According to the analysis performed on the microbiome dataset, it was determined that L. delbrueckii has increased abundance (significant) in low stress cohorts compared to moderate stress cohorts (male + female) and B. bifidum has increased abundance (significant) in females low stress cohorts compared to female moderate stress cohorts. In the female cohort L. delbrueckii also has increased abundance in the low to moderate stress category.

[0012] With this surprising discovery, a composition comprising L. delbrueckii and / or B. bifidum and treatment program for male and female humans was developed for reduction of stress in male and females.

[0013] In some aspects, the technology described herein relates to a method of reducing stress in a subject, the method including consumption by, or administration to, the subject of a probiotic composition including Lactobacillus delbrueckii and Bifidobacterium bifidum.

[0014] In some aspects, the technology described herein relate to a composition for use in reducing stress in a human subject, the composition including a live, freeze-dried probiotic composition including a combination of Lactobacillus delbrueckii and Bifidobacterium bifidum; wherein the Lactobacillus delbrueckii and Bifidobacterium bifidum are each present in an amount of up to 5 billion CFUs.

[0015] Other features and advantages of the invention will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG.1 is a schematic representation of an exemplary process for making a probiotic composition described herein.

[0017] FIG.2 is a graph showing the log fold change (natural log of fold change) in the abundance of L. delbruekii in moderate stress (right hand, lighter bar) and high stress (left hand, darker bar) subjects compared to subjects reporting low stress levels.

[0018] FIG.3 is a graph showing the log fold change (natural log of fold change) in the abundance of L. delbruekii in moderate stress (right hand, lighter bar) and high stress (left hand, darker bar) female subjects compared to female subjects reporting low stress levels.Attorney Docket No. BHMP106WO

[0019] FIG.4 is a graph showing the log fold change (natural log of fold change) in the abundance B.bifidum in moderate stress (right hand, lighter bar) and high stress (left hand, darker bar) female subjects compared to female subjects reporting low stress levels.

[0020] FIG.5 is a graph showing the log fold change (natural log of fold change) in the abundance of S. moorei in moderate stress (right hand, lighter bar) and high stress (left hand, darker bar) male and female subjects compared to male and female subjects reporting low stress levels.

[0021] FIG.6 is a photo of multiple experimental wells to determine MIC of S. moorei in B. bifidum and L. delbrueckii subsp. bulgaricus.

[0022] FIG.7 is a graph of optical density over time of fructose oligosaccharide (FOS) effect on growth promotion of B. bifidum. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention is based, in part, upon the discovery that a composition or a dosage form comprising both non-pathogenic bacteria and fungi improves nutrient (for example, protein and / or vitamin) absorption by mammalian (for example, human) intestinal epithelial cells. Also provided herein is the discovery that it is possible to prevent or disrupt biofilms comprising pathogenic bacteria and / or fungi, and optionally archaea and / or protozoa, in a subject by administering to the subject a composition or dosage form comprising both isolated and viable non-pathogenic bacteria and non-pathogenic fungi, which when administered promotes a more balanced microbiome and / or promotes the restoration of the natural microbiome in the subject. Unless the context dictates otherwise, the terms composition and dosage form can be used interchangeably herein, where a dosage form is a composition and vice versa. For example, it is contemplated that the composition consumed by, or administered to, a subject can be consumed or administered as a dosage form, for example, as a unit (for example, a spoonful) of the composition. Similarly, it is understood that a dosage form can, for example, comprise a composition described herein. For example, a dosage form can comprise, for example, a composition in the form of a powder described herein, or, for example, a capsule that contains such a composition.

[0024] Lactobacillus delbrueckii (“L. delbrueckii”) is a gram-positive, non-spore forming bacteria that plays a vital role in the fermentation of dairy products, particularly in the production of yogurts and other fermented milk products.Attorney Docket No. BHMP106WO

[0025] L. delbrueckii, has been used as a probiotic to support gut health and gastrointestinal microbiome balance. Bifidobacterium Bifidum (“B. bifidum”) is a gram-positive, anaerobic bacteria that is commonly found in the human gastrointestinal tract, particularly in infants and breast-fed babies. B. bifidum plays a role in the fermentation of carbohydrates and fibers to produce beneficial short-chain fatty acids which are associated with overall colon and gut health.

[0026] In an embodiment, the stress-reduction composition can also include non-pathogenic bacteria and fungi that improves nutrient (for example, protein, vitamin, mineral, and co-factor) absorption by mammalian (for example, human) intestinal epithelial cells.

[0027] Testing and development formulation components begin with in vitro assays. Growth promotion assays will be performed to identify an appropriate prebiotic fiber to optimize the growth of L. delbrueckii and / or B. bifidum. Minimum inhibitory concentration (MIC) assays were performed to determine whether the select organisms are antagonistic to the opportunistic pathogen Solobacteria moorei. Further in vivo assays in mice can be conducted to study the effect on stress, gut permeability, inflammation and other gastrointestinal effects.

[0028] Live, freeze-dried powdered probiotic organisms of L. delbrueckii and / or B. bifidum can be selected for direct supplementation in the formulation. Additional supplementation with a probiotic / enzyme blend, particularly Lactobacillus acidophilus, Lactobacillus rhamnosus, Saccharomyces boulardii, Bifidobacterium breve and amylase); Lactobacillus plantarum DR7 may also be used in combination.

[0029] In an embodiment, the composition comprises L. delbrueckii alone for stress treatment. In particular, this may be administered to a male patient. In an embodiment, the composition comprises B. bifidum alone for stress treatment. In particular, this may be administered to a female patient. In an embodiment, the composition comprises L. delbrueckii and B. bifidum for stress treatment. This may be administered to a male or female patient. In this embodiment, the composition may be in a tablet form with each tablet having a mass of 10 to 120 mg, such as, for example 20 to 60 mg, or 30 mg to 40 mg. CFU counts of L. delbrueckii and B. bifidum may be a total of 1 to 5 billion, such as 1 to 2.5 billion, or 2 billion to 3 billion. The particular strains of L. delbrueckii and B. bifidum may be L. delbrueckii subsp. Bulgaricus (BGBX-19) and B. bifidum (SGBX-17).

[0030] In an embodiment either L. delbrueckii or B. bifidum or both can be combined with Lactobacillus acidophilus, Lactobacillus rhamnosus, Saccharomyces boulardii, Bifidobacterium breve and amylase for stress treatment.Attorney Docket No. BHMP106WO

[0031] In an embodiment, the composition can comprise L. delbrueckii and a fiber source for stress treatment. In an embodiment, the composition can comprise B. bifidum and a fiber source for stress treatment. In an embodiment, the composition can comprise B. bifidum, L. delbrueckii and a fiber source for stress treatment. Optionally, any of these compositions can additionally comprise Lactobacillus acidophilus, Lactobacillus rhamnosus, Saccharomyces boulardii, Bifidobacterium breve and amylase for stress treatment.

[0032] In any embodiment described above Lactobacillus plantarum, such as Lactobacillus plantarum DR7 can be added to the composition for stress treatment.

[0033] In addition to the above combinations, the composition may also include combinations of enzymes (bromelain, cellulase(es), lipase(es), papain, protease(es) amylase(es), galactosidase) in the 10-250 mg range, such as, for example, 25 to 200 mg, or 50 to 150 mg. Bacopa monnieri or Bacopa monnieri extracts can be present in a 10-500 mg range, such as 25 to 300 mg, or 100 to 200 mg. In addition, Lions Mane mushroom or Lion’s Mane mushroom extracts can be present in a 10-500 mg range, such as 25 to 300 mg, or 100 to 200 mg.

[0034] Dried powdered form of L. delbrueckii and B. bifidum can each be present in an amount of 20 mg to 1000 mg, such as, for example, 50 mg to 500 mg, or 100 mg to 250 mg; or alternatively, CFU 1 billion to 10 billion, such as, for example, CFU 2 Billion to 5 Billion, or CFU 1.5 billion to 7 billion.

[0035] Other additives can be present in a total amount of 1 to 15,000 mg, such as 100 mg to 10,000, or 1,500 to 5,000 mg. Total fiber content can be present in a range of 500 mg to 5,000 mg), such as, for example, 1,000 mg to 4,000 mg, or 2,000 mg to 3,000 mg. The blend of Lactobacillus acidophilus, Lactobacillus rhamnosus, Saccharomyces boulardii, Bifidobacterium breve and amylase can be present in a total amount of CFU 1 billion to 30 billion, such as, for example, CFU 2 billion to CFU 10 billion, or CFU 3 to 5 billion. Golden Kiwi Fiber can be present in an amount of 500 mg to 5,000 mg, such as, for example, 1,000 mg to 4,000 mg, or 2,000 mg to 3,000 mg.

