Methods of Synbiotic Treatment to Improve Health

Abstract

Synbiotic compositions are provided that include both a prebiotic component and a probiotic component, the prebiotic component comprising at least one compound that can be converted into a bioactive metabolite by microbial strains present in the healthy human gut microbiome, and the probiotic component comprising a rationally defined and assembled consortium of microbial strains.

Description

[Technical field]

[0001] The present disclosure relates generally to microbial biologics for pediatric use, and in particular to rationally defined microbial consortia that are effective in imparting health across organ systems in human children by modulating the natural gut microbiota and host tissue function. [Background technology]

[0002] Human gut microbiome The human gut microbial community is a complex network of bacteria, archaea, viruses, fungi, and even protozoa that reside primarily in the host's colon. This gut microbial community is comparable in cell number to the human body, but contributes more than 100-fold more genetic coding capacity.

[0003] Due to its important role in human health, the microbiome may be considered an organ of the human body, and like other organs, this microbial organ evolves over time in terms of its microbial numbers and complexity. Although microbial DNA has been detected at fetal stages, the intestine may be considered sterile or at best sparsely populated at birth. Further colonization of the intestine depends on the birth, environment, and type of first food, i.e., breast milk or formula. During the first few days of life, the intestinal microbiome is dominated by aerobic bacteria and facultative anaerobes, such as the enterobacteria Enterococcus, Staphylococcus, and Streptococcus. These microorganisms proliferate in the intestine until the intestinal oxygen concentration is sufficiently reduced to allow the proliferation of obligate anaerobes, such as Bifidobacterium species, which then become the dominant colonizers during the first few weeks of life. In some infants, Bacteroides species, another type of facultative anaerobe, may also predominate. As more foods are introduced into the infant's diet around 6 months of age, infants acquire a more diverse range of bacteria, resulting in a more complex microbiome by 12 months of age. During this period, the number of clostridia increases in proportion to the increased need to digest carbohydrates accessible to more diverse microbiota than reach the colon, resulting in a higher diversity and maturity of the infant gut microbiome. By about 3 years of age, the child's gut microbiome resembles that of an adult. Members of the mature gut microbial community are primarily derived from five major phyla: Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Verrucomicrobia, with the former two constituting approximately 90% of the population.

[0004] Gut bacteria co-evolve with their human host to extract energy for their own existence while also nurturing the host. The gut microbiota plays an important role in nutrient digestion, digestive tract maturation, immune activation, protection against pathogens, production of vitamins not normally produced by the host, and even brain health. Dysbiosis in the gut microbiota is associated with gut-related diseases, such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS); immune diseases, such as asthma, atopic dermatitis, and rheumatoid arthritis; and lifestyle-related metabolic diseases, such as obesity and diabetes. Gut bacteria are also increasingly associated with brain-related problems, such as mood, anxiety, and neurodegenerative disorders.

[0005] Intestinal commensals are adept at cooperatively extracting energy from organic material that evades host metabolism and reaches the colon. Thus, undigested food remnants, mainly plant dietary fibers and polyphenols, as well as glycoproteins shed from the intestinal mucosa, are metabolized by gut bacteria to produce acidic metabolites. The most important of these are the organic acid lactic acid, and short-chain fatty acids (SCFAs), such as acetic acid, propionic acid, and butyric acid.

[0006] Acetic acid and lactic acid are produced in the earlier steps of bacterial carbohydrate decomposition. Then, through a process of symbiotic resource sharing, these metabolites serve as carbon sources for propionic acid and butyric acid producing strains, which can therefore be called "secondary" feeders. Acetic acid is produced by most enterobacteria, while lactic acid is produced by fewer bacteria, especially Bifidobacterium species and lactic acid bacteria (Lactobacillales).

[0007] SCFAs play a major role in regulating host metabolism, immune defense, and even brain health. Acetate, a three-carbon molecule, has beneficial effects on host energy and metabolism through appetite modulation and fat oxidation. Propionate is known to increase the levels of plasma glucagon, fatty acid binding protein 4 (FABP4) and norepinephrine, leading to insulin resistance and compensatory hyperinsulinemia. Butyrate provides most of the fuel required for colonocytes and plays a key role in maintaining the intestinal barrier by modulating tight junction proteins. Butyrate also acts as a histone deacetylase inhibitor and affects signaling through several G protein-coupled receptors that mediate anti-inflammatory activity. Barrier maintenance is energy intensive, and butyrate produced by microorganisms is the most important source of this energy. Taken together, butyrate has beneficial effects on gut homeostasis and energy metabolism, and its anti-inflammatory effects enhance gut barrier function and mucosal immunity.

[0008] The presence of balanced SCFAs shows an essential role in preventing chronic inflammatory conditions, inhibiting the production of proinflammatory cytokines, regulating T cells, and protecting colonic epithelial cells. The effects of SCFAs extend beyond the digestive tract, as these metabolites are associated with the respiratory system by stimulating airway hyperresponsiveness, inhibiting skin pathogens through the gut-skin axis, and inhibiting the function of pulmonary innate lymphoid cells (ILC2), regulators of bone mass and osteoclast metabolism. Furthermore, SCFAs also act through the gut-brain axis to regulate mood, sleep, and neuronal health.

[0009] The Gut Microbiome and Health in Children The relationship between the gut microbiome and health is becoming increasingly clear. One of the earliest associations was well explained by the "hygiene hypothesis," which suggested that infections early in life due to "unhygienic contacts" (e.g., resulting from living on a farm, keeping pets, etc.) prevented allergies. Clear links have been found between the gut microbiome and the development of diseases in children.

[0010] There are many factors that influence the development and maturation of the gut microbiome in children. These factors include mode of birth, first food (i.e., breast milk vs. formula), household size, and environment (location, urban vs. rural, presence of pets, etc.). Non-limiting examples of associations between gut microbiome status and disease in children include the association between allergies and reduced species diversity; the association between autism spectrum disorder and increased Clostridium species and / or Sutterella and Desulfovibrio species; the association between colic and reduced microbial diversity and / or increased anaerobic bacterial populations; the association between onset of eczema and early colonization by opportunistic species; the association between nutritional disorders and excess of gut pathogenicity with anaerobic depletion, early dysbiosis, and / or reduced bacterial diversity; prematurity, and prostate cancer. Associations between an increase in Theobacteria and / or a decrease in microbial diversity; associations between sepsis and changes in the structure and composition of the microbiota before the onset of disease; associations between type I diabetes and an increase in the Bacteriodetes:Firmicutes ratio, an increase in Clostridia species, a decrease in butyrate-producing bacteria, a decrease in bacterial diversity, and / or a decrease in the stability of the microbial community; associations between type II diabetes and an increase in the Firmicutes:Bacteroidetes ratio and / or an increase in SCFAs.

[0011] Probiotics It has been over a century since the correlation between increased life span and consumption of yogurt containing lactic acid-producing bacteria was first observed, and since Bifidobacterium species were first detected in the stool of breast-fed infants but not in formula-fed infants suffering from diarrhea. These early "germs for health" concepts led to the widely adapted use of the term "probiotics," which means "live microorganisms that, when administered in adequate amounts, confer a health benefit to the host."

[0012] Probiotics historically encompassed the genera Bifidobacterium and Lactobacillus. Over 250 Lactobacillus species have been identified, which appear to be highly genetically distinct from one another and metabolically, ecologically, and functionally diverse. These observations have led to the recent reclassification of Lactobacillus into 25 genera, including a revised Lactobacillus (now encompassing the host-compatible organisms referred to as the Lactobacillus delbrueckii group), Paralactobacillus, and 23 novel genera.

[0013] The National Institutes of Health Human Microbiome Project showed a large variation in microbial diversity and abundance among healthy individuals, indicating that a universally healthy microbiome does not exist. Despite this variety of health states, important correlations between health and disease states and the presence or abundance of specific microbial species have been established, raising the possibility that manipulation of these communities can prevent and treat disease. This concept has prompted the isolation of specific microbiota members from healthy individuals for the development of so-called live biological therapeutic products (LBPs). These have been added to probiotic products based on classical Lactobacillus and Bifidobacterium with the goal of beneficially influencing disease states. Notably, gut microbiota interventions not only provide health benefits to the local environment, but also have distant effects, for example through the gut-skin, gut-heart, and gut-brain axes.

[0014] Prebiotics A prebiotic is defined by the International Probiotic and Prebiotic Science Society as "a substance selectively utilized by the host microorganism that confers a health benefit." For a substance to be considered a prebiotic under this definition, it must meet three criteria: (1) it is not digested or utilized by the host's system; (2) it is selectively utilized by the host microorganisms and results in a favorable change in the microbial balance or metabolite formation that benefits the host; and (3) the benefit to the host is demonstrated by clinical studies and is the result of microbial alterations in the host's gut.

[0015] The main type of dietary prebiotics are certain microbiota-fermentable dietary fibers. Some plant-derived polyphenols may also demonstrate prebiotic effects due to their ability to selectively modulate bacterial growth and undergo microbial biotransformation into a range of metabolites, with beneficial effects on the host.

[0016] Dietary fiber prebiotics are polymeric and oligomeric carbohydrates that are utilized by gut microbes, leading to the production of beneficial metabolites through a multi-step fermentation process. Currently, most commercial prebiotics are in this category, and common examples include fructooligosaccharides (FOS), galactooligosaccharides (GOS), and inulin. The number of fructose molecules linked together (i.e., the degree of polymerization, or DP), and the branching of these molecules play a key role in the fermentability of these prebiotics. In general, low DP (DP of 1 or 2) fructans are more easily and more rapidly destroyed by gut bacteria in laboratory models of the gut, while large DP fructans (DP of 3 to 60) show a slower and more constant rate of fermentation by stool bacteria. Therefore, higher DP inulin may be able to remain intact long enough to be subjected to the forces of intestinal peristalsis and pushed more distally into the colon, extending the period of microbial activity.

[0017] Symbiotics When both probiotics and prebiotics are combined in a product, the product may be referred to as a "synbiotic". Thus, a synbiotic may be defined as a mixture containing live organisms and substances selectively utilized by the host microorganisms that confer a health benefit to the host. Such formulations may be complementary in their mechanism of action, i.e., the prebiotic targets the native commensal organism, or the prebiotic substance is synergistic in that it is selectively utilized by the co-administered probiotic. Delivery of synbiotics to targeted niches in the human gastrointestinal tract is difficult due to the harsh and variable conditions of the gut, problems associated with maintaining the activity and efficacy of probiotics, and masking the unpleasant flavors of prebiotics. Synbiotic delivery technology has been widely researched by the functional food and supplement industry. Summary of the Invention

[0018] In an embodiment of the present disclosure, a method for treating a disease or achieving a physiological objective in a human subject comprises administering to the subject a therapeutically effective amount of a synbiotic composition, the synbiotic composition comprising: a prebiotic component comprising at least one compound that can be converted into a bioactive metabolite by a microbial strain present in a healthy human gut microbiota; and a probiotic component comprising a consortium of microbial strains, the consortium being selected from the group consisting of: (i) Lacticaseibacillus rhamnosus SD-GG-BE; (ii) Ligilactobacillus salivarius SD-LS1-IT; (iii) Bifidobacterium breve SD-BR632-IT; (iv) Bifidobacterium breve SD-BR03-IT; (v) Bifidobacterium longum SD-GG-BE; (vi) Lacticaseibacillus casei SD-CECT9104-SP; and (vii) Bifidobacterium lactis SD-CECT8145-SP.

[0019] In embodiments, the disease is adrenoleukodystrophy, AGE-induced genomic damage, Alexander's disease, alopecia areata, Alpers' disease, Alzheimer's disease, amyotrophic lateral sclerosis, angina, arthritis, asthma, Baro's concentric sclerosis, Behcet's disease, bullous pemphigoid, Canavan disease, heart failure including left ventricular dysfunction, central nervous system vasculitis, Charcot-Marie-Tooth disease, childhood ataxia with central nervous system hypomyelination, chronic idiopathic peripheral neuropathy, chronic obstructive pulmonary disease, Crohn's disease, cutaneous lupus, dermatitis (contact, acute and chronic), diabetic retinopathy, graft versus host disease, granulomas, hepatitis C virus infection, herpes simplex virus infection, herpes zoster virus infection, hepatitis C virus infection, herpes simplex virus infection, herpes zoster virus infection, In some embodiments, the present invention may be selected from the group consisting of human immunodeficiency virus infection, Huntington's disease, irritable bowel disorder, ischemia, Krabbe disease, lichen planus, macular degeneration, mitochondrial encephalomyopathy, unilateral upper limb muscular atrophy, multiple sclerosis, myocardial infarction, neurodegeneration with cerebral iron accumulation, neuromyelitis optica, neurosarcoidosis, NF-κB mediated disease, optic neuritis, paraneoplastic syndromes, Parkinson's disease, Pelizaeus-Merzbacher disease, pemphigus, primary lateral sclerosis, progressive supranuclear palsy, psoriasis, pyoderma gangrenosum, reperfusion injury, retinitis pigmentosa, sarcoidosis, Schilder's disease, subacute necrotizing myelopathy, Susac syndrome, transplant rejection, transverse myelitis, tumor, ulcerative colitis, and Zellweger syndrome.

[0020] In an embodiment, the disease may be a digestive disease or an infectious disease.The disease may, but need not be, selected from the group consisting of irritable bowel syndrome, COVID-19, and constipation.The disease may, but need not be, antibiotic-induced dysbiosis of the intestinal microbiota of the subject.

[0021] In an embodiment, the disease may be selected from the group consisting of metabolic syndrome, type 2 diabetes, and pre-diabetes.

[0022] In embodiments, the physiological objective may be selected from the group consisting of improving cardiovascular health, maintaining or reducing weight, reducing glycated hemoglobin levels, normalizing glucose or insulin response, treating acne or otherwise improving skin health, and improving cognitive function.

[0023] In embodiments, the method may further include co-administering or administering to a human subject one or more other pharmaceutical agents in combination with the synbiotic composition, where the co-administration or administration in combination may improve the therapeutic effect of the one or more other pharmaceutical agents compared to administration of the synbiotic composition and the one or more other pharmaceutical agents independently.

[0024] In an embodiment, the physiological objective may be selected from the group consisting of increasing the Bristol Fecal Form Scale (BSFS), decreasing the duration of bowel movements, reducing abdominal pain, reducing abdominal bloating, reducing heartburn, reducing acid regurgitation, reducing digestive disorders, maintaining or increasing the diversity of the gastrointestinal microbiota, and improving health-related quality of life as measured by the KINDL questionnaire score.

[0025] In an embodiment, the synbiotic composition may be administered as an ingestible formulation. The ingestible formulation may be, but need not be, in the form of a flowable powder provided in a single-serving sachet. The sachet may, but need not be, in the form of a single-serving sachet, such as from about 1 mg to about 12 g, or from about 250 mg to about 11.75 g, or from about 500 mg to about 11.5 g, or from about 750 mg to about 11.25 g, or from about 1 g to about 11 g, or from about 1.25 g to about 10.75 g, or from about 1.5 g to about 10.5 g, or from about 1.75 g to about 10.25 g, or from about 2 g to about 10 g, or from about 2.25 g to about 9.75 g, or from about 2.5 g to about 9.5 g, or from about 2.75 g to about 9.25 g, or from about 3 g to about 5 g. g to about 9 g, or about 3.25 g to about 8.75 g, or about 3.5 g to about 8.5 g, or about 3.75 g to about 8.25 g, or about 4 g to about 8 g, or about 4.25 g to about 7.75 g, or about 4.5 g to about 7.5 g, or about 4.75 g to about 7.25 g, or about 5 g to about 7 g, or about 5.25 g to about 6.75 g, or about 5.5 g to about 6.5 g, or about 5.75 g to about 6.25 g, or about 6 g of the prebiotic component. The sachet may, but need not, contain the consortium of microbial strains in an amount of about 62.5 million AFU to about 312.5 billion AFU, about 625 million AFU to about 250 billion AFU, about 1.25 billion AFU to about 125 billion AFU, about 6.25 billion AFU to about 62.5 billion AFU, about 12.5 billion AFU to about 60 billion AFU, about 25 billion AFU to about 55 billion AFU, about 40 billion AFU to about 50 billion AFU, or about 45 billion AFU. The material of the sachet may, but need not, have a water vapor transmission rate of less than about 0.01 grams per square meter per day at 23° C. and 50% relative humidity. A dose of the synbiotic composition may, but need not, be administered at least once a day, and a dose may, but need not, contain the contents of one sachet. The sachet may, but need not, further comprise at least one pharma- ceutically acceptable vehicle.

