Synbiotic treatment method for improving health

Abstract

A synbiotic composition comprising both a prebiotic component and a probiotic component is provided. The prebiotic component comprises at least one compound that can be converted into a biologically active metabolite by a microbial strain present in a healthy human gut microbiota, and the probiotic component comprises a reasonably defined and assembled microbial strain complex.

Description

Technical Field

[0001] The present disclosure relates generally to microbial biologics for pediatric use, and in particular to rationally defined microbial communities that effectively confer health in organ systems of human children by modulating the function of native gut microbiota and host tissues. Background Art

[0002] Human gut microbiome

[0003] The human gut microbiome is a complex network of bacteria, archaea, viruses, fungi and even protozoa that reside primarily in the host's colon. This gut microbiome is comparable in cell number to that of the human body, but has more than 100 times the genetic coding capacity of the human body.

[0004] Due to its important role in human health, the microbiome is sometimes regarded as an organ of the human body, and much like other organs, the microbial organ evolves over time in terms of the number and complexity of its microorganisms. Although microbial DNA has been detected in the fetal stage, it can be considered that the intestine is sterile or at most sparsely colonized at birth. Further colonization of the intestine depends on the type of birth, environment and first food, i.e., breast milk or formula. In the first few days of life, the intestinal microbiome is dominated by aerobic bacteria and facultative anaerobes such as enterobacteria, enterococci, staphylococci and streptococci. These microorganisms proliferate in the intestine, thereby fully reducing the intestinal oxygen concentration to proliferate obligate anaerobes such as Bifidobacterium spp., which then become the main colonizers in the first few weeks. In some infants, another facultative anaerobic bacterium, Bacteroides spp., can also dominate. As more foods are introduced into the infant's diet at about six months of age, the infant acquires a variety of bacteria, resulting in a more complex microbiome by the age of twelve months. During this period, Clostridia increase in number, commensurate with the increased need to digest carbohydrates available to the more diverse microbiota reaching the colon, leading to greater diversity and maturation of the infant gut microbiome. By about three years of age, the child's gut microbiome resembles that of an adult. Members of the mature gut microbiome were primarily from five major phyla—Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Verrucomicrobia—with the first two accounting for about 90% of the population.

[0005] Gut bacteria have co-evolved with their human hosts to extract energy for their own survival while also nourishing the host. The gut microbiota plays a key role in the digestion of nutrients, maturation of the gastrointestinal tract, immune activation, defense against pathogens, production of vitamins that the host doesn’t produce, and even brain health. Dysbiosis in the gut microbiota has been linked to a number of groups: gut-related conditions, such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS); immune conditions, such as asthma, atopic dermatitis, and rheumatoid arthritis; and lifestyle-related metabolic diseases, such as obesity and diabetes. Gut bacteria are also increasingly being linked to brain-related issues, such as mood, anxiety, and neurodegenerative diseases.

[0006] Intestinal symbionts are good at cooperatively extracting energy from organic matter that escapes host metabolism and reaches the colon. Therefore, undigested food residues (mainly plant dietary fiber and polyphenols, as well as glycoproteins shed from the intestinal mucosa) are metabolized by intestinal bacteria to generate acidic metabolites. The main acid metabolites are lactic acid (an organic acid) and short-chain fatty acids (SCFA) such as acetic acid, propionic acid and butyric acid.

[0007] Acetic acid and lactic acid are produced in the early steps of bacterial carbohydrate degradation. These metabolites then serve as carbon sources for propionic acid and butyric acid producers through a process of mutualistic resource sharing, and these producers can therefore be referred to as "secondary" consumers. While acetic acid is produced by most intestinal bacteria, lactic acid is produced by a small fraction of bacteria, particularly Bifidobacterium and Lactobacilli (Lactobacilliales).

[0008] SCFAs play a major role in regulating host metabolism, immune defense, and even brain health. The three-carbon molecule acetic acid beneficially affects host energy and metabolism via appetite regulation and fat oxidation. Propionic acid is known to increase plasma glucagon, fatty acid binding protein 4 (FABP4), and norepinephrine levels, leading to insulin resistance and compensatory hyperinsulinemia. Butyric acid provides most of the fuel required for colonocytes and plays a key role in maintaining the intestinal barrier by regulating tight junction proteins. Butyric acid also acts as a histone deacetylase inhibitor and affects signal transduction through several G protein-coupled receptors that mediate anti-inflammatory activity. Barrier maintenance is energy-intensive, and butyric acid produced by microorganisms is the main source of this energy. In general, butyric acid has a beneficial effect on intestinal homeostasis and energy metabolism, and its anti-inflammatory effect enhances intestinal barrier function and mucosal immunity.

[0009] The balanced presence of SCFAs plays an important role in preventing chronic inflammatory states, inhibiting the production of proinflammatory cytokines, regulating T cells, and protecting colonic epithelial cells. The impact of SCFAs extends beyond the gastrointestinal tract; these metabolites are associated with the respiratory system by inhibiting the function of pulmonary innate lymphoid cells (ILC2) that stimulate airway hyperresponsiveness, inhibiting skin pathogens through the gut-skin axis, and are regulators of bone mass and osteoclast metabolism. In addition, SCFAs also regulate mood, sleep, and neuronal health via the gut-brain axis.

[0010] Gut Microbiome and Child Health

[0011] The link between the gut microbiome and health is becoming increasingly clear. One of the earliest associations was well described by the "hygiene hypothesis," which suggested that infections early in life caused by "unhygienic exposure" (e.g., due to living on a farm, having pets, etc.) protected against allergies. There is a clear association between the gut microbiome and the development of childhood disease.

[0012] There are many factors that influence the development and maturation of a child's gut microbiome. These include mode of birth, first food (i.e., breast milk vs. formula), family 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 an association between allergies and reduced species diversity; an association between autism spectrum disorder and increased Clostridium species and / or Sutterella and Dephosphomonus species; an association between colic and reduced microbial diversity and / or increased anaerobic populations; an association between the development of eczema and early colonization by opportunistic species; an association between malnutrition and anaerobic depletion, early dysbiosis, and / or an excess of enteric pathogens and reduced bacterial diversity; an association between preterm birth and increased Proteobacteria and / or reduced microbial diversity; an association between sepsis and altered microbiota structure and composition prior to disease onset; an association between type I diabetes and increased Bacteroidetes:Firmicutes ratio, increased Clostridium species, decreased butyrate-producing bacteria, reduced bacterial diversity, and / or reduced microbial community stability; and an association between type II diabetes and increased Firmicutes:Bacteroidetes ratio and increased SCFAs.

[0013] Probiotics

[0014] More than a century has passed since the first observed correlation between increased lifespan and consumption of yogurt containing lactic acid-producing bacteria, and since Bifidobacterium species were first detected in the feces of breastfed infants but not in formula-fed infants and those with diarrhea. These early concepts of “healthy bacteria” eventually converged into the widely adopted term “probiotic,” meaning “live microorganisms that confer a health benefit on the host when administered in adequate amounts.”

[0015] Probiotics historically encompass Bifidobacterium and Lactobacillus. More than 250 Lactobacillus species have been identified, which appear to be highly genetically distinct from one another, but also metabolically, ecologically, and functionally distinct. These observations have led to the recent reclassification of the Lactobacillus genus into 25 genera, including the revised Lactobacillus genus (which encompasses host-adapted organisms, now known as the Lactobacillus delbrueckii group), Paralactobacillus genus, and 23 new genera.

[0016] The National Institutes of Health Human Microbiome Project has shown that there is a wide variation in the diversity and abundance of microorganisms in healthy individuals, suggesting that there is no universally healthy microbiome. Despite the diversity 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 facilitated the isolation of specific microbiota members from healthy individuals for the development of so-called live biotherapeutic products (LBPs). These products, which are added to classic Lactobacillus and Bifidobacterium-based probiotics, are designed to beneficially affect disease states. Notably, gut microbiota interventions not only bring health benefits to the local environment, but also have far-reaching effects through, for example, the gut-skin, gut-heart, and gut-brain axes.

[0017] Prebiotics

[0018] Prebiotics have been defined by the International Scientific Association of Probiotics and Prebiotics as "substrates that are selectively utilized by the host microorganisms to confer a health benefit." For a substrate 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, resulting in a favorable change in microbial balance or the formation of metabolites that benefit the host; and (3) the benefit to the host has been demonstrated through clinical studies and is the result of changes in the host's intestinal microorganisms.

[0019] The main types of dietary prebiotics are dietary fibers that are fermentable by certain microbiota. Some plant-derived polyphenols can also exhibit 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.

[0020] Dietary fiber prebiotics are polymeric and oligomeric carbohydrates that are utilized by intestinal microorganisms, resulting in the production of beneficial metabolites through a multi-step fermentation process. Most commercial prebiotics today belong to this category; common examples include oligofructose (FOS), oligogalactose (GOS), and inulin. The number of fructose molecules linked to each other (i.e., the degree of polymerization or DP) and the branching of these molecules play an important role in the fermentability of these prebiotics. In general, in laboratory models of the intestine, low DP (DP of 1 or 2) fructans are more easily and faster broken down by intestinal bacteria, while high DP fructans (DP of 3 to 60) exhibit a slower, more stable rate of fermentation by fecal bacteria. Therefore, higher DP inulin can remain intact long enough to withstand intestinal peristalsis and be pushed farther into the colon, thereby being able to prolong the duration of microbial activity.

[0021] Biostime

[0022] 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 living organisms and substrates selectively utilized by the host microorganisms that impart a health benefit to the host". Such formulations may be complementary in their mechanism of action, i.e., prebiotics target native symbionts, or synergistic in that the prebiotic substrate is selectively utilized by the co-administered probiotics. Delivery of synbiotics to targeted niches in the human gastrointestinal tract is challenging due to the harsh and changing conditions of the intestine, problems associated with maintaining the activity and efficacy of probiotics, and masking the unpleasant taste of prebiotics. Synbiotic delivery technologies have been and continue to be extensively researched by the functional food and supplement industry. SUMMARY OF THE INVENTION

[0024] In aspects of the present disclosure, a method for treating a disease or achieving a physiological purpose in a human subject comprises administering to the subject a therapeutically effective amount of a synbiotic composition comprising a prebiotic component and a probiotic component, wherein the prebiotic component comprises at least one compound that can be converted into a bioactive metabolite by a microbial strain present in a healthy human intestinal microbiota; the probiotic component comprises a consortium of microbial strains, the consortium comprising at least two microbial strains selected from the group consisting of: (i) Lactobacillus rhamnosus SD-GG-BE; (ii) Lactobacillus salivarius SD-LS1-IT; (iii) Bifidobacterium breve SD-BR632-IT; (iv) Bifidobacterium breve SD-BR03-IT; (v) Bifidobacterium longum SD-CECT7347-SP; (vi) Lactobacillus casei SD-CECT9104-SP; and (vii) Bifidobacterium lactis SD-CECT8145-SP.

[0025] In an embodiment, the disease can be selected from the group consisting of adrenoleukodystrophy, age-induced genomic damage, Alexander disease, alopecia areata, Alpers disease, Alzheimer's disease, amyotrophic lateral sclerosis, angina, arthritis, asthma, Balo concentric sclerosis, Behcet's disease, bullous pemphigus, Canavan disease, cardiac insufficiency (including left ventricular dysfunction), central nervous system vasculitis, Charcot-Marie-Tooth disease, childhood ataxia with central nervous system demyelination, 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 immunodeficiency virus infection, Huntington's disease, irritable bowel disease, ischemia, Krabbe disease, lichen planus, macular degeneration, mitochondrial encephalomyopathy, monomelic amyotrophy, multiple sclerosis, myocardial infarction, neurodegeneration with brain iron accumulation, neuromyelitis, neurosarcoidosis, NF-κB-mediated diseases, optic neuritis, carcinoid syndrome, Parkinson's disease, Pelizaeus-Merzbacher disease, pemphigus, primary lateral sclerosis, progressive supranuclear palsy, psoriasis, pustular gangrene, reperfusion injury, retinitis pigmentosa, sarcoidosis, Sheldon's disease, subacute necrotizing myelopathy, susac syndrome, transplant rejection, transverse myelitis, tumors, ulcerative colitis, and Zellweger syndrome.

[0026] In embodiments, the disease may be a gastrointestinal 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 subject's intestinal microbiota.

[0027] In an embodiment, the disease may be selected from the group consisting of metabolic syndrome, type 2 diabetes, and prediabetes.

[0028] In an embodiment, the physiological goal can be selected from the group consisting of improving cardiovascular health, maintaining or reducing body weight, lowering glycated hemoglobin levels, normalizing glucose or insulin response, treating acne or otherwise improving skin health, and improving cognitive function.

[0029] In embodiments, the method may further comprise co-administering or combining one or more other pharmaceutical agents with the synbiotic composition to the human subject, and the co-administration or combining administration may improve the therapeutic efficacy of the one or more other pharmaceutical agents relative to independent administration of the synbiotic composition and the one or more other pharmaceutical agents.

[0030] In an embodiment, the physiological goal may be selected from the group consisting of increasing the Bristol Stool Shape Scale (BSFS), reducing bowel movement duration, relieving abdominal pain, relieving bloating, relieving heartburn, relieving acid reflux, relieving indigestion, maintaining or increasing the diversity of the gastrointestinal microbiota, and improving health-related quality of life as measured by the KINDL questionnaire score.

[0031] In an embodiment, the synbiotic composition can be administered as an ingestible formulation. The ingestible formulation can be, but not necessarily, in the form of a free-flowing powder provided in a single-serving pouch. The pouch can, but not necessarily, contain the following amounts of the prebiotic component: about 1 mg to about 12 g, or about 250 mg to about 11.75 g, or about 500 mg to about 11.5 g, or about 750 mg to about 11.25 g, or about 1 g to about 11 g, or about 1.25 g to about 10.75 g, or about 1.5 g to about 10.5 g, or about 1.75 g to about 10.25 g, or about 2 g to about 10 g, or about 2.25 g to about 9.75 g, or about 2.5 g to about 9.5 g, or about From about 2.75g to about 9.25g, or from about 3g to about 9g, or from about 3.25g to about 8.75g, or from about 3.5g to about 8.5g, or from about 3.75g to about 8.25g, or from about 4g to about 8g, or from about 4.25g to about 7.75g, or from about 4.5g to about 7.5g, or from about 4.75g to about 7.25g, or from about 5g to about 7g, or from about 5.25g to about 6.75g, or from about 5.5g to about 6.5g, or from about 5.75g to about 6.25g, or about 6g. The pouch may, but need not, contain a 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 pouch may, but need not, have a water vapor permeability of less than about 0.01 g / m2 / day at 23°C and 50% relative humidity. The dose of the synbiotic composition may, but need not, be administered at least once a day, and the dose may, but need not, contain the contents of a pouch. The pouch may, but need not, further contain at least one pharmaceutically acceptable vehicle.