[0036] In another aspect, the composition or method of treatment can comprise a nutrient in combination with the probiotic composition. The combination does not require that the nutrient and the probiotic composition be present in a single composition. In certain embodiments, the composition comprising a nutrient comprises both the nutrient and the probiotic composition.

[0037] In certain embodiments, the consumption by, or administration to the subject of the composition comprising a nutrient is contemporaneous with the consumption or administrationAttorney Docket No. BHMP106WO of the probiotic composition. In certain embodiments, the consumption by, or administration to the subject of the composition comprising a nutrient is subsequent to or prior the consumption by, or administration of the probiotic composition.

[0038] In certain embodiments, a nutrient is included in the composition. In an embodiment, the nutrient is a protein. The protein may be selected from whey protein, soy protein, milk protein, egg protein, animal muscle protein, fish protein, brown rice protein, pea protein, hemp protein, cranberry protein, and artichoke protein. In certain embodiments, the protein is casein. In certain embodiments, the protein is a protein hydrolysate. In certain embodiments, the protein is comprised in a natural food source. In certain embodiments, the protein is an isolated and / or purified protein.

[0039] The subject may be a mammal (e.g., human, a companion animal (e.g., dog, cat, or rabbit), or a livestock animal (for example, cow, sheep, pig, goat, horse, donkey, and mule, buffalo, oxen, or camel)). In certain embodiments, the subject is a healthy subject. In other embodiments, the probiotic composition further comprises an enzyme, for example, an enzyme capable of disrupting a biofilm that may block or otherwise retard nutrient absorption. The inclusion of such an enzyme is particularly useful if subject has a biofilm of pathogenic bacteria and / or fungi. The microorganisms within the biofilm that are disrupted by the composition may comprise any type of pathogenic microorganism, e.g., bacteria (for example, Gram-positive bacteria and Gram-negative bacteria), fungi (for example, yeast and mold), archaea, and protozoa.

[0040] In certain embodiments, the probiotic composition comprises at least two different non- pathogenic bacterial strains. In certain embodiments, the probiotic composition comprises three different non-pathogenic bacterial strains. In certain embodiments, the non-pathogenic bacterial strain is selected from: Lactobacillus rhamnosus, Bifidobacterium breve, Lactobacillus acidophilus, Bifidobacterium bifidum, and Lactobacillus reuteri. In certain embodiments, the non-pathogenic bacterial strain is selected from: Lactobacillus rhamnosus LB 20, Bifidobacterium breve S 46, and Lactobacillus acidophilus RP 32. In certain embodiments, the probiotic composition comprises Lactobacillus rhamnosus LB 20, Bifidobacterium breve S 46, and Lactobacillus acidophilus RP 32. In certain embodiments, the non-pathogenic fungal strain is selected from: Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomycetes sp HZ178, Saccharomyces bayanus, Pichia burtonii, Pichia jadinii, Pichia kudriavzevii, Pichia onychis, Pichia sp., and Picoa juniper. In certain embodiments, the non-pathogenic fungal strain is Saccharomyces boulardii SB 48.Attorney Docket No. BHMP106WO

[0041] In certain embodiments, the probiotic composition further comprising an enzyme capable of disrupting a biofilm in a subject. In certain embodiments, the enzyme is selected from the group consisting of amylase, cellulase, hemicellulase, lysozyme, pectinase, DNase I, Serratia peptidase or Serratiopeptidase, hemicellulase / pectinase complex, β-1,3-glucanase, acid protease, alkaline protease, glucoamylase, endoglucanase, xylanase, lipase, lysozyme, protease / peptidase complex, dipeptidyl peptidase IV (DPP-IV), chitosanase, bromelain, papain, kiWi protease actinidi, a plant-derived protease, and phytase. In certain embodiments, the enzyme is an amylase selected from the group consisting of Bacillus stearothermophilus amylase, Bacillus amyloliquefaciens amylase, Bacillus subtilis amylase, Bacillus licheniformi amylase, Aspergillus niger amylase, and Aspergillus oryzae amylase. In certain embodiments, the probiotic composition comprises 100 to 5,000 SKB units of amylase, 200 to 4,000 SKB units of amylase, from 300 to 2,000 SKB units of amylase, or 400 to 1,000 SKB units of amylase. In certain embodiments, the composition comprises 500 SKB units of amylase.

[0042] In certain embodiments, the probiotic composition comprises 1 to 30, 3 to 25, 10 to 20, or 15 billion colony forming units of Bifidobacterium breve, about 10 billion colony forming units of Lactobacillus rhamnosus, 0.5 to 10, 1 to 8, 2 to 5 or 3.5 billion colony forming units of Saccharomyces boulardii, 0.5 to 5, 1 to 3.5, 1.25 to 2, or 1.5 billion colony forming units of Lactobacillus acidophilus, and 100 to 1,000, 250 to 750, 450 to 550 or 500 SKP of amylase. In certain embodiments, the probiotic composition comprises an admixture (blend) of one or more isolated and viable non-pathogenic bacterial strains and one or more isolated and viable non- pathogenic fungal strains is in the form of a powder. In certain embodiments, the probiotic composition comprises a powder of one or more isolated and viable non-pathogenic bacterial strains and one or more isolated and viable non-pathogenic fungal strains that is coated with functional coating (for example, coated with a controlled release coating) or a non-functional coating (for example, aesthetic coating). In certain embodiments, the probiotic composition is in an oral dosage form. In certain embodiments, the oral dosage form is a capsule, for example, a capsule that optionally is coated with a functional or non-functional coating.

[0043] In certain embodiments, the probiotic composition is formulated as a powdered blend, where the powdered blend optionally is coated, for example, coated with a functional coating such as a controlled release coating. Furthermore, in certain embodiments, the powdered blend of one or more isolated and viable non-pathogenic bacterial strains and one or more isolated and viable non-pathogenic fungal strains is freeze-dried or spray-dried.

[0044] The enzyme in the composition or dosage form disrupts extracellular matrix polysaccharides (EPSs) present in the biofilm making the pathogenic organisms more open toAttorney Docket No. BHMP106WO challenge by the non-pathogenic bacterial and fungal strains in the dosage forms. In certain embodiments, upon administration of the composition, the non-pathogenic bacterial strains (i) displace the pathogenic bacteria in the biofilm, (ii) interfere with the attachment of the pathogenic bacteria or fungi to a substratum of the biofilm, (iii) displace the pathogenic bacteria / fungi from an extracellular polymeric matrix present in the biofilm, (iv) prevent filamentation of the pathogenic fungi in the biofilm, or (v) a combination of any of the foregoing. In certain embodiments, administration of the composition permits restoration of the natural microbiome of the region in the subject and reduces the growth and / or development of pathogenic bacteria and / or pathogenic fungi.

[0045] In another aspect, the present invention provides a beverage product comprising a probiotic composition comprising the probiotic and enzyme components discussed above. The beverage can be a protein-rich beverage, e.g., comprising at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of protein. The probiotic composition can include one or more of the compositions or dosage forms described herein.

[0046] In another aspect, the invention provides a method of identifying a subject suitable for receiving the composition disclosed herein. The method comprises: (a) quantifying the number or density of organisms present in a tissue or body fluid sample harvested from the region of the test subject, e.g., a fecal sample; and (b) comparing the abundance levels or number of organisms quantified in the sample against the number or density of corresponding organisms present in a corresponding sample of a corresponding region of a healthy cohort. More particularly, the number or density of organisms can be compared between the patient sample and data on a healthy cohort who report normal or low stress levels. When the number or density of organisms in the sample from the test subject is less than the number or density of organisms present in the corresponding region of the healthy subject, the test subject is suitable for receiving the composition or dosage form.

[0047] In one aspect, the present invention provides a composition for use in treating stress in a subject in need thereof, the composition comprising any of those disclosed above comprising either L. delbrueckii, B. bifidum, or both.