[0026] In embodiments, the synbiotic composition may be administered at least once a day for at least about seven days.

[0027] In an embodiment, the at least one compound that can be converted into bioactive metabolites by microbial strains present in healthy human gut microbiome may include at least one fructan. The at least one fructan may, but does not necessarily include at least one polysaccharide fructan. The at least one polysaccharide fructan may, but does not necessarily include inulin. The inulin may, but does not necessarily include at least one plant selected from the group consisting of agave, asparagus, banana, barley, burdock, barley, chicory, cornflower, costus, dandelion, elegans, garlic, artichoke, Jerusalem artichoke, jicama, leek, hareweed, mugwort, onion, psyllium, wheat, yacon, and yam. The at least one fructan may, but does not necessarily include fructooligosaccharide. The fructooligosaccharides may, but need not, be derived from or extracted from at least one plant selected from the group consisting of agave, asparagus, banana, barley, burdock, barley, chicory, cornflower, costus, dandelion, elegans, garlic, artichoke, Jerusalem artichoke, jicama, leek, hareweed, mugwort, onion, psyllium, wheat, yacon, and yam.

[0028] In embodiments, the prebiotic component may consist essentially of about 50 wt% inulin and about 50 wt% fructooligosaccharides.

[0029] In embodiments, the consortium may include at least three, at least four, at least five, at least six, or all of (i) through (vii).

[0030] In an embodiment, the consortium may further comprise at least one microbial strain selected from the group consisting of (viii) Lactobacillus acidophilus SD-NCFM-US; and (ix) Bifidobacterium animalis subsp. lactis SD-BI07-US. The consortium may, but need not, comprise both (viii) and (ix). The consortium may, but need not, comprise all of (i) through (vii), and both (viii) and (ix). The consortium may, but need not, consist essentially of (i) through (vii), (viii), and (ix).

[0031] Although specific embodiments and applications have been illustrated and described, the disclosure is not limited to the precise configuration and components described herein. Various modifications, changes and variations which will be apparent to those skilled in the art may be made in the arrangement, operation and details of the methods and systems disclosed herein without departing from the spirit and scope of the entire disclosure.

[0032] As used herein, unless otherwise specified, terms such as "about", "approximately", and the like, when used in relation to a numerical limit or range, mean that the recited limit or range may vary by up to 10%. As a non-limiting example, "about 750" may mean as little as 675 or as little as 825, or any value therebetween. When used in relation to a ratio or relationship between two or more numerical limits or ranges, terms such as "about", "approximately", and the like, mean that each of the limits or ranges may vary by up to about 10%, and as a non-limiting example, a statement that two quantities are "approximately equal" may mean that the ratio between the two quantities is as little as 0.9:1.1 or as little as 1.1:0.9 (or any value therebetween), a statement that a four-way ratio is "about 5:3:1:1" may mean that the first number in the ratio may be any value of at least 4.5 and no more than 5.5, the second number in the ratio may be any value of at least 2.7 and no more than 3.3, and so on.

[0033] The embodiments and configurations described herein are not complete or exhaustive, and it will be appreciated that other embodiments may utilize, alone or in combination, one or more of the features set forth above or described in detail below. [Brief description of the drawings]

[0034] [Figure 1-1] FIG. 1A is a graph of the change in OD600 of probiotic strains according to the present disclosure with various prebiotic substances. [Figure 1-2] FIG. 1B is a graph of the change in OD600 of probiotic strains according to the present disclosure with various prebiotic substances. [Figure 1-3] FIG. 1C is a graph of the change in OD600 of probiotic strains according to the present disclosure with various prebiotic substances. [Figure 1-4] FIG. 1D is a graph of the change in OD600 of probiotic strains according to the present disclosure with various prebiotic substances. [Figure 1-5]FIG. 1E is a graph of the change in OD600 of probiotic strains according to the present disclosure with various prebiotic substances. [Figure 1-6] FIG. 1F is a graph of the change in OD600 of probiotic strains according to the present disclosure with various prebiotic substances. [Figure 1-7] FIG. 1G is a graph of the change in OD600 of probiotic strains according to the present disclosure with various prebiotic substances. [Figure 1-8] FIG. 1H is a graph of the change in OD600 of probiotic strains according to the present disclosure with various prebiotic substances. [Figure 1-9] FIG. 1I is a graph of the change in OD600 of probiotic strains according to the present disclosure with various prebiotic substances. [Diagram 2] FIG. 2 is a plot of fluorescence activated cell sorting (FACS) based determination of cell viability of probiotic strains according to the present disclosure. [Diagram 3] Figure 3 is a schematic diagram of the standard setup of the Simulator of Human Gut Microbial Ecosystems (SHIME). [Figure 4] FIG. 4 is a general schematic diagram of the adapted SHIME system used to study the survival of probiotic strains according to the present disclosure in the gastrointestinal tract (GIT). [Figure 5-1] FIG. 5A is a graph of the pH profile during a survival experiment under fasted and fed conditions. [Figure 5-2] FIG. 5B is a graph of the pH profile during the survival experiment under fasted and fed conditions. [Figure 6-1] FIG. 6A is a graph of the mean surviving bacterial population during passage through the upper GIT under fasted and fed conditions, respectively, of naked probiotic strains according to the present disclosure. [Figure 6-2] FIG. 6B is a graph of the mean surviving bacterial population during passage through the upper GIT under fasted and fed conditions, respectively, of naked probiotic strains according to the present disclosure. [Figure 7-1]FIG. 7A is a graph of the mean surviving bacterial population during passage through the upper GIT under fasted and fed conditions, respectively, of microencapsulated probiotic strains according to the present disclosure. [Figure 7-2] FIG. 7B is a graph of the mean surviving bacterial population during passage through the upper GIT under fasted and fed conditions, respectively, of microencapsulated probiotic strains according to the present disclosure. [Figure 8-1] FIG. 8A is a graph of the mean pH change during short-term colonic incubation of a predigested blank control or a microencapsulated probiotic composition according to the present disclosure when supplemented with a non-sterile (FIG. 8A) inoculum in healthy children aged 6-10 years. [Figure 8-2] FIG. 8B is a graph of the mean pH change during short-term colonic incubation of predigested blank control or a microencapsulated probiotic composition according to the present disclosure when supplemented with a sterile (FIG. 8B) inoculum in healthy children aged 6-10 years. [Figure 9-1] FIG. 9A is a graph of the average lactate concentrations obtained during short-term colonic incubation of a predigested blank control or a microencapsulated probiotic composition according to the present disclosure when supplemented with a non-sterile (FIG. 9A) inoculum in healthy children aged 6 to 10 years. [Figure 9-2] FIG. 9B is a graph of the average lactate concentrations obtained during short-term colonic incubation of a predigested blank control or a microencapsulated probiotic composition according to the present disclosure when supplemented with a sterile (FIG. 9B) inoculum in healthy children aged 6 to 10 years. [Figure 10-1] FIG. 10A is a graph of average short chain fatty acid (SCFA) production during short-term colonic incubation of predigested blank control or a microencapsulated probiotic composition according to the present disclosure when supplemented with non-sterile (FIG. 10A) inoculum in healthy children aged 6-10 years. [Figure 10-2]FIG. 10B is a graph of average short chain fatty acid (SCFA) production during short-term colonic incubation of predigested blank control or a microencapsulated probiotic composition according to the present disclosure when supplemented with a germ-free (FIG. 10B) inoculum in healthy children aged 6-10 years. [Figure 11-1] FIG. 11A is a graph of the average acetate production during short-term colonic incubation of a predigested blank control or a microencapsulated probiotic composition according to the present disclosure when supplemented with a non-sterile (FIG. 11A) inoculum in healthy children aged 6-10 years. [Figure 11-2] FIG. 11B is a graph of the average acetate production during short-term colonic incubation of predigested blank control or a microencapsulated probiotic composition according to the present disclosure when supplemented with a sterile (FIG. 11B) inoculum in healthy children aged 6-10 years. [Figure 12-1] FIG. 12A is a graph of the average propionic acid production during short-term colonic incubation of a predigested blank control or a microencapsulated probiotic composition according to the present disclosure when supplemented with a non-sterile (FIG. 12A) inoculum in healthy children aged 6-10 years. [Figure 12-2] FIG. 12B is a graph of the average propionic acid production during short-term colonic incubation of predigested blank control or a microencapsulated probiotic composition according to the present disclosure when supplemented with a sterile (FIG. 12B) inoculum in healthy children aged 6-10 years. [Figure 13-1] FIG. 13A is a graph of the average butyrate production during short-term colonic incubation of predigested blank control or a microencapsulated probiotic composition according to the present disclosure when supplemented with non-sterile (FIG. 13A) inoculum in healthy children aged 6-10 years. [Figure 13-2] FIG. 13B is a graph of the average butyric acid production during short-term colonic incubation of predigested blank control or a microencapsulated probiotic composition according to the present disclosure when supplemented with a sterile (FIG. 13B) inoculum in healthy children aged 6-10 years. [Figure 14-1]FIG. 14A is a graph of mean branched short chain fatty acid (bSCFA) production during short-term colonic incubation of predigested blank control or a microencapsulated probiotic composition according to the present disclosure when supplemented with non-sterile (FIG. 14A) inoculum in healthy children aged 6-10 years. [Figure 14-2] FIG. 14B is a graph of average branched short chain fatty acid (bSCFA) production during short-term colonic incubation of predigested blank control or a microencapsulated probiotic composition according to the present disclosure when supplemented with a sterile (FIG. 14B) inoculum in healthy children aged 6-10 years. [Figure 15-1] FIG. 15A is a graph of the mean viable bacterial population during passage of Culturelle Kids Probiotic Composition through the upper GIT. [Figure 15-2] FIG. 15B is a graph of the mean viable bacterial population during passage of Garden of Life® Raw Probiotics Kids compositions through the upper GIT. [Figure 15-3] FIG. 15C is a graph of the mean viable bacterial population during passage of Optibac Probiotics® Babies & Children composition through the upper GIT. [Figure 15-4] FIG. 15D is a graph of the mean viable bacterial population during passage of the Life-Space® Probiotic Powder for Children composition through the upper GIT. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art.All patents, applications, published applications, and other publications referenced herein are incorporated by reference in their entirety.If there are multiple definitions for a term in this specification, the definition provided in the abstract shall prevail unless otherwise stated.

[0036] "CRISPR" (clustered regularly interspaced short palindromic repeats) loci refer to certain loci that code for components of DNA cleavage systems, used, for example, by bacterial and archaeal cells to destroy foreign DNA. CRISPR loci can consist of CRISPR arrays that contain short direct repeats (CRISPR repeats) separated by short variable DNA sequences (called spacers), which can be flanked by diverse Cas (CRISPR-associated) genes. An example of a pathway unknown to science before the age of DNA sequencing, the CRISPR-Cas system is now understood to confer bacteria and archaea with acquired immunity against phages and viruses. Intensive research over the past decade has revealed the biochemistry of this system. The CRISPR-Cas system consists of Cas proteins, which are involved in the acquisition, targeting and cleavage of foreign DNA or RNA, as well as CRISPR arrays that contain direct repeats flanking short spacer sequences that guide the Cas proteins to their targets. Class 2 CRISPR-Cas are simplified versions in which a single RNA-bound Cas protein is responsible for binding and cleaving the target sequence. The programmable nature of these minimal systems has facilitated their use as a versatile technology that is revolutionizing the field of genome engineering.

[0037] As used herein, "effector" or "effector protein" refers to a protein that encompasses activities including recognizing, binding to, and / or cleaving or nicking a polynucleotide target. Effector or effector protein may also be an endonuclease. The "effector complex" of the CRISPR system includes the crRNA and Cas proteins that are involved in target recognition and binding. Some of the component Cas proteins may additionally include domains that are involved in target polynucleotide cleavage.

[0038] The term "Cas protein" refers to a polypeptide encoded by a Cas (CRISPR-associated) gene. Cas proteins include proteins encoded by genes in the cas locus, including adaptive and interference molecules. Interference molecules of bacterial adaptive immune complexes include endonucleases. Cas endonucleases described herein include one or more nuclease domains. Cas endonucleases include, but are not limited to, novel Cas-alpha proteins disclosed herein, Cas9 proteins, Cpf1 (Cas12) proteins, C2c1 proteins, C2c2 proteins, C2c3 proteins, Cas3, Cas3-HD, Cas5, Cas7, Cas8, Cas10, or combinations or complexes thereof. Cas proteins may be "Cas endonucleases" or "Cas effector proteins" and can recognize, bind to, and optionally nick or cleave all or part of a specific polynucleotide target sequence when complexed with a suitable polynucleotide component.

[0039] CRISPR-Cas systems have been classified according to the sequence and structural analysis of the components. Several CRISPR / Cas systems have been described, including class 1 systems with multi-subunit effector complexes (including types I, III, and IV), and class 2 systems with single protein effectors (including types II, V, and VI). CRISPR-Cas systems minimally contain a CRISPR RNA (crRNA) molecule and at least one CRISPR-associated (Cas) protein to form a crRNA ribonucleoprotein (crRNP) effector complex. The CRISPR-Cas locus contains a DNA target spacer that encodes the crRNA component, and a series of identical repeats interspersed with operon-like units of cas genes that encode the Cas protein component. The resulting ribonucleoprotein complex recognizes polynucleotides in a sequence-specific manner. The crRNA serves as a guide RNA for sequence-specific binding of the effector (protein or complex) to the double-stranded DNA sequence, forming a so-called R-loop by base pairing with the complementary DNA strand while displacing the non-complementary strand. The RNA transcript (pre-crRNA) of the CRISPR locus is specifically cleaved at the repeat sequence by CRISPR-associated (Cas) endoribonuclease in type I and type III systems, or by RNaseIII in type II systems. The number of CRISPR-associated genes in a given CRISPR locus can vary between species.

[0040] Different cas genes, encoding proteins with different domains, are present in different CRISPR systems. The cas operon contains genes encoding one or more effector endonucleases and other Cas proteins. Some domains may serve more than one purpose, e.g., Cas9 contains a domain for endonuclease functionality and a domain for target cleavage. Cas endonucleases are guided by a single CRISPR RNA (crRNA) through direct RNA-DNA base pairing to recognize DNA target sites in the vicinity of a protospacer adjacent motif (PAM). Class I CRISPR-Cas systems include types I, III, and IV. A distinctive feature of class I systems is the presence of an effector endonuclease complex instead of a single protein. The Cascade complex contains an RNA recognition motif (RRM) and a nucleic acid binding domain that is the core fold of the diverse RAMP (repeat-associated mysterious protein) protein superfamily.

[0041] Type I CRISPR-Cas systems minimally contain a complex of effector proteins called Cascade (CRISPR-associated complex for antiviral defense), including Cas5 and Cas7. The effector complex functions together with a single CRISPR RNA (crRNA) and Cas3 to defend against invading viral DNA. Type I systems are divided into seven subtypes.

[0042] Type III CRISPR-Cas systems, which contain multiple cas7 genes, target either ssRNA or ssDNA and function as either an RNase and a target RNA-activated DNA nuclease. Type IV systems contain typical type I cas5 and cas7 domains in addition to a cas8-like domain, but may lack the CRISPR array characteristic of most other CRISPR-Cas systems.