[0032] In embodiments, the synbiotic composition may be administered at least once daily for at least about 7 days.

[0033] In embodiments, at least one compound that can be converted into a bioactive metabolite by a microbial strain present in a healthy human intestinal microbiota can include at least one fructan. At least one fructan may, but need not, include at least one polysaccharide fructan. At least one polysaccharide fructan may, but need not, include inulin. Inulin 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, Camellia sinensis, chicory, rudbeckia rutaecarpa, costus, dandelion, scutellaria baicalensis, garlic, artichoke, Jerusalem artichoke, yam, leek, leopard 's bane, mugwort, onion, plantain, wheat, Jerusalem artichoke and yam. At least one fructan may, but need not, include at least one oligofructose. Oligofructose can, but need not, be derived from or extracted from at least one plant selected from the group consisting of agave, asparagus, banana, barley, burdock, Camellia, chicory, coneflower, costus, dandelion, elecampane, garlic, artichoke, Jerusalem artichoke, yam, leek, leopard's bane, mugwort, onion, plantain, wheat, Jerusalem artichoke, and yam.

[0034] In embodiments, the prebiotic component may consist essentially of about 50 wt% inulin and about 50 wt% fructo-oligosaccharides.

[0035] In an embodiment, the consortium may include at least three, at least four, at least five, at least six, or all of (i) to (vii).

[0036] 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) to (vii) and both (viii) and (ix). The consortium may, but need not, consist essentially of (i) to (vii), (viii) and (ix).

[0037] Although specific embodiments and applications have been shown and described, the present disclosure is not limited to the precise configurations and components described herein. Various modifications, changes and variations that are obvious to those skilled in the art may be made to the arrangement, operation and details of the methods and systems disclosed herein without departing from the spirit and scope of the entire disclosure.

[0038] As used herein, unless otherwise indicated, the terms "about," "approximately," and the like, when used in connection with a numerical limit or range, mean that the limit or range can vary by up to 10%. As a non-limiting example, "about 750" can mean as little as 675 or as much as 825, or any value therebetween. When used in connection with a ratio or relationship between two or more numerical limits or ranges, the terms "about," "approximately," and the like mean that each limit or range can vary by up to about 10%; as a non-limiting example, a statement that two quantities are "approximately equal" can mean that the ratio between the two quantities is as low as 0.9:1.1 or as high as 1.1:0.9 (or any value therebetween), and a statement that a four-way ratio is "about 5:3:1:1" can mean that the first number in the ratio can be any value of at least 4.5 and not more than 5.5, the second number in the ratio can be any value of at least 2.7 and not more than 3.3, and so on.

[0039] The embodiments and configurations described herein are neither complete nor exhaustive.As will be appreciated, other embodiments may utilize one or more of the features set forth above or described in detail below, alone or in combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1A , 1B , 1C, 1D, 1E, 1F, 1G, 1H and 1I are the OD values ​​of the probiotic strains according to the present disclosure using various prebiotic substrates. 600 Changes in the graph.

[0041] Figure 2 is a graph of the determination of fluorescence activated cell sorting (FACS) based cell viability of probiotic strains according to the present disclosure.

[0042] Figure 3 Schematic diagram of the standard setup of the Human Gut Microbial Ecosystem Simulator (SHIME).

[0043] Figure 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).

[0044] Figure 5A and 5B Plots of the pH profiles during survival experiments under fasting and fed conditions, respectively.

[0045] Fig. 6A and 6B Graph of the average viable bacterial population during passage through the upper GIT under fasting and fed conditions, respectively, for naked probiotic strains according to the present disclosure.

[0046] Fig. 7A and 7BGraph of the average viable bacterial population during passage through the upper GIT under fasting and fed conditions, respectively, for microencapsulated probiotic strains according to the present disclosure.

[0047] Fig. 8A and 8B is when supplemented with non-sterile ( Fig. 8A ) or sterile ( Figure 8B ) inoculum during short-term colonic incubation of a pre-digested blank control or a microencapsulated probiotic composition according to the present disclosure.

[0048] Fig.9A and 9B is when supplemented with non-sterile ( Fig.9A ) or sterile ( Fig. 9B ) inoculum during short-term colonic incubation of a pre-digested blank control or a microencapsulated probiotic composition according to the present disclosure.

[0049] Fig. 10A and 10B is when supplemented with non-sterile ( Fig. 10A ) or sterile ( Fig. 10B ) inoculum during short-term colonic incubation of a pre-digested blank control or a microencapsulated probiotic composition according to the present disclosure.

[0050] Fig.11A and 11B is when supplemented with non-sterile ( Fig.11A ) or sterile ( Fig. 11B ) inoculum during short-term colonic incubation of a pre-digested blank control or a microencapsulated probiotic composition according to the present disclosure.

[0051] Fig. 12A and 12B is when supplemented with non-sterile ( Fig. 12A ) or sterile ( Fig. 12B ) inoculum during short-term colonic incubation of a pre-digested blank control or a microencapsulated probiotic composition according to the present disclosure.

[0052] Fig.13A and 13B is when supplemented with non-sterile ( Fig.13A ) or sterile ( Fig. 13B ) inoculum during short-term colonic incubation of a pre-digested blank control or a microencapsulated probiotic composition according to the present disclosure.

[0053] Fig.14Aand 14B is when supplemented with non-sterile ( Fig.14A ) or sterile ( Fig. 14B ) inoculum during short-term colonic incubation of a pre-digested blank control or a microencapsulated probiotic composition according to the present disclosure.

[0054] Fig.15A , 15B , 15C and 15D are Children's probiotic composition, Garden of Original Probiotics Kids Combination, Optibac Baby & Child Compositions and Plot of the average viable bacterial population of children's probiotic powder compositions during passage through the upper GIT. DETAILED DESCRIPTION OF THE INVENTION

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. All patents, applications, published applications and other publications cited herein are incorporated by reference in their entirety. If a term in this article has multiple definitions, the definition provided in the Summary of the Invention shall prevail unless otherwise stated.

[0057] "CRISPR" (Clustered Regularly Interspaced Short Palindromic Repeats) loci refer to certain genetic loci encoding components of a DNA cutting system, used, for example, by bacterial and archaeal cells to destroy foreign DNA. A CRISPR locus can consist of a CRISPR array, which contains short direct repeats (CRISPR repeats) separated by short variable DNA sequences (called spacers), which can be flanked by different Cas (CRISPR-associated) genes. The CRISPR-Cas system is an example of a pathway unknown to science before the era of DNA sequencing, and is now understood to confer acquired immunity to bacteria and archaea against phages and viruses. In-depth 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 cutting of foreign DNA or RNA) and CRISPR arrays (which include direct repeats flanking short spacer sequences that guide Cas proteins to their targets). Class 2 CRISPR-Cas is a streamlined version in which a single Cas protein bound to RNA is responsible for binding and cutting the targeting sequence. The programmable nature of these minimal systems facilitates their use as a versatile technology that is revolutionizing the field of genome manipulation.

[0058] As used herein, "effector" or "effector protein" is a protein that encompasses activities including recognition, binding and / or cleavage or nicking of a polynucleotide target. An effector or effector protein may also be an endonuclease. The "effector complex" of the CRISPR system includes Cas proteins that participate in crRNA and target recognition and binding. Some component Cas proteins may additionally include domains that participate in target polynucleotide cleavage.

[0059] The term "Cas protein" refers to a polypeptide encoded by a Cas (CRISPR-related) gene. Cas proteins include proteins encoded by genes in the cas locus, and include adaptive molecules and interfering molecules. Interfering molecules of bacterial adaptive immune complexes include endonucleases. The Cas endonucleases described herein comprise one or more nuclease domains. Cas endonucleases include, but are not limited to, novel Cas-α 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 of these. Cas proteins can be "Cas endonucleases" or "Cas effector proteins", which, when compounded with suitable polynucleotide components, are capable of recognizing, binding to, and optionally nicking or cutting all or part of a specific polynucleotide target sequence.

[0060] CRISPR-Cas systems have been classified according to the sequence and structural analysis of components. A variety of CRISPR / Cas systems have been described, including a class 1 system (including type I, type III and type IV) with a multi-subunit effector complex and a class 2 system (including type II, type V and type VI) with a single protein effector. The CRISPR-Cas system at least includes CRISPRRNA (crRNA) molecules and at least one CRISPR-related (Cas) protein to form a crRNA ribonucleoprotein (crRNP) effector complex. The CRISPR-Cas locus includes a series of identical repeats of DNA targeting spacers interspersed with crRNA components and operon-like units of cas genes encoding Cas protein components. The resulting ribonucleoprotein complex recognizes polynucleotides in a sequence-specific manner. By forming base pairs with complementary DNA strands, while displacing non-complementary strands to form so-called R loops, crRNA is used as a guide RNA for sequence-specific binding of effectors (proteins or complexes) to double-stranded DNA sequences. The RNA transcripts (pre-crRNA) of the CRISPR loci are specifically cleaved within the repetitive sequences by CRISPR-associated (Cas) endonucleases in type I and III systems or by RNase III in type II systems. The number of CRISPR-associated genes at a given CRISPR locus can vary between species.

[0061] 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 can be used for more than one purpose, for example, Cas9 contains domains for endonuclease function and for target cutting, etc. Cas endonucleases are guided by a single CRISPR RNA (crRNA) through direct RNA-DNA base pairing to recognize DNA target sites adjacent to the protospacer adjacent motif (PAM). Class I CRISPR-Cas systems include type I, type III, and type IV. Class I systems are characterized by the presence of effector endonuclease complexes rather than single proteins. The Cascade complex contains an RNA recognition motif (RRM) and a nucleic acid binding domain, which is a core fold of a diverse RAMP (repeat-associated mysterious protein) protein superfamily.

[0062] Type I CRISPR-Cas systems contain a complex of effector proteins, called Cascade (CRISPR-associated complex for antiviral defense), which contains at least Cas5 and Cas7. The effector complex works with a single CRISPR RNA (crRNA) and Cas3 to defend against invading viral DNA. Type I systems are divided into seven subtypes.

[0063] Type III CRISPR-Cas systems, which contain multiple cas7 genes, target either ssRNA or ssDNA and function as RNases as well as target RNA-activated DNA nucleases. Type IV systems, although containing typical type I cas5 and cas7 domains in addition to a cas8-like domain, may lack the CRISPR array that is characteristic of most other CRISPR-Cas systems.

[0064] Class II CRISPR-Cas systems include Type II, Type V, and Type VI. Class II systems are characterized by the presence of a single Cas effector protein rather than an effector complex. Type II and Type V Cas proteins include 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 Cas endonucleases to their DNA targets. The crRNA includes a spacer region complementary to one strand of the double-stranded DNA target and a region that base pairs with tracrRNA (trans-activating CRISPR RNA) to form an RNA duplex that guides the Cas endonuclease to cut the DNA target, leaving a flat end. The spacer region is obtained by a process that is not yet fully understood involving Cas1 and Cas2 proteins. In addition to the cas9 gene, the Type II CRISPR / Cas locus generally also includes cas1 and cas2 genes. The Type II CRISR-Cas locus can encode tracrRNA, which is partially complementary to the repeat sequence within the corresponding CRISPR array, and can include other proteins such as Csn1 and Csn2. The presence of cas9 near cas1 and cas2 genes is a hallmark of type II loci. Type V CRISPR / Cas systems include single Cas endonucleases, including Cpf1 (Cas12), which are active RNA-guided endonucleases that, unlike Cas9, do not necessarily require additional trans-activated CRISPR (tracr) RNA for target cutting. Type VI CRISPR-Cas systems include cas13 genes encoding nucleases with two HEPN (higher eukaryotic and prokaryotic nucleotide binding) domains but without HNH or RuvC domains, and are independent of tracrRNA activity. Most HEPN domains include conserved motifs that constitute metal-independent endoRNase active sites. Due to this feature, it is believed that type VI systems act on RNA targets rather than DNA targets shared with other CRISPR-Cas systems.

[0065] To comply with the written description and implementation requirements, the following patent publications are incorporated herein by reference below: 2014 / 0349405 to Sontheimer; 2014 / 0377278 to Elinav; 2014 / 0068797 to Doudna; 20200190494 to Hou et al.; and 2020 / 0199555 to Zhang.

[0066] To further comply with applicable written description and enabling requirements, U.S. Patent Application Publication No. 2020 / 0138722, published on May 7, 2020 by Kabadi et al., entitled “Targeted Gastrointestinal Delivery of Probiotic Organisms and / or Therapeutic Agents,” is incorporated herein by reference.

[0067] To further satisfy applicable written description and enabling requirements, the following references are incorporated herein by reference:

[0068] PCT application PCT / US2021 / 015103, filed on January 26, 2021, and titled “Probiotic compositions and microbiota for improving the health of human organ systems.”

[0069] PCT application PCT / US2021 / 015107, filed on January 26, 2021, and entitled “Methods and compositions for precise release of probiotics to improve human health”

[0070] U.S. Provisional Patent Application 63 / 141,874, titled “Probiotic Therapeutic Methods for Improving Human Health,” filed on January 26, 2021.

[0071] As used herein, unless otherwise indicated, the term "animal" refers to any organism in the kingdom Animalia, including but not limited to humans.

[0072] As used herein, unless otherwise indicated, the term "disease" refers to a disease, disorder or condition, or a symptom thereof.

[0073] As used herein, unless otherwise indicated, the term "ingestible formulation" refers to a composition of matter suitable for or configured to be consumed by an animal by orally taking the composition into the gastrointestinal tract (e.g., by eating or drinking). The term "ingestible composition" as used herein can be provided in a form selected from the group consisting of: ampoule, aqueous solution, aqueous suspension, capsule, drops, granules, liquid, mist, powder, sachet, syrup, tablet, functionalized food, beverage, toothpaste, sublingual preparation, etc.