[0048] Biofilms may exist in the GI tract and can block nutrient absorption and effectiveness of probiotics. They potentially may contribute to stress. In certain embodiments, composition or dosage forms described herein can be used to disrupt a biofilm comprising, for example, pathogenic bacteria and fungi within the gastrointestinal (GI) tract (for example, esophagus, stomach, upper intestine, and / or the lower intestine) of a subject. In certain embodiments, theAttorney Docket No. BHMP106WO preselected region comprises the duodenum, the jejunum, and / or the ileum of the upper GI tract, whereas in other embodiments, the preselected region comprises the appendix, the proximal colon, and / or the rectum of the lower GI tract.

[0049] In certain embodiments, a composition or dosage form described herein is used to disrupt a biofilm disposed within the GI tract of a subject with an elevated relative abundance of pathogenic bacterial species such as Solobacteria moorei, E. coli, Serratia marcescens, and Ruminococcus gnavus in the biofilm disposed within the GI tract of the subject when compared to levels typically found in healthy subjects. In certain embodiments, a composition or dosage form described herein comprises non-pathogenic bacterial strains for disrupting or replacing a biofilm containing two pathogenic bacterial species. For example, the biofilm targeted for disruption or replacement may contain E. coli and a Bacteroides spp. (e.g., Bacteroides coprophilus, Bacteroides eggerthii, Bacteroides ovatus, Bacteroides fragilis, Bacteroides plebeius, or Bacteroides uniformis), or Solobacteria moorei. S. moorei was found to be significantly differentially abundant in high stress cohorts (male + female) compared to low stress cohort (male + female).

[0050] In certain embodiments, the abundance of pathogenic fungus Candida tropicalis in a biofilm disposed within the GI tract of a subject is elevated when compared to levels typically found in healthy subjects. In certain embodiments, biofilms within the GI tract containing C. tropicalis or C. Albicans combined with E. coli and / or S. marcescens are enriched in fungal hyphae, a form of growth associated with pathogenic conditions. Fungal filamentation can sometimes be a virulence factor used by Candida to damage host tissues and to trigger a specific host immune response. In certain embodiments, the dosage form of the invention is used to disrupt a biofilm disposed within the GI tract of a subject with an elevated relative abundance of bacterial species such as species of the Firmicutes phylum. In certain embodiments, the dosage form of the invention is used to disrupt a biofilm disposed within the GI tract of a subject with an elevated relative abundance of fungal species such as species of the Ascomycota phylum and species of the Zygomycota phylum and species of the Basidiomycota phylum.

[0051] The bacteria within the biofilm exist in intimate contact with the fungus but may differ in their specific interactions with the fungus. In certain embodiments, the pathogenic bacteria (e.g., E. coli) may be fused to the fungal cells within the biofilm. Alternatively or in addition, the pathogenic bacteria and pathogenic fungi disposed within the biofilm may form a “digestive plaque,” where the bacteria and fungi are protected from antimicrobial drugs and host’s immune system. It is understood that digestive plaque can disrupt the normal or healthy microbiome of the GI tract, and cause or be otherwise associated with a GI disease or disorder.Attorney Docket No. BHMP106WO

[0052] Compositions or dosage forms are provided herein that can moderate or improve stress. In certain embodiments, the composition is formulated as a powdered blend of the components described above that optionally is coated with a functional coating (e.g., controlled release coating) or a non-functional coating (e.g., aesthetic coating). In certain embodiments, the powder blend of the composition is freeze-dried or spray-dried. In certain embodiments, the freeze-dried or spray-dried blend of the composition is further blended with an enzyme (for example, amylase).

[0053] In certain embodiments, the composition of the present invention comprises (i) one or more enzymes capable of disrupting a biofilm in a preselected region of the subject, (ii) one or more non-pathogenic fungal strains that are viable (e.g., with or without replication) in the region of the subject, and (iii) one or more non-pathogenic bacterial strains that are viable (e.g., with or without replication) in the region of the subject. In certain embodiments, the composition comprises (i) one or more enzymes capable of disrupting the biofilm in the target region (e.g., gastrointestinal tract, urinary tract, reproductive tract, upper respiratory tract, lower respiratory tract, biliary tract, mouth, eye, nose, ear, or skin) of a subject, (ii) one or more non- pathogenic fungal strains that are viable (e.g., with or without replication) in the region of the subject, and (iii) one or more non-pathogenic bacterial strains that are viable being viable (e.g., with or without replication) in the region of the subject. The subject may be a mammal (e.g., human, a companion animal (e.g., dog, cat, or rabbit), or livestock animal (for example, cow, sheep, pig, goat, horse, donkey, and mule, buffalo, oxen, or camel)). (i) Non-Pathogenic Bacteria

[0054] In addition to the L. delbrueckii and / or B. bifidum the composition or dosage form may comprise one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) different non-pathogenic bacterial strains.

[0055] In certain embodiments, exemplary bacterial strains to be included in the composition or dosage form of the present invention may comprise bacterial strains of any one or more of the following bacterial species: Agrococcus jenensis, Alistipes indistinctus, Alistipes massiliensis, Alkalibacterium iburiense, Anoxybacillus kestanbolensis, Bacillus cereus, Bacillus clausii, Bacillus Coagulans, Bacteroides coprophilus, Bacteroides eggerthii, Bacteroides ovatus, Bacteroides fragilis, Bacteroides plebeius, Bacteroides uniformis, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium pseudolongum, Blautia obeum, Blautia product, Candidatus azobacteroides, Candidatus portiere, Candidatus Portiera, Clostridium celatum, Clostridium hiranonis, Clostridium neonatale, Clostridium perfringens, Clostridium tyrobutyricum, Collinsella aerofaciens,Attorney Docket No. BHMP106WO Collinsella stercoris, Coprococcus eutactus, Corynebacterium stationis, Desulfosporosinus meridiei, Desulfovibrio D168, Dorea formicigenerans, Eggerthella lenta, Erwinia oleae, Faecalibacterium prausnitzii, Lactobacillus agilis, Lactobacillus reuteri, Lactobacillus ruminis, Lactobacillus salivarius, Lactobacillus zeae, Listeria weihenstephanensis, Paenibacillus mucilaginosus, Parabacteroides distasonis, Pediococcus acidilactici, Peptostreptococcus anaerobius, Prevotella copri, Prevotella melaninogenica, Prevotella stercorea, Propionibacterium acnes, Pseudoramibacter eubacterium, Roseburia faecis, Rothia dentocariosa, Rothia mucilaginosa, Ruminococcus bromii, Ruminococcus callidus, Ruminococcus flavefaciens, Ruminococcus lavefaciens, Ruminococcus gnavus, Ruminococcus torques, Salinibacillus aidingensis, Staphylococcus sciuri, Streptococcus anginosus, Streptococcus sobrinus, Tissierella soehngenia, Veillonella dispar, and Veillonella parvula.

[0056] In certain embodiments, one or more of the bacterial strains listed in TABLE 1 below are included in the composition or dosage form of the present invention.Attorney Docket No. BHMP106WO TABLE 1 Bifidobacterium adolescentis Bacillus clausii Bifidobacterium animalis Agrococcus jenensis

[0057] E 2 below are included in the composition or dosage form of the present invention. TABLE 2 Bifidobacterium adolescentis Eggerthella lentaAttorney Docket No. BHMP106WO

[0058] In certain embodiments, the composition or dosage form comprises one, two or three different bacterial strains (e.g., strains listed in TABLE 1 or 2) or one, two or three of Lactobacillus rhamnosus, Bifidobacterium breve, Lactobacillus reuteri, and Lactobacillus acidophilus) capable of replicating in the region of the mammal.

[0059] In certain embodiments, the composition or dosage form comprises non-pathogenic bacterial strains for disrupting a biofilm containing one or more of C. tropicalis, E. coli and / or S. marcescens in the GI tract. For example, the GI tract biofilm may comprise: Candida tropicalis and Escherichia coli; Candida tropicalis and Serratia marcescens; Candida tropicalis, Escherichia coli, and Serratia marcescens; other Candida yeasts, Klebsiella oxytoca, or Solobacteria moorei.