[0043] Class II CRISPR-Cas systems include types II, V, and VI. A distinctive feature of class II systems is the presence of a single Cas effector protein instead of an effector complex. Type II and V Cas proteins contain a RuvC endonuclease domain that adopts an RNase H fold. Type II CRISPR / Cas systems use crRNA and tracrRNA (trans-activating CRISPR RNA) to guide the Cas endonuclease to its DNA target. The crRNA contains a spacer region that is complementary to one strand of the double-stranded DNA target and a region that base pairs with the tracrRNA (trans-activating CRISPR RNA), forming an RNA duplex and directing the Cas endonuclease to cleave the DNA target, leaving a blunt end. The spacer is acquired through a not fully understood process that involves Cas1 and Cas2 proteins. Type II CRISPR / Cas loci typically contain cas1 and cas2 genes in addition to the cas9 gene. Type II CRISR-Cas loci can encode tracrRNA that is partially complementary to the repeats in individual CRISPR arrays and can include other proteins such as Csn1 and Csn2. The presence of cas9 in the vicinity of the cas1 and cas2 genes is a hallmark of type II loci. Type V CRISPR / Cas systems contain a single Cas endonuclease, including Cpf1 (Cas12), an active RNA-guided endonuclease that, unlike Cas9, does not necessarily require additional trans-activating CRISPR (tracr)RNA for target cleavage. Type VI CRISPR-Cas systems contain the cas13 gene, which encodes a nuclease that has two HEPN (higher eukaryotic and prokaryotic nucleotide binding) domains but does not have HNH or RuvC domains and is independent of tracrRNA activity. The majority of the HEPN domain contains a conserved motif that constitutes a metal-independent endoRNase active site. Because of this property, the Type VI system is thought to act on RNA targets instead of the DNA targets common to other CRISPR-Cas systems.

[0044] Incorporated herein by reference for purposes of disclosure and enablement are the following patent publications: 2014 / 0349405 to Sontheimer; 2014 / 0377278 to Elinav; 2014 / 0068797 to Doudna; 20200190494 to Hou et al.; and 2020 / 0199555 to Zhang.

[0045] Incorporated herein by reference for purposes of further complying with applicable written description and enablement requirements is U.S. Patent Application Publication No. 2020 / 0138722, published May 7, 2020, to Kabadi et al., entitled "Targeted Gastrointestinal Delivery of Probiotic Organisms and / or Therapeutic Agents."

[0046] To further comply with applicable written description and enablement requirements, the following references are incorporated herein by reference: PCT Application No. PCT / US2021 / 015103, filed on January 26, 2021, entitled "Probiotic Compositions and Microbial Consortia for Improving Human Organ System Health." PCT Application No. PCT / US2021 / 015107, filed on January 26, 2021, entitled "Methods and Compositions for Precision Release of Probiotics to Improve Human Health." U.S. Provisional Patent Application No. 63 / 141,874, filed January 26, 2021, entitled "Method of Probiotic Treatment for Improving Human Health."

[0047] As used herein, unless otherwise specified, the term "animal" refers to any organism in the animal kingdom, including but not limited to humans.

[0048] As used herein, unless otherwise specified, the term "disease" refers to a disease, disorder, or condition, or a symptom thereof.

[0049] As used herein, unless otherwise specified, the term "ingestible formulation" refers to a composition of matter adapted or configured to be ingested by an animal by taking the composition through the mouth and into the digestive tract, e.g., by eating or drinking. An "ingestible composition", as that term is used herein, may be provided in a form selected from the group consisting of ampoules, aqueous solutions, aqueous suspensions, capsules, drops, granules, liquids, mists, powders, sachets, syrups, tablets, functional foods, beverages, toothpastes, sublingual articles, and the like.

[0050] As used herein, unless otherwise specified, the term "Lactobacillus", when used without further elaboration, refers to any organism that was classified in the family Lactobacillaceae prior to the 2020 reclassification of the genus Lactobacillus into 25 separate genera.

[0051] As used herein, unless otherwise specified, the term "patient" refers to a mammal, including, as a non-limiting example, a human.

[0052] As used herein, unless otherwise specified, the term "pharmaceutical acceptable" means approved or approvable by a regulatory agency of the Federal or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, for use in animals, and more specifically, in humans.

[0053] As used herein, unless otherwise specified, the term "pharmaceutically acceptable vehicle" refers to a pharmaceutically acceptable diluent, a pharmaceutically acceptable adjuvant, a pharmaceutically acceptable excipient, a pharmaceutically acceptable carrier, or a combination of any of the foregoing, with which a substance provided by the present disclosure may be administered to a patient, which does not destroy its pharmacological activity, and which is non-toxic when administered in a dosage sufficient to provide a therapeutically effective amount of the substance.

[0054] As used herein, unless otherwise specified, the term "pharmaceutical formulation" refers to a therapeutically active agent and at least one pharma- ceutically acceptable vehicle with which the agent is administered to a patient.

[0055] As used herein, unless otherwise specified, the term "prebiotic" refers to a substance selectively utilized by microorganisms that confers a health benefit in the microbial host.

[0056] As used herein, unless otherwise specified, the term "probiotic" refers to live microorganisms that, when administered in appropriate amounts, confer a health benefit to the host.

[0057] As used herein, unless otherwise specified, the term "synbiotic" refers to a combination or mixture of probiotics and prebiotics that beneficially affect the host by improving survival and engraftment of live microbial supplements in the gastrointestinal tract through selective stimulation of the growth and / or metabolic activation of one or more health-promoting microorganisms.

[0058] As used herein, the terms "treating" and "treatment" refer to reversing, alleviating, arresting, ameliorating, or preventing a disease or at least one clinical symptom of the disease, reducing the risk of acquiring a disease or at least one clinical symptom of the disease, inhibiting the progression of a disease or at least one clinical symptom of the disease, or reducing the risk of developing a disease or at least one clinical symptom of the disease. "Treatment" or "treatment" also refers to inhibiting a disease physically (e.g., stabilizing a recognizable symptom), physiologically (e.g., stabilizing a physical parameter), or both, and inhibiting at least one physical parameter that may or may not be recognizable to the patient. In certain embodiments, "treating" or "treatment" refers to delaying the onset of a disease or at least one or more symptoms thereof in a patient who may be exposed to or predisposed to the disease, even if the patient has not yet experienced or presented symptoms of the disease.

[0059] As used herein, unless otherwise specified, the term "therapeutically effective amount" refers to the amount of a substance that, when administered to a subject for treating a disease or at least one of the clinical symptoms of the disease, is sufficient to affect such treatment of the disease or its symptoms. "Therapeutically effective amount" may vary, for example, depending on the substance, the disease and / or symptoms of the disease, the severity of the disease and / or symptoms of the disease or disorder, the age, weight, and / or health of the patient to be treated, and the judgment of the prescribing physician. The appropriate amount in any given case can be ascertained by one skilled in the art or can be determined by routine experimentation.

[0060] As used herein, unless otherwise specified, the term "therapeutically effective dose" refers to a dose that provides effective treatment of a disease or disorder in a patient. Therapeutically effective doses may vary from substance to substance and from patient to patient, and may depend on factors such as the condition of the patient and the route of delivery. Therapeutically effective doses may be determined according to routine pharmacological procedures known to those skilled in the art.

[0061] The present disclosure provides rationally defined and assembled microbial consortia to confer health across organ systems by initially modulating the native gut microbiota and host tissue function. More specifically, the present disclosure provides designed oral prophylactic probiotic strain mixtures to maintain and improve host health at local and distant body sites.

[0062] In an embodiment, the present disclosure provides one or more consortia of live microorganisms with a range of benefits that have been validated in humans. The viability of the synbiotic composition of the present disclosure throughout production and administration has been validated by comprehensive assessment of active fluorescent units (AFU) in a simulated human gut environment, as described in more detail in the examples provided below. In addition to extensive mechanistic and genetic characterization, the consortium includes strains that have been clinically evaluated in strain-specific double-blind placebo-controlled human studies for clinical outcomes including improved digestion, modulation of the gut-skin axis, low-density lipoprotein modulation, gut immunological response, epithelial barrier regulation, and micronutrient synthesis.

[0063] microbial strains The development of next-generation sequencing (NGS) technologies has introduced new and much cheaper methods for prokaryotic whole genome sequencing. Combined with the falling prices for the computational resources required for bioinformatics analysis, this has led to the publication of a large number of new bacterial genomes. An increasing number of "non-bioinformatics" laboratories are now sequencing their own prokaryotic genomes and are faced with problems such as which sequencing platform to choose, how many libraries to generate, and which assembly method to use for the libraries.

[0064] An embodiment of the present disclosure includes a synbiotic composition comprising Lacticaseibacillus rhamnosus SD-GG-BE. This bacterial strain may have any one or more of several beneficial effects on the health of the host. As a first non-limiting example, Lacticaseibacillus rhamnosus SD-GG-BE may reduce toll-like receptor mRNA levels on antigen-presenting cells (APCs), reduce CD16 expression on macrophages and CD11 expression on monocytes, and / or induce type 1 immune response polarization as observed from elevated production of IL-12 and TNF-α. As a second non-limiting example, Lacticaseibacillus rhamnosus SD-GG-BE may reduce abdominal pain in children with functional dyspepsia, irritable bowel syndrome, and / or functional abdominal pain. As a third non-limiting example, Lacticaseibacillus rhamnosus SD-GG-BE may improve bloody stools, diarrhea, irritability, abdominal distension, mucus in the stool, and / or vomiting in children with cow's milk protein allergies. As a fourth non-limiting example, Lacticaseibacillus rhamnosus SD-GG-BE may shorten the duration of diarrhea and / or reduce the number of daily bowel movements in children with diarrhea. As a fifth non-limiting example, Lacticaseibacillus rhamnosus SD-GG-BE may reduce the effect of antibiotic treatment on the gut microbiome of children. As a sixth non-limiting example, Lacticaseibacillus rhamnosus SD-GG-BE may play a role in preventing or treating infant colic.

[0065] An embodiment of the present disclosure includes a synbiotic composition comprising Ligilactobacillus salivarius SD-LS1-IT. This bacterial strain may have any one or more of several beneficial effects on the health of the host. As a first non-limiting example, Ligilactobacillus salivarius SD-LS1-IT has been shown to improve clinical parameters of atopic dermatitis (especially in children) as measured by the SCORAD and Dermatological Quality of Life (DLQ) indices, reduce microbial translocation and / or immune activation, and / or enhance the expression of T helper cell (Th)17 / regulatory T cell (T reg ) and improve the Th1 / Th2 ratio. As a second non-limiting example, Ligilactobacillus salivarius SD-LS1-IT may reduce staphylococcal counts in host feces. As a third non-limiting example, Ligilactobacillus salivarius SD-LS1-IT may improve clinical parameters of atopic dermatitis as measured by an itch index that may persist after cessation of synbiotic administration. As a fourth non-limiting example, Ligilactobacillus salivarius SD-LS1-IT may reduce staphylococcal load and / or reduce production of Th2 cytokines, as well as maintain stable production of Th1 cytokines. As a fifth non-limiting example, Ligilactobacillus salivarius SD-LS1-IT may reduce pro-inflammatory cytokines, increase anti-inflammatory cytokines, inhibit reactive oxygen species production, restore cell membrane integrity, and / or inhibit pathogenic E. coli and Klebsiella pneumoniae.

[0066] The embodiments of the present disclosure include a synbiotic composition comprising Bifidobacterium breve SD-B632-IT. This bacterial strain may have any one or more of several beneficial effects on the health of the host. As a first non-limiting example, Bifidobacterium breve SD-B632-IT may improve the composition of the stool microbiota in children with celiac disease, particularly by increasing the Firmicutes / Bacteroidetes ratio and / or increasing the abundance of Actinobacteria. As a second non-limiting example, Bifidobacterium breve SD-B632-IT may modulate serum TNF-α and / or stool short-chain fatty acids. As a third non-limiting example, Bifidobacterium breve SD-B632-IT may improve insulin sensitivity and blood glucose levels in children with obesity and insulin resistance. As a fourth non-limiting example, Bifidobacterium breve SD-B632-IT may play a role in preventing or treating infantile colic. As a fifth non-limiting example, Bifidobacterium breve SD-B632-IT may help restore microbial balance in children with celiac disease on a gluten-free diet. As a sixth non-limiting example, Bifidobacterium breve SD-B632-IT may support the growth of healthy gut bacteria in children with celiac disease. As a seventh non-limiting example, Bifidobacterium breve SD-B632-IT may support healthy carbohydrate metabolism and insulin sensitivity in adolescents.As an eighth non-limiting example, Bifidobacterium breve SD-B632-IT may improve allergic airway responses and / or otherwise support healthy respiratory function in patients with asthma, seasonal allergies, etc., support healthy responses to seasonal allergens, and / or improve rescue of respiratory distress during seasonal allergies.

[0067] An embodiment of the present disclosure includes a synbiotic composition comprising Bifidobacterium breve SD-BR03-IT. This bacterial strain may have any one or more of several beneficial effects on the health of the host. As a first non-limiting example, Bifidobacterium breve SD-BR03-IT may improve the host's bowel movement frequency, stool consistency, and ease of evacuation, and may alleviate symptoms of intestinal discomfort such as abdominal bloating, itching, burning, or pain. As a second non-limiting example, Bifidobacterium breve SD-BR03-IT may improve clinical parameters of atopic dermatitis as measured by the SCORAD and Dermatological Quality of Life (DLQ) indices, reduce microbial translocation and / or immune activation, and / or promote T helper cell (Th)17 / regulatory T cell (T reg) and improve the Th1 / Th2 ratio. As a third non-limiting example, Bifidobacterium breve SD-BR03-IT can sustain the host's gut microbiota after cessation of synbiotic administration, which can correct the dysbiotic gut microbiota of atopic dermatitis patients. As a fourth non-limiting example, Bifidobacterium breve SD-BR03-IT can inhibit the growth of multiple E. coli biotypes, including pathogenic E. coli O157:H7. As a fifth non-limiting example, Bifidobacterium breve SD-BR03-IT may improve the composition of the stool microbiota in children with celiac disease, particularly by increasing the Firmicutes / Bacteroidetes ratio and / or increasing the abundance of Actinobacteria. As a sixth non-limiting example, Bifidobacterium breve SD-BR03-IT may modulate serum TNF-α and / or stool short-chain fatty acids. As a seventh non-limiting example, Bifidobacterium breve SD-BR03-IT may improve insulin sensitivity and blood glucose levels in children with obesity and insulin resistance. As an eighth non-limiting example, Bifidobacterium breve SD-BR03-IT may play a role in preventing or treating infant colic.

[0068] The present disclosure includes a synbiotic composition comprising Bifidobacterium longum SD-CECT7347-SP. This bacterial strain may have any one or more of several beneficial effects on the health of the host. As a first non-limiting example, Bifidobacterium longum SD-CECT7347-SP, in combination with Lacticaseibacillus casei SD-CECT9104-SP and Bifidobacterium lactis SD-CECT8145-SP, may reduce the SCORAD index and the use of topical steroid treatments to treat erythema in chronic dermatological diseases. As a second non-limiting example, Bifidobacterium longum SD-CECT7347-SP may reduce alterations in the expression of cellular proteins involved in cytoskeleton disruption, inflammation, and apoptosis in Caco-2 cells. As a third non-limiting example, Bifidobacterium longum SD-CECT7347-SP may suppress the pro-inflammatory cytokine pattern of peripheral blood mononuclear cells (PBMCs) and increase IL-10 production in hosts with celiac disease. As a fourth non-limiting example, Bifidobacterium longum SD-CECT7347-SP may affect the phenotype and functional maturation of monocyte-derived dendritic cells. As a fifth non-limiting example, Bifidobacterium longum SD-CECT7347-SP can reduce cellular expression of toxic amino acid sequences and reduce expression of NF-κB, TNF-α, and IL-1β.

[0069] An embodiment of the present disclosure includes a synbiotic composition comprising Lacticaseibacillus casei SD-CECT9104-SP. This bacterial strain may have any one or more of several beneficial effects on the health of the host. As a non-limiting example, Lacticaseibacillus casei SD-CECT9104-SP, in combination with Bifidobacterium longum SD-CECT7347-SP and Bifidobacterium lactis SD-CECT8145-SP, may reduce the SCORAD index and the use of topical steroid treatments to treat erythema in chronic dermatological diseases.