[0074] As used herein, unless otherwise specified, the term "lactobacillus," when used without further elaboration, refers to any organism classified as the Lactobacillaceae family prior to the 2020 reclassification of the Lactobacillus genus into 25 distinct genera.

[0075] As used herein, unless otherwise indicated, the term "patient" refers to mammals, including humans as a non-limiting example.

[0076] As used herein, unless otherwise indicated, the term "pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0077] As used herein, unless otherwise indicated, 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, which can be administered to a patient together with a substance provided by the present disclosure, does not destroy the pharmacological activity of the substance, and is non-toxic when administered in a dose sufficient to provide a therapeutically effective amount of the substance.

[0078] As used herein, unless otherwise indicated, the term "pharmaceutical formulation" refers to a therapeutically active substance and at least one pharmaceutically acceptable vehicle with which the substance is administered to a patient.

[0079] As used herein, unless otherwise specified, the term "prebiotic" refers to a substrate that is selectively utilized by a microorganism to confer a health benefit to the microbial host.

[0080] As used herein, unless otherwise specified, the term "probiotic" refers to live microorganisms that confer a health benefit on the host when administered in adequate amounts.

[0081] As used herein, unless otherwise specified, the term "synbiotic" refers to a combination or mixture of probiotics and prebiotics that beneficially affects the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract by selectively stimulating the growth and / or activating metabolism of one or more health-promoting microorganisms.

[0082] As used herein, the terms "treating" and "treatment" refer to reversing, alleviating, preventing, ameliorating or preventing a disease or at least one clinical symptom of a disease, reducing the risk of acquiring a disease or at least one clinical symptom of a disease, inhibiting the progression of a disease or at least one clinical symptom of a disease, or reducing the risk of developing a disease or at least one clinical symptom of a disease. "Treatment" also refers to inhibiting a disease physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of a physical parameter), or both, and inhibiting at least one physical parameter discernible or indiscernible to a 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 the disease or susceptible to the disease, even if the patient has not yet experienced or exhibited symptoms of the disease.

[0083] As used herein, unless otherwise indicated, the term "therapeutically effective amount" refers to an amount of a substance that is sufficient to affect such treatment of a disease or its symptoms when it is administered to a subject to treat at least one clinical symptom of a disease. A "therapeutically effective amount" may vary depending on, for example, the severity of the substance, the disease and / or disease symptoms, the disease and / or symptoms of the disease or condition, 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 may be determined by those skilled in the art or can be determined by routine experimentation.

[0084] As used herein, unless otherwise indicated, the term "therapeutically effective dose" refers to a dose that provides effective treatment of a disease or condition in a patient. The therapeutically effective dose may vary depending on the substance and the patient, and may vary depending on factors such as the patient's condition and the route of delivery. The therapeutically effective dose may be determined according to conventional pharmacological procedures known to those skilled in the art.

[0085] The present disclosure provides rationally defined and assembled microbial consortia to bring health to organ systems by first modulating the function of native intestinal microbiota and host tissues. More specifically, the present disclosure provides designed oral preventive probiotic strain mixtures to maintain and improve the host health status of local and remote body sites.

[0086] In embodiments, the present disclosure provides one or more live microbial consortia with a range of benefits validated in humans. The activity of the synbiotic compositions of the present disclosure throughout the production and administration process has been validated by comprehensive evaluation of active fluorescent units (AFU) in a simulated human intestinal environment, as further described in detail in the Examples provided below. In addition to extensive mechanistic and genetic characterization, the consortium also includes strains that have been clinically evaluated for clinical outcomes (including digestive improvement, gut-skin axis regulation, low-density lipoprotein regulation, intestinal immune response, epithelial barrier regulation, and micronutrient synthesis) in strain-specific, double-blind, placebo-controlled human studies.

[0087] Microbial strains

[0088] The development of next generation sequencing (NGS) technology has introduced new and much cheaper methods for prokaryotic whole genome sequencing. Combined with the falling price of computing resources required for bioinformatics analysis, this has led to the disclosure of a large number of new bacterial genomes. More and more "non-bioinformatics" laboratories are now sequencing their own prokaryotic genomes and are faced with questions such as which sequencing platform to choose, how many libraries should be generated, and which assembly method should be used for the libraries.

[0089] Embodiments of the present disclosure include synbiotic compositions comprising Lactobacillus rhamnosus SD-GG-BE. The bacterial strain may have any one or more of a number of beneficial effects on the health of the host. By way of a first non-limiting example, Lactobacillus rhamnosus SD-GG-BE may reduce toll-like receptor mRNA levels on antigen presenting cells (APCs), reduce CD16 expression in macrophages and CD11 expression in monocytes, and / or induce type 1 immune response polarization, as observed from the increased production of IL-12 and TNF-α. By way of a second non-limiting example, Lactobacillus rhamnosus SD-GG-BE may relieve abdominal pain in children with functional dyspepsia, irritable bowel syndrome, and / or functional abdominal pain. By way of a third non-limiting example, Lactobacillus rhamnosus SD-GG-BE may improve bloody stools, diarrhea, irritability, bloating, mucous stools, and / or vomiting in children with milk protein allergy. By way of a fourth non-limiting example, Lactobacillus rhamnosus SD-GG-BE can shorten the duration of diarrhea and / or reduce the number of daily stools in children with diarrhea. By way of a fifth non-limiting example, Lactobacillus rhamnosus SD-GG-BE can reduce the effects of antibiotic treatment on the intestinal microbiota of children. By way of a sixth non-limiting example, Lactobacillus rhamnosus SD-GG-BE can play a role in preventing or treating infantile colic.

[0090] Embodiments of the present disclosure include synbiotic compositions comprising Lactobacillus salivarius SD-LS1-IT. The bacterial strain may have any one or more of a number of beneficial effects on the health of the host. By way of a first non-limiting example, Lactobacillus salivarius SD-LS1-IT may improve clinical parameters of atopic dermatitis (particularly in children) as measured by SCORAD and Dermatology Life Quality (DLQ) index, reduce microbial translocation and / or immune activation, and / or improve helper T cells (Th) 17 / regulatory T cells (T reg ) and Th1 / Th2 ratio. By a second non-limiting example, Lactobacillus saliva SD-LS1-IT can reduce Staphylococcus counts in host feces. By a third non-limiting example, Lactobacillus saliva SD-LS1-IT can improve clinical parameters of atopic dermatitis as measured by the itch index, which can persist after cessation of synbiotic administration. By a fourth non-limiting example, Lactobacillus saliva SD-LS1-IT can reduce Staphylococcus load and / or reduce the production of Th2 cytokines and maintain stable production of Th1 cytokines. By a fifth non-limiting example, Lactobacillus saliva SD-LS1-IT can reduce pro-inflammatory cytokines, increase anti-inflammatory cytokines, inhibit reactive oxygen species production, restore cell membrane integrity and / or inhibit pathogenic Escherichia coli and Klebsiella pneumoniae.

[0091] Embodiments of the present disclosure include synbiotic compositions comprising Bifidobacterium breve SD-B632-IT. The bacterial strain may have any one or more of a number of beneficial effects on the health of the host. By a first non-limiting example, Bifidobacterium breve SD-B632-IT may improve the composition of the fecal microbiota in children with diarrheal celiac disease, particularly by increasing the Firmicutes / Bacteroidetes ratio and / or increasing the abundance of Actinobacteria. By a second non-limiting example, Bifidobacterium breve SD-B632-IT may regulate serum TNF-α and / or fecal short-chain fatty acids. By 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. By a fourth non-limiting example, Bifidobacterium breve SD-B632-IT may play a role in preventing or treating infantile colic. By a fifth non-limiting example, Bifidobacterium breve SD-B632-IT may help restore microbial balance in children with diarrheal celiac disease on a gluten-free diet. By way of a sixth non-limiting example, Bifidobacterium breve SD-B632-IT can support the growth of healthy intestinal bacteria in children with diarrheal celiac disease. By way of a seventh non-limiting example, Bifidobacterium breve SD-B632-IT can support healthy carbohydrate metabolism and insulin sensitivity in adolescents. By way of an eighth non-limiting example, Bifidobacterium breve SD-B632-IT can improve allergic airway responses in patients with asthma, seasonal allergies, etc., and / or otherwise support healthy airway function, support healthy responses to seasonal allergens, and / or improve rescue of respiratory distress during seasonal allergies.

[0092] Embodiments of the present disclosure include synbiotic compositions comprising Bifidobacterium breve SD-BR03-IT. The bacterial strain may have any one or more of a number of beneficial effects on the health of the host. By way of a first non-limiting example, Bifidobacterium breve SD-BR03-IT may improve the host's bowel movement frequency, stool consistency, and ease of excretion, and may relieve symptoms of intestinal discomfort such as bloating, itching, burning, or pain. By way of a second non-limiting example, Bifidobacterium breve SD-BR03-IT may improve clinical parameters of atopic dermatitis as measured by SCORAD and the Dermatology Life Quality (DLQ) Index, reduce microbial translocation and / or immune activation, and / or improve helper T cells (Th) 17 / regulatory T cells (T reg) and Th1 / Th2 ratio. By a third non-limiting example, Bifidobacterium breve SD-BR03-IT can persist in the host's intestinal microbiota after cessation of synbiotic administration, which can correct the dysbiotic intestinal microbiota of patients with atopic dermatitis. By a fourth non-limiting example, Bifidobacterium breve SD-BR03-IT can inhibit the growth of multiple Escherichia coli biotypes, including pathogenic Escherichia coli O157:H7. By a fifth non-limiting example, Bifidobacterium breve SD-BR03-IT can improve the composition of fecal microbiota in children with diarrheal celiac disease, particularly by increasing the Firmicutes / Bacteroidetes ratio and / or increasing the abundance of Actinomycetes. By a sixth non-limiting example, Bifidobacterium breve SD-BR03-IT can regulate serum TNF-α and / or fecal short-chain fatty acids. By a seventh non-limiting example, Bifidobacterium breve SD-BR03-IT can improve insulin sensitivity and blood glucose levels in children with obesity and insulin resistance. By way of an eighth non-limiting example, Bifidobacterium breve SD-BR03-IT may play a role in preventing or treating infantile colic.

[0093] Embodiments of the present disclosure include synbiotic compositions comprising Bifidobacterium longum SD-CECT7347-SP. The bacterial strain may have any one or more of the many beneficial effects on the health of the host. By a first non-limiting example, Bifidobacterium longum SD-CECT7347-SP may be combined with Lactobacillus casei SD-CECT9104-SP and Bifidobacterium lactis SD-CECT8145-SP to reduce the SCORAD index, and to treat the onset of chronic skin diseases using external steroid therapy. By a second non-limiting example, Bifidobacterium longum SD-CECT7347-SP may reduce the altered expression of cellular proteins involved in cytoskeletal destruction, inflammation, and apoptosis in Caco-2 cells. By a third non-limiting example, Bifidobacterium longum SD-CECT7347-SP may suppress proinflammatory cytokine patterns and increase the production of IL-10 in peripheral blood mononuclear cells (PBMCs) in hosts with diarrheal celiac disease. By way of a fourth non-limiting example, Bifidobacterium longum SD-CECT7347-SP can affect the phenotypic and functional maturation of monocyte-derived dendritic cells. By way of a fifth non-limiting example, Bifidobacterium longum SD-CECT7347-SP can reduce the cellular display of toxic amino acid sequences and reduce the expression of NF-κB, TNF-α and IL-1β.

[0094] Embodiments of the present disclosure include synbiotic compositions comprising Lactobacillus casei SD-CECT9104-SP. The bacterial strain may have any one or more of many beneficial effects on the health of the host. By way of non-limiting example, Lactobacillus casei SD-CECT9104-SP may be combined with Bifidobacterium longum SD-CECT7347-SP and Bifidobacterium lactis SD-CECT8145-SP to reduce the SCORAD index and treat the onset of chronic skin diseases using external steroid therapy.

[0095] Embodiments of the present disclosure include synbiotic compositions comprising Bifidobacterium lactis SD-CECT8145-SP. The bacterial strain may have any one or more of a number of beneficial effects on the health of the host. By way of non-limiting example, Bifidobacterium lactis SD-CECT8145-SP may be combined with Bifidobacterium longum SD-CECT7347-SP and Lactobacillus casei SD-CECT9104-SP to reduce the SCORAD index and treat the onset of chronic skin disease using external steroid therapy.

[0096] Embodiments of the present disclosure include synbiotic compositions comprising Lactobacillus acidophilus SD-NCFM-US. The bacterial strain can have any one or more of a number of beneficial effects on the health of the host. By way of non-limiting example, Lactobacillus acidophilus SD-NCFM-US can reduce the incidence and duration of fever, rhinorrhoea, cough, antibiotic prescriptions, and days missed from school due to illness in children. By way of a second non-limiting example, Lactobacillus acidophilus SD-NCFM-US can reduce stool frequency and / or improve stool consistency in children with diarrhea.

[0097] Embodiments of the present disclosure include synbiotic compositions comprising Bifidobacterium animalis subsp. lactis SD-BI07-US. The bacterial strain may have any one or more of a number of beneficial effects on the health of the host. By way of non-limiting example, Bifidobacterium animalis subsp. lactis SD-BI07-US may reduce the incidence and duration of fever, rhinorrhoea, cough, antibiotic prescriptions, and days missed from school due to illness in children. By way of a second non-limiting example, Bifidobacterium animalis subsp. lactis SD-BI07-US may reduce stool frequency and / or improve stool consistency in children with diarrhea.

[0098] Aspects of the present disclosure allow for rational and systematic screening, and selection of bacterial strains of interest for synbiotic compositions. In particular, as described throughout the present disclosure, bacterial strains of interest can be screened for a variety of functional attributes, and in preferred embodiments are screened, including but not limited to upregulating Nrf2 transcription factors, increasing the production of short-chain fatty acids (SCFAs) in the intestine, and / or improving intestinal epithelial barrier function. As a result of this screening, a single strain or a combination of strains can be included in a synbiotic composition to provide a synergistic effect on host health, each of which has one or more of these desired functional attributes, and shares most or all of the desired attributes. By way of non-limiting example, in some embodiments, upregulation of Nrf2, increase in SCFA production, and improvement in epithelial barrier function can enhance each other in the intestinal environment of the host, and thus can result in an overall host health improvement greater than any one or both of these functional results; therefore, strains can be screened for these attributes, and are reasonably screened to be included in the synbiotic composition of the present disclosure as a result of this screening.