[0060] In certain embodiments, the biofilm in addition to or alternatively to Candida tropicalis may comprise: (i) Candida albicans and Escherichia coli, (ii) Candida albicans and Serratia marcescens, or (iii) Candida albicans, Escherichia coli, and Serratia marcescens; Candida yeasts, Klebsiella oxytoca, or Solobacteria moorei

[0061] In certain embodiments, the composition or dosage form comprises non-pathogenic bacterial strains for disrupting or replacing a biofilm containing two bacterial species. For example, the biofilm may contain E. coli and a Bacteroides spp. (e.g., Bacteroides coprophilus, Bacteroides eggerthii, Bacteroides ovatus, Bacteroides fragilis, Bacteroides plebeius, or Bacteroides uniformis).

[0062] In certain embodiments, the non-pathogenic bacterial strains included in the composition or dosage forms of the present invention may comprise a combination of any two of Lactobacillus rhamnosus LB 20, Bifidobacterium breve S 46, and Lactobacillus acidophilus RP 32. In certain embodiments, the non-pathogenic bacterial strains included in the composition or dosage form may comprise a combination of Lactobacillus rhamnosus LB 20 and Bifidobacterium breve S 46. In certain embodiments, the non-pathogenic bacterial strains included in the composition or dosage form may comprise a combination of Lactobacillus rhamnosus LB 20 and Lactobacillus acidophilus RP 32. In certain embodiments, the non- pathogenic bacterial strains included in the composition or dosage form may comprise a combination of Bifidobacterium breve S 46 and Lactobacillus acidophilus RP 32. In certain embodiments, the non-pathogenic bacterial strains included in the composition or dosage form of the present invention may comprise a combination of Lactobacillus rhamnosus LB 20, Bifidobacterium breve S 46, and Lactobacillus acidophilus RP 32.Attorney Docket No. BHMP106WO

[0063] In certain embodiments, the composition or dosage form of the invention comprises 10 billion to 40 billion, e.g., 15 billion to 40 billion, 20 billion to 40 billion, 10 billion to 30 billion, 15 billion to 30 billion, or 20 billion to 30 billion colony forming units of the non-pathogenic bacterial strain(s). (ii) Non-Pathogenic Fungi

[0064] In certain embodiments, exemplary fungal strains to be included in the composition or dosage form of the present invention may comprise any one or more of the following fungal species: Albatrellus syringae, Alternaria alli, Alternaria daturicola, Ambispora granatensis, Amyloathelia crassiuscula, Amylomyces rouxii, species of Ascomycota, Ascosphaera apis, Aspergillus flavus, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus sp, Aspergillus terreus, Aspergillus versicolor, Bettsia alvei, Botryosphaeria mamane, Botryotinia fuckeliana, Candida arabinofermentans, Candida ernobii, Candida ethanolica, Candida glabrata, Candida humilis, Candida intermedia, Candida parapsilosis, Candida piceae, Candida quercitrusa, Candida tartarivorans, Candida temnochilae, Candida zemplinina, Candida zeylanoides, Chamonixia caespitosa, Cladonia polystomata, Cladosporium cladosporioides, Cladosporium halotolerans, Cladosporium sp JP67, Cladosporium sphaerospermum, Clavicorona taxophila, Clavispora lusitaniae, Craterellus sp, Dactylellina phymatopaga, Debaryomyces hansenii, Debaryomyces marasmus, Debaryomyces sp, Debaryomyces subglobosus, Dipodascus australiensis, Ectomycorrhizal, Emericella nidulans, Epulorhiza sp, Eremascus fertilis, Eremothecium gossypii, Eupenicillium cinnamopurpureum, Eurotium amstelodami, Eurotium cristatum, Exophiala dermatitidis, Filobasidiella neoformans, Fonsecaea monophora, Fusarium solani, Fusarium dimerum, Fusarium oxysporum, Fusarium sp, Galactomyces geotrichum, Galactomyces sp, Geomyces sp, Geotrichum cucujoidarum, Geotrichum sp, Glomus mosseae, Hanseniaspora sp, Helicobasidium longisporum, Helicostylum pulchrum, Hyphodontia flavipora, Hypochnicium cystidiatum, Inocybe sp, Kazachstania africana, Kazachstania unispora, Kluyveromyces lactis, Kluyveromyces yarrowii, Kodamaea ohmeri, Leptosphaeria biglobosa, Leptosphaerulina chartarum, Leucoagaricus sp, Lewia infectoria, Lichtheimia ramose, Macrophomina phaseolina, Monacrosporium coelobrochum, Mucor circinelloides, Mucor flavus, Mucor fuscus, Mucor sp, Myrothecium sp, Myxozyma melibiosi, Neocallimastix frontalis, Neocallimastix sp, Omphalotus nidiformis, Ophiocordyceps filiformis, Ophiocordyceps sinensis, Penicillium carneum, Penicillium chrysogenum, Penicillium concentricum, Penicillium crustosum, Penicillium digitatum, Penicillium granulatum, Penicillium griseofulvum, Penicillium griseoroseum, Penicillium polonicum, Penicillium psychrosexualis, Penicillium pulvillorum, Penicillium roqueforti, Penicillium sclerotigenum, Penicillium sp, PenicilliumAttorney Docket No. BHMP106WO spinulosum, Penicillium verrucosum, Pezizomycetes sp genotype 323, Phaeophyscia exornatula, Phialocephala lagerbergii, Phlebia radiata, Phlebia sp, Phomopsis sp, Physcia stellaris, Physoderma maydis, Pichia burtonii, Pichia jadinii, Pichia kudriavzevii, Pichia onychis, Pichia sp, Picoa juniperi, Pilaira cesatii, Pilaira sp, Pirella circinans, Preussia sp, Reddellomyces donkii, Rhizomucor pusillus, Rhizopus oryzae, Rhodosporidium kratochvilovae, Rhodosporidium sp, Rhodotorula sp, Rinodina milvina, Saccharomyces bayanus, Saccharomyces bulderi, Saccharomyces cerevisiae, Saccharomyces mikatae, Saccharomyces sp, Saccharomycetes sp, Scleroderma sp, Sclerotinia sp, Scutellospora nodose, Sebacinales sp, Sporopachydermia sp, Stenocarpella maydis, Thamnidium elegans, Torulaspora delbrueckii, Trichosporon chiarellii, Tuber oligospermum, Umbelopsis isabellina, Wallemia sebi, Wallemia sp, and Zygoascus meyerae.

[0065] In certain embodiments, one or more of the fungal strains listed in TABLE 3 below may be included in the composition or dosage form of the present invention. TABLE 3 Saccharomycetes sp HZ178 Pilaira sp Pi 3

[0066] ce a e o e s, e co pos o o osage o ay co p se a on- pathogenic fungal strain selected from Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomycetes sp HZ178, Saccharomyces bayanus, strains belonging to the Pichia genus (for example, including but limited to Pichia burtonii, Pichia jadinii, Pichia kudriavzevii, Pichia onychis, and Pichia juniper).Attorney Docket No. BHMP106WO

[0067] In certain embodiments, the composition or dosage form comprises a composition comprising a non-pathogenic fungal strain for disrupting a biofilm containing C. tropicalis. For example, the GI tract biofilm may comprise: (i) Candida tropicalis and Escherichia coli, (ii) Candida tropicalis and Serratia marcescens, or (iii) Candida tropicalis, Escherichia coli, and Serratia marcescens. In certain embodiments, the composition or dosage form comprises a composition comprising a non-pathogenic fungal strain for disrupting a biofilm containing C. albicans. In certain embodiments, the biofilm may comprise: (i) Candida albicans and Escherichia coli, (ii) Candida albicans and Serratia marcescens, or (iii) Candida albicans, Escherichia coli, and Serratia marcescens.

[0068] In certain embodiments, the composition or dosage form of the invention comprises one or more fungal strains listed in TABLE 3. In certain embodiments, the composition or dosage form of the invention comprises one or more fungal strains listed in TABLE 3 and one or more of the bacterial strains listed in TABLE 1 or TABLE 2.

[0069] In certain embodiments, the composition or dosage form comprises a composition comprising a non-pathogenic fungal strain Saccharomyces boulardii SB 48. In certain embodiments, the composition or dosage form of the invention comprises a non-pathogenic fungal strain Saccharomyces boulardii SB 48 and one or more of the bacterial strains listed in TABLE 1 or TABLE 2.

[0070] In certain embodiments, the composition or dosage form of the invention comprises 1 billion to 10 billion, e.g., 2 billion to 8 billion, from 3 billion to 6 million colony forming units of the non-pathogenic fungal strain(s).