[0070] The present disclosure includes a synbiotic composition comprising Bifidobacterium lactis SD-CECT8145-SP. This bacterial strain may have any one or more of several beneficial effects on the health of the host. As a non-limiting example, Bifidobacterium lactis SD-CECT8145-SP, in combination with Bifidobacterium longum SD-CECT7347-SP and Lacticaseibacillus casei SD-CECT9104-SP, may reduce the SCORAD index and the use of topical steroid treatments to treat erythema in chronic dermatological diseases.

[0071] An embodiment of the present disclosure includes a synbiotic composition comprising Lactobacillus acidophilus SD-NCFM-US. This bacterial strain may have any one or more of several beneficial effects on the health of the host. As a non-limiting example, Lactobacillus acidophilus SD-NCFM-US may reduce the incidence and duration of fever, rhinorrhea, cough, antibiotic prescriptions, and days of school absence due to illness in children. As a second non-limiting example, Lactobacillus acidophilus SD-NCFM-US may reduce stool frequency and / or improve stool consistency in children with diarrhea.

[0072] An embodiment of the present disclosure includes a synbiotic composition comprising Bifidobacterium animalis subsp. lactis SD-BI07-US. This bacterial strain may have any one or more of several beneficial effects on the health of the host. As a non-limiting example, Bifidobacterium animalis subsp. lactis SD-BI07-US may reduce the incidence and duration of fever, rhinorrhea, cough, antibiotic prescriptions, and days of school absence due to illness in children. As a second non-limiting example, Bifidobacterium animalis subsp. lactis SD-BI07-US may reduce bowel movement frequency and / or improve stool consistency in children with diarrhea.

[0073] Aspects of the present disclosure allow for rational and systematic screening and selection of bacterial strains of interest for use in synbiotic compositions.In particular, as described throughout this disclosure, bacterial strains of interest can be, and in preferred embodiments are, screened for a variety of functional properties, including but not limited to upregulation of Nrf2 transcription factor, increased short chain fatty acid (SCFA) production in the intestine, and / or improved epithelial barrier function in the intestine.As a result of this screening, individual strains or combinations of strains, each with one or more of these desired functional properties, and collectively with most or all of the desired properties, can be included in a synbiotic composition to provide synergistic effects on host health. As a non-limiting example, upregulation of Nrf2, increased SCFA production, and improved epithelial barrier function may, in some embodiments, mutually reinforce each other in the host's intestinal environment and thus result in a greater improvement in overall host health than any one or two of these functional outcomes; therefore, strains may be screened for these attributes and, as a result of this screening, rationally selected for inclusion in the synbiotic compositions of the present disclosure.

[0074] Prebiotics It should be expressly understood that any compound that can be converted into a bioactive metabolite by a microbial strain present in a healthy human gut microbiota can be provided in the synbiotic composition according to the present disclosure. As a first non-limiting example, the prebiotic component may include one or more microbial-fermentable dietary fibers, i.e., polymeric and oligomeric carbohydrates, such as fructooligosaccharides (FOS), galactooligosaccharides (GOS), and inulin. As a second non-limiting example, the prebiotic component may include one or more punicalagins, which act as powerful antioxidants in the human body and can be metabolized by certain gut bacteria into a class of dibenzopyran-6-ones known as urolithins, including but not necessarily limited to urolithin-A. As a third non-limiting example, the prebiotic component may include one or more glucosinolates, which can be metabolized by the gut microbiota into isothiocyanates, which in turn have been shown to have anti-cancer properties and other useful roles in human health. As a fourth non-limiting example, the prebiotic component may include one or more catechins, which may be metabolized by microorganisms in the colon to γ-valerolactone and hippuric acid, and then biotransformed in the human liver into metabolites useful for human health. As a fifth non-limiting example, the prebiotic component may include one or more polyphenols, many of which are known to be metabolized by the gut microbiota into compounds that are beneficial to human health. These and other embodiments are within the scope of the present disclosure. In some embodiments, the compound may be a compound that is otherwise biologically "inactive" from the host's perspective (i.e., can only be metabolized by one or more strains in the gut microbiota, and not by the host), while in other embodiments, the compound can be metabolized by both the host and the gut microbiota.

[0075] Pharmaceutical preparations Pharmaceutical formulations provided by the present disclosure may include a therapeutically effective amount of the synbiotic composition together with a suitable amount of one or more pharma- ceutically acceptable vehicles to provide the formulation for proper administration to a patient. Suitable pharmaceutical vehicles are described in the art.

[0076] In certain embodiments, the synbiotic composition may be incorporated into a pharmaceutical preparation that is orally administered. Oral administration of such a pharmaceutical preparation may result in the release and / or uptake of the synbiotic composition throughout the intestine. Such oral preparations may be prepared in a manner known in the pharmaceutical art, and include the synbiotic composition and at least one pharmaceutically acceptable vehicle. Oral pharmaceutical preparations may include a therapeutically effective amount of the synbiotic composition and a suitable amount of a pharmaceutically acceptable vehicle to provide a suitable form for administration to patients.

[0077] The synbiotic composition may also be incorporated into pharmaceutical formulations administered by any other suitable systemic route of administration, including intramuscular, intravenous and oral.

[0078] The embodiments of the present disclosure generally include ingestible formulations that include a synbiotic composition. The embodiments of the present disclosure also include methods of administration of such formulations to a subject, preferably a human, and even more preferably a child, to treat a disease or condition and / or achieve a physiological purpose. In particular, in some embodiments, the synbiotic composition is co-administered or administered in combination with one or more other pharmaceutical preparations to achieve an adjunctive or synergistic effect, i.e., an improved effect compared to the independent administration of the synbiotic composition and one or more other pharmaceutical preparations. As a first non-limiting example, the synbiotic composition according to the present disclosure may be co-administered with one or more pharmaceutical preparations effective for treating inflammatory bowel disease to improve the effective response rate of the pharmaceutical preparation in treating inflammatory bowel disease. As a second non-limiting example, the synbiotic composition according to the present disclosure may be co-administered with one or more cancer immunotherapeutic agents (e.g., CTLA-4 antagonists and / or PD-1 antagonists) to improve the effective response rate of the cancer immunotherapeutic agent.

[0079] The ingestible formulation of the present invention can be provided in any suitable form and physical manifestation. As a non-limiting example, the ingestible formulation can be administered to a subject as a dietary supplement, a medical food, a nutraceutical composition, and a pharmaceutical composition. As a further non-limiting example, the ingestible composition can be provided in any suitable physical form for oral administration, such as an ampoule, an aqueous solution or suspension (e.g., an infused drink such as an energy drink or an energy "shot"), a capsule (which may or may not be chewable), a drop, a granule, a liquid, a mist, a powder, a sachet, a syrup, a tablet (e.g., a chewable, saliva-soluble tablet, and / or an oral tablet), a functional food (e.g., an energy or nutrition bar, a cookie, a gum, a candy, etc.), a toothpaste, a sublingual article, etc. In some embodiments, the composition can be provided in a form, such as a powder, which can be applied to food (similar to seasoning or condiments, etc.) or mixed with a drink. The ingestible compositions of the present invention may therefore include any suitable pharma- ceutically acceptable excipients, binders, and / or fillers, and may also include active pharmaceutical or therapeutic agents other than the synbiotic compositions disclosed herein.

[0080] In embodiments, the ingestible formulation of the present invention may be provided in the form of a flowable powder. The powder may be adapted to be mixed with or dissolved or suspended in a beverage (e.g., water, milk, etc.) or other liquid, and then the subject drinks or otherwise receives it orally. In certain embodiments, the ingestible formulation in the form of a powder may be packaged in a sachet, for example, a single-use sachet containing a single daily dose, which may be adapted to be immersed in liquid (similar to a tea bag) to allow the ingestible formulation to diffuse into the liquid, to be torn open and pour the contents into the liquid, etc. This may be preferred for packaging materials, in some embodiments, where the sachet is made with a relatively low water vapor transmission rate, for example, a water vapor transmission rate of less than about 0.01 grams per square meter per day at 23°C and 50% relative humidity, to extend the shelf life of the ingestible formulation.

[0081] The pharmaceutical preparations containing the synbiotic composition can be manufactured by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, encapsulating, or lyophilizing processes. The pharmaceutical preparations can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants, which facilitate the processing of the synbiotic composition and one or more pharma-ceutically acceptable vehicles into a pharma-ceutically usable preparation. The appropriate formulation depends on the route of administration selected. The pharmaceutical preparations provided by the present disclosure can take the form of sustained release formulations suitable for administration to patients.

[0082] The pharmaceutical preparations provided by the present disclosure can be formulated into unit dosage forms. Unit dosage forms refer to physically separate units suitable as unit dosages for patients undergoing treatment, each unit containing a predetermined amount of the synbiotic composition calculated to produce the intended therapeutic effect. The unit dosage forms can be a single dose per day for administration, a double dose per day, or multiple doses per day, for example, three or more doses per day. When multiple doses per day are used, the unit dosage forms can be the same or different for each dose. One or more dosage forms can contain a dose, which can be administered to a patient at a single time point or during a time interval.

[0083] In certain embodiments, the oral dosage form provided by the present disclosure may be a controlled release dosage form. Controlled delivery technology can improve the absorption of the active ingredient in a specific area or areas of the digestive tract. A controlled active ingredient delivery system can be designed to deliver the active ingredient in such a way that the level of the active ingredient is maintained within a therapeutically effective window and effective and safe blood levels are maintained for a period of time as long as the system continues to deliver the active ingredient with a specific release profile in the digestive tract. Controlled active ingredient delivery can produce a substantially constant blood level of the active ingredient over a period of time, compared to the fluctuations observed with immediate release dosage forms. For some applications, maintaining a constant blood and tissue concentration throughout the course of treatment is the most desirable mode of treatment. An immediate release of the active ingredient can cause blood levels to peak above the level required to induce the desired response, which can waste the active ingredient and cause or exacerbate toxic side effects. Controlled active ingredient delivery can result in optimal treatment and can reduce the frequency of dosing, as well as reduce the severity of side effects. Examples of controlled release dosage forms include dissolution-controlled systems, diffusion-controlled systems, ion exchange resins, osmotic pressure-controlled systems, erodible matrix systems, pH-independent formulations, gastric retention systems, and the like.

[0084] The appropriate oral dosage form for a particular pharmaceutical formulation provided by the present disclosure may depend, at least in part, on the gastrointestinal absorbability of the active ingredient and / or the stability of the active ingredient in the gastrointestinal tract, the pharmacokinetics of the active ingredient, and the intended therapeutic profile. An appropriate controlled release oral dosage form may be selected for a particular ingredient or combination of ingredients. For example, a gastric retention oral dosage form may be appropriate for an active ingredient that is absorbed primarily from the upper gastrointestinal tract, and a sustained release oral dosage form may be appropriate for an active ingredient that is absorbed primarily from the lower gastrointestinal tract. Certain active ingredients are absorbed primarily from the small intestine. In general, the active ingredient passes through the entire length of the small intestine in about 3-5 hours. For active ingredients that are not readily absorbed or readily dissolved by the small intestine, the window for absorption of the active agent in the small intestine may be too short to provide the desired therapeutic effect.

[0085] In certain embodiments, the pharmaceutical formulations provided by the present disclosure may be implemented in dosage forms adapted to provide sustained release of the synbiotic composition upon oral administration. Sustained release oral dosage forms may be used to release active ingredients over an extended period of time and are useful when it is desired that the active ingredient be delivered to the lower gastrointestinal tract, including the colon. Sustained release oral dosage forms include any oral dosage form that maintains therapeutic concentrations of the active ingredient in biological fluids, such as plasma, blood, cerebrospinal fluid, or in tissues or organs for an extended period of time. Sustained release oral dosage forms include diffusion-controlled systems, such as reservoir and matrix devices, dissolution-controlled systems, osmotic systems, and erosion-controlled systems. Sustained release oral dosage forms and methods for preparing them are well known in the art.

[0086] In certain embodiments, the pharmaceutical composition provided by the present disclosure may include any enteric-coated sustained release oral dosage form for administering the synbiotic composition.In one embodiment, the enteric-coated oral dosage form is administered to the patient at a dosage frequency of three times a day.In another embodiment, the enteric-coated oral dosage form is administered to the patient at a dosage frequency of two times a day.In yet another embodiment, the enteric-coated oral dosage form is administered to the patient at a dosage frequency of once a day.

[0087] In certain embodiments, the pharmaceutical formulation provided by the present disclosure may include any non-enteric coated sustained release oral dosage form for administering synbiotic composition.In one embodiment, the non-enteric coated oral dosage form is administered to the patient at a dosage frequency of three times a day.In another embodiment, the non-enteric coated oral dosage form is administered to the patient at a dosage frequency of two times a day.In yet another embodiment, the non-enteric coated oral dosage form is administered to the patient at a dosage frequency of once a day.

[0088] In certain embodiments, the pharmaceutical formulation provided by the present disclosure may include any oral dosage form for administering the synbiotic composition.In one embodiment, the oral dosage form is administered to the patient at a dosage frequency of three times a day.In another embodiment, the oral dosage form is administered to the patient at a dosage frequency of two times a day.In yet another embodiment, the oral dosage form is administered to the patient at a dosage frequency of once a day.

[0089] In certain embodiments, the pharmaceutical formulation provided by the present disclosure can include any suitable dosage form that achieves the aforementioned in vitro release profile.Such dosage form can be any systemic dosage form, including sustained release enteric coated oral dosage form, and sustained release enteric coated or non-enteric coated oral dosage form.Examples of suitable dosage forms are described herein.Those skilled in the art of formulation can develop any number of acceptable dosage forms by considering the dosage forms described in the examples as starting points.

[0090] The appropriate dose of the synbiotic composition can be determined according to any one of several well-established protocols. For example, animal studies, such as studies using mice, rats, dogs, and / or monkeys, can be used to determine the appropriate dose of the pharmaceutical compound. Results from animal studies can be extrapolated to determine doses for use in other species, such as humans.

[0091] use The methods and formulations disclosed herein can be used to treat patients suffering from diseases, disorders, conditions and symptoms for which synbiotic compositions are known to provide or are subsequently found to provide therapeutic benefits.The formulations disclosed herein can be used to treat adrenoleukodystrophy, AGE-induced genomic damage, Alexander's disease, alopecia areata, Alpers' disease, Alzheimer's disease, amyotrophic lateral sclerosis, angina pectoris, arthritis, asthma, Baro's concentric sclerosis, Behcet's disease, bullous pemphigoid, Canavan's disease, heart failure including left ventricular dysfunction, central nervous system vasculitis, Charcot-Marie-Tooth disease, childhood ataxia with central nervous system hypomyelination, chronic idiopathic peripheral neuropathy, chronic obstructive pulmonary disease, Crohn's disease, cutaneous lupus, dermatitis (contact, acute and chronic), diabetic retinopathy, graft-versus-host disease, granulomas, hepatitis C virus infection, herpes simplex virus infection, varicella-zoster virus infection, human immunodeficiency It can be used to treat a disease selected from viral infections, Huntington's disease, irritable bowel disorder, ischemia, Krabbe disease, lichen planus, macular degeneration, mitochondrial encephalomyopathy, unilateral upper limb muscular atrophy, multiple sclerosis, myocardial infarction, neurodegeneration with brain iron accumulation, neuromyelitis optica, neurosarcoidosis, NF-κB mediated disease, optic neuritis, paraneoplastic syndromes, Parkinson's disease, Pelizaeus-Merzbacher disease, pemphigus, primary lateral sclerosis, progressive supranuclear palsy, psoriasis, pyoderma gangrenosum, reperfusion injury, retinitis pigmentosa, sarcoidosis, Schilder's disease, subacute necrotizing myelopathy, Susac syndrome, transplant rejection, transverse myelitis, tumors, ulcerative colitis, and Zellweger syndrome.

[0092] The methods of treating a disease in a patient provided by the present disclosure include administering a therapeutically effective amount of a synbiotic composition of the present disclosure to a patient in need of such treatment. These methods and pharmaceutical formulations provide a therapeutic or prophylactic amount of a prebiotic compound and / or a probiotic strain after administration to the patient. The synbiotic composition may be administered in an amount and using a dosing schedule as appropriate for the treatment of the particular disease.

