阅读说明：本技术 产生短链脂肪酸的益生菌菌株和包含该益生菌菌株的组合物 (Probiotic bacterial strain producing short chain fatty acids and compositions comprising same ) 是由 M·J·贾努什 A·M·普勒查蒂 于 2020-04-28 设计创作，主要内容包括：本文公开了产生短链脂肪酸(SCFA)诸如丁酸盐的益生菌菌株或菌种,以及包含所述益生菌菌株或菌种的组合物。所述细菌菌株或由此制备的组合物用于制备食品、补充剂、组合物和其他消费品以便为多种障碍,包括代谢障碍、免疫障碍、肠障碍和炎性障碍提供健康有益效果,包括治疗应用。因此,本文还公开了用包含本文所公开的益生菌菌株或菌种的组合物治疗患有诸如代谢障碍、免疫障碍、肠障碍或炎性障碍等障碍的受试者的方法。本发明还公开了细菌菌株或菌种以及包含所述细菌菌株或菌种的组合物在营养产品或药物中用于改善受试者的健康或预防或治疗受试者的多种障碍的用途。(Disclosed herein are probiotic strains or strains that produce Short Chain Fatty Acids (SCFAs), such as butyrate, and compositions comprising the same. The bacterial strains or compositions prepared therefrom are useful in the preparation of foods, supplements, compositions and other consumer products to provide health benefits, including therapeutic applications, for a variety of disorders, including metabolic disorders, immune disorders, intestinal disorders and inflammatory disorders. Thus, also disclosed herein are methods of treating a subject having a disorder, such as a metabolic disorder, an immune disorder, an intestinal disorder, or an inflammatory disorder, with a composition comprising a probiotic strain or species disclosed herein. The invention also discloses the use of a bacterial strain or species and compositions comprising the bacterial strain or species in a nutritional product or medicament for improving the health of a subject or preventing or treating a variety of disorders in a subject.)

1. A composition, comprising:

(a) at least one human isolate, or a mixture thereof, of a Short Chain Fatty Acid (SCFA) -producing bacterium, wherein the bacterium comprises a 16S ribosomal RNA (16S-rRNA) encoding gene sequence having at least 80% identity to any one of the nucleotide sequences of SEQ ID NOS: 1-23, or a DNA sequence having at least 80% identity to any one of the nucleotide sequences of SEQ ID NOS: 24-35, and

(b) excipients, carriers and/or diluents.

2. The composition of claim 1, wherein the bacteria are selected from the group consisting of: agrobacterium recalis, anaerobic Corynebacterium faecalis (Anaerobiosis caccae), Anaerobiosis hadrus, Butyricoccusfaeci, Clostridium butyricum (Clostridium butyricum), Clostridium curosum (Clostridium cochleariae), Clostridium innocuous (Clostridium innoculum), Coprococcus (Coprococcus comatus), Flavonociceptor platinii, Ralstonia faecalis (Roseburia faecalis), Roseburia hominis (Roseburia hominis), Roseburia intestinalis, and Gluconobacter oxydans (Roseburia inivorans).

3. The composition of claim 1 or claim 2, wherein the excipient is a cryoprotectant.

4. The composition of claim 3, wherein the cryoprotectant comprises a sugar or sugar alcohol.

5. The composition of claim 4, wherein the cryoprotectant further comprises any one or more of propyl gallate, sodium caseinate, sodium citrate, sodium glutamate, cysteine, ascorbic acid, and/or maltodextrin.

6. The composition of claim 3, wherein the cryoprotectant comprises:

(a) 1% to 25% sucrose;

(b) 1% to 25% trehalose;

(c) 0.1% to 5% sorbitol;

(d) 0.05% to 1.0% propyl gallate;

(e) 0.5% to 10% sodium caseinate;

(f) 0.1% to 5% sodium citrate;

(g) 1% to 15% sodium glutamate;

(h) 0.01% to 2.0% cysteine;

(i) 0.005% to 5.0% ascorbic acid;

(j) 1% to 20% maltodextrin; or

(k) Any combination of any one or more of (a) - (j).