[0071] In certain embodiments, in addition to L. delbrueckii and / or B. bifidum the composition or dosage form of the invention comprises a non-pathogenic fungal species Saccharomyces boulardii and non-pathogenic bacterial species of one or more of Lactobacillus rhamnosus, Bifidobacterium breve, and Lactobacillus acidophilus. In certain embodiments, the composition or dosage form comprises a non-pathogenic fungal species Saccharomyces boulardii and non- pathogenic bacterial species Lactobacillus rhamnosus and Bifidobacterium breve. In certain embodiments, the composition or dosage form comprises a non-pathogenic fungal species Saccharomyces boulardii and non-pathogenic bacterial species Lactobacillus rhamnosus and Lactobacillus acidophilus. In certain embodiments, the composition or dosage form comprises a non-pathogenic fungal species Saccharomyces boulardii and non-pathogenic bacterial species Bifidobacterium breve and Lactobacillus acidophilus. In certain embodiments, the composition or dosage form comprises a non-pathogenic fungal species Saccharomyces boulardii and theAttorney Docket No. BHMP106WO non-pathogenic bacterial species Lactobacillus rhamnosus, Bifidobacterium breve, and Lactobacillus acidophilus.

[0072] In certain embodiments, the composition or dosage form of the invention comprises a non-pathogenic fungal strain Saccharomyces boulardii SB 48 and non-pathogenic bacterial strains of one or more of Lactobacillus rhamnosus LB 20, Bifidobacterium breve S 46, and Lactobacillus acidophilus RP 32. In certain embodiments, the composition or dosage form comprises a non-pathogenic fungal strain Saccharomyces boulardii SB 48 and non-pathogenic bacterial strains Lactobacillus rhamnosus LB 20 and Bifidobacterium breve S 46. In certain embodiments, the composition or dosage form comprises a non-pathogenic fungal strain Saccharomyces boulardii SB 48 and non-pathogenic bacterial strains Lactobacillus rhamnosus LB 20 and Lactobacillus acidophilus RP 32. In certain embodiments, the composition or dosage form comprises a non-pathogenic fungal strain Saccharomyces boulardii SB 48 and non- pathogenic bacterial strains Bifidobacterium breve S 46 and Lactobacillus acidophilus RP 32. In certain embodiments, the composition or dosage form comprises a non-pathogenic fungal strain Saccharomyces boulardii SB 48 and the non-pathogenic bacterial strains Lactobacillus rhamnosus LB 20, Bifidobacterium breve S 46, and Lactobacillus acidophilus RP 32. (iii) Enzymes

[0073] In addition to non-pathogenic bacteria and fungi, the composition or dosage form further comprises an enzyme capable of disrupting the biofilm or improving nutrient absorption. For example, the enzyme preferably digests or otherwise disrupts / breaks down the EPS matrix of the biofilm.

[0074] The enzyme can be selected from amylase, cellulase, hemicellulase, lysozyme, pectinase, DNase I, Serratia peptidase, Serratiopeptidase, hemicellulase / pectinase complex, β- 1,3-glucanase, acid protease, alkaline protease, glucoamylase, endoglucanase, xylanase, lipase, lysozyme, protease / peptidase complex, dipeptidyl peptidase IV (DPP-IV), chitosanase, bromelain, papain, kiWi protease actinidi, a plant-derived protease, phytase, zymolase and nuclease. The enzyme may be chosen depending upon the type of biofilm and the microorganisms disposed therein. For example, an amylase enzyme may be used to degrade or otherwise disrupt carbohydrate components of the biofilm, and a nuclease such a DNase I may be used for digest or otherwise disrupt DNA in the biofilm.

[0075] In certain embodiments, the composition or dosage form comprises two or more (e.g., 2, 3, 4, 5, or more) different enzymes selected from amylase, cellulase, hemicellulase, lysozyme, pectinase, DNase I, Serratia peptidase, Serratiopeptidase, hemicellulase / pectinase complex, β-Attorney Docket No. BHMP106WO 1,3-glucanase, acid protease, alkaline protease, glucoamylase, endoglucanase, xylanase, lipase, lysozyme, protease / peptidase complex, dipeptidyl peptidase IV (DPP-IV), chitosanase, bromelain, papain, kiWi protease actinidi, a plant-derived protease, phytase, zymolase and nuclease.

[0076] In certain embodiments, a composition or dosage form described hereinabove comprises an amylase selected from Bacillus stearothermophilus amylase, Bacillus amyloliquefaciens amylase, Bacillus subtilis amylase, Bacillus licheniformi amylase, Aspergillus niger amylase, and Aspergillus oryzae amylase.

[0077] In certain embodiments, the composition or dosage form comprises an amylase, for example, 100 to 5,000 SKB units of amylase, from 200 to 4,000 SKB units of amylase, from 300 to 2,000 SKB units of amylase or from 400 to 1,000 SKB units of amylase. An SKB or Sandstedt, Kneen, and Blish unit refers to the amount of amylase to catalyze 1 µmole substrate per minute. In certain embodiments, composition or dosage form comprises a cellulose, for example, and comprises 100 to 300 CU (Cellulase unit) units per unit composition or dosage form, for example, 200 CU. In certain embodiments, composition or dosage form comprises a hemicellulose / pectinase complex, and comprises 60 to 100 HSU (Hemicellulose Specific Units) units per unit composition or dosage form, for example, 80 HSU. In certain embodiments, the composition or dosage form comprises a β-gluconase, and comprises 6 to 10 BGU (Beta Glucanase Unit), units per unit composition or dosage form, for example, about 8 BGU. In certain embodiments, the composition or dosage form comprises an acid protease, and comprises 15 to 25 SAP (Shrimp Alkaline Phosphatase) units per unit composition or dosage form, for example, about 20 SAP units. In certain embodiments, the composition or dosage form comprises alkaline protease, and comprises 15 to 25 HUT (hemoglobin unit Tyrosine base) units per unit composition or dosage form, for example, about 20 HUT units.

[0078] In certain embodiments, total amount of cellulase administered, for example, by the administration of one or more units of the composition or dosage form, may be 1 to 10,000 CU, the total amount of hemicellulase / pectinase complex administered, for example, by the administration of one or more units of the composition or dosage form, may be 1 to 8,000 HSU, the total amount of β-gluconase, for example, by the administration of one or more dosage units, may be 1 to 1000 BGU, the total amount of acid protease, for example, by the administration of one or more dosage units, may range be 1 to 10,000 SAP, and the total amount of alkaline protease, for example, by the administration of one or more dosage units, may be 1 to 40,000 HUT.Attorney Docket No. BHMP106WO

[0079] In certain embodiments, the composition or dosage form comprises about 500 SKB units of amylase, selected from α–Amylase, an endo-hydrolase that catalyzes the hydrolysis of internal α-1, 4-glycosidic linkages in starch to yield products like glucose and maltose, β- Amylase, an exo-hydrolase enzyme that hydrolyses α -1, 4-glucan linkages to yield successive maltose units, and γ-Amylase, which cleaves α -1, 6-glycosidic linkages, in addition to cleaving the last α -1, 4-glycosidic linkages to yield glucose, or a combination thereof. (iv) Exemplary Compositions

[0080] In addition to the L. delbrueckii and / or B. bifidum, the composition of the present invention may include bacteria (B. breve, L. acidophilus, L. rhamnosus), fungi (S. boulardii), and an enzyme (e.g., amylase), and can be used as a probiotic which breaks down digestive plaque and achieves total gut balance. The probiotic composition can be delivered in powder form comprising one or more isolated and viable non-pathogenic bacterial strains and one or more isolated and viable non-pathogenic fungal strains that is optionally coated with a functional coating, which is optionally further blended with an enzyme, and contained in a capsule. In certain embodiments, 30 – 50 of such capsules (e.g., 42 capsules) can be consumed over a period of 2-8 weeks (e.g., 6 weeks). In certain embodiments, the product can be consumed by a subject for up to one month (e.g., 30 days), or repeated cycles of one month. In each cycle, a single serving size (e.g., 1 scoop) may be consumed per day.