[0093] The daily dose of the prebiotic component compound of the synbiotic composition may range from about 0.01 mg / kg to about 200 mg / kg, about 0.1 mg / kg to about 175 mg / kg, about 1 mg / kg to about 150 mg / kg, about 5 mg / kg to about 125 mg / kg, and in certain embodiments, about 100 mg / kg. In certain embodiments, the prebiotic component compound may be administered over time at a dose of about 1 mg to about 12 g per day, about 10 mg to about 11 g per day, in certain embodiments, about 20 mg to about 2 g per day, in certain embodiments, about 100 mg to about 1 g per day, in certain embodiments, about 150 mg to about 650 mg per day, in certain embodiments, about 250 mg to about 550 mg per day, in certain embodiments, about 350 mg to about 450 mg per day, and in certain embodiments, about 400 mg per day.

[0094] In certain embodiments, the microbial strains of the probiotic components may be administered over time at doses of about 125 million AFU to about 625 billion AFU per day, in certain embodiments, about 1.25 billion AFU to about 500 billion AFU per day, in certain embodiments, about 2.5 billion AFU to about 250 billion AFU per day, in certain embodiments, about 12.5 billion AFU to about 125 billion AFU per day, in certain embodiments, about 25 billion AFU to about 100 billion AFU per day, in certain embodiments, about 37.5 billion AFU to about 75 billion AFU per day, and in certain embodiments, about 50 billion AFU to about 62.5 billion AFU per day.

[0095] In some embodiments, the probiotic component may include Lacticaseibacillus rhamnosus SD-GG-BE at least about 40 million AFU per dose, and in certain embodiments, about 7.5 billion AFU per dose. In some embodiments, the probiotic component may include Ligilactobacillus salivarius SD-LS1-IT at least about 2 billion AFU per dose, and in certain embodiments, about 7.5 billion AFU per dose. In some embodiments, the probiotic component may include Bifidobacterium breve SD-B632-IT at least about 7.5 billion AFU per dose, and in certain embodiments, about 7.5 billion AFU per dose. In some embodiments, the probiotic component may include Bifidobacterium breve SD-BR03-IT at least about 5 billion AFU per dose, and in certain embodiments, about 7.5 billion AFU per dose. In some embodiments, the probiotic component may include Bifidobacterium longum SD-CECT7347-SP at least about 420 million AFU per dose, and in certain embodiments, about 7.5 billion AFU per dose. In some embodiments, the probiotic component may include Lacticaseibacillus casei SD-CECT9104-SP at least about 360 million AFU per dose, and in certain embodiments, about 7.5 billion AFU per dose. In some embodiments, the probiotic component may include at least about 420 million AFU per dose, and in certain embodiments, about 7.5 billion AFU per dose of Bifidobacterium lactis SD-CECT8145-SP.In some embodiments, the probiotic component may include Lactobacillus acidophilus SD-NCFM-US at least about 7.5 billion AFU per dose, and in certain embodiments, about 7.5 billion AFU per dose. In some embodiments, the probiotic component may include Bifidobacterium animalis subsp. lactis SD-BI07-US at least about 7.5 billion AFU per dose, and in certain embodiments, about 7.5 billion AFU per dose. In some embodiments, the absolute AFU count of any one or more strains present in a dose of the probiotic component may vary, so long as the AFU ratio between any two or more selected microbial strains remains approximately the same as a given ratio, e.g., a ratio shown above (e.g., an AFU ratio between Lacticaseibacillus rhamnosus SD-GG-BE and Ligilactobacillus salivarius SD-LS1-IT of about 40 million:2 billion, or an AFU ratio between Lactobacillus acidophilus SD-NCFM-US and Bifidobacterium lactis SD-BI07-US of about 7.5 billion:7.5 billion).

[0096] An appropriate dose of the synbiotic composition can be determined based on several factors, including, for example, the weight and / or condition of the patient being treated, the severity of the disease being treated, the incidence and / or severity of side effects, the manner of administration, and the judgment of the prescribing physician. Appropriate dosage ranges can be determined by methods known to those of skill in the art.

[0097] The synbiotic composition may be assayed in vitro and in vivo for the desired therapeutic or prophylactic activity prior to use in humans. For example, in vivo assays using appropriate animal models may also be used to determine whether administration of the synbiotic composition will be therapeutically effective.

[0098] In certain embodiments, a therapeutically effective dose of the synbiotic composition can provide therapeutic benefits without causing substantial toxicity, including adverse side effects. The toxicity of the synbiotic composition and / or its metabolites can be determined using standard pharmaceutical procedures and confirmed by those skilled in the art. The dose ratio between toxicity and therapeutic effect is the therapeutic index. The dose of the synbiotic composition can be within a range that can establish and maintain therapeutically effective circulating plasma and / or blood concentrations of, for example, prebiotic components, with little or no toxicity.

[0099] Administration of the synbiotic composition may be useful in treating a variety of conditions, including adrenoleukodystrophy, AGE-induced genomic damage, Alexander's disease, alopecia areata, Alpers' disease, Alzheimer's disease, amyotrophic lateral sclerosis, angina, arthritis, asthma, Baro's concentric sclerosis, Behcet's disease, bullous pemphigoid, Canavan disease, heart failure including left ventricular dysfunction, central nervous system vasculitis, Charcot-Marie-Tooth disease, childhood ataxia with central nervous system hypomyelination, chronic idiopathic peripheral neuropathy, chronic obstructive pulmonary disease, Crohn's disease, cutaneous lupus, dermatitis (contact, acute and chronic), diabetic retinopathy, graft versus host disease, granulomas, hepatitis C virus infection, herpes simplex virus infection, herpes zoster virus infection, human immune deficiency virus infection, and the like. The present invention may be used to treat diseases selected from the group consisting of immunodeficiency virus infection, Huntington's disease, irritable bowel disorder, ischemia, Krabbe's disease, lichen planus, macular degeneration, mitochondrial encephalomyopathy, unilateral upper limb muscular atrophy, multiple sclerosis, myocardial infarction, neurodegeneration with brain iron accumulation, neuromyelitis optica, neurosarcoidosis, NF-κB-mediated disease, optic neuritis, paraneoplastic syndromes, Parkinson's disease, Pelizaeus-Merzbacher disease, pemphigus, primary lateral sclerosis, progressive supranuclear palsy, psoriasis, pyoderma gangrenosum, reperfusion injury, retinitis pigmentosa, sarcoidosis, Schilder's disease, subacute necrotizing myelopathy, Susac's syndrome, transplant rejection, transverse myelitis, tumors, ulcerative colitis, and Zellweger syndrome. The underlying etiology of any of the aforementioned diseases to be treated may have multiple origins. Additionally, in certain embodiments, a therapeutically effective amount of the synbiotic composition may be administered to a patient, such as a human, as a preventative measure against the aforementioned diseases and disorders.Therefore, a therapeutically effective amount of the synbiotic composition may be used to treat a variety of conditions, including but not limited to adrenoleukodystrophy, AGE-induced genomic damage, Alexander's disease, alopecia areata, Alpers' disease, Alzheimer's disease, amyotrophic lateral sclerosis, angina pectoris, arthritis, asthma, Baro's concentric sclerosis, Behcet's disease, bullous pemphigoid, Canavan disease, heart failure including left ventricular dysfunction, central nervous system vasculitis, Charcot-Marie-Tooth disease, childhood ataxia with central nervous system hypomyelination, chronic idiopathic peripheral neuropathy, chronic obstructive pulmonary disease, Crohn's disease, cutaneous lupus, dermatitis (contact, acute and chronic), diabetic retinopathy, graft versus host disease, granulomas, hepatitis C virus infection, herpes simplex virus infection, varicella-zoster virus infection, human immunodeficiency virus infection, and pulmonary fibrosis. It may also be administered as a prophylactic measure to patients with a predisposition to and / or history of pan-viral infections, Huntington's disease, irritable bowel disorder, ischemia, Krabbe disease, lichen planus, macular degeneration, mitochondrial encephalomyopathy, unilateral upper limb muscular atrophy, multiple sclerosis, myocardial infarction, neurodegeneration with cerebral iron accumulation, neuromyelitis optica, neurosarcoidosis, NF-κB mediated disease, optic neuritis, paraneoplastic syndromes, Parkinson's disease, Pelizaeus-Merzbacher disease, pemphigus, primary lateral sclerosis, progressive supranuclear palsy, psoriasis, pyoderma gangrenosum, reperfusion injury, retinitis pigmentosa, sarcoidosis, Schilder's disease, subacute necrotizing myelopathy, Susac syndrome, transplant rejection, transverse myelitis, tumors, ulcerative colitis, and Zellweger syndrome.

[0100] In some embodiments, the methods and compositions of the present disclosure may be effective in improving metagenomic stability and metabolic output of the gut microbiota in subjects with irritable bowel syndrome.

[0101] In some embodiments, the methods and compositions of the disclosure may be effective in improving, repairing, or restoring function of the gut and airway microbiome in subjects with mild to moderate COVID-19 symptomatology.

[0102] In some embodiments, the methods and compositions of the present disclosure may be effective in preserving or improving the integrity of the intestinal barrier and / or the composition of the gut microbiota in subjects suffering from irritable bowel syndrome (IBS). In particular, the methods and compositions disclosed herein are intended to (1) maintain or increase the diversity of the gastrointestinal microbiota in a subject suffering from constipation-predominant IBS (IBS-C); (2) increase the representation of at least one of Bifidobacterium longum, Prevotella intermedia, Bacteroides helcogenes, Akkermansia muciniphila, Alistipes finegoldii, and Faecalibacterium prausnitzii, and / or Blautia obeum, in the gastrointestinal microbiota in a subject suffering from mixed / alternating stool pattern IBS (IBS-M). (3) improving any one or more of an individual's IBS symptoms selected from the group consisting of abdominal pain, abdominal bloating, difficulty in defecation, and stool consistency, (4) increasing the frequency of complete spontaneous bowel movements (CSBM) in subjects suffering from IBS-C, and (5) improving the visceral sensitivity index (VSI). In some embodiments, the methods and compositions of the present disclosure may be effective for any one or more of these purposes after a subject has been treated with an antibiotic, e.g., rifaximin.

[0103] In some embodiments, the methods and compositions of the present disclosure may be effective in ameliorating, preventing, or treating constipation, supporting the integrity of the intestinal barrier following acute alcohol exposure, and / or supporting vaginal health.

[0104] The present disclosure is further described by reference to the following non-limiting examples, which illustrate various aspects of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the disclosure. EXAMPLES

[0105] [Example 1] Use of prebiotics Individual strains of nine probiotic bacterial strains were streaked from frozen stocks onto De Man, Rogosa and Sharpe (MRS) solid medium containing 0.5 g / L L-cysteine ​​and incubated anaerobically at 37°C for 24 h under static conditions. The nine bacterial strains were Lacticaseibacillus rhamnosus SD-GG-BE, Ligilactobacillus salivarius SD-LS1-IT, Bifidobacterium breve SD-B632-IT, Bifidobacterium breve SD-BR03-IT, Bifidobacterium longum SD-CECT7347-SP, Lacticaseibacillus casei SD-CECT9104-SP, and Bifidobacterium lactis SD-CECT7347-SP. lactis SD-CECT8145-SP, Lactobacillus acidophilus SD-NCFM-US, and Bifidobacterium animalis subsp. lactis SD-BI07-US (hereinafter, these nine strains may be collectively referred to as "PDS-08 strains" and a composition containing the nine strains may be referred to as the "PDS-08 composition" or simply as "PDS-08". The first seven of these strains listed, namely, Lactobacillus acidophilus SD-NCFM-US and Bifidobacterium animalis subsp. lactis SD-BI07-US, were The nine PDS-08 strains, excluding PDS-08 (SEQ ID NO: 13) and PDS-07 (SEQ ID NO: 14) are sometimes collectively referred to as "MXP271 strains," and the composition containing the seven strains is sometimes referred to as the "MXP271 composition," or simply "MXP271."A single colony of each PDS-08 strain was selected and inoculated into MRS broth with 0.5 g / L L-cysteine ​​and then incubated anaerobically at 37° C. overnight under stationary conditions to generate experimental cultures. The resulting stationary phase cultures were supplemented with 5 g / L soluble starch, 2 g / L pectin, 1 g / L guar gum, 4 g / L mucin, 2 g / L xylan, 2 g / L arabinogalactan, 3 g / L casein, 5 mL / L of 0.15% (w / v) peptone water, 5 g / L tryptone, 0.4 g / L bile salts, 4.5 g / L yeast extract, 10 mL / L glycerol, 0.5 g ... The cells were subcultured (1:100) into 96-well plates containing medium mimicking the colonic environment, containing 0.005 g / L FeSO4·7H2O, 4.5 g / L NaCl, 4.5 g / L KCl, 0.5 g / L KH2PO4, 1.25 g / L MgSO4·7H2O, 0.15 g / L CaCl2·6H2O, 1.5 g / L NaHCO3, 0.8 g / L L-cysteine, 0.05 g / L hemin, and 1 mL / L Tween 80. Some samples were supplemented with one of three commercially available prebiotic compositions at 1 wt%: Orafti® Synergy 1 (approximately 50 wt% inulin, 50% fructooligosaccharides), Glucidex® (maltodextrin), and waxy maize (a complex starch mixture with high amylopectin content), while others were not, to serve as controls. Plates were incubated for 24 hours during which time the optical density at 600 nanometers (OD. 600 ) was measured every 30 min using a microplate reader. Three to nine biological replicates were performed with three technical replicates.

[0106] FIG. 1A illustrates the growth of Lacticaseibacillus casei SD-CECT9104-SP, FIG. 1B illustrates the growth of Lacticaseibacillus rhamnosus SD-GG-BE, FIG. 2C illustrates the growth of Bifidobacterium breve SD-BR03-IT, FIG. 1D illustrates the growth of Bifidobacterium lactis SD-CECT8145-SP, FIG. 1E illustrates the growth of Ligilactobacillus salivarius SD-LS1-IT, and FIG. 1F illustrates the growth of Bifidobacterium longum SD-GG-BE. FIG. 1G illustrates the growth of Lactobacillus acidophilus SD-NCFM-US, FIG. 1H illustrates the growth of Bifidobacterium animalis subsp. lactis SD-Bi-07-US, and FIG. 1I illustrates the growth of Bifidobacterium breve SD-B632-IT. OD 600Comparison of the changes in the IL-10 expression level reveals that the Orafti® prebiotic composition significantly enhanced the growth of Lacticaseibacillus casei SD-CECT9104-SP (Figure 1A), Bifidobacterium breve SD-BR03-IT (Figure 1C), Ligilactobacillus salivarius SD-LS1-IT (Figure 1E), Bifidobacterium longum SD-CECT7347-SP (Figure 1F), and Bifidobacterium breve SD-B632-IT (Figure 1I) compared with the control group. For the other four tested strains, a trend towards increased growth is observed in the presence of the Orafti® composition compared to the control group, but this trend is not statistically significant. Notably, the Orafti® composition stimulates the growth of all strains, except Lacticaseibacillus casei SD-CECT9104-SP, better than both the Glucidex® and waxy maize compositions, and in fact, specifically in the case of Bifidobacterium animalis subsp. lactis SD-Bi-07-US (Figure 1H), where the Glucidex® and waxy maize compositions appeared to inhibit growth. (Significance was determined using paired one-way ANOVA with Dunnett's multiple comparisons (parametric data) or Kruskal-Wallis (non-parametric data) tests to determine OD 0.01 for each test substance.) 600 The results were determined by comparing the changes in the mean mean and mean mean (Figures 1A to 1I); the results are indicated in Figures 1A to 1I as "ns" if not significant and "significant at P < 0.05". * ", and " if significant at P<0.01 ** ", and " if significant at P<0.001 *** " and " if significant at P < 0.0001 ****"). Taken together, these data show significant growth enhancement for at least five of the PDS-08 strains by the Orafti® prebiotic composition, while growth of the other four strains was not negatively affected. This provides a rationale for the use of an inulin / fructooligosaccharide prebiotic composition, such as Orafti® Synergy 1, in the formulation of the PDS-08 synbiotic composition.