7. The composition of claim 3, wherein the cryoprotectant comprises:

(a) 15% to 20% sucrose, 0.05% to 1.0% propyl gallate, 4% to 8% sodium caseinate, and 0.2% to 1.0% sodium citrate;

(b) 15% to 20% sucrose, 0.5% to 1.5% sorbitol, 5% to 12% sodium glutamate and 0.1% to 1.5% sodium citrate;

(c) 5% to 20% trehalose, 3% to 15% sodium glutamate, 0.01% to 1.0% cysteine; or

(d) 5% to 20% trehalose, 3% to 15% sodium glutamate, 0.01% to 2% ascorbic acid and 2% to 18% maltodextrin.

8. The composition of claim 3, wherein the cryoprotectant comprises:

(a) 17.8% of sucrose, 0.2% of propyl gallate, 6.4% of sodium caseinate and 0.6% of sodium citrate;

(b) 17.5% sucrose, 0.9% sorbitol, 8.5% sodium glutamate and 0.6% sodium citrate;

(c) 15% trehalose, 8.5% sodium glutamate and 0.1% cysteine; or

(d) 10% trehalose, 8.5% sodium glutamate, 0.2% ascorbic acid and 10% maltodextrin.

9. The composition of claim 6, wherein within 12 months after lyophilization in the cryoprotectant, the bacteria survive less than 1 log unit CFU reduction, less than 2 log unit CFU reduction, less than 3 log unit CFU reduction, less than 4 log unit CFU reduction, less than 5 log unit CFU reduction, or less than 6 log unit CFU reduction of viable bacteria.

10. The composition of any one of the preceding claims, wherein the composition comprises from 1 × E3 Colony Forming Units (CFU) to 1 × E11 CFU of the SCFA-producing bacteria and mixtures thereof.

11. The composition according to any one of the preceding claims, wherein the composition further comprises a prebiotic and/or a resistant starch.

12. The composition of any one of the preceding claims, wherein the composition further comprises at least one additional bacterium that degrades resistant starch.

13. The composition according to claim 12, wherein the bacteria degrading resistant starch is Bifidobacterium adolescentis (Bifidobacterium adolescentis), Ruminococcus branchii (Ruminococcus braunii), bacteroides thetaiotaomicron (Bacteriodes thetaiotaomicron), bacteroides ovatus (Bacteriodes ovatus), Bifidobacterium breve (Bifidobacterium brave) or Roseburia intestinalis (rosebria intestinalis).

14. A method of increasing Short Chain Fatty Acids (SCFA) in the gastrointestinal tract of a subject, comprising administering to the subject an effective amount of a composition according to claim 1.

15. A method for reducing or maintaining glucose levels and/or body weight in a subject in need thereof, comprising administering to the subject an effective amount of a composition according to any one of claims 1 to 18.

Technical Field

The present disclosure relates to probiotic strains or strains that produce Short Chain Fatty Acids (SCFAs), such as butyrate, for their delivery to the colon where the SCFAs are endogenously produced at their site of action, thereby providing health benefits to a variety of disorders, including metabolic, immune, intestinal and inflammatory disorders, and to compositions comprising such bacterial strains or strains. In certain embodiments, the bacterial strains or compositions are used in the preparation of foods, supplements, and other consumer products to provide health benefits, including therapeutic applications, for a variety of disorders, including metabolic disorders, immune disorders, intestinal disorders, and inflammatory disorders.

Sequence listing is incorporated by reference

This application contains as a separate part of the disclosure a sequence listing in computer readable form (file name: 53257_ seqlikting. txt; size: 60,978,131 bytes; created 2019 on 4/24), which is incorporated herein by reference in its entirety.

Background

Obesity and its associated cardiovascular disorders are a significant health care problem, and the importance of the gut microbiota and its relationship to cardiovascular disorders is increasingly recognized. Increased consumption of dietary fiber is associated with decreased appetite and weight loss. The gut microbiota ferments non-absorbable dietary fibers and produces Short Chain Fatty Acids (SCFAs) important for gut health and metabolic function (Canforda et al, Nature Reviews Endocrinology11: 577-. Health benefits of SCFAs, particularly butyrate, include maintenance of the intestinal epithelial barrier by acting as an energy source for colonic epithelial cells (Ping et al, J.Nutr.139: 1619-. In addition, SCFA have been shown to have anti-inflammatory activity in vitro and in vivo by increasing the number and function of anti-inflammatory T regulatory cells (Smith et al, Science 341:569-73, 2013).