[0081] For example, one capsule can be consumed per day, with or without the simultaneous consumption of food. The probiotic composition preferably does not contain an allergen, artificial ingredient, or sweetener, and / or can be stored at room or ambient temperature, without refrigeration. (v) Other Additives

[0082] In certain embodiments, the composition can be naturally sweetened, for example, with monk fruit, and preferably does not contain an allergen, artificial ingredient, or sweetener, and / or can be stored at room or ambient temperature, without refrigeration. The composition can be an engineered formulation that combines fruits, vegetables and herbal extracts with probiotics, prebiotics and enzymes to break down digestive plaque and achieve total gut balance. Depending upon the circumstances, the organic super green composition can further comprise organic plant extract (e.g., spirulina, barley grass, alfalfa leaf, wheat grass, chlorella, dulse, spinach leaf, broccoli (whole plant), parsley leaf, kale leaf, Echinacea angustifolia root, licorice root, milk thistle seed, Siberian Eleuthero root, beet root, rose hips, acai (fruit), green tea leaf, raspberry leaf, blueberry (fruit), goji berry, bilberry (fruit), ashwagandha root, rhodiola root, reishi mushroom, maca root, bee pollen, nettle leaf, ginko biloba (4:1 leaf extract), royal jelly (3xAttorney Docket No. BHMP106WO concentrate), grape seed (2:1 extract), ginger, plant-based protein (e.g., pea protein), and fiber (e.g., sunflower lecithin, apple (fruit), brown rice bran, inulin). A nutritional ingredient may be selected from one or more of: a prebiotic (e.g., inulin), collagen peptides, deglycyrrhizinated licorice (DGL), marshmallow root, vitamin A (e.g., retinyl form), vitamin C, and vitamin D3.

[0083] In certain embodiments, the composition comprises 100 mg to 2,000 mg of a prebiotic, such as inulin (e.g., 200 mg to 1500 mg, or 300 to 1000 mg). In certain embodiments, the composition comprises 1,000 mg to 20,000 mg of collagen peptides (e.g., 1,000 mg to 15,000 mg, or 2,000 to 10,000 mg). In certain embodiments, the composition comprises 100 mg to 1,000 mg of DGL or marshmallow root (e.g., 150 mg to 700 mg, or 200 to 500 mg).

[0084] In certain embodiments, the composition comprises 1,000 IU to 10,000 IU of vitamin A (e.g., 1,500 to 7,000, or 3,000 to 6,000 IU). In certain embodiments, the composition comprises 500 IU to 1,500 IU of vitamin D3 (e.g., 700 to 1,250 IU, or 850 to 1,100 IU). In certain embodiments, the composition comprises 100 mg to 1,000 mg of vitamin C (e.g., 200 mg to 800 mg, or 250 to 600 mg).

[0085] In certain embodiments, the composition comprises 1,000 mg to 20,000 mg of a plant- based protein, such as pea protein (e.g., 1,200 mg to 15,000 mg, or 2,000 mg to 5,000 mg). In certain embodiments, the composition comprises about 10 mg to about 200 mg of grape seed extract (e.g., 20 mg to 150 mg, 45 mg to 100 mg).

[0086] In certain embodiments, the composition comprises 100 mg to 1,000 mg of ginger (e.g., 125 mg to 750 mg, or 250 mg to 550 mg).

[0087] In certain embodiments, a composition or dosage form of the composition may further comprises one or more of the following ingredients, including spirulina, chlorella, dulse, barley grass, alfafa juice concentrate, wheat grass, spinach leaf, kale leaf, beet root, rose hips, acai fruit, raspberry fruit, blueberry fruit, goji berry, bilberry fruit extract, green tea leaf extract, ginkgo biloba, Echinacea purpurea root, licorice root extract, milk thistle extract, stinging nettle root, royal jelly, grape seed extract, bee pollen, maca root, reishi mushroom, rhodiola root, ashwagandha root, and / or fiber (e.g., sunflower lecithin, apple fiber, brown rice bran). In certain embodiments, a composition of the present invention can be flavored using stevia or monk fruit, or another natural flavor (e.g., natural blueberry flavor).

[0088] The compositions or dosage forms described herein, can be administered or consumed individually or in combination. For example, a composition or dosage form that includes L.Attorney Docket No. BHMP106WO delbrueckii and / or B. bifidum may be administered or consumed individually as such, or may be co-administered or concurrently consumed with other nutrients or supplements. (vi) Manufacturing Process

[0089] In certain embodiments, a composition of the present invention may be manufactured according to the following process: The L. delbrueckii and / or B. bifidum and in an embodiment, the non-pathogenic fungal (e.g., S. boulardii) and non-pathogenic bacterial strains (e.g., B. breve, L. acidophilus, and L. rhamnosus), available from commercial sources, are each cultured separately in a small scale fermenter. For each strain, a culture sample is then used to inoculate a corresponding large scale production fermenter. The cultured fungal and bacterial strains are harvested via filtration and / or centrifugation, and can then be dried, for example, freeze dried. The dried organisms can then optionally be coated, for example, spray coated with a polymer such as hydroxylpropylmethyl cellulose (HPMC). The coated strains, for example, HPMC- coated strains, can then be subjected to further drying, such as, low temperature drying (e.g., room temperature). The resulting strains are then blended together at the appropriate concentrations for the specific formulation being manufactured. At this blending stage, if desired, other ingredients specific to the formulation (e.g., dried amylase) can be added. If desired, the blended composition can then be encapsulated, which can then be packaged into an appropriately labeled container. An exemplary manufacturing process is depicted in Fig.1. Oral Dosage Form

[0090] In certain embodiments, the composition or dosage form may be consumed orally by the subject. In certain embodiments, the composition is consumed as a powder or product containing the powder. In other embodiments, the composition can be in the form of an oral dosage form, for example, where the composition in included, for example, within a capsule, cachet, pill, tablet, lozenge, powder, granule, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles, each containing the requisite number of colony forming units of the non- pathogenic bacteria and non-pathogenic fungus, and optionally the appropriate amount of enzyme.

[0091] In certain embodiments, the composition is disposed in a capsule or in a tablet. In certain embodiments, the composition is formulated as a tablet. In certain embodiments, the capsule is a vegetable cellulose capsule. In certain embodiments, the dosage form, for example, capsule or tablet, is coated with a coating, for example, a non-functional aesthetic coating or aAttorney Docket No. BHMP106WO functional coating, for example, a controlled release coating. The capsule or tablet may be formulated so as to provide slow or controlled release of the ingredients disposed therein.

[0092] In certain embodiment, the composition or dosage form comprises additives such as calcium carbonate, xylitol, cetyl alcohol, citric acid, natural flavor, monk fruit. In certain embodiments, the composition comprises additives such as cascara sagrada bark, psyllium husk, senna leaf, Flaxseed, aloe vera leaf, licorice root, medium chain triglyceride (MCT) oil. In certain embodiments, the composition or dosage form comprises additives such as a dietary fiber, e.g., inulin (fructooligosaccharides FOS) and apple pectin. In certain embodiments, the composition or dosage form comprises additives such as a blend of spirulina, barley grass, alfalfa leaf, wheat grass, chlorella, dulse, spinach leaf, broccoli, parsley leaf, kale leaf, echinacea angustifolia root, licorice root, milk thistle seed, Siberian eleuthero root, beet root, rose hips, acai (fruit), green tea leaf, raspberry leaf, blueberry (fruit), goji berry, bilberry (fruit), ashwagandha root, rhodiola root, reishi mushroom, maca root, bee pollen, nettle leaf, ginko biloba (leaf extract), royal jelly (3x concentrate), grape seed. In certain embodiments, the composition further comprises a vitamin, such as, vitamin C. Coatings

[0093] Depending upon the mode of delivery, the composition or dosage form of the present invention may be prepared as a powdered blend of organisms where the powder can be coated with a non-functional coating (e.g., aesthetic coating) or a functional coating (e.g., controlled release coating to modulate the release of the organisms in, for example, a time- and / or pH- dependent manner). Similarly, the composition or dosage form may be a capsule containing a powdered blend of organisms, where the capsule is coated with a non-functional (e.g., aesthetic coating) or a functional coating (e.g., a controlled release coating to modulate the release of the organisms in, for example, a time- and / or pH-dependent manner). In certain embodiments, controlled release coating is hydroxypropyl methylcellulose (HPMC).