[0099] Prebiotics

[0100] It must be clearly understood that any composition that can be converted into active metabolites by microbial strains present in the healthy human intestinal microbiota can be provided in a synbiotic composition according to the present disclosure. By way of a first non-limiting example, the prebiotic component can include one or more microbially fermentable dietary fibers, i.e., polymeric or oligomeric carbohydrates, such as, for example, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), and inulin. By way of a second non-limiting example, the prebiotic component can include one or more punicalagins, which act as potent antioxidants in the human body and can be metabolized by certain intestinal bacteria into a class of dibenzopyran-6-ones called urolithins, including, but not necessarily limited to, urolithin-A. By way of a third non-limiting example, the prebiotic component can include one or more glucosinolates, which can be metabolized by the intestinal microbiota into isothiocyanates, which in turn have been shown to have anti-cancer properties and other beneficial effects in human health. By way of a fourth non-limiting example, the prebiotic component may include one or more catechins, which may be metabolized by microorganisms in the colon into gamma-valerolactone and hippuric acid, which in turn may be bioconverted in the human liver into metabolites useful for human health. By way of a fifth non-limiting example, the prebiotic component may include one or more polyphenols, many of which are known to be metabolized by the intestinal 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 an otherwise biologically "inert" compound from the perspective of the host (i.e., may be metabolized only by one or more strains in the intestinal microbiota and not by the host), whereas in other embodiments, the compound may be metabolized by both the host and the intestinal microbiota.

[0101] Pharmaceutical preparations

[0102] Pharmaceutical formulations provided by the present disclosure may include a therapeutically effective amount of a synbiotic composition and a suitable amount of one or more pharmaceutically acceptable vehicles so as to provide a formulation for proper administration to a patient. Suitable pharmaceutical vehicles are described in the art.

[0103] In certain embodiments, the synbiotic composition may be incorporated into a pharmaceutical formulation to be orally administered. Oral administration of such a pharmaceutical formulation may result in release and / or uptake of the synbiotic composition throughout the intestinal tract. Such oral formulations may be prepared in a manner known in the pharmaceutical art and include a synbiotic composition and at least one pharmaceutically acceptable vehicle. An oral pharmaceutical formulation may include an effective amount of a synbiotic composition and an appropriate amount of a pharmaceutically acceptable vehicle to provide a suitable form for administration to a patient.

[0104] The synbiotic composition may be incorporated into a pharmaceutical formulation to be administered by any other appropriate route of systemic administration, including intramuscular, intravenous and oral.

[0105] Embodiments of the present disclosure generally include ingestible formulations comprising synbiotic compositions. Embodiments of the present disclosure also include methods of applying such formulations to subjects (preferably humans, and more preferably children) to treat diseases or conditions and / or achieve physiological purposes. In particular, in some embodiments, the synbiotic composition is co-administered or combined with one or more other pharmaceutical preparations to achieve an auxiliary or synergistic effect, that is, an improved effect relative to the independent administration of the synbiotic composition and one or more other pharmaceutical preparations. By way of a first non-limiting example, the synbiotic composition according to the present disclosure can be co-administered with one or more pharmaceutical preparations that are effective in treating inflammatory bowel disease to improve the efficacy response rate of the pharmaceutical preparation in treating inflammatory bowel disease. By way of a second non-limiting example, the synbiotic composition according to the present disclosure can be co-administered with one or more cancer immunotherapy drugs (e.g., CTLA-4 antagonists and / or PD-1 antagonists) to improve the efficacy response rate of cancer immunotherapy drugs.

[0106] The ingestible preparation of the present invention can be provided in any suitable form and physical manifestation. By non-limiting example, the ingestible preparation can be applied to a subject as a dietary supplement, a medicinal feed, a nutritional composition and a pharmaceutical composition. By another non-limiting example, the ingestible composition can be provided in any suitable physical form for oral administration, for example, an ampoule, an aqueous solution or a suspension (for example, an infused beverage, for example, an energy drink or an energy "short drink"), a capsule (which may or may not be chewable), drops, granules, liquids, mists, powders, pouches, syrups, tablets (for example, chewable, saliva soluble and / or swallowable tablets), functionalized foods (for example, energy or nutritional bars, biscuits, chewing gum, candy, etc.), toothpaste, sublingual products, etc. In some embodiments, the composition can be provided in the form of, for example, a powder, which can be applied to food (similar to a condiment or seasoning, etc.) or mixed with a beverage. Therefore, the ingestible composition of the present invention can include any suitable pharmaceutically acceptable additives, adhesives and / or fillers, and can also include active drugs or therapeutic agents other than the synbiotic composition disclosed herein.

[0107] In embodiments, the ingestible preparation of the present invention can be provided in the form of a free-flowing powder. The powder can be suitable for mixing with a beverage (e.g., water, milk, etc.) or other liquids, or dissolved or suspended therein, and then drunk by the subject or orally in other ways. In certain embodiments, the ingestible preparation in powder form can be packaged in a pouch, for example, a disposable pouch containing a single daily dose, which can be suitable for immersion in a liquid (similar to a tea bag) to allow the ingestible preparation to diffuse into the liquid, tear open so that the contents are poured into the liquid, etc. In some embodiments, the packaging material for preparing the pouch preferably has a relatively low water vapor transmission rate (e.g., at 23 ° C and 50% relative humidity, the water vapor transmission rate is less than about 0.01 g / m2 / day) to extend the shelf life of the ingestible preparation.

[0108] Pharmaceutical preparations comprising synbiotic compositions can be prepared by conventional mixing, dissolving, granulating, coating, polishing, emulsifying, encapsulating, embedding or lyophilizing processes. Pharmaceutical preparations can be prepared 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 pharmaceutically acceptable vehicles into a pharmaceutically acceptable preparation. The appropriate formulation depends on the selected route of administration. The pharmaceutical preparations provided in the present disclosure can take the form of a sustained release preparation suitable for administration to a subject.

[0109] The pharmaceutical preparations provided by the present disclosure can be formulated in unit dosage forms. Unit dosage forms refer to physically discrete units suitable for use as single doses in patients undergoing treatment, wherein each unit contains a predetermined amount of synbiotic composition calculated to produce a desired therapeutic effect. The unit dosage form can be for a single daily dose, for administration twice a day or one of multiple daily doses, for example, 3 times a day or more. When multiple daily doses are used, the unit dosage form of each dose can be the same or different. One or more dosage forms can include a dose, which can be administered to a patient at a single time point or during a time interval.

[0110] In certain embodiments, the oral dosage form provided by the present disclosure can be a controlled release dosage form. Controlled delivery technology can improve the absorption of active ingredients in a specific area or multiple areas of the gastrointestinal tract. Controlled active ingredient delivery systems can be designed to deliver active ingredients in such a way that the level of active ingredients remains within the treatment effective window, and as long as the system continues to deliver active ingredients with a specific release spectrum in the gastrointestinal tract, effective and safe blood levels can be maintained for a period of time. Compared with the fluctuations observed with immediate release dosage forms, controlled active ingredient delivery can produce substantially constant blood levels of active ingredients over a period of time. For some applications, maintaining constant blood and tissue concentrations throughout the treatment process is the most ideal treatment mode. The immediate release of active ingredients can cause blood levels to reach a peak value higher than the level required to induce the desired reaction, which may waste active ingredients and may cause or aggravate toxic side effects. Controlled active ingredient delivery can lead to optimal treatment, and not only can reduce the frequency of administration, but also can reduce the severity of side effects. Examples of controlled release dosage forms include: dissolution control systems, diffusion control systems, ion exchange resins, permeation control systems, erodible matrix systems, pH-independent preparations, gastric retention systems, etc.

[0111] The suitable oral dosage form of the specific drug formulation provided by the present disclosure may depend at least in part on: the gastrointestinal absorption characteristics of the active ingredient and / or the stability of the active ingredient in the gastrointestinal tract, the pharmacokinetics of the active ingredient and the expected therapeutic spectrum. Suitable controlled release oral dosage forms may be selected for specific ingredients or ingredient combinations. For example, gastric retention oral dosage forms may be applicable to active ingredients mainly absorbed from the upper gastrointestinal tract, and sustained release oral dosage forms may be applicable to active ingredients mainly absorbed from the lower gastrointestinal tract. Some active ingredients are mainly absorbed from the small intestine. Typically, the active ingredient passes through the length of the small intestine in about 3 to 5 hours. For active ingredients that are not easily absorbed by the small intestine or are not easily soluble, the window of active agent absorption in the small intestine may be too short to provide the desired therapeutic effect.

[0112] In certain embodiments, the pharmaceutical preparations provided by the present disclosure can be practiced with a dosage form suitable for providing a sustained release of a synbiotic composition when orally administered. Sustained release oral dosage forms can be used to release active ingredients over an extended period of time, and when it is desired that the active ingredient be delivered to the lower gastrointestinal tract (including the colon), sustained release oral dosage forms are useful. Sustained release oral dosage forms are included in any oral dosage form for a time period in which the therapeutic concentration of the active ingredient can be maintained in a biological fluid (e.g., plasma, blood, cerebrospinal fluid) or in a tissue or organ for an extended period of time. Sustained release oral dosage forms include diffusion control systems (e.g., reservoir devices and matrix devices), dissolution control systems, permeation systems, and erosion control systems. Sustained release oral dosage forms and methods for preparing them are well known in the art.

[0113] In certain embodiments, the pharmaceutical compositions 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 dosing frequency of three times per day. In another embodiment, the enteric coated oral dosage form is administered to the patient at a dosing frequency of twice per day. In another embodiment, the enteric coated oral dosage form is administered to the patient at a dosing frequency of once per day.

[0114] In certain embodiments, the pharmaceutical formulations provided by the present disclosure may include any non-enteric coated sustained release oral dosage form for administering the synbiotic composition. In one embodiment, the non-enteric coated oral dosage form is administered to the patient at a dosing frequency of three times a day. In another embodiment, the non-enteric coated oral dosage form is administered to the patient at a dosing frequency of twice a day. In another embodiment, the non-enteric coated oral dosage form is administered to the patient at a dosing frequency of once a day.

[0115] In certain embodiments, the pharmaceutical formulations 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 frequency of three times per day. In another embodiment, the oral dosage form is administered to the patient at a frequency of twice per day. In another embodiment, the oral dosage form is administered to the patient at a frequency of once per day.

[0116] In certain embodiments, the pharmaceutical preparation provided by the present disclosure may include any suitable dosage form that realizes the above-mentioned in vitro release profile. Such dosage form may 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. Given the dosage form described in the example as a starting point, the technical staff of the formulation field can develop any number of acceptable dosage forms.

[0117] The appropriate dosage of the synbiotic composition can be determined according to any of several well-established protocols. For example, animal studies, e.g., studies using mice, rats, dogs, and / or monkeys, can be used to determine the appropriate dosage of the pharmaceutical compound. Results from animal studies can be extrapolated to determine dosages for other species (e.g., such as humans).

[0118] use

[0119] The methods and formulations disclosed herein can be used to treat patients with diseases, disorders, conditions and symptoms for which the synbiotic composition is known to provide a therapeutic benefit or is later found to provide a therapeutic benefit. The formulations disclosed herein can be used to treat a disease selected from the group consisting of adrenoleukodystrophy, age-induced genomic damage, Alexander disease, alopecia areata, Alpers disease, Alzheimer's disease, amyotrophic lateral sclerosis, angina pectoris, arthritis, asthma, Balo concentric sclerosis, Behcet's disease, blistering pemphigus, Canavan disease, cardiac insufficiency (including left ventricular insufficiency), central nervous system vasculitis, Charcot-Marie-Tooth disease, childhood ataxia with decreased demyelination of the central nervous system, 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 immunodeficiency virus infection, Huntington's disease, irritable bowel disease, ischemia, Krabbe disease, lichen planus, macular degeneration, mitochondrial encephalomyopathy, monomelic amyotrophy, multiple sclerosis, myocardial infarction, neurodegeneration with brain iron accumulation, neuromyelitis, neurosarcoidosis, NF-κB-mediated diseases, optic neuritis, carcinoid syndrome, Parkinson's disease, Pelizaeus-Merzbacher disease, pemphigus, primary lateral sclerosis, progressive supranuclear palsy, psoriasis, pustular gangrene, reperfusion injury, retinitis pigmentosa, sarcoidosis, Sheldon's disease, subacute necrotizing myelopathy, Susac syndrome, transplant rejection, transverse myelitis, tumors, ulcerative colitis, or Zellweger's syndrome.

[0120] The present disclosure provides methods for treating a patient's disease, comprising 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 a patient. The synbiotic composition can be administered at an appropriate dose and using an appropriate dosing regimen for the treatment of a particular disease.

[0121] The daily dose range of the compound of the prebiotic component of the synbiotic composition can be 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, the daily dose is about 100 mg / kg. In certain embodiments, the compound of the prebiotic component can 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.

[0122] In certain embodiments, the microbial strains of the probiotic component may be administered over time at a dosage 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.