SCFA have been shown to stimulate the production of the enteroendocrine peptides glucagon-like peptide 1(GLP-1) and yy (pyy), which increases satiety, stimulates insulin secretion from the pancreas, increases energy expenditure and increases central appetite regulation, leading to potential metabolic benefits (canforta (supra); Yadav et al, j.biol.chem.288: 25088-. The obese phenotype and insulin resistance can be transferred from obese mice to lean mice by fecal bacteria (Turnbaugh et al, Nature 444:1027-31, 2006). In humans, stool transplantation from a lean donor to a subject with metabolic syndrome improves insulin sensitivity and is associated with an increase in butyrate producing bacteria (Vrieze et al, Gastroenterology 143:913-6,2012; Kootte et al, Cell Metab.26:611-9,2017).

Butyrate content varies greatly between individuals and can be increased by the consumption of fermentable fiber (McOrist et al, J.Nutr.141:883-9, 2011). Oral butyrate supplements also exist on the market, but this approach is not desirable because of the rancid smell of butyrate, requiring multiple capsules to deliver an effective dose and rapid absorption in the upper gastrointestinal tract (Pituch et al, Przeglad gastroenterology 8: 295-. Administration of SCFA-producing probiotic bacterial strains delivers butyrate-producing organisms to the colon where SCFAs may be endogenously produced at their site of action, thereby providing health benefits for a variety of metabolic and immune disorders, including but not limited to healthy glucose control and weight management.

Disclosure of Invention

The present disclosure provides novel probiotic bacterial strains that produce Short Chain Fatty Acids (SCFA) important to intestinal health and metabolic function. The present disclosure includes a composition comprising at least one human isolate, or a mixture thereof, of a SCFA-producing bacterium comprising a 16S ribosomal RNA (16S-rRNA) encoding gene sequence having at least about 80% identity to any of the nucleotide sequences of SEQ ID NOs 1-23, or a DNA sequence having at least about 80% identity to any of the nucleotide sequences of SEQ ID NOs 24-35, and an excipient, carrier, and/or diluent. In some aspects, the bacterium is selected from the group consisting of: agrobacterium recalis, anaerobic Corynebacterium faecalis (Anaerobiosis caccae), Anaerobiosis hadrus, Butyricoccus faecalis, Clostridium butyricum (Clostridium butyricum), Clostridium curosum (Clostridium cochleariae), Clostridium innocuum (Clostridium innoculum), Coprococcus (Coprococcus comatus), Flavonociceptor platii, Ralstonia faecalis (Roseburia faecalis), Rourella anthropogonis (Roseburia hominis), Rourella enterica (Roseburia intestinalis), and Gluconobacter acidilans (Roseburia inivorans). In some aspects, the bacteria are lyophilized. In some aspects, the excipient is a cryoprotectant. In some aspects, the cryoprotectant comprises a sugar or sugar alcohol. In some aspects, the cryoprotectant further comprises any one or more of propyl gallate, sodium caseinate, sodium citrate, sodium glutamate, cysteine, ascorbic acid, and/or maltodextrin.

In some aspects, the cryoprotectant comprises about 1% to about 25% sucrose; about 1% to about 25% trehalose; about 0.1% to about 5% sorbitol; from about 0.05% to about 1.0% propyl gallate; from about 0.5% to about 10% sodium caseinate; from about 0.1% to about 5% sodium citrate; from about 1% to about 15% sodium glutamate; about 0.01% to about 2.0% cysteine; from about 0.005% to about 5.0% ascorbic acid; about 1% to about 20% maltodextrin; or a combination of any one or more of the above components.

In some aspects, the cryoprotectant comprises about 15% to about 20% sucrose, about 0.05% to about 1.0% propyl gallate, about 4% to about 8% sodium caseinate, and about 0.2% to about 1.0% sodium citrate; about 15% to about 20% sucrose, about 0.5% to about 1.5% sorbitol, about 5% to about 12% sodium glutamate, and about 0.1% to about 1.5% sodium citrate; about 5% to about 20% trehalose, about 3% to about 15% sodium glutamate, about 0.01% to about 1.0% cysteine; or about 5% to about 20% trehalose, about 3% to about 15% sodium glutamate, about 0.01% to about 2% ascorbic acid, and about 2% to about 18% maltodextrin.

In some aspects, the cryoprotectant comprises about 17.8% sucrose, about 0