[0094] Controlled release coatings can facilitate the continuous release, gradual release, prolonged release, and / or programmed release (e.g., pH-dependent release) of the microorganisms in the compositions or dosage forms disclosed herein. Topical Dosage Form

[0095] In certain embodiments, the composition or dosage form may be formulated for topical delivery, for example, as a liquid, emulsion, suspension, ointment, cream, gel, lotion, or powder. In certain embodiments, upon application to a skin of a subject, the composition or dosage form may form a patch. Depending up the concentration of non-pathogenic organisms and theAttorney Docket No. BHMP106WO particular delivery device, the composition or dosage form may be formulated to deliver from 1.0 mL / 5 cm2to 1.0 mL / 50 cm2, or from 1.0 mL / 5 cm2to 50 mL / 50 cm2, or from 1.0 mL / 5 cm2to 100 mL / 50 cm2.

[0096] The topical dosage form may also include a pharmaceutically acceptable carrier. Suitable carriers that may be useful in topical formulations include, but are not limited to, solubilizers such as C2-C8, straight and branched chain alcohols, diols and triols, moisturizers and humectants such as glycerin, amino acids and amino acid derivatives, polyaminoacids and derivatives, pyrrolidone carboxylic acids and its salts and derivatives, surfactants such as sodium lauryl sulfate, sorbitan monolaurate, emulsifiers such as cetyl alcohol, stearyl alcohol, thickeners such as methyl cellulose, ethyl cellulose, hydroxymethylcellulose, hydroxypropylcellulose, polyvinylpyrollidone, polyvinyl alcohol and acrylic polymers. Spray and Inhalation Dosage Forms

[0097] In certain embodiments, the composition or dosage form may be formulated for nasal or oral administration in the form of a nasal spray and inhalation solution, and suspension, that is administered for example, by inhalation through the nose. The dose can be delivered to the nasal cavity via a spray pump or as nasal drops for local and / or systemic effects.

[0098] In certain embodiments, the composition or dosage form may be an inhalation solution and / or suspension for delivery to the upper and lower respiratory tract (e.g. oropharynx, lungs) by oral inhalation for local and / or systemic effects and can be used with a specified nebulizer. (III) DOSE

[0099] The size of the dose delivered or consumed, will depend upon, among other things, the size and age of the subject, the indication or condition to be treated, and the mode of delivery of the composition or dosage form. In certain embodiments, for example, oral composition or dosage forms may be 1 mg to 1,000 mg or 50 mg to 1,000 mg of the active ingredients (for example, the non-pathogenic bacteria, the non-pathogenic fungus, and / or the enzyme). In certain embodiments, the composition or dosage form may be 1 mg to 900 mg, 1 mg to 800 mg, 1 mg to 700 mg, 1 mg to 600 mg, 1 mg to 500 mg, 1 mg to 400 mg, 1 mg to 300 mg, 1 mg to 200 mg, 1 mg to 100 mg, 1 mg to 500 mg, 10 mg to 1000 mg, 20 mg to 1000 mg, 30 mg to 1000 mg, 40 mg to 1000 mg, 50 mg to 1000 mg, 100 mg to 1000 mg, 200 mg to 1000 mg, 300 mg to 1000 mg, 400 mg to 1000 mg, 500 mg to 1000 mg, 600 mg to 1000 mg, 700 mg to 1000 mg, 800 mg to 1000 mg, or 900 mg to 1000 mg of the active ingredients (for example, the L. delbrueckii and / or B. bifidum, non-pathogenic bacteria, the non-pathogenic fungus, and / or the enzyme).Attorney Docket No. BHMP106WO The composition or dosage forms (either one or multiple units (for example 2, 3, 4, or 5 units, for example, capsules or tablets) may be administered once, twice or three times a day during the treatment period, for example, until the biofilm of interest has been disrupted, prevented from forming, and / or the subject’s normal microbiome has been restored which could take, for example, one week, two weeks, one month, two months, or three months and one year. In certain embodiments, one capsule, such as one enteric-coated capsule, that comprises about 30 billion colony forming units of non-pathogenic fungal strain(s) and non-pathogenic bacterial strain(s) and an enzyme, is administered once, twice, or three times a day during the treatment period, for example, until the biofilm of interest has been disrupted and / or the subject’s normal microbiome has been restored which could take, for example, one week, two weeks, one month, two months, or three months and one year. METHOD OF TESTING SUITABILITY OF A SUBJECT FOR TREATMENT

[0100] The invention also provides a method of identifying a test subject suitable for treatment with a composition or dosage form described herein. The method comprises: (a) quantifying the number or density and / or abundance of organisms of at least one non-pathogenic bacterial strain; and (b) comparing the number or density of organisms quantified in the sample against the number or density of corresponding organisms present in a corresponding sample of a corresponding region of a healthy subject or a cohort of healthy subjects, or a cohort of subjects that report low stress levels. When the number or level of organisms in the sample from the test subject is less than the number or level of organisms present in the corresponding region of the healthy subject, e.g., at least 10% less than, at least 25% less than, or at least 50% less than the test subject is suitable for treatment with the dosage form. In certain embodiments, the non- pathogenic bacteria that are quantified may include Lactobacillus delbrueckii and Bifidobacterium bifidum. Incorporation by Reference Other compositions, components, concentrations, and dosages that can be combined with the teachings herein are disclosed in U.S.2019 / 0381119 and U.S.2021 / 0030818, incorporated herein by reference. (XI) DEFINITIONS

[0101] The following definitions are included for the purpose of understanding the present subject matter and for constructing the appended patent claims.

[0102] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having,Attorney Docket No. BHMP106WO including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0103] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0104] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present disclosure, whether explicit or implicit herein. For example, where reference is made to a particular composition, that composition can be used in the various embodiments of such compositions of the present invention and / or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.

[0105] It should be understood that the expression “at least one of” includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0106] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0107] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.Attorney Docket No. BHMP106WO

[0108] Where a molecular weight is provided and not an absolute value, for example, of a polymer, then the molecular weight should be understood to be an average molecule weight, unless otherwise stated or understood from the context.

[0109] As a general matter, compositions specifying a percentage are by weight unless otherwise specified.

[0110] As used herein, the term “nutritional ingredient” refers to one or more ingredient(s) added to a composition of the present disclosure, which can be a natural ingredient having no known adverse physiological effect in a human, or known to be beneficial or necessary for optimal physiology and health of a human.

[0111] As used herein, the terms “treat,” “treating,” or “treatment,” and other grammatical equivalents as used herein, include alleviating, abating, ameliorating, or preventing a disease, condition or symptoms, preventing additional symptoms, ameliorating or preventing the underlying causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition, and are intended to include prophylaxis.

[0112] As used herein, the terms, “subject,” “patient,” “subject in need thereof,” and “patient in need thereof” are used interchangeably herein, and refer to a living organism, including animals and humans, suffering from or prone to a disease or condition that can be treated by the methods and compositions provided herein. The subject can be a human or non-human animal.

[0113] The foregoing examples are presented herein for illustrative purposes only, and should not be construed as limiting the invention in any way. EXAMPLES

[0114] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.

[0115] EXAMPLE 1

[0116] A bioinformatics database was developed from patient fecal samples that were examined for various microorganisms. Data was developed with questionnaire data correlating to the various patients based on their various symptoms. By comparing the microbiomes of individuals within the cohort to the comprehensive database, deficiencies were identified and correlated to the higher stress cohort (broken up into moderate and high stress).Attorney Docket No. BHMP106WO

[0117] EXAMPLE 2

[0118] The analysis revealed a differential abundance of several probiotic organisms of interest that are associated with lower stress cohorts when compared to higher stress cohorts. The symptom of stress was evaluated and attempts were made to correlate it to the microorganisms present or absent in the microorganism data for the patient. Microorganisms that were not in a normal range were identified as L. delbrueckii and / or B. bifidum in the patients. Male patients suffering from stress were more likely to have a deficiency in their levels of L. delbrueckii and female patients were more likely to have a deficiency in their levels of B. bifidum.

[0119] Fig.2 is a graph showing the log fold change (natural log of fold change) in the abundance of L. delbruekii in moderate stress (right hand, lighter bar) and high stress (left hand, darker bar) subjects compared to subjects reporting low stress levels. The numbers are reported as the log of the average change. The total number for the low stress cohort was 703, the total number for the moderate stress cohort was 2846, and the total number for the high stress cohort was 1779 individuals. The negative values indicate a deficiency in the moderate and high stress cohort compared to the low stress cohort. The unit of measurement in this example is “normalized abundance” as seen by the ANCOM BC2 method. The differences noted by an asterisk in the figure were determined to be statistically significant.