[0123] In some embodiments, the probiotic component may include at least about 40 million AFU of Lactobacillus rhamnosus SD-GG-BE per dose, and in specific embodiments, about 7.5 billion AFU of Lactobacillus rhamnosus SD-GG-BE per dose. In some embodiments, the probiotic component may include at least about 2 billion AFU of Lactobacillus salivarius SD-LS1-IT per dose, and in specific embodiments, about 7.5 billion AFU of Lactobacillus salivarius SD-LS1-IT per dose. In some embodiments, the probiotic component may include at least about 7.5 billion AFU of Bifidobacterium breve SD-B632-IT per dose, and in specific embodiments, about 7.5 billion AFU of Bifidobacterium breve SD-B632-IT per dose. In some embodiments, the probiotic component may include at least about 5 billion AFU of Bifidobacterium breve SD-B632-IT per dose, and in specific embodiments, about 7.5 billion AFU of Bifidobacterium breve SD-B632-IT per dose. In some embodiments, the probiotic component may include at least about 420 million AFU Bifidobacterium longum SD-CECT7347-SP per dose, and in specific embodiments, about 7.5 billion AFU Bifidobacterium longum SD-CECT7347-SP per dose. In some embodiments, the probiotic component may include at least about 360 million AFU Lactobacillus casei SD-CECT9104-SP per dose, and in specific embodiments, about 7.5 billion AFU Lactobacillus casei SD-CECT9104-SP per dose. In some embodiments, the probiotic component may include at least about 420 million AFU Bifidobacterium lactis SD-CECT8145-SP per dose, and in specific embodiments, about 7.5 billion AFU Bifidobacterium lactis SD-CECT8145-SP per dose. In some embodiments, the probiotic component may include at least about 7.5 billion AFU Lactobacillus acidophilus SD-NCFM-US per dose, and in specific embodiments, about 7.5 billion AFU Lactobacillus acidophilus SD-NCFM-US per dose. In some embodiments, the probiotic component may include at least about 7.5 billion AFU of Bifidobacterium animalis lactis subsp. SD-BI07-US per dose, and in particular embodiments, about 7.5 billion AFU of Bifidobacterium animalis lactis subsp. SD-BI07-US 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, as long as the AFU ratio between any two or more selected microbial strains remains approximately equal to a predetermined ratio, e.g., a ratio as described above (e.g., an AFU ratio of about 40 million:2 billion between Lactobacillus rhamnosus SD-GG-BE and Lactobacillus salivarius SD-LS1-IT, or an AFU ratio of about 7.5 billion:7.5 billion between Lactobacillus acidophilus SD-NCFM-US and Bifidobacterium lactis SD-BI07-US).

[0124] The appropriate dosage of the synbiotic composition can be determined based on many 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 mode of administration, and the judgment of the prescribing physician. Suitable dosage ranges can be determined by methods known to those skilled in the art.

[0125] Synbiotic compositions can 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 can also be used to determine whether administration of a synbiotic composition is therapeutically effective.

[0126] In certain embodiments, a therapeutically effective dose of a synbiotic composition can provide therapeutic benefit without causing substantial toxicity (including adverse side effects). The toxicity of a synbiotic composition and / or its metabolites can be determined using standard pharmaceutical procedures and can be determined by one skilled in the art. The dose ratio between toxicity and therapeutic effect is the therapeutic index. The dose of a synbiotic composition can be within a range capable of establishing and maintaining, for example, a therapeutically effective circulating plasma and / or blood concentration of a prebiotic component that exhibits little or no toxicity.

[0127] Administration of the synbiotic composition can be used to treat a disease selected from the group consisting of adrenoleukodystrophy, age-induced genomic damage, Alexander disease, alopecia areata, Alpers disease, Alzheimer's disease, amyotrophic lateral sclerosis, angina pectoris, arthritis, asthma, Balo concentric sclerosis, Behcet's disease, bullous pemphigus, Canavan disease, cardiac insufficiency (including left ventricular dysfunction), central nervous system vasculitis, Charcot-Marie-Tooth disease, childhood ataxia with central nervous system demyelination, 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 immunodeficiency virus infection, Huntington's disease, irritable bowel disease, ischemia, Krabbe disease, lichen planus, macular degeneration, mitochondrial encephalomyopathy, monomelic amyotrophy, multiple sclerosis, myocardial infarction, neurodegeneration with brain iron accumulation, neuromyelitis, neurosarcoidosis, NF-κB-mediated diseases, optic neuritis, carcinoid syndrome, Parkinson's disease, Pelizaeus-Merzbacher disease, pemphigus, primary lateral sclerosis, progressive supranuclear palsy, psoriasis, pustular gangrene, reperfusion injury, retinitis pigmentosa, sarcoidosis, Sheldon's disease, subacute necrotizing myelopathy, Susac syndrome, transplant rejection, transverse myelitis, tumors, ulcerative colitis, or Zellweger's syndrome. The underlying cause of any of the aforementioned diseases being treated may be of various origins. In addition, in certain embodiments, a therapeutically effective amount of a synbiotic composition may be administered to a patient, e.g., a human, as a preventative measure against the aforementioned diseases and conditions.Thus, a therapeutically effective amount of the synbiotic composition can be administered as a preventive measure to a patient who has a predisposition to and / or a history of the following diseases: adrenoleukodystrophy, age-induced genomic damage, Alexander disease, alopecia areata, Alpers disease, Alzheimer's disease, amyotrophic lateral sclerosis, angina pectoris, arthritis, asthma, Balo concentric sclerosis, Behcet's disease, bullous pemphigus, Canavan disease, cardiac insufficiency (including left ventricular dysfunction), central nervous system vasculitis, Charcot-Marie-Tooth disease, childhood ataxia with central nervous system demyelination, chronic idiopathic peripheral neuropathy, chronic obstructive pulmonary disease, diseases, Crohn's disease, lupus cutanea, 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 immunodeficiency virus infection, Huntington's disease, irritable bowel disease, ischemia, Krabbe disease, lichen planus, macular degeneration, mitochondrial encephalomyopathy, monomelic amyotrophy, multiple sclerosis, myocardial infarction, neurodegeneration with brain iron accumulation, neuromyelitis, neurosarcoidosis, NF-κB-mediated diseases, optic neuritis, carcinoid syndrome, Parkinson's disease, Pelizaeus-Merzbacher disease disease, pemphigus, primary lateral sclerosis, progressive supranuclear palsy, psoriasis, pustular gangrene, reperfusion injury, retinitis pigmentosa, sarcoidosis, Sheldon's disease, subacute necrotizing myelopathy, Susac syndrome, transplant rejection, transverse myelitis, tumors, ulcerative colitis, or Zellweger's syndrome.

[0128] In some embodiments, the methods and compositions of the present disclosure may be effective in improving the metagenomic stability and metabolic output of the gut microbiota in a subject suffering from irritable bowel syndrome.

[0129] In some embodiments, the methods and compositions of the present disclosure may be effective in improving, remediating, or restoring the function of the intestinal and airway microbiomes in subjects with mild to moderate COVID-19 symptoms.

[0130] In some embodiments, the methods and compositions of the present disclosure can effectively maintain or improve the intestinal barrier integrity and / or intestinal microbiota composition of subjects with irritable bowel syndrome (IBS). In particular, the methods and compositions of the present disclosure can be effectively used for any one or more of the following: (1) maintaining or increasing the diversity of the gastrointestinal microbiota of subjects with constipation-type IBS (IBS-C), (2) increasing the representation of at least one of the following: Bifidobacterium longum, Prevotella intermedia, Bacteroides ulcerans, Akkermansia muciniphila, Alternaria finschaeger, and Faecalibacterium prausnitzii, and / or reducing the representation of Eubacterium muciniphila in the gastrointestinal microbiota of patients with mixed / alternating stool pattern IBS (IBS-M), (3) improving any one or more individual IBS symptoms selected from the group consisting of abdominal pain, bloating, dyspnea, and stool consistency, (4) increasing the frequency of complete spontaneous bowel movements (CSBM) in subjects with 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 (eg, rifaximin).

[0131] In some embodiments, the methods and compositions of the present disclosure may be effective in improving, preventing, or treating constipation, supporting intestinal barrier integrity following acute alcohol exposure, and / or supporting vaginal health.

[0132] The present disclosure is further described by reference to the following non-limiting examples. The examples illustrate various aspects of the present disclosure. It will be apparent to those skilled in the art that many modifications of materials and methods may be practiced without departing from the scope of the present disclosure.

[0133] Example 1

[0134] Prebiotic Utilization

[0135] Individual strains of nine probiotic 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 stable conditions. The nine bacterial strains were: Lactobacillus rhamnosus SD-GG-BE, Lactobacillus salivarius SD-LS1-IT, Bifidobacterium breve SD-B632-IT, Bifidobacterium breve SD-BR03-IT, Bifidobacterium longum SD-CECT7347-SP, Lactobacillus casei SD-CECT9104-SP, Bifidobacterium lactis SD-CECT8145-SP, Lactobacillus acidophilus SD-NCFM-US, and Bifidobacterium animalis subsp. SD-BI07-US. (Hereinafter, these nine strains may be collectively referred to as "PDS-08 strains", and the composition containing these nine strains may be referred to as "PDS-08 composition" or simply "PDS-08". The list of the first seven strains (i.e., the 9 strains except Lactobacillus acidophilus SD-NCFM-US and Bifidobacterium animalis subspecies SD-BI07-US) may be collectively referred to as "MXP271 strains", and the composition containing these seven strains may be referred to as "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 overnight at 37°C under stable conditions to produce experimental cultures. The obtained stationary phase culture was subcultured (1:100) into a 96-well plate containing a medium simulating the colon environment, which contained 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 0.15% (w / v) peptone water, 5 g / L tryptone, 0.4 g / L bile salts, 4.5 g / L yeast extract, and 0.005 g / L FeSO4. 4 7H 2 O, 4.5g / LNaCl, 4.5g / L KCl, 0.5g / L KH 2 PO 4 、1.25g / LMgSO 4 7H 2 O, 0.15 g / L CaCl 2 6H 2 O, 1.5 g / L NaHCO 3 , 0.8 g / L L-cysteine, 0.05 g / L heme and 1 mL / L Tween 80. Some samples were spiked with 1 wt% of three commercially available prebiotic compositions ( Synergy1 (about 50wt% inulin, 50% oligofructose), (maltodextrin) and waxy maize (a complex starch mixture with high amylopectin content), whereas the other samples were not spiked as controls. The plates were incubated for 24 hours, during which time the optical density at 600 nm (OD 600 ). Three to nine biological replicates and three technical replicates were performed.

[0136] Figure 1A shows the growth of Lactobacillus casei SD-CECT9104-SP, Figure 1B shows the growth of Lactobacillus rhamnosus SD-GG-BE, Figure 2 C shows the growth of Bifidobacterium breve SD-BR03-IT, Figure 1D shows the growth of Bifidobacterium lactis SD-CECT8145-SP, Figure 1E shows the growth of Lactobacillus salivarius SD-LS1-IT, Figure 1F shows the growth of Bifidobacterium longum SD-CECT7347-SP, Figure 1G shows the growth of Lactobacillus acidophilus SD-NCFM-US, Figure 1H shows the growth of Bifidobacterium animalis subsp. SD-Bi-07-US, and Fig. 1I The growth of Bifidobacterium breve SD-B632-IT is shown. OD 600 Comparison of the changes revealed that The prebiotic composition significantly enhanced the growth of Lactobacillus casei SD-CECT9104-SP ( Figure 1A ), Bifidobacterium breve SD-BR03-IT( Figure 1C ), saliva combined with Lactobacillus SD-LS1-IT ( Figure 1E )、Bifidobacterium longum SD-CECT7347-SP( Figure 1F ) and Bifidobacterium breve SD-B632-IT( Fig. 1I ) growth. For the other four tested strains, it was observed that relative to the control group, There was an increase in the growth trend in the presence of the composition, although this trend was not statistically significant. Composition ratio and waxy corn compositions stimulated the growth of all strains better (except Lactobacillus casei SD-CECT9104-SP); in fact, especially in Bifidobacterium animalis subsp. lactis SD-Bi-07-US ( Figure 1H ), and waxy corn compositions appeared to inhibit growth. (Comparison of OD values ​​for each substrate tested was performed using one-way ANOVA (parametric data) or Kruskal-Wallis (nonparametric data) test with Dunnett's multiple comparisons. 600 Changes to determine significance ( FIG. 1A to FIG. 1I ); if not significant, the result is FIG. 1A to FIG. 1I If the significance reaches P < 0.05, it is marked as “ns”; if the significance reaches P < 0.05, it is marked as “*”; if the significance reaches P < 0.01, it is marked as “**”; if the significance reaches P < 0.001, it is marked as “***”; if the significance reaches P < 0.0001, it is marked as “****”). In summary, these data show that The prebiotic composition significantly enhanced the growth of at least five PDS-08 strains, while the growth of the other four strains was not negatively affected. This provides a basis for the use of inulin / oligofructose prebiotic compositions (e.g., Synergy1) provides the basic principles.

[0137] Example 2

[0138] Microbial activity in synbiotic products

[0139] A synbiotic composition was formulated containing 6.3 grams of Synergy1 (prebiotic component) and 5.75·10 per dose 10 The synbiotic composition was formulated as a free-flowing powder in which the only ingredients were the prebiotic and probiotic components. After the synbiotic composition was formulated, a sample of the probiotic component was obtained and quantitatively analyzed by flow cytometry analysis using fluorescence activated cell sorting (FACS) for total bacteria, live bacteria, and dead bacteria. Specifically, 0.01 mM SYTO TM 10-fold serial dilutions of the samples were double-stained with 24 green fluorescent nucleic acid stain and 3 μM propidium iodide for 15 minutes at 37°C in the dark under anaerobic conditions. The samples were then analyzed on a BD FACSVerse machine using a high flow rate setting, and bacteria were separated from medium debris and signal noise by applying a threshold level of 200 on the SYTO channel. Flow cytometry data were analyzed using FlowJo version 10.5.2, and total bacterial counts are reported in logarithmic units.

[0140] Reference now Figure 2, FACS analysis revealed that 79.0% of the cells in the formulated synbiotic composition belonged to the active fraction, whereas 15.8% appeared to be dead, and the remaining minor fraction (about 5.2%) was identified as alive but unculturable. This high survival rate justifies the classification of the PDS-08 microbial component of the composition as a true probiotic, and therefore the composition as a whole can conceptually be classified as a true synbiotic.