[0107] [Example 2] Viability of microorganisms in synbiotic products 6.3 grams of Orafti® Synergy 1 (prebiotic ingredient) per dose, and 5.75 10 10 A synbiotic composition was formulated that contains the combined potency of AFU's PDS-08 microbial strain (probiotic component). The synbiotic composition was formulated as a free-flowing powder, where the only components were the prebiotic and probiotic components. After formulation of the synbiotic composition, samples of the probiotic components were obtained, and quantification of total, live and dead bacteria was performed by the flow cytometry analysis technique of fluorescence-activated cell sorting (FACS). Specifically, 10-fold serial dilutions of the samples were double-stained with 0.01 mM SYTO™ 24 green fluorescent nucleic acid dye and 3 μM propidium iodide for 15 minutes at 37° C. under dark anaerobic conditions. The samples were then analyzed on a BD FACSVerse instrument using a high flow rate setting, and bacteria were separated from medium debris and signal noise by applying a threshold level of 200 in the SYTO channel. Flow cytometry data were analyzed using FlowJo version 10.5.2 and reported as total bacterial counts in log units.

[0108] Referring now to Figure 2, FACS analysis revealed that 79.0% of the cells in the formulated synbiotic composition were in the viable fraction, while 15.8% appeared to be dead, and the remaining minor fraction (approximately 5.2%) was identified as viable, although not culturable. This high viability justifies the classification of the PDS-08 microbial component of the composition as a true probiotic, and therefore the composition as a whole can be conceptually classified as a true synbiotic.

[0109] [Example 3] Survival of probiotic strains along the upper gastrointestinal tract The synbiotic composition may be tested to confirm the survival of the microbial consortium of the probiotic components using a simulator of human intestinal microbial ecosystem (SHIME) 400 as illustrated in FIG. 3. In particular, SHIME 400 includes a stomach receptacle 410, a small intestine receptacle 420, an ascending colon receptacle 430, a transverse colon receptacle 440, and a descending colon receptacle 450. The stomach receptacle 410 receives and mixes the test composition 401 and gastric acid 402 via a pump along with a supply of nitrogen gas. The output from the stomach receptacle 410 is pumped to the small intestine receptacle 420 where it is mixed with pancreatic juice 411 received via a pump. The output from the small intestine receptacle 420 is pumped to the ascending colon receptacle 430, which is pH controlled to simulate the human ascending colon. The output from the ascending colon receptacle 430 is pumped to the transverse colon receptacle 440, which is pH controlled to simulate the human transverse colon. The output from the transverse colon vessel 440 is pumped to the descending colon vessel 450, which is pH controlled to simulate the human descending colon. The output from the descending colon vessel 450 is finally pumped to a waste tank 451. In this way, SHIME 400 reproduces physiological and biological conditions representative of the human gastrointestinal tract (e.g., food intake, peristalsis, digestive enzymes, pancreatic and bile acids, residence time, etc.). (Further details regarding the SHIME model can be found in K. Molly et al., “Development of a 5-step multi-chamber reactor as a simulation of the human intestinal microbial ecosystem,” 39 Applied Microbiology and Biotechnology 254 (May 1993), the contents of which are incorporated herein by reference.)

[0110] Now referring to FIG. 4, an adapted SHIME system 500 was used to study the survival of probiotic strains in the upper gastrointestinal tract. The adapted SHIME system 500 includes a single reactor 505 and a shaker 560, with these components representing physiological conditions of the stomach and small intestine in a single reactor 505 over time. To mimic fed or fasted conditions, a specific gastric suspension containing the test composition 501 is added to the reactor 505, and the reactor 505 is agitated using the shaker 560 to simulate gastric conditions 510. Standardized enzymes and bile juice are then added to the reactor 505, and the reactor 505 is agitated using the shaker 560 to simulate small intestinal conditions 520. The incubation conditions are optimized to resemble in vivo conditions in different regions of the gastrointestinal tract for fasted or fed conditions.

[0111] To simulate gastric conditions 510 in the fed state, the gastric suspension contains, in addition to the test composition 501, pepsin (supplied with an activity that is standardized by measuring the increase in absorbance at 280 nanometers of the trichloroacetic acid soluble products upon digestion of a hemoglobin reference protein), phosphatidylcholine, arabinogalactan, pectin, xylan, starch, glucose, yeast extract, peptone, mucin, and L-cysteine ​​hydrochloride, as well as additional levels of salt (sodium chloride and potassium chloride) as recommended by Alan Mackie and Neil Rigby, “InfoGest Consensus Method,” The Impact of Food Bioactives on Health 13 (2015) (hereinafter “Mackie,” which is incorporated herein by reference in its entirety). The gastric suspension was incubated in reactor 505 for 2 hours at 37° C. with mixing via agitation using shaker 560, with a sigmoidal decrease in pH profile from 4.6 to 3.0, as illustrated in FIG 5A. Samples of the gastric suspension were taken at the beginning (t=0, “ST0 / product”, FIG 5A) and end (t=120 min, “ST end”, FIG 5A) of the incubation period.

[0112] To simulate gastric conditions 510 in the fasted state, the gastric suspension contains, in addition to the test composition 501, pepsin and phosphatidylcholine in amounts equal to one-quarter of the amounts used in the suspension in the fed state, as well as the mucin and salts of the fed suspension. The gastric suspension is incubated in reactor 505 at 37° C. for 45 minutes with mixing via agitation using shaker 560 at a constant pH of 2.0, as illustrated in FIG. 5B. Samples of the gastric suspension were obtained at the beginning (t=0, “ST0 / product”, FIG. 5B) and end (t=45 min, “ST end”, FIG. 5B) of the incubation period.

[0113] To simulate small intestinal conditions 520 in the fed state, raw animal pancreatic extract (pancreatin) containing enzymes described by Mackie is added to the reactor 505, as is 10 mM bovine bile extract (closer to human bile than porcine bile in terms of taurocholate and glycocholate content). The pH is rapidly increased to 6.5 and held constant at 6.5 for 27 minutes ("duodenal phase"), then increased stepwise to 7.5 over 63 minutes ("jejunal phase"), and finally held constant at 7.5 for 90 minutes ("ileal phase"). The temperature is maintained at 37°C throughout the small intestinal conditions 520, and the suspension is mixed via agitation using a shaker 560. Samples are obtained at the end of each of the duodenal, jejunal, and ileal stages (i.e., in FIG. 5A, t=147 min / "end of duodenal", t=210 min / "end of jejunal", and t=300 min / "end of ileal", respectively).

[0114] To simulate small intestinal conditions 520 in the fasted state, pancreatin was added in an amount equal to one-fifth the amount used in the fed state, and bile salts were added in an amount equal to one-third the amount used in the fed state (i.e., 3.33 mM bovine bile extract). The pH was rapidly increased to 6.5 and held constant at 6.5 for 27 minutes ("duodenal phase"), then increased stepwise to 7.5 over 63 minutes ("jejunal phase"), and finally held constant at 7.5 for 90 minutes ("ileal phase"). The temperature was maintained at 37°C throughout the small intestinal conditions 520, and the suspension was mixed via agitation using a shaker 560. Samples are obtained at the end of each of the duodenal, jejunal, and ileal stages (i.e., in FIG. 5B, t=72 min / "end of duodenal", t=135 min / "end of jejunal", and t=225 min / "end of ileal", respectively).

[0115] From each sample, the number of viable microbial cells was determined by the flow cytometry analysis technique of fluorescence activated cell sorting (FACS). Specifically, a 10-fold dilution series of each sample was prepared in anaerobic phosphate buffered saline (PBS). The viable population in each sample was evaluated by staining the dilutions with SYTO™ 24 dye and propidium iodide. Samples were analyzed in a BD Accuri C6 Plus flow cytometer using a high flow rate. Bacterial cells were separated from medium debris and signal noise by applying two thresholds: a primary forward scatter height (FSC-H) threshold of 500, and a secondary FL-1 threshold of 700. Samples were analyzed in triplicate, and the results are reported as the mean logarithm of counts per reactor ± 1 standard deviation.

[0116] For each of the fed and fasted conditions, triplicates of two different synbiotic compositions (hereinafter referred to as the "naked strain" composition and the "microencapsulated strain" composition) were tested as test composition 501, and a single "blank" control was also tested. The naked strain composition was 3.0·10 10 AFU non-microencapsulated MXP271 bacterial strain, 5.0·10 9AFU non-microencapsulated Bifidobacterium animalis subsp. lactis SD-BI07-US, 3.0·10 10 The microencapsulated strain composition consisted of 3.0·10 AFU of non-microencapsulated Lactobacillus acidophilus SD-NCFM-US and 6.025 g of Orafti® Synergy 1 prebiotic, encapsulated in a vegetable oil and lipid matrix, based on the reactor. 10 AFU MXP271 bacterial strain, 5.0·10 9 AFU non-microencapsulated Bifidobacterium animalis subsp. lactis SD-BI07-US, 3.0·10 10 The study consisted of 100 g of non-microencapsulated Lactobacillus acidophilus SD-NCFM-US AFU, and 6.025 g of Orafti® Synergy 1 prebiotic.

[0117] Now referring to Figures 6A and 6B, it was observed that the viability of the strains in the naked strain composition was negatively affected by the gastric and small intestinal conditions in both fasted and fed states. However, the reduction in viability was more pronounced under fasted conditions, and while not wishing to be bound by any particular theory, we hypothesize that this difference was caused by a different pH profile (i.e., lower pH gastric conditions in the fasted state than in the fed state). The reduction in viability continued during the duodenal stage, but this reduction was not statistically significant in the fasted state. Bile salts added at the beginning of the duodenal stage can be highly toxic to bacterial cells, especially at relatively low pH. As pH increased in the jejunal and ileal stages, no further reduction in viability was observed, and in fact the opposite (i.e., increased cell viability) was observed during the ileal stage in both fasted and fed states, but this increase was only statistically significant in the fed state. At the end of the ileal phase, fasting conditions were observed 10 min after the start of gastric incubation. 10.91 =8.15 10 10 The last 10 AFU / reactor to ileum stage 7.97 =1.20 10 8 The fed conditions resulted in a reduction in viable population density by 99.85% (0.15% survival) up to AFU / reactor, but not by 10% at the start of stomach incubation. 10.91 =8.15 10 10 The last 10 minutes of the jejunum stage from the AFU / reactor 10.19 =1.57 10 10 AFU / reactor, resulting in a reduction in viable population density of 80.67% (19.33% survival). (Statistically significant reductions in viability from one stage to another are marked with an asterisk in Figures 6A and 6B. Statistical significance was determined by Student's t-test with a 95% confidence interval.)

[0118] Now, referring to Figures 7A and 7B, it was again observed that the viability of the strains in the microencapsulated strain composition was negatively affected by the stomach and small intestine conditions in the fasted state.In the fed state, on the other hand, the viability remained largely unaffected, and, without being bound to any particular theory, the inventors again believe that this difference between the fasted and fed states is due to the difference in pH profile during gastric incubation.During the duodenal stage, both the fasted and fed states resulted in a significant reduction in viable population density, and, without wishing to be bound to any particular theory, the inventors again believe that this is due to toxicity or other negative effects of bile salts added at the beginning of the duodenal stage. The reduction in viable population density in the fasted state, however, was less pronounced for the microencapsulated strain formulation (FIG. 7A) than for the naked strain formulation (FIG. 6A) and, without wishing to be bound by any particular theory, the inventors hypothesize that this difference is the result of a slower release of the microencapsulated strain compared to the naked strain, which may have a protective effect during incubation in the stomach and at least part of the duodenal stage. After the duodenal stage, no further reduction in viable population was observed and, for the naked strain composition, an increase in viability was further observed during the ileal stage in the fed state. At the end of the ileal stage, conditions in the fasted state were significantly lower than those at the start of the gastric incubation at 10 10.87 =7.61 10 10 The last 10 AFU / reactor to ileum stage 8.80 =6.70 10 8 Up to AFU / reactor resulted in a reduction in viable population density of 99.12% (0.88% survival), whereas the fed condition was significantly lower than the 10 AFU / reactor at the start of stomach incubation. 10.87 =7.61 10 10 The last 10 AFU / reactor to ileum stage 10.43 =2.72 10 10AFU / reactor, resulting in a reduction in viable population density of 64.27% (35.73% survival). (Statistically significant reductions in viability from one stage to another are marked with an asterisk in Figures 7A and 7B. Statistical significance was determined by Student's t-test with a 95% confidence interval.)

[0119] It can be concluded that administration of microencapsulated strain composition may generally be preferable in terms of survival of probiotic organisms to administration of naked strain composition.Without wishing to be bound by any particular theory, the inventors hypothesize that this may indicate that microencapsulation provides a protective effect for the PDS-08 probiotic strain during transit through the stomach and small intestine.In addition, administration of the synbiotic composition under fed conditions results in higher survival compared to administration under fasting conditions, indicating that administration in the fed state (e.g., when the subject has recently eaten) may be the recommended approach to deliver the highest number of viable bacterial cells to the site of probiotic activity (i.e., distal ileum and colon).

[0120] [Example 4] Short-term colonic batch incubation Following the experiments of Example 3, the predigested upper GIT suspension subjected to treatment in the adapted SHIME system 500 was used in short-term single-stage colonic incubations. In these experiments, a representative dose of the predigested upper GIT suspension was combined with a representative bacterial inoculum under conditions characteristic of the proximal large intestine. This bacterial inoculum could be derived from an already "in vitro adapted" microbial community (e.g., from the ascending colon container 430 of the SHIME system 400) or from a fresh or cryopreserved stool sample. In this example, two types of colonic incubations were performed: a non-sterile incubation, where the bacterial inoculum was derived from a fresh stool sample of a 6-10 year old child and added directly to the colonic reactor, and a sterile incubation, where the bacterial inoculum was first filter-sterilized to remove the bacterial background. In the sterile incubation, the use of a filter-sterilized inoculum allows recreation of the metabolic background caused by the probiotic strains in the colon.

[0121] At the start of the short-term colonic incubation, the predigested upper GIT suspension (obtained as the end product from the experiment in Example 3) and fresh or filter-sterilized bacterial inoculum were added to SHIME nutrient medium (9.27 g / L K2HPO4, 29.06 g / L KH2PO4, 3.57 g / L NaHCO3, 3.57 g / L yeast extract, 3.57 g / L peptone, 1.78 g / L mucin, 0.89 g / L L-cysteine-HCl, and 3.57 mL / L Tween 80) containing the basic nutrients present in the colon. Orafti® Synergy 1 prebiotic was added to each colonic reactor at a concentration of 4.3 g / L, and in each case the volume of predigested suspension was selected to achieve a total prebiotic concentration in each reactor of 5 g / L or less (the maximum concentration that can be tested in a short-term colonic incubation experiment; higher concentrations would result in excessive acidification). Incubation was carried out over the course of 48 hours at a temperature of 37° C., under anaerobic conditions with shaking (90 rpm). Table 1 below shows details for each of the six different experimental setups, each of which was carried out in triplicate (18 replicates in total). Samples were collected for analysis after 0, 1, 24, and 48 hours of incubation.

[0122] In Figures 8A to 14B, the reported statistics are the average of triplicates for each experimental setup. Statistically significant differences for pH change (Figures 8A, 8B), lactate production (Figures 9A, 9B), and total SCFA production (Figures 10A, 10B) are determined over the course of the entire 48-h incubation period. For both germ-free and non-germ-free incubations, significant differences between fed and fasted test products relative to each other are indicated by different lowercase letters (e.g., "a" vs. "b"). For non-germ-free incubations only, significant differences between the microencapsulated strain test products (whether under fasted or fed conditions) and the corresponding blank / control are indicated by an asterisk. All tests for statistical significance were performed by Student's t-test with 95% confidence intervals.