[0120] Fig.3 is a graph showing the log fold change (natural log of fold change) in the abundance of L. delbruekii in moderate stress (right hand, lighter bar) and high stress (left hand, darker bar) female subjects compared to female subjects reporting low stress levels. The negative values indicate a deficiency in the moderate and high stress cohort compared to the low stress cohort. The differences noted by an asterisk in the figure were determined to be statistically significant.

[0121] Fig.4 is a graph showing the log fold change (natural log of fold change) in the abundance of B.bifidum in moderate stress (right hand, lighter bar) and high stress (left hand, darker bar) of female subjects compared to female subjects reporting low stress levels. The negative values indicate a deficiency in the moderate and high stress cohort compared to the low stress cohort. The differences noted by an asterisk in the figure were determined to be statistically significant.

[0122] Fig.5 is a graph showing the log fold change (natural log of fold change) showing change in abundances in high stress vs low stress (left-hand, darker column) and medium stress vs low stress cohorts (right-hand, lighter column). The asterisk indicates statistical significance.Attorney Docket No. BHMP106WO The cohort comprised of both male and female subjects. The differences noted by an asterisk in the figure were determined to be statistically significant. The abundance of S. Moorei in the high stress cohort over the low stress cohort, may indicate a way of diagnosing stress.

[0123] It should be noted that Bulleidia moorei and Solobacterium moorei, as used herein, are the same species. Both are used herein due to a recent taxonomic change.

[0124] EXAMPLE 3

[0125] Minimum inhibitory concentration (MIC) assays were preformed to determine whether the select organisms L. delbrueckii subsp. Bulgaricus and B. Bifidum are antagonistic to opportunistic pathogens, such as Solobacteria moorei. As shown in Fig.6 several experimental wells including S. moorei, varying in concentration of L. delbrueckii subsp. bulgaricus and B. bifidum in reinforced clostridial media with 5% goat serum were observed for growth.10 μL of pathogenic cultures (1x105CFU / mL) were added to serially diluted concentrated cell free supernatants (ranging from 1:2 to 1:128). The wells were subjected to anaerobic conditions at 37˚C for 24 hrs.

[0126] Determination of the MIC (Minimum Inhibitory Concentration) was done by finding the concentration resulting in 50% growth compared to the positive control (PC)(no spent media). A comparison of the MIC of spent media (PC) to unspent media (NC) was also made. It was determined that Solobacterium moorei population of about 1x105was inhibited: 93.7% by Lactobacillus delbrueckii subsp. Bulgaricus and Bifidobacterium bifidum showing strong inhibition at about 1:16 ratio of either L. delbrueckii subsp. Bulgaricus or B. Bifidum to S. moorei.

[0127] EXAMPLE 4

[0128] Fig.7 is a graph showing optical density over time of fructose oligosaccharide (FOS) effect on growth promotion of B. bifidum. 0.5% to 2.0% FOS showed about the same level of growth promotion and improvement over the negative control (sterility control media with no inoculum). The positive control indicated bacterial growth in the media containing dextrose as a carbohydrate. The other examples with B. bifidum were added into carbohydrate free media. While not as effective as dextrose, dextrose is not conducive to healthful supplement and is known to grow other undesirable yeast and bacteria.

[0129] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference.Attorney Docket No. BHMP106WO

[0130] What has been described above includes examples of one or more embodiments. It is, of course, not possible to describe every conceivable modification and alteration of the above devices or methodologies for purposes of describing the aforementioned aspects, but one of ordinary skill in the art can recognize that many further modifications and permutations of various aspects are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims. Furthermore, to the extent that the term “includes” is used in either the detailed description or the claims, such term is intended to be inclusive in a manner similar to the term “comprising” as “comprising” is interpreted when employed as a transitional word in a claim. The term “consisting essentially” as used herein means the specified materials or steps and those that do not materially affect the basic and novel characteristics of the material or method. Unless the context indicates otherwise, all percentages and averages are by weight. If not specified above, the properties mentioned herein may be determined by applicable ASTM standards, or if an ASTM standard does not exist for the property, the most commonly used standard known by those of skill in the art may be used. The articles “a,” “an,” and “the,” should be interpreted to mean “one or more” unless the context indicates the contrary.

Claims

Attorney Docket No. BHMP106WO WHAT IS CLAIMED IS:

1. A method of reducing stress in a subject, the method comprising consumption by, or administration to, the subject of a probiotic composition comprising Lactobacillus delbrueckii and Bifidobacterium bifidum.

2. The method of claim 1, further comprising an enzyme.

3. The method of claim 1, wherein the probiotic composition further comprises Lactobacillus acidophilus, Lactobacillus rhamnosus, Saccharomyces boulardii, and Bifidobacterium breve.

4. The method of claim 1, wherein the probiotic composition further comprises Lactobacillus plantarum.

5. The method of claim 1, wherein the Lactobacillus delbrueckii is subsp. Bulgaricus.

6. The method of claim 1, wherein the probiotic composition further comprises a prebiotic fiber that improves growth of Lactobacillus delbrueckii and Bifidobacterium bifidum.

7. The method of claim 2, wherein the enzyme is an amylase.

8. The method of claim 1, wherein the probiotic composition further comprises a protein or vitamin.

9. The method of claim 1, wherein the probiotic composition is freeze dried or spray dried.

10. The method of claim 1, wherein one or more organisms in the probiotic composition are antagonistic to pathogen Solobacteria moorei.

11. A composition for use in reducing stress in a human subject, the composition comprising a live, freeze-dried probiotic composition comprising a combination of Lactobacillus delbrueckii and Bifidobacterium bifidum; wherein the Lactobacillus delbrueckii and Bifidobacterium bifidum are each present in an amount of 1 billion to 5 billion CFUs.

12. The composition of claim 11, further comprising (i) an isolated and viable non-pathogenic fungal strain and (ii) an additional isolated and viable non-pathogenic bacterial strain.Attorney Docket No. BHMP106WO 13. The composition of claim 12, wherein the non-pathogenic bacterial strain comprises Lactobacillus rhamnosus, Bifidobacterium breve, Lactobacillus acidophilus, and Bifidobacterium bifidum.

14. The composition of claim 12, wherein the non-pathogenic fungal strain is selected from Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomycetes sp HZ178, Saccharomyces bayanus, Pichia burtonii, Pichia jadinii, Pichia kudriavzevii, Pichia onychis, Pichia sp., and Picoa juniper.

15. The composition of claim 11, wherein the composition is in a dosage form comprising 50 mg to 1,000 mg of the composition.

16. The composition of claim 11, further comprising an enzyme.

17. The composition of claim 11, wherein the composition further comprises Lactobacillus plantarum.

18. The composition of claim 11, wherein Lactobacillus delbrueckii is subsp. Bulgaricus.

19. The composition of claim 11, wherein the probiotic composition further comprises a prebiotic fiber that improves growth of Lactobacillus delbrueckii, Bifidobacterium bifidum, or Lactobacillus delbrueckii, Bifidobacterium bifidum, and any combination thereof.

20. The composition of claim 16, wherein the enzyme is an amylase selected from the group consisting of Bacillus stearothermophilus amylase, Bacillus amyloliquefaciens amylase, Bacillus subtilis amylase, Bacillus licheniformi amylase, Aspergillus niger amylase, Aspergillus oryzae amylase, and any combination thereof.

21. The composition of claim 11, wherein the probiotic composition comprises protein and the protein is a protein hydrolysate.

22. The composition of claim 11 further comprising at least one digestive enzyme and a nutritional ingredient.

23. The composition of claim 23, wherein the nutritional ingredient is present and is selected from inulin, collagen peptides, deglycyrrhizinated licorice (DGL), marshmallow root, grape seed extract, ginger, a vitamin, and any combination thereof.Attorney Docket No. BHMP106WO 24. The composition of claim 11, further comprising Bacopa monnieri or Bacopa monnieri extracts in a 10 to 500 mg or Lions Mane mushroom or Lion’s Mane mushroom extracts in a range of 10 to 500 mg.

25. The composition of claim 11, further comprising fructose oligosaccharide.

26. The method of claim 1, wherein the subject has a lower level of L. delbrueckii or B. bifidum relative to an average cohort reporting low stress.

27. The method of claim 1, wherein the subject has a higher level of S. Moorei relative to an average cohort reporting low stress.

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