[0141] Example 3

[0142] Survival of probiotic strains along the upper digestive tract

[0143] Using the Human Intestinal Microbial Ecosystem (SHIME) 400 simulator, synbiotic compositions can be tested to confirm the survival of the microbial consortium of the probiotic components, such as Figure 3 As shown. In particular, SHIME 400 includes: gastric blood vessel 410, small intestinal blood vessel 420, ascending colon blood vessel 430, transverse colon blood vessel 440 and descending colon blood vessel 450. Gastric blood vessel 410 receives and mixes test composition 401 and gastric acid 402 via pump and nitrogen supply. Output from gastric blood vessel 410 is pumped to small intestinal blood vessel 420, where it mixes with pancreatic juice 411 received via pump. Output from small intestinal blood vessel 420 is pumped to ascending colon blood vessel 430, which is pH controlled to simulate human ascending colon. Output from ascending colon blood vessel 430 is pumped to transverse colon blood vessel 440, which is pH controlled to simulate human transverse colon. Output from transverse colon blood vessel 440 is pumped to descending colon blood vessel 450, which is pH controlled to simulate human descending colon. Output from descending colon blood vessel 450 is finally pumped to overflow tank 451. Thus, SHIME 400 reproduces physiological and biological conditions representative of the human gastrointestinal tract (e.g., food intake, motility, digestive enzymes, pancreatic and bile acids, residence time, etc.). (Further details about the SHIME model can be found in K. Molly et al., "Development of a 5-step multi-chamber reactor as asimulation of the human intestinal microbial ecosystem," 39 Applied Microbiology and Biotechnology 254 (May 1993), the entire contents of which are incorporated herein by reference.)

[0144] Reference now Figure 4, the modified SHIME system 500 is used to study the survival of probiotic strains in the upper gastrointestinal tract. The modified SHIME system 500 contains a single reactor 505 and an oscillator 560, and these components are used to represent the physiological conditions of the stomach and small intestine over time within the single reactor 505. To simulate fed or fasted conditions, a specific gastric suspension containing a test composition 501 is added to the reactor 505, and the reactor 505 is agitated using an oscillator 560 to simulate gastric conditions 510. Subsequently, standardized enzymes and bile liquids are added to the reactor 505, and the reactor 505 is agitated using an oscillator 560 to simulate small intestinal conditions 520. The incubation conditions are optimized to resemble the in vivo conditions of different regions of the gastrointestinal tract in the fasted or fed state.

[0145] To simulate gastric conditions 510 in a fed state, in addition to the test composition 501, the gastric suspension also contains: pepsin (provided as standardized activity by measuring the increase in absorbance of trichloroacetic acid soluble products at 280 nm when digesting hemoglobin reference protein), phosphatidylcholine, arabinogalactan, pectin, xylan, starch, glucose, yeast extract, peptone, mucin and L-cysteine ​​hydrochloride and more levels of salt (sodium chloride and potassium chloride), as recommended in Alan Mackie and Neil Rigby, "InfoGest Consensus Method," The Impact of Food Bioactives on Health 13 (2015) (hereinafter referred to as "Mackie," the entire contents of which are incorporated herein by reference). The gastric suspension was incubated in the reactor 505 at 37° C. for 2 hours while mixing by stirring using an oscillator 560, and the pH profile decreased in an S-shape from 4.6 to 3.0, as shown in FIG. Figure 5A As shown. At the beginning of the incubation period (t = 0, Figure 5A "ST0 / product") and end (t = 120 minutes, Figure 5A The gastric suspension was sampled at "ST end" ("end of ST" in the figure).

[0146] To simulate fasting gastric conditions 510, the gastric suspension contained, in addition to the test composition 501, pepsin and phosphatidylcholine in an amount equal to one-fourth of that used in the fed state suspension, plus mucin and salts for the fed state suspension. The gastric suspension was incubated in the reactor 505 at 37°C for 45 minutes while mixing by stirring using an oscillator 560, with a constant pH of 2.0, as Figure 5B As shown. At the beginning of the incubation period (t = 0, Figure 5B "ST0 / product") and end (t = 45 minutes, Figure 5B The gastric suspension was sampled at "ST end" ("end of ST" in the figure).

[0147] To simulate the small intestinal conditions 520 under the fed state, raw animal pancreatic extract (pancreatin) containing enzymes as described by Mackie and 10 mM ox bile extract (which is closer to human bile than pig bile in terms of taurocholate and glycocholate content) were added to the reactor 505. The pH increased rapidly to 6.5 and remained constant at 6.5 for 27 minutes ("duodenal phase"), then gradually increased to 7.5 over 63 minutes ("jejunal phase"), and finally remained constant at 7.5 for 90 minutes ("ileal phase"). Throughout the small intestinal conditions 520, the temperature was maintained at 37°C, and the suspension was mixed by stirring using an oscillator 560. Samples were collected at the end of each of the duodenal phase, jejunal phase, and ileal phase (i.e., at Figure 5A , they are t=147 min / “DUO end”, t=210 min / “JEJ end” and t=300 min / “ILE end”).

[0148] To simulate the small intestinal conditions 520 in the fasting state, pancreatic enzymes were added in an amount equal to one-fifth of the amount used in the fed state, and bile salts (i.e., 3.33 mM ox bile extract) were added in an amount equal to one-third of the amount used in the fed state. The pH increased rapidly to 6.5 and remained constant at 6.5 for 27 minutes ("duodenal phase"), then gradually increased to 7.5 over 63 minutes ("jejunal phase"), and finally remained constant at 7.5 for 90 minutes ("ileal phase"). Throughout the small intestinal conditions 520, the temperature was maintained at 37°C, and the suspension was mixed by stirring using an oscillator 560. Samples were collected at the end of each of the duodenal phase, jejunal phase, and ileal phase (i.e., at Figure 5B , they are t=72 minutes / "DUO end", t=135 minutes / "JEJ end" and t=225 minutes / "ILE end").

[0149] From each sample, the number of viable microbial cells was determined by flow cytometry analysis using fluorescence activated cell sorting (FACS). Specifically, a 10-fold dilution series of each sample was prepared in anaerobic phosphate buffered saline (PBS). TM 24 stain and propidium iodide stain dilution to assess the live population in each sample. Samples were analyzed on a BD Accuri C6 Plus flow cytometer using a high flow rate. Bacterial cells were separated from culture 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 were reported as the mean logarithm of each reactor count ± 1 standard deviation.

[0150] For each of the fed and fasted states, two different synbiotic compositions were tested in triplicate as test composition 501 (hereinafter referred to as the "naked strain" composition and the "microencapsulated strain" composition); a single "blank" control was also tested. On a per reactor basis, the naked strain composition consisted of 3.0·10 10 AFU non-microencapsulated MXP271 bacterial strain, 5.0·10 9 AFU non-microencapsulated Bifidobacterium lactis subsp. SD-BI07-US, 3.0·10 10 AFU non-microencapsulated Lactobacillus acidophilus SD-NCFM-US and 6.025g Synergy1 prebiotic composition. On a per reactor basis, the microencapsulated strain composition consisted of 3.0·10 10 AFU MXP271 bacterial strain, 5.0·10 9 AFU non-microencapsulated Bifidobacterium lactis subsp. SD-BI07-US, 3.0·10 10 AFU non-microencapsulated Lactobacillus acidophilus SD-NCFM-US and 6.025g Synergy1 prebiotic composition.

[0151] Reference now Fig. 6A and 6B , it was observed that the viability of strains in the naked strain composition was negatively affected by gastric and small intestinal conditions in both the fasted and fed states. However, the reduction in viability was more pronounced in the fasted state; without wishing to be bound by any particular theory, the inventors hypothesize that this difference is caused by differences in the pH profile (i.e., gastric conditions of lower pH in the fasted state than in the fed state). The reduction in viability persisted during the duodenal phase, although this reduction was not statistically significant in the fasted state. The bile salts added at the beginning of the duodenal phase may be highly toxic to bacterial cells, especially at relatively low pH. No further reduction in viability was observed as the pH increased during the jejunal and ileal phases, and in fact, the opposite was observed during the ileal phase in both the fasted and fed states (i.e., an increase in cell viability), although this increase was only statistically significant in the fed state. By the end of the ileal phase, conditions in the fasted state resulted in a 99.85% reduction in viable population density (0.15% survival), from 10 at the beginning of gastric incubation. 10.91 =8.15·10 10 AFU / reactor to the end of the ileal phase 10 7.97 =1.20·10 8 AFU / reactor, however, the fed condition resulted in an 80.67% decrease in viable population density (19.33% survival rate) from 10 10.91=8.15·10 10 AFU / reactor to the end of the ileal phase 10 10.19 =1.57·10 10 AFU / reactor. (In Fig. 6A and 6B Statistically significant decreases in viability from one stage to another are marked with an asterisk. Statistical significance was determined by Student's t-test with a 95% confidence interval.

[0152] Reference now Fig. 7A and 7B , again observing that the viability of the strains in the microencapsulated strain composition was negatively affected by gastric and small intestinal conditions in the fasted state. On the other hand, in the fed state, viability was essentially unaffected; without being bound by any particular theory, the inventors again attribute this difference between the fasted and fed states to differences in the pH profile during gastric incubation. During the duodenal phase, both the fasted and fed states resulted in a significant decrease in viable population density; without wishing to be bound by any particular theory, the inventors again attribute this to the toxicity or other negative effects of the bile salts added at the beginning of the duodenal phase. However, in the fasted state, the microencapsulated strain preparation ( Fig. 7A ) was not as effective as the naked strain preparation ( Fig. 6A ) was evident; without wishing to be bound by any particular theory, the inventors hypothesize that this difference is the result of a more delayed 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 phase. After the duodenal phase, no further reduction in the viable population was observed, and, for the naked strain composition, an increase in viability was even observed during the ileal phase in the fed state. By the end of the ileal phase, conditions in the fasted state resulted in a 99.12% reduction in viable population density (0.88% survival), from 10% at the start of the gastric incubation. 10.87 =7.61·10 10 AFU / reactor to the end of the ileal phase 10 8.80 =6.70·10 8 AFU / reactor, however, the fed condition resulted in a 64.27% decrease in viable population density (35.73% survival rate) from 10 10.87 =7.61·10 10 AFU / reactor to the end of the ileal phase 10 10.43 =2.72·10 10 AFU / reactor. (In Fig. 7A and 7B Statistically significant decreases in viability from one stage to another are marked with an asterisk. Statistical significance was determined by Student's t-test with a 95% confidence interval.

[0153] It can be concluded that administration of microencapsulated strain compositions may generally be superior to administration of naked strain compositions in terms of probiotic organism survival. Without wishing to be bound by any particular theory, the inventors hypothesize that this may indicate that microencapsulation provides a protective effect to the PDS-08 probiotic strain during passage through the stomach and small intestine. Furthermore, administration of the synbiotic composition under fed conditions resulted in a higher survival rate relative to administration of the synbiotic composition under fasting conditions, suggesting that administration in the fed state (e.g., when the subject has recently eaten) may be a recommended method for delivering the maximum number of live bacterial cells to the probiotic active sites (i.e., the distal ileum and colon).

[0154] Example 4

[0155] Short-term colonic batch incubation

[0156] After the experiment of Example 3, the predigested upper GIT suspension subjected to treatment in the adjusted SHIME system 500 was used for short-term single-stage colon incubation. In these experiments, under conditions characterized by the proximal large intestine, a representative dose of the predigested upper GIT suspension was combined with a representative bacterial inoculum. This bacterial inoculum can 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 frozen fecal sample. In this embodiment, two types of colon incubations were performed: non-sterile incubations, in which the bacterial inoculum was derived from a fresh fecal sample of a 6-10 year old child and added directly to the colon reactor, and sterile incubations, in which the bacterial inoculum was first filter-sterilized to remove the bacterial background. In sterile incubations, the use of the filter-sterilized inoculum allows reconstruction of the metabolic background encountered by the probiotic strains in the colon.

[0157] At the beginning of the short-term colon incubation, the predigested upper GIT suspension (obtained as the final product of the experiment in Example 3) and fresh or filter-sterilized bacterial inoculum were added to SHIME nutrient medium containing the basal nutrients present in the colon (9.27 g / L K 2 HPO 4 , 29.06g / LKH 2 PO 4 、3.57g / L NaHCO 3 , 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). Synergy1 prebiotics were added to each colon reactor at a concentration of 4.3 g / L, and in each case a volume of predigestion suspension was chosen to achieve a total prebiotic concentration of no more than 5 g / L in each reactor (the maximum concentration that could be tested in short-term colon incubation experiments; higher concentrations resulted in over-acidification). Incubation was carried out for 48 hours at a temperature of 37°C with shaking (90 rpm) under anaerobic conditions. Table 1 below lists the details of each of the six different experimental setups, each performed in triplicate (18 replicates in total). Samples were collected for analysis after 0, 1, 24 and 48 hours of incubation.

[0158] exist Figures 8A to 14B The statistics reported are the average of three replicates for each experimental setup. The changes in pH ( Fig. 8A , 8B ), lactic acid production ( Fig.9A , 9B ) and total SCFA production ( Fig. 10A , 10B ). For both sterile and non-sterile incubations, the significant differences between the fed and fasted test products relative to each other are indicated by different lowercase letters (e.g., "a" vs. "b"). For non-sterile incubations only, the significant differences between the microencapsulated strain test products (whether under fed or fasted conditions) and the corresponding blank / control are indicated with asterisks. All tests for statistical significance were performed by Student's t-test with a 95% confidence interval.

[0159] Table 1

[0160] Container No. Upper GIT suspension Inoculum 1，2，3 Blank / control, fed Fresh 4，5，6 Blank / control, fasting Fresh 7，8，9 Microencapsulation, ingestion Fresh 10，11，12 Microencapsulation, ingestion Filter sterilized 13，14，15 Microencapsulation, fasting Fresh 16，17，18 Microencapsulation, fasting Filter sterilized

[0161] In the colon incubation experiment of the present embodiment, three relevant markers of microbial metabolic activity were recorded: changes in pH, production of short-chain fatty acids (SCFAs), and changes in lactic acid 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 period, and therefore gives an indication of metabolic rate. The production of SCFA is an assessment of the microbial metabolism of carbohydrates (to produce acetic acid, propionic acid, and butyric acid) or proteins (to produce branched SCFAs), and can be compared with the typical fermentation pattern of normal gastrointestinal microflora. Finally, since the intestine contains bacteria that produce lactic acid (e.g., lactic acid bacteria) and bacteria that consume lactic acid, changes in lactic acid concentration can indicate the balance between these organisms. Lactic acid can also reduce the pH of the environment, act as an antimicrobial agent, and can be quickly converted into other simple SCFAs (e.g., acetic acid, butyric acid, propionic acid) by other microorganisms.