[0123] [Table 1]

[0124] In the colonic incubation experiments of this example, three relevant markers of microbial metabolic activity were recorded: changes in pH, short chain fatty acid (SCFA) production, and changes in lactate concentration. The degree of acidification during the experiment is a measure of the intensity of bacterial metabolism (fermentation) at any given point during the incubation, and therefore gives an indication of the speed of metabolism. SCFA production is an assessment of the microbial metabolism of carbohydrates (producing acetate, propionate, and butyrate) or proteins (producing branched SCFAs) and can be compared to the typical fermentation pattern for normal gut microbiota. Finally, since the intestine harbors both lactate-producing bacteria (e.g., Lactobacillus subtilis) and lactate-consuming bacteria, changes in lactate concentration can indicate the balance between these organisms. Lactate also reduces the pH of the environment, acts as an antibacterial agent, and can be rapidly converted by other microorganisms to other simple SCFAs (e.g., acetate, butyrate, propionate).

[0125] pH change Monitoring pH during short-term colonic incubation was used to measure the levels of SCFAs, lactate, and ammonium (NH + ) production. Generally, a pH drop is observed during the first 24 hours of incubation due to the formation of SCFAs and / or lactic acid. This pH drop is often accompanied by, among other things, the production of NH4 + An increase in pH follows during the second 24 hours of incubation due to proteolytic fermentation resulting in the production of lactic acid, and due to the conversion of stronger acids to weaker acids (e.g., lactic acid to propionic acid and / or butyric acid) by syntrophy.

[0126] Referring now to Figures 8A and 8B, for the blank / control suspension, a reduction in pH over the 48-h incubation period was observed under both fasted and fed conditions, but this reduction was more pronounced under fed conditions (Figure 8A, third column, ΔpH=-0.23) than under fasted conditions (Figure 8A, first column, ΔpH=-0.13), and without wishing to be bound by any particular theory, we hypothesize that this can be explained by a higher nutrient availability originating from differences in gastric fluid composition under fed conditions. The pH reduction was particularly pronounced from 1 to 24 hours of incubation, indicating that fermentation of available substrates occurred primarily during this time interval. Overall, we conclude that the gut microbial community of the pediatric stool donors was able to ferment available substrates in the colonic medium and predigested upper GIT suspensions.

[0127] For non-sterile incubations of the predigested microencapsulated strain composition, the pH strongly decreased over the entire course of colonic incubation (i.e., from 0 to 48 h). Similar to the blank / control suspension, this decrease was especially evident from 1 to 24 h of incubation. However, a slight increase was observed during the last 24 h under fed conditions (Figure 8A, fourth column, ΔpH=+0.03 from 24 h to 48 h), indicating a shift in carbohydrate metabolism towards protein degradation. For both fasted and fed conditions, the overall pH decrease in non-sterile incubations was significantly greater than the corresponding blank controls (Figure 8A: ΔpH=-0.60 vs. -0.13 under fasted conditions, -0.67 vs. -0.23 under fed conditions), indicating that the supplemented prebiotics were efficiently fermented by the newly established gut microbial community. Similar to the blank control, the pH decrease was more pronounced under fed conditions in the non-sterile incubations; without wishing to be bound to any particular theory, we hypothesize that this may be explained at least in part by a higher nutrient availability and at least in part by a higher survival rate of the probiotic strains during passage through the upper GIT.

[0128] For axenic incubation of the predigested microencapsulated strain composition, the pH decrease was again more pronounced under fed conditions (FIG. 8B, right column, ΔpH=−0.74) than under fasted conditions (FIG. 8B, left column, ΔpH=−0.16), which again could be explained by higher nutrient availability and / or higher probiotic survival. In addition, the pH decrease under both conditions confirms that the probiotic strains were able to ferment available nutrients, including those of the prebiotic. Interestingly, supplementation of the feeding with predigested upper GIT suspension resulted in a similar pH decrease in both the germ-free incubation (FIG. 8B, right column, ΔpH=-0.74) and the non-germ-free incubation (FIG. 8A, fourth column, ΔpH=-0.67), whereas under fasting conditions the pH decrease was much more pronounced in the non-germ-free incubation (FIG. 8A, second column, ΔpH=-0.60) than in the germ-free incubation (FIG. 8B, left column, ΔpH=-0.16). As the survival rate of the probiotic strains was much higher under feeding conditions, we conclude, without wishing to be bound by any particular theory, that the large number of viable probiotic cells in the feeding suspension were likely to colonize the donor's gut microbial community to a higher extent and therefore contributed more to the metabolic activity of the newly established gut microbial community.

[0129] lactic acid concentration The human intestine harbors both lactic acid-producing and lactic acid-consuming bacteria. Lactic acid bacteria produce lactic acid, especially during primary substrate degradation, and reduce the pH of the intestinal environment. Particularly at low pH values, lactic acid can exhibit strong antibacterial effects against pathogens. Another beneficial effect of lactic acid is that it can be efficiently converted to butyric acid and / or propionic acid, and therefore, as different microbial species produce or consume lactic acid, an increase in lactic acid concentration can result from either or both increased production and decreased consumption.

[0130] 9A and 9B, for the blank control, lactate concentrations initially increased during the first hour of colony incubation, indicating that lactate was produced by the donor gut microbial community more rapidly than it was consumed. This increase, however, was very limited under both fasted (FIG. 9A, first column, lactate concentration Δ=+0.14 mM) and fed (FIG. 9A, third column, lactate concentration Δ=+0.20 mM) conditions. As incubation progressed, lactate levels subsequently decreased (Δ=-0.37 mM under fasted conditions, -0.45 mM under fed conditions), indicating either decreased lactate production or that lactate was more efficiently converted to other metabolites.

[0131] A similar effect was observed for non-sterile incubation of the predigested microencapsulated strain composition, i.e., an initial increase during the first hour of incubation (Figure 9A, second and fourth columns: Δ=0.20 mM under fasted conditions, 0.72 under fed conditions) was followed by a decrease from 1 to 24 hours (Δ=-0.56 mM under fasted conditions, -2.45 mM under fed conditions). Under both fasted and fed conditions, the overall decrease from 0 to 48 hours was significantly greater for the microencapsulated strain composition than for the blank control (-0.36 vs. -0.23 under fasted conditions, -1.73 vs. -0.25 under fed conditions). Both the initial increase and the subsequent decrease were more pronounced under fed conditions. Because the concentrations of prebiotic nutrients were similar under both sets of conditions, the inventors do not wish to be bound by any particular theory, but conclude that the probiotic strains may provide an initial boost to lactic acid production and improve the conversion of lactic acid to other metabolic products (e.g., propionate, butyrate) during the subsequent stages of incubation.

[0132] For the axenic incubation of the predigested microencapsulated strain composition, a relatively high level of lactic acid was produced. This was especially true under fed conditions (Figure 9B, right column, total lactic acid concentration Δ=+33.5 mM), which may be associated with a higher nutrient availability (originating from differences in gastric juice composition) and higher survival rate of the probiotic strains. The strong increase in lactic acid concentration indicates that the probiotic strains were able to ferment available nutrients (originating from both the background medium and the prebiotic composition), resulting in the accumulation of lactic acid. Notably, unlike the non-axenic incubation (Figure 9A, second and fourth columns), no net consumption of lactic acid was observed during the last 24 h of incubation under axenic conditions (Figure 9B), probably due to the absence of syntrophic interactions with propionate- and / or butyrate-producing strains in the background microbial community.

[0133] Total SCFA production SCFA production results from carbohydrate metabolism in the colon and is associated with various health effects. The SCFAs produced in the highest amounts in the colon are acetate, propionate, and butyrate, each of which has a beneficial effect on the host's health: acetate can be used as an energy source for the host and a potential substrate for lipid synthesis, propionate improves metabolic homeostasis by reducing cholesterol and fatty acid synthesis in the liver, and butyrate is the main energy source for colonocytes and is differentially induced in these cells (an effect related to cancer prevention). Branched SCFAs (bSCFAs), in contrast, are the result of proteolytic microbial activity, which is also associated with the formation of toxic compounds such as p-cresol. Thus, high production of acetate, propionate, and butyrate in the colon is associated with beneficial health effects, while high bSCFA production in the colon is associated with adverse health effects.

[0134] Now referring to Figures 10A and 10B, total SCFA production reflects the overall fermentation of the gut microbiota. Fasted and fed blank controls (Figure 10A, first and third columns) resulted in total SCFA production of 32.9 and 44.6 mM, respectively. Therefore, the donor gut microbiota was able to ferment the nutrients available in the colonic medium. The higher values ​​obtained under fed conditions could again be related to a higher nutrient availability originating from differences in gastric juice composition. Under both fasted and fed conditions, the strongest SCFA production occurred during the first 24 hours of incubation, which corresponded to the pH decrease observed during this time interval (Figure 8A).

[0135] For non-sterile incubation of the predigested microencapsulated strain composition, significantly higher total SCFA concentrations (Figure 10A, second and fourth columns: total SCFA = 58.8 mM fasted, 68.0 mM fed) were obtained compared to the blank control, with the fed condition resulting in slightly higher total SCFA production. Since the concentration of prebiotics was similar under both conditions, the enhanced production under fed conditions could be the result of a higher survival rate of the microbial strains, which could increase SCFA production either directly (by fermenting available nutrients themselves) or indirectly (by stimulating the fermentation of available nutrients by the spontaneous gut microbiota). Similar to the blank control, the strongest increase in total SCFA production occurred in the first 24 hours.

[0136] For axenic incubation of the predigested microencapsulated strain composition, almost no SCFAs were produced under fasted conditions (FIG. 10B, left column), whereas under fed conditions, total SCFA production was only 8.5 mM (FIG. 10B, right column). These results confirm that the probiotic strains directly contribute to SCFA production, i.e., convert available nutrients into SCFAs by themselves.

[0137] Acetogenesis Acetic acid is one of the important metabolites formed during primary substrate fermentation and can be produced by a wide range of gut microorganisms, including Bacteroides species and Bifidobacterium species. Now, referring to Figures 11A and 11B, the fasted and fed blank controls (Figure 11A, first and third columns) resulted in an overall acetate production of 21.0 and 27.3 mM, respectively, indicating that the majority of total SCFA production consisted of acetate. Similar to total SCFA production (Figure 10A, first and third columns), higher acetate levels were obtained under fed conditions, with acetate levels increasing most significantly during the first 24 hours of incubation.

[0138] For non-sterile incubation of the predigested microencapsulated strain composition, significantly higher acetate levels were obtained under both fasted ( FIG. 11A , second column, 41.2 mM) and fed ( FIG. 11A , fourth column, 40.6 mM) conditions compared to the control. Although no significant differences were observed between fasted and fed conditions, the inventors believe that this may indicate that the spontaneous gut microbiota was primarily responsible for acetate production, although they do not wish to be bound by any particular theory.

[0139] For the axenic incubation of the predigested microencapsulated strain composition, a very small amount of acetic acid was produced under fasting conditions (FIG. 11B, left column), whereas under fed conditions, the total acetic acid production was only 8.6 mM (FIG. 10B, right column). Based on these results, we conclude that the SCFA activity of the probiotic strains mainly results in the production of acetic acid, which is typically that of Lactobacillus and Bifidobacterium strains. The more significant increase in acetic acid concentration under fed conditions may be due to higher nutrient availability and survival of higher probiotic strains existing in the small intestine. In addition, the much lower acetic acid levels produced in axenic incubation compared to non-axenic incubation confirm that administration of the microencapsulated strain composition stimulated the production of acetic acid by the donor's spontaneous gut microbiota.

[0140] Propionate production Like acetate, propionate can be produced by a wide range of gut microbes, with the most abundant propionate producers in the gut microbiome being Bacteroides species, Akkermansia muciniphila, and Veillonellaceae species.

[0141] 12A and 12B, the overall propionate production levels in the blank control under fasted and fed conditions (FIG. 12A, first and third columns) were 5.8 and 8.8 mM, respectively. Like acetate, propionate was produced primarily during the first 24 hours of incubation and in greater amounts under fed conditions than under fasted conditions. Thus, propionate production clearly contributed to total SCFA production in the control, although to a much lesser extent than acetate.

[0142] For non-sterile incubations of the pre-digested microencapsulated strain compositions, significantly higher propionic acid levels were obtained compared to the blank controls (Figure 12A, second and fourth columns: 13.34 mM under fasted conditions, 16.99 mM under fed conditions). The significantly higher propionic acid production under fed conditions is again associated with higher nutrient availability and / or higher survival rate of the probiotic strains.

[0143] For the axenic incubation of the predigested microencapsulated strain composition, the probiotic strain was unable to produce significant amounts of propionic acid under either fasting (FIG. 12B, left column, 0.00 mM) or fed (FIG. 12B, right column, -0.16 mM) conditions. Based on these results, the inventors do not wish to be bound by any particular theory, but conclude that at least one of the probiotic microbial strains and the prebiotic composition indirectly increases propionic acid production by stimulating the spontaneous gut microbial community. This may be the result of a symbiotic mechanism, whereby the end product of metabolic activity of certain bacterial species can be used as a substrate by other bacterial species. In this case, certain gut microbiota may be capable of converting lactate to propionic acid.

[0144] Butyrate production Butyrate is produced by members of Clostridium clusters IV and XIVa. By syntrophy, these microorganisms are able to convert acetate and / or lactate, among other substrates, to butyrate.

[0145] 13A and 13B, the blank controls (FIG. 13A, first and third columns) resulted in butyrate production of 2.5 and 3.5 mM, respectively. Similar to acetate and propionate, butyrate was primarily produced during the first 24 hours of incubation, with slightly higher production obtained under fed than fasted conditions.

[0146] Significantly higher butyric acid levels were obtained for non-sterile incubations of the predigested microencapsulated strain compositions compared to the corresponding blank controls (Figure 13A, second and fourth columns: 4.41 mM under fasted conditions, 9.70 mM under fed conditions). The highest values ​​were again obtained under fed conditions, which may be related to higher nutrient availability and / or higher survival rate of the probiotic strains.

[0147] For the axenic incubation of the predigested microencapsulated strain composition, the probiotic strain was unable to produce significant amounts of propionic acid under either fasting ( FIG. 13B , left column, −0.30 mM) or fed ( FIG. 11B , right column, 0.01 mM) conditions. Based on these results, the inventors do not wish to be bound by any particular theory, but conclude that at least one of the probiotic microbial strains and the prebiotic composition indirectly increases butyric acid production by stimulating the spontaneous gut microbial community. This may be the result of a symbiotic mechanism, in which, similar to propionic acid, certain gut microbiota may be able to convert lactic acid to butyric acid.

[0148] Branched SCFAs Branched SCFAs (bSCFAs) are produced as a result of proteolytic fermentation and are associated with the formation of toxic compounds such as p-cresol. Thus, high bSCFA levels may be an indicator of harmful proteolytic activity in the colon, and reducing levels is believed to be beneficial for health.

[0149] Now, referring to Figures 14A and 14B, the blank control resulted in 2.03 and 3.38 mM overall bSCFA production under fasting and fed conditions, respectively (Figure 14A, first and third columns). bSCFA production was slightly higher under fed conditions, which again may be associated with higher nutrient availability. When compared to acetate, propionate, and butyrate, a distinct increase in bSCFA production was observed during the last 24 hours of incubation, indicating that the gut microbiota initially uses carbohydrates as substrates, and then shifts metabolic activity toward protein degradation later in the incubation.

[0150] For non-sterile incubations of the predigested microencapsulated strain compositions, significantly lower bSCFA concentrations were obtained compared to the corresponding blank controls (Figure 14A, second and fourth columns: 0.92 mM under fasted conditions, 0.36 mM under fed conditions). In addition, the lowest values ​​were obtained under fed conditions. Thus, administration of the synbiotic composition led to an increase in polysaccharide fermentation, likely due to the presence of prebiotics.

[0151] For sterile incubation of the predigested microencapsulated strain compositions, the probiotic strains were unable to produce significant amounts of bSCFAs under either fasted (FIG. 14B, left column, 0.00 mM) or fed (FIG. 14B, right column, 0.03 mM) conditions.

[0152] [Example 5] Comparative survival of probiotic strains along the upper GIT Survival of microbial strains in commercially available pediatric probiotic products was investigated by subjecting the probiotic products to treatment in an adapted SHIME system 500, as illustrated in FIG. 4, as described above in Example 3. The probiotic products were added to the reactor vessel 505 in amounts corresponding to the recommended daily dosage of each product at the start of the upper GIT experiment under fed conditions. Four commercially available pediatric probiotic products were tested: Culturelle Kids Probiotic composition (1 sachet / reactor), Garden of Life Raw Probiotics Kids composition (1.6 g / reactor), Optibac Probiotics Babies & Children composition (1 sachet / reactor), and Life-Space Probiotic Powder for Children composition (1 g / reactor).