[0162] pH changes

[0163] Monitoring pH during short-term colonic incubations provided information on SCFA, lactate, and ammonium (NH 4 + ) production. Typically, a pH drop is observed during the first 24 h of incubation due to the formation of SCFA and / or lactic acid. During the second 24 h of incubation, a pH drop is observed due to proteolytic fermentation (which in particular leads to NH 4 + This pH drop is usually followed by a pH increase due to the conversion of stronger acids to weaker acids through cross-feeding (e.g., conversion of lactic acid to propionic and / or butyric acid).

[0164] Reference now Fig. 8A and 8B For the blank / control suspension, a decrease in pH over the 48 h incubation period was observed under both fasting and fed conditions, but this decrease was not observed under fed conditions ( Fig. 8A , third column, ΔpH = -0.23) than under fasting conditions ( Fig. 8A , first column, ΔpH=-0.13); without wishing to be bound by any particular theory, the inventors hypothesize that this can be explained by a higher nutrient availability under fed conditions, originating from differences in gastric juice composition. The pH decrease was particularly pronounced between 1 and 24 hours of incubation, suggesting that fermentation of available substrates occurred primarily during this time interval. In summary, the inventors conclude that the intestinal microflora of pediatric stool donors is able to ferment available substrates in both colonic culture medium and predigested upper GIT suspensions.

[0165] For the non-sterile incubation of the pre-digested microencapsulated strain composition, the pH decreased strongly throughout the colonic incubation (i.e., from 0 h to 48 h). As with the blank / control suspension, this decrease was particularly evident between 1 h and 24 h of incubation. However, a slight increase was observed during the last 24 h under fed conditions ( Fig. 8A , fourth column, ΔpH from 24h to 48h = +0.03), indicating a shift in carbohydrate metabolism to proteolysis. For both fasting and fed conditions, the overall pH decrease in non-sterile incubations was significantly greater than that in the corresponding blank controls ( Fig. 8A : under fasting conditions, ΔpH = -0.60 vs. -0.13, under fed conditions, ΔpH = -0.67 vs. -0.23), which indicates that the supplemented prebiotics are effectively fermented by the newly established intestinal microflora. As with the blank control, in the non-sterile incubations, the pH reduction was more pronounced under fed conditions; without wishing to be bound by any particular theory, the inventors hypothesize that this can be explained at least in part by higher nutrient availability and at least in part by higher survival of the probiotic strains during passage through the upper GIT.

[0166] For aseptic incubation of pre-digested microencapsulated strain compositions, the pH under fed conditions decreased ( Figure 8B , right column, ΔpH = -0.74) again lower than the pH under fasting conditions ( Figure 8B , left column, ΔpH = -0.16) is more pronounced, which can again be explained by higher nutrient availability and / or higher probiotic survival. In addition, the pH reduction under both conditions confirms that the probiotic strains are able to ferment available nutrients, including those of prebiotics. Interestingly, in the aseptic incubation ( Figure 8B , right column, ΔpH = -0.74) and non-sterile incubation ( Fig. 8A , fourth column, ΔpH = -0.67), supplementation with a predigested upper GIT suspension resulted in a similar pH decrease, whereas under fasting conditions, the pH decrease in non-sterile incubations ( Fig. 8A , second column, ΔpH = -0.60) lower than the pH in sterile incubation ( Figure 8B , left column, ΔpH=-0.16). Since the survival rate of the probiotic strains under fed conditions is much higher, the inventors conclude, without wishing to be bound by any particular theory, that the higher number of viable probiotic cells in the fed suspension most likely colonized the donor's intestinal microflora to a greater extent and therefore contributed more significantly to the metabolic activity of the newly established intestinal microflora.

[0167] Lactate concentration

[0168] The human intestine contains bacteria that produce and consume lactic acid. In particular, lactic acid bacteria produce lactic acid during the degradation of primary substrates and reduce the pH of the intestinal environment. Lactic acid can exert a strong antimicrobial effect against pathogens, especially at low pH values. Another beneficial effect of lactic acid is that it can be effectively converted into butyric acid and / or propionic acid; therefore, when different microbial species produce or consume lactic acid, an increase in lactic acid concentration can be caused by either or both of increased production and reduced consumption.

[0169] Reference now Fig.9A and 9B For the blank control, lactate concentrations initially increased during the first hour of colon incubation, suggesting that the donor's gut microbiota produced lactate faster than it was consumed. However, this increase Fig.9A , first column, lactate concentration Δ = +0.14 mM) and feeding ( Fig.9A , third column, lactate concentrations were very limited under both fasting and fed conditions (Δ = +0.20 mM). As the incubation progressed, lactate levels subsequently decreased (Δ = -0.37 mM under fasting conditions and Δ = -0.45 mM under fed conditions), suggesting that lactate production was reduced or that lactate was more efficiently converted to other metabolites.

[0170] Similar effects were observed for non-sterile incubations of pre-digested microencapsulated strain compositions, i.e., an initial increase during the first hour of incubation ( Fig.9A , second and fourth columns: Δ = 0.20 mM under fasting conditions, 0.72 under fed conditions), followed by a decrease between 1 hour and 24 hours (Δ = -0.56 mM under fasting conditions, -2.45 mM under fed conditions). The overall reduction from 0 hours to 48 hours for the microencapsulated strain composition was significantly greater than that for the blank control under both fasting and fed conditions (-0.36 vs. -0.23 under fasting conditions, -1.73 vs. -0.25 under fed conditions). Both the initial increase and subsequent decrease were more pronounced under fed conditions. Since the concentrations of prebiotic nutrients were similar under both sets of conditions, the inventors concluded, without wishing to be bound by any particular theory, that the probiotic strains can provide both an initial boost in lactate production and an improved conversion of lactate to other metabolites (e.g., propionate, butyrate) in the later stages of incubation.

[0171] For aseptic incubations of pre-digested microencapsulated strain compositions, relatively high levels of lactic acid were produced. This was particularly true under fed conditions ( Fig. 9B , right column, total lactate concentration Δ = +33.5 mM), which may be related to the higher nutrient availability (derived from the difference in gastric juice composition) and the higher survival rate of the probiotic strains. The strong increase in lactate concentration indicates that the probiotic strains were able to ferment the available nutrients (derived from both the background medium and the prebiotic composition), which led to the accumulation of lactate. In particular, compared with non-sterile incubations ( Fig.9A , second and fourth columns), no net consumption of lactate was observed during the last 24 h of incubation under sterile conditions ( Fig. 9B ), which may be due to the absence of cross-feeding interactions with propionate- and / or butyrate-producing bacterial strains in the background microbial community.

[0172] Total SCFA production

[0173] The production of SCFAs is due to carbohydrate metabolism in the colon and is associated with various health effects. The SCFAs produced in the largest amounts in the colon are acetic acid, propionic acid, and butyric acid, each of which has a beneficial effect on the health of the host - acetic acid can be used as an energy source for the host and a potential substrate for lipid synthesis, propionic acid improves metabolic homeostasis by reducing cholesterol and fatty acid synthesis in the liver, and butyric acid is the main energy source for colonocytes and induces differentiation of these cells (an effect associated with cancer prevention). In contrast, branched-chain SCFAs (bSCFAs) are the result of proteolytic microbial activity, which is also associated with the formation of toxic compounds (e.g., p-cresol). Therefore, high production of acetic acid, propionic acid, and butyric acid in the colon is associated with beneficial health effects, while high bSCFA production in the colon is associated with harmful health effects.

[0174] Reference now Fig. 10A and 10B , total SCFA production reflects the total fermentation of the gut microbiota. Fig. 10A , first and third columns) resulted in a total SCFA production of 32.9 mM and 44.6 mM, respectively. Therefore, the intestinal microbiota of the donor was able to ferment the nutrients available in the colonic culture medium. The higher values ​​obtained under fed conditions could again be related to a higher nutrient availability originating from differences in the composition of the gastric juice. The strongest SCFA production occurred during the first 24 h of incubation under both fasting and fed conditions, which is consistent with the pH decrease observed during this time interval ( Fig. 8A ) are consistent.

[0175] For non-sterile incubation of the pre-digested microencapsulated strain composition, significantly higher total SCFA concentrations were obtained compared to the blank control ( Fig. 10A , second and fourth columns: total SCFA = 58.8 mM fasting, 68.0 mM fed), where the fed condition resulted in slightly higher total SCFA production. Since the concentration of prebiotics was similar under both conditions, the increased production under fed conditions may be the result of higher survival of the microbial strains, which can increase SCFA production directly (by fermenting available nutrients themselves) or indirectly (by stimulating fermentation of available nutrients by native intestinal microbiota). As with the blank control, the strongest increase in total SCFA production occurred in the first 24 hours.

[0176] For the aseptic incubation of the pre-digested microencapsulated strain composition, almost no SCFAs were produced under fasting 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 the production of SCFAs, i.e. they convert available nutrients into SCFAs by themselves.

[0177] Acetic acid production

[0178] Acetate is one of the key metabolites formed during the fermentation of primary substrates and can be produced by a variety of gut microorganisms, including Bacteroides and Bifidobacterium. Fig.11A and 11B , fasting and fed blank controls ( Fig.11A , first and third columns) resulted in total acetate production of 21.0 mM and 27.3 mM, respectively, indicating that total SCFA production was mainly composed of acetate. Fig. 10A , first and third columns), higher acetate levels were obtained under fed conditions, and acetate levels increased most significantly during the first 24 hours of incubation.

[0179] For non-sterile incubation of pre-digested microencapsulated strain compositions, Fig.11A , second column, 41.2 mM) and fed ( Fig.11A , fourth column, 40.6 mM) conditions, which obtained significantly higher acetate levels than the control. No significant differences were observed between fasting and fed conditions, which the inventors believe, without wishing to be bound by any particular theory, may indicate that the native gut microbiota is primarily responsible for acetate production.

[0180] For the aseptic incubation of the pre-digested microencapsulated strain composition, only very little acetic acid was produced under fasting conditions ( Fig. 10B , left column), whereas under fed conditions, total acetate production was only 8.6 mM ( Fig. 10B , right column). Based on these results, the inventors concluded that the SCFA activity of the probiotic strains mainly resulted in the production of acetic acid, which is typical of Lactobacillus and Bifidobacterium strains. The more significant increase in acetic acid concentration under fed conditions may be due to higher nutrient availability and higher survival of the probiotic strains leaving the small intestine. In addition, the much lower levels of acetic acid produced in the sterile incubation compared to the non-sterile incubation confirm that the administration of the microencapsulated strain composition stimulated the production of acetic acid by the donor's native intestinal microbiota.

[0181] Propionate production

[0182] Like acetate, propionate can be produced by a variety of gut microorganisms, with the most abundant propionate producers in the gut microbiome being Bacteroides , Akkermansia muciniphila , and Veillonella .

[0183] Reference now Fig. 12A and 12B , blank control under fasting and feeding conditions ( Fig. 12A, first and third columns) were 5.8 mM and 8.8 mM, respectively. Like acetate, propionate was produced primarily in the first 24 hours of incubation and was produced more under fed conditions than under fasted conditions. Therefore, propionate production clearly contributes to total SCFA production in the control, although to a significantly lesser extent than acetate.

[0184] For non-sterile incubation of pre-digested microencapsulated strain compositions, compared with the blank control ( Fig. 12A , second and fourth columns: 13.34 mM under fasting conditions, 16.99 mM under fed conditions). The significantly greater propionic acid production under fed conditions is again associated with higher nutrient availability and / or higher survival of the probiotic strain.

[0185] For the aseptic incubation of the pre-digested microencapsulated strain composition, the probiotic strains were fasted ( Fig. 12B , left column, 0.00 mM) or fed ( Fig. 12B , right column, -0.16mM) conditions failed to produce significant amounts of propionic acid. Based on these results, without wishing to be bound by any particular theory, the inventors conclude that at least one of the probiotic microbial strains and prebiotic composition indirectly increases propionic acid production by stimulating the native intestinal microbial community. This may be the result of a cross-feeding mechanism, by which the end products of the metabolic activity of certain bacterial species can be used as substrates by other bacterial species. In this case, certain intestinal microbiota may be able to convert lactic acid into propionic acid.

[0186] Butyrate production

[0187] Butyrate is produced by members of Clostridium clusters IV and XIVa. Through cross-feeding, these microorganisms can convert acetate and / or lactate, as well as other substrates, into butyrate.

[0188] Reference now Fig.13A and 13B , blank control ( Fig.13A , first and third columns) resulted in butyrate production of 2.5 mM and 3.5 mM, respectively. Like acetate and propionate, butyrate was produced primarily in the first 24 hours of incubation, and slightly higher production was obtained under fed conditions than under fasted conditions.

[0189] For non-sterile incubation of pre-digested microencapsulated strain compositions, compared with the corresponding blank controls ( Fig.13A , second and fourth columns: 4.41 mM under fasting conditions, 9.70 mM under fed conditions), which obtained significantly higher butyrate levels. The highest values ​​were again obtained under fed conditions, which may be related to higher nutrient availability and / or higher survival rate of probiotic strains.

[0190] For the aseptic incubation of the pre-digested microencapsulated strain composition, the probiotic strains were fasted ( Fig. 13B , left column, -0.30 mM) or fed ( Fig. 11B , right column, 0.01 mM) conditions failed to produce significant amounts of propionic acid. Based on these results, without wishing to be bound by any particular theory, the inventors conclude that at least one of the probiotic microbial strain and the prebiotic composition indirectly increases butyrate production by stimulating the native intestinal microbial community. This may be the result of a cross-feeding mechanism; in this case, as with propionic acid, certain intestinal microbiota may be able to convert lactic acid into butyric acid.

[0191] Branched-chain SCFA

[0192] Branched-chain SCFAs (bSCFAs) are produced by proteolytic fermentation and are associated with the production of toxic compounds (e.g., p-cresol). Therefore, high bSCFA levels may indicate detrimental proteolytic activity in the colon, and reduced levels are considered beneficial for health.

[0193] Reference now Fig.14A and 14B , the blank control resulted in 2.03mM and 3.38mM total bSCFA production under fasting and fed conditions, respectively ( Fig.14A , first and third columns). Under fed conditions, bSCFA production was slightly higher, which may again be related to higher nutrient availability. A clear increase in bSCFA production was observed during the last 24 h of incubation compared to acetate, propionate, and butyrate, suggesting that the gut microbiota initially used carbohydrates as substrates and then shifted metabolic activity towards proteolysis in the later stages of incubation.