[0153] Referring now to FIG. 15A, administration of the Culturelle Kids Probiotic composition, whose microbial strains consisted primarily of Lactobacillus rhamnosus GG cells, revealed that the viability of the probiotic strains was not significantly affected during passage through the fed stomach (10 per reactor at both the beginning and end of the gastric conditions). 10.47 The Lactobacillus rhamnosus GG strain present in this probiotic composition (a) thus proved to be resistant to the pH profile in the stomach (i.e. a sigmoidal decrease in pH from 4.6 to 3.0 over 2 hours). Upon entering the duodenal stage, however, the viability of the probiotic strain decreased significantly (10 per reactor). 10.47 =2.97 10 10 From 10 9.02 =1.06 10 9As described above in Example 3, bile salts added at the beginning of the duodenal stage can be highly toxic to bacterial cells, especially at the relatively low pH. As the pH increased throughout the jejunal and ileal stages, no further reduction in viability was observed (10 at the end of the jejunal and ileal stages, respectively). 9.02 and 10 9.03 Therefore, from the beginning to the end of the upper GIT, the number of viable cells was 10 per reactor. 10.47 =2.95 10 10 From 10 9.03 =1.10 10 9 The viable cells were reduced by 96.27% (3.73% viability).

[0154] Referring now to Figure 15B, administration of the Garden of Life® Raw Probiotics Kids composition, whose microbial strains consist mainly of Lactobacillus and Bifidobacterium species, revealed that the microbial strains present in the probiotic product were not negatively affected by the environmental conditions caused during the stomach transit under fed conditions. Indeed, at the end of the stomach transit, the viable cell count was significantly higher than at the start (10 per reactor). 10.48 =3.02 10 10 vs 10 10.37 =2.36 10 10 Although the in vivo extrapolation of this finding may be questionable, the growth of the probiotic strains during their transit through the stomach is highly unlikely and therefore the measured effect may be an artifact of the lower solubility of the test composition in aqueous buffer compared to the gastric juice simulating medium. The viability of the probiotic strains was then significantly reduced at the duodenal stage (10 per reactor). 10.16 =1.45 10 10 No significant reduction in viability was observed in the jejunum and ileum stages. At the end of the ileum stage, 10 10.13=1.35 10 10 The viable population density of viable cells was observed to show a decrease of 42.82% (57.18% viable) compared to the starting population, and 55.33% (44.67% viable) when compared to the viable population at the end of the stomach stage.

[0155] Referring now to FIG. 15C, administration of the Optibac Probiotics® Babies & Children composition, whose microbial strains consist primarily of Lactobacillus acidophilus Rosell-52, Bifidobacterium infantis Rosell-33, and Bifidobacterium bifidum Rosell-71, increased the survival rate of the strains by 10 per reactor. 10.20 =1.59 10 10 From 10 10.08 =1.20 10 10 We found that the passage through the stomach was negatively affected, with a significant reduction in viable cells, indicating that these strains are less robust when faced with the harsh pH conditions in the stomach. The viable population density further decreased throughout the small intestine stage, but only at the end of the ileum stage did this reduction (10 per reactor). 9.48 =3.49 10 9 The decrease in viability (to viable cells) was statistically significant. Therefore, these strains could also be negatively affected by high bile salt concentrations and low pH values ​​at the duodenal stage, although to a lesser extent than for the Culturelle Kids Probiotic composition (FIG. 15A). Overall, passage through the upper GIT resulted in a reduction in viability of 78.04% (21.96% survival) for this composition.

[0156] Now referring to FIG. 15D, administration of the Life-Space® Probiotic Powder for Children composition, whose microbial strains consist primarily of Lactobacillus and Bifidobacterium species, increased the survival rate of these strains by 10 per reactor. 10.32 =2.07 10 10 From 10 10.21 =1.63 10 10 A significant reduction in viable cell count to viable cells revealed a negative impact during passage through the stomach. Similar to the Optibac Probiotics® Babies & Children composition (Figure 15C), this reduction was most likely a result of the probiotic strain's low robustness to the harsh pH conditions in the stomach. The viable population density further decreased at the duodenal stage (10 per reactor). 10.08 =1.21 10 10 The results showed that these strains were also negatively affected by the addition of bile salts (up to viable cells). However, no further reduction in viability was observed in the jejunum stage, and in fact an increase in viability was observed in the ileum stage (10 per reactor at the end of the jejunum stage). 10.10 =1.26 10 10 10 per reactor at the end of the ileum stage from viable cells 10.21 =1.61 10 10 Overall, passage through the upper GIT resulted in a reduction in viability of 22.31% (77.69% viable) for this composition.

[0157] A comparison of viable cell viability after transient passage through the upper GIT for the four commercial compositions tested in this example and the two compositions of the present disclosure tested in Example 3 (naked and microencapsulated strain compositions) is provided in Table 2 below. In Table 2, viability is simply the number of colony forming units (CFU) obtained at the end of the ileum stage divided by the number of CFU present in the probiotic composition at the start of the experiment, "MXP271+" refers to the compositions of the present disclosure tested in Example 3 (i.e., seven MXP271 strains and PDS-08 strains including Lactobacillus acidophilus SD-NCFM-US and Bifidobacterium animalis subsp. lactis SD-BI07-US), and "ME" stands for "microencapsulated."

[0158] [Table 2]

[0159] As shown in Table 2, the viability of Culturelle Kids Probiotic compositions is generally too low to provide a sufficiently high number of viable cells to the site of their probiotic activity (i.e., the distal ileum and colon). Although a similar percentage of cells of the Optibac Probiotics Babies & Children composition survives in the upper GIT as in the Naked Strain composition of the present disclosure, the low number of cells provided in the recommended dosage form results in a very low number of viable cells delivered to the distal ileum and colon. The high starting viable cell density of the Naked Strain composition also resulted in a number of viable cells at the end of the ileum stage that was higher than that of the Garden of Life Raw Probiotics Kids composition and comparable to that of the Life-Space Probiotic Powder for Children composition. Finally, the microencapsulated strain composition of the present disclosure delivered a significantly higher number of viable cells at the end of the ileum stage than any of the currently commercially available pediatric probiotic compositions tested.

[0160] The concepts illustratively disclosed herein may suitably be practiced in the absence of any element not specifically disclosed herein, however, it will be apparent to those skilled in the art that many modifications, variations, alterations, other uses, and applications of the present disclosure are possible, and modifications, variations, alterations, other uses, and applications that do not depart from the spirit and scope of the present disclosure are deemed to be encompassed by the present disclosure.

[0161] The foregoing discussion has been presented for purposes of illustration and description. The foregoing is not intended to limit the disclosure to the form or forms disclosed herein. In the foregoing detailed description, for example, various features are grouped together in one or more embodiments for the purpose of streamlining the disclosure. Features of the embodiments may be combined in alternative embodiments other than those discussed above. This method of disclosure should not be interpreted as reflecting an intention that the claims require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. As such, the following claims are hereby incorporated into this detailed description, with each claim standing on its own as a separate embodiment.

[0162] Also, while the present disclosure includes a description of one or more embodiments and certain variations and modifications, other variations, combinations and modifications are within the scope of the present disclosure, for example, as would be within the skill and knowledge of one of ordinary skill in the art after understanding the present disclosure. It is intended to be entitled to include alternative embodiments to the extent permitted, including alternative, interchangeable and / or equivalent structures, functions, ranges or steps to those set forth in the claims, without intending to dedicate to the public any patentable subject matter, whether or not such alternative, interchangeable and / or equivalent structures, functions, ranges or steps are disclosed herein.

Claims

1. 1. A method for treating a disease or achieving a physiological objective in a human subject, comprising administering to said subject a therapeutically effective amount of a synbiotic composition, said synbiotic composition comprising: A prebiotic component comprising at least one compound that can be converted into a bioactive metabolite by a microbial strain present in a healthy human gut microbiome; and A probiotic composition comprising a consortium of microbial strains, said consortium comprising: (i) Lacticaseibacillus rhamnosus SD-GG-BE; (ii) Ligilactobacillus salivarius SD-LS1-IT; (iii) Bifidobacterium breve SD-BR632-IT; (iv) Bifidobacterium breve SD-BR03-IT; (v) Bifidobacterium longum SD-CECT7347-SP; (vi) Lacticaseibacillus casei SD-CECT9104-SP; and (vii) Bifidobacterium lactis SD-CECT8145-SP A probiotic composition comprising at least two microbial strains selected from the group consisting of: A method comprising:

2. The diseases include adrenoleukodystrophy, AGE-induced genomic damage, Alexander's disease, alopecia areata, Alpers' disease, Alzheimer's disease, amyotrophic lateral sclerosis, angina, arthritis, asthma, Baro's concentric sclerosis, Behcet's disease, bullous pemphigoid, Canavan's disease, heart failure including left ventricular dysfunction, central nervous system vasculitis, Charcot-Marie-Tooth disease, childhood ataxia with central nervous system hypomyelination, chronic idiopathic peripheral neuropathy, chronic obstructive pulmonary disease, Crohn's disease, cutaneous lupus, dermatitis (contact, acute and chronic), diabetic retinopathy, graft versus host disease, granulomas, hepatitis C virus infection, herpes simplex virus infection, varicella-zoster virus infection, human immunodeficiency virus, 10. The method of claim 1, wherein the tumor is selected from the group consisting of myelopathy, encephalomyopathy, rhusiopathies, Huntington's disease, irritable bowel disorder, ischemia, Krabbe disease, lichen planus, macular degeneration, mitochondrial encephalomyopathy, unilateral upper limb muscular atrophy, multiple sclerosis, myocardial infarction, neurodegeneration with brain iron accumulation, neuromyelitis optica, neurosarcoidosis, NF-κB mediated disease, optic neuritis, paraneoplastic syndromes, Parkinson's disease, Pelizaeus-Merzbacher disease, pemphigus, primary lateral sclerosis, progressive supranuclear palsy, psoriasis, pyoderma gangrenosum, reperfusion injury, retinitis pigmentosa, sarcoidosis, Schilder's disease, subacute necrotizing myelopathy, Susac syndrome, transplant rejection, transverse myelitis, tumors, ulcerative colitis, and Zellweger syndrome.

3. 2. The method of claim 1, wherein the disease is a gastrointestinal disease or an infectious disease.

4. 4. The method of claim 3, wherein the disease is selected from the group consisting of irritable bowel syndrome, COVID-19, and constipation.

5. 4. The method of claim 3, wherein the disease is antibiotic-induced dysbiosis of the subject's intestinal microbiota.

6. 2. The method of claim 1, wherein the disease is selected from the group consisting of metabolic syndrome, type 2 diabetes, and pre-diabetes.

7. 2. The method of claim 1, wherein the physiological objective is selected from the group consisting of improving cardiovascular health, maintaining or reducing weight, reducing glycated hemoglobin levels, normalizing glucose or insulin response, treating acne or otherwise improving skin health, and improving cognitive function.

8. Co-administration or administration of one or more other pharmaceutical agents to a human subject in combination with said synbiotic composition. Further comprising:

2. The method of claim 1, wherein the co-administration or administration in combination improves the therapeutic effect of the one or more other pharmaceutical agents compared to administration of the synbiotic composition and the one or more other pharmaceutical agents independently.

9. 2. The method of claim 1, wherein the physiological objective is selected from the group consisting of increasing the Bristol Fecal Form Scale (BSFS), decreasing the duration of bowel movements, reducing abdominal pain, reducing abdominal bloating, reducing heartburn, reducing acid regurgitation, reducing digestive disorders, maintaining or increasing the diversity of the gastrointestinal microbiota, and improving health-related quality of life as measured by the KINDL questionnaire score.

10. The method of claim 1 , wherein the synbiotic composition is administered as an ingestible formulation.

11. 11. The method of claim 10, wherein the ingestible formulation is in the form of a free-flowing powder provided in a unit-serving sachet.

12. the sachet has a weight of from about 1 mg to about 12 g, or from about 250 mg to about 11.75 g, or from about 500 mg to about 11.5 g, or from about 750 mg to about 11.25 g, or from about 1 g to about 11 g, or from about 1.25 g to about 10.75 g, or from about 1.5 g to about 10.5 g, or from about 1.75 g to about 10.25 g, or from about 2 g to about 10 g, or from about 2.25 g to about 9.75 g, or from about 2.5 g to about 9.5 g, or from about 2.75 g to about 9.25 g, or from about 3 g to about 9 g, 12. The method of claim 11, wherein the prebiotic component is in an amount of from about 3.25 g to about 8.75 g, or from about 3.5 g to about 8.5 g, or from about 3.75 g to about 8.25 g, or from about 4 g to about 8 g, or from about 4.25 g to about 7.75 g, or from about 4.5 g to about 7.5 g, or from about 4.75 g to about 7.25 g, or from about 5 g to about 7 g, or from about 5.25 g to about 6.75 g, or from about 5.5 g to about 6.5 g, or from about 5.75 g to about 6.25 g, or about 6 g.

13. 12. The method of claim 11, wherein the pouch comprises the consortium of microbial strains in an amount of about 62.5 million AFU to about 312.5 billion AFU, about 625 million AFU to about 250 billion AFU, about 1.25 billion AFU to about 125 billion AFU, about 6.25 billion AFU to about 62.5 billion AFU, about 12.5 billion AFU to about 60 billion AFU, about 25 billion AFU to about 55 billion AFU, about 40 billion AFU to about 50 billion AFU, or about 45 billion AFU.

14. 12. The method of claim 11, wherein the pouch material has a water vapor transmission rate of less than about 0.01 grams per square meter per day at 23°C and 50% relative humidity.

15. 12. The method of claim 11, wherein the dose of the synbiotic composition is administered at least once a day, and the dose comprises the contents of one sachet.

16. The method of claim 11, wherein the sachet further comprises at least one pharma- ceutically acceptable vehicle.

17. 10. The method of claim 1, wherein the synbiotic composition is administered at least once a day for at least about seven days.

18. 2. The method of claim 1, wherein the at least one compound that can be converted into a biologically active metabolite by a microbial strain present in the healthy human gut microbiome comprises at least one fructan.

19. 20. The method of claim 18, wherein the at least one fructan comprises at least one polysaccharide fructan.

20. 20. The method of claim 19, wherein the at least one polysaccharide fructan comprises inulin.

21. 21. The method of claim 20, wherein the inulin is derived from or extracted from at least one plant selected from the group consisting of agave, asparagus, banana, barley, burdock, barley, chicory, cornflower, costus, dandelion, elegans, garlic, artichoke, Jerusalem artichoke, jicama, leek, hareweed, mugwort, onion, psyllium, wheat, yacon, and yam.

22. 20. The method of claim 18, wherein the at least one fructan comprises a fructooligosaccharide.

23. 23. The method of claim 22, wherein the fructooligosaccharides are derived from or extracted from at least one plant selected from the group consisting of agave, asparagus, banana, barley, burdock, barley, chicory, cornflower, costus, dandelion, elegans, garlic, artichoke, Jerusalem artichoke, jicama, leek, hareweed, mugwort, onion, psyllium, wheat, yacon, and yam.

24. 2. The method of claim 1, wherein the prebiotic component consists essentially of about 50 wt% inulin and about 50 wt% fructooligosaccharides.

25. 2. The method of claim 1, wherein the consortium comprises at least three, at least four, at least five, at least six, or all of (i) through (vii).

26. The consortium, (viii) Lactobacillus acidophilus SD-NCFM-US; and (ix) Bifidobacterium animalis subsp. lactis SD-BI07-US 2. The method of claim 1, further comprising at least one microbial strain selected from the group consisting of:

27. 27. The method of claim 26, wherein the consortium comprises both (viii) and (ix).

28. 28. The method of claim 27, wherein the consortium comprises all of (i) through (vii), and both (viii) and (ix).

29. 30. The method of claim 28, wherein the consortium consists essentially of (i) through (vii), (viii), and (ix).

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