[0194] For non-sterile incubation of pre-digested microencapsulated strain compositions, compared with the corresponding blank controls ( Fig.14A , second and fourth columns: 0.92 mM under fasting conditions, 0.36 mM under fed conditions), which obtained significantly lower bSCFA concentrations. In addition, the lowest values ​​were obtained under fed conditions. Therefore, the administration of the synbiotic composition resulted in increased saccharolytic fermentation, which may be due to the presence of prebiotics.

[0195] For the aseptic incubation of the pre-digested microencapsulated strain composition, the probiotic strains were fasted ( Fig. 14B , left column, 0.00 mM) or fed ( Fig. 14B , right column, 0.03 mM) conditions failed to produce significant amounts of bSCFA.

[0196] Example 5

[0197] Comparative survival of probiotic strains along the upper GIT

[0198] By making probiotic products Figure 4 500 to study the survival of microbial strains in commercial pediatric probiotic products, as described above in Example 3. Under fed conditions, at the beginning of the upper GIT experiment, the probiotic products were added to the reactor vessel 505 in amounts corresponding to the recommended daily dose of each product. Four commercial pediatric probiotic products were tested: Children's probiotic composition (1 bag / reactor), Garden of Original Probiotics Children's Composition (1.6g / reactor), Optibac Infant and child composition (1 bag / reactor) and Children's probiotic powder composition (1 gram / reactor).

[0199] Reference now Fig.15A , Administration of a probiotic composition for children, whose microbial strains consisted mainly of Lactobacillus rhamnosus GG cells, showed that the viability of the probiotic strains was substantially unaffected during passage through the fed stomach (10 cells per reactor at the beginning and end of gastric conditions). 10.47 Thus, the Lactobacillus rhamnosus GG strain (a) present in these probiotic compositions proved to be resistant to the pH profile in the stomach (i.e., an S-shaped decrease in pH from 4.6 to 3.0 within 2 hours). However, upon entering the duodenal phase, the viability of the probiotic strain decreased significantly (the number of viable cells per reactor decreased from 10 to 2.5). 10.47 =2.97·10 10 Up to 10 9.02 =1.06·10 9 ); As described in Example 3 above, 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 throughout the jejunal and ileal stages, no further decrease in viability was observed (the number of viable cells at the end of the jejunal and ileal stages was 10 9.02 and 10 9.03 ). Therefore, the number of viable cells decreased by 96.27% (3.73% survival rate) from the beginning to the end of the upper GIT, and the number of viable cells in each reactor increased from 10 10.47 =2.95·10 10 Up to 10 9.03 =1.10·10 9 .

[0200] Reference now Fig. 15B , Garden of Administration of the original probiotic children's composition, whose microbial strains consisted mainly of Lactobacillus and Bifidobacterium, showed that the microbial strains present in the probiotic product were not negatively affected by the environmental conditions encountered during transit through the stomach under fed conditions. In fact, at the end of transit through the stomach, the viable cell counts were significantly higher than at the beginning (10 viable cells per reactor). 10.48 =3.02·10 10 vs.10 10.37 =2.36·10 10 ), although the in vivo significance of this finding may be questionable; growth of the probiotic strain during transit through the stomach is quite unlikely, and therefore the measured effect may be an artifact of the lower solubility of the test composition in aqueous buffer compared to that in gastric fluid-simulating medium. Then, the viability of the probiotic strain was significantly reduced in the duodenal stage (the number of viable cells per reactor dropped to 10 10.16 =1.45·10 10 ), which may again be related to the toxicity of bile salts at low pH. No significant decrease in viability was observed during the jejunal and ileal phases. At the end of the ileal phase, the number of viable cells per reactor was observed to be 10 10.13 =1.35·10 10 The live population density was reduced by 42.82% (57.18% survival rate) compared to the initial population and by 55.33% (44.67% survival rate) when compared to the live population at the end of the gastric phase.

[0201] Reference now Fig. 15C Optibac Administration of a composition for infants and children, whose microbial strains consisted mainly of Lactobacillus acidophilus Rosell-52, Bifidobacterium infantis Rosell-33 and Bifidobacterium bifidum Rosell-71, showed that the viability of the strains was negatively affected during passage through the stomach, with the number of viable cells per reactor decreasing from 10 10.20 =1.59·10 10 Significantly reduced to 10 10.08 =1.20·10 10 This suggests that these strains are less robust in the face of the harsh pH conditions in the stomach. The viable population density further decreased throughout the small intestinal phase, although this decrease was only observed at the end of the ileal phase (where the number of viable cells per reactor dropped to 10 9.48 =3.49·10 9 ) was statistically significant. Therefore, these strains may also be negatively affected by high bile salt concentrations and low pH during the duodenal phase, although to a lesser extent than Children's probiotic composition ( Fig.15A). Overall, delivery through the upper GIT resulted in a 78.04% decrease in viability of the composition (21.96% survival rate).

[0202] Reference now Fig.15D , The administration of a probiotic powder composition for children (whose microbial strains mainly consisted of Lactobacillus and Bifidobacterium) showed that the viability of these strains was negatively affected during transit through the stomach, with the viable cell count decreasing from 10 viable cells per reactor to 10 10.32 =2.07·10 10 Significantly reduced to 10 10.21 =1.63·10 10 . With Optibac Infant and child compositions ( Fig. 15C ), this reduction is likely the result of the probiotic strains being less robust to the harsh pH conditions in the stomach. The viable population density was further reduced in the duodenum phase (to 10 viable cells per reactor). 10.08 =1.21·10 10 ), indicating that these strains were also negatively affected by the addition of bile salts. However, no further decrease in viability was observed during the jejunal stage, and in fact, an increase in viability was observed during the ileal stage (from 1.26 10 viable cells per reactor at the end of the jejunal stage to 10 At the end of the ileal phase, the number of viable cells per reactor was 10 10.21 =1.61·10 10 ). Overall, delivery through the upper GIT resulted in a 22.31% decrease in viability of the composition (77.69% survival).

[0203] A comparison of viable cell survival after transit through the upper GIT for the four commercially available compositions tested in this example and the two compositions of the present disclosure tested in Example 3 (a naked strain composition and a microencapsulated strain composition) is provided below in Table 2. In Table 2, survival is simply the number of colony forming units (CFU) obtained at the end of the ileal phase divided by the number of CFU present in the probiotic composition at the beginning of the experiment, "MXP271+" refers to the composition of the present disclosure tested in Example 3 (i.e., PDS-08 strain, which contains 7 strains of MXP271 strain plus Lactobacillus acidophilus SD-NCFM-US and Bifidobacterium animalis subsp. lactis SD-BI07-US), and "ME" means "microencapsulated".

[0204] Table 2

[0205]

[0206] As shown in Table 2, The viability of probiotic compositions for children is generally too low to provide sufficiently high numbers of viable cells to their probiotic active sites (i.e., the distal ileum and colon). The proportion of cells from the infant and child compositions surviving in the upper GIT was similar to that of the naked bacterial strain compositions of the present disclosure, with the lower number of cells provided in the recommended dosage form resulting in a lower number of viable cells delivered to the distal ileum and colon. The higher initial viable cell density in the naked bacterial strain composition also resulted in a higher number of viable cells at the end of the ileal phase than in the Garden of Original probiotics children's composition, and with Children's probiotic powder compositions were comparable. Finally, the microencapsulated strain compositions of the present disclosure delivered significantly greater numbers of viable cells to the end of the ileal stage than any currently commercially available pediatric probiotic composition tested.

[0207] The concepts disclosed illustratively herein can be suitably practiced without any element not specifically disclosed herein. However, it will be apparent to those skilled in the art that many changes, variations, modifications, other uses and applications of the present disclosure are possible, and changes, variations, modifications, other uses and applications that do not depart from the spirit and scope of the present disclosure are considered to be covered by the present disclosure.

[0208] The foregoing discussion has been presented for the purpose of illustration and description. The foregoing is not intended to limit the present disclosure to one or more forms disclosed herein. In the foregoing detailed description, for example, various features are combined together in one or more embodiments for the purpose of simplifying the present disclosure. The features of the embodiments may be combined in alternative embodiments other than those discussed above. These disclosed methods should not be interpreted as reflecting the intention that the claims require more features than those explicitly stated in each claim. On the contrary, as reflected in the following claims, the creative aspects lie in less than all the features of a single foregoing disclosed embodiment. Therefore, the following claims are hereby incorporated into the detailed description, wherein each claim exists independently as a separate embodiment.

[0209] In addition, although the present disclosure has included descriptions of one or more embodiments and certain variations and modifications, other variations, combinations and modifications within the scope of the present disclosure (e.g., within the skill and knowledge of those skilled in the art) are possible after understanding the present disclosure. It is intended to obtain rights including alternative embodiments to the extent permitted, including those claimed alternative, interchangeable and / or equivalent structures, functions, scopes or steps, whether or not these alternative, interchangeable and / or equivalent structures, functions, scopes or steps are disclosed herein, and it is not intended to publicly contribute any patentable subject matter.

Claims

1. A method for treating a disease or achieving a physiological purpose in a human subject, the method comprising administering to the subject a therapeutically effective amount of a 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 the healthy human intestinal microbiota; and A probiotic component comprising a combination of microbial strains, the combination comprising at least two microbial strains selected from the group consisting of: (i) Lactobacillus rhamnosus SD-GG-BE; (ii) salivary combined Lactobacillus SD-LS1-IT; (iii) Bifidobacterium breve SD-BR632-IT; (iv) Bifidobacterium breve SD-BR03-IT; (v) Bifidobacterium longum SD-CECT7347-SP; (vi) Lactobacillus casei SD-CECT9104-SP; and (vii) Bifidobacterium lactis SD-CECT8145-SP.

2. The method of claim 1, wherein the disease is selected from the group consisting of adrenoleukodystrophy, age-induced genomic damage, Alexander disease, alopecia areata, Alpers disease, Alzheimer's disease, amyotrophic lateral sclerosis, angina pectoris, arthritis, asthma, Balo concentric sclerosis, Behcet's disease, vesicular pemphigus, Canavan disease, heart failure (including left ventricular dysfunction), central nervous system vasculitis, Charcot-Marie-Tooth disease, childhood ataxia with central nervous system demyelination, chronic idiopathic peripheral neuropathy, chronic obstructive pulmonary disease, Crohn's disease, cutaneous lupus, skin inflammation (contact, acute and chronic), diabetic retinopathy, graft-versus-host disease, granulomas, hepatitis C virus infection, herpes simplex virus infection, herpes zoster virus infection, human immunodeficiency virus infection, Huntington's disease, irritable bowel disease, ischemia, Krabbe disease, lichen planus, macular degeneration, mitochondrial encephalomyopathy, monomelic amyotrophy, multiple sclerosis, myocardial infarction, neurodegeneration with brain iron accumulation, neuromyelitis, neurosarcoidosis, NF-κB-mediated diseases, optic neuritis, carcinoid syndrome, Parkinson's disease, Pelizaeus-Merzbacher disease, pemphigus, primary lateral sclerosis, progressive supranuclear palsy, psoriasis, pustular gangrene, reperfusion injury, retinitis pigmentosa, sarcoidosis, Sheldon's disease, subacute necrotizing myelopathy, susac syndrome, transplant rejection, transverse myelitis, tumors, ulcerative colitis and Zellweger syndrome.

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

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

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

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

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

8. The method of claim 1, further comprising: co-administering or combining one or more other pharmaceutical agents with the synbiotic composition to a human subject, wherein the co-administration or combined administration improves the therapeutic effect of the one or more other pharmaceutical agents relative to independent administration of the synbiotic composition and the one or more other pharmaceutical agents.

9. The method of claim 1, wherein the physiological purpose is selected from the group consisting of increasing the Bristol Stool Shape Scale (BSFS), reducing bowel movement duration, relieving abdominal pain, relieving bloating, relieving heartburn, relieving acid reflux, relieving indigestion, maintaining or increasing the diversity of 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. The method of claim 10, wherein the ingestible formulation is in the form of a free-flowing powder provided in a single-serving sachet.

12. The method of claim 11, wherein the pouch comprises the following amount of the prebiotic component: 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. .5g, or about 2.75g to about 9.25g, or about 3g to about 9g, or about 3.25g to about 8.75g, or about 3.5g to about 8.5g, or about 3.75g to about 8.25g, or about 4g to about 8g, or about 4.25g to about 7.75g, or about 4.5g to about 7.5g, or about 4.75g to about 7.25g, or about 5g to about 7g, or about 5.25g to about 6.75g, or about 5.5g to about 6.5g, or about 5.75g to about 6.25g, or about 6g.

13. The method of claim 11, wherein the pouch comprises a 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. The method of claim 11, wherein the material of the pouch has a water vapor transmission rate of less than about 0.01 g / m2 / day at 23°C and 50% relative humidity.

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

16. The method of claim 11, wherein the pouch further comprises at least one pharmaceutically acceptable vehicle.

17. The method of claim 1, wherein the synbiotic composition is administered at least once a day for at least about 7 days.

18. The method of claim 1, wherein the at least one compound that can be converted into a bioactive metabolite by a microbial strain present in the healthy human intestinal microbiota comprises at least one fructan.

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

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

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, Camelina, chicory, coneflower, costus, dandelion, elecampane, garlic, artichoke, Jerusalem artichoke, yam bean, leek, leopard's bane, mugwort, onion, plantain, wheat, Jerusalem artichoke, and yam.

22. The method of claim 18, wherein the at least one fructan comprises fructooligosaccharides.

23. The method of claim 22, wherein the oligofructose is derived from or extracted from at least one plant selected from the group consisting of agave, asparagus, banana, barley, burdock, Camellia, chicory, coneflower, costus, dandelion, elecampane, garlic, artichoke, Jerusalem artichoke, yam, leek, leopard's bane, mugwort, onion, plantain, wheat, Jerusalem artichoke, and yam.

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

25. 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) to (vii).

26. The method of claim 1, wherein the consortium further comprises 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.

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

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

29. The method of claim 28, wherein the combination consists essentially of (i) to (vii), (viii) and (ix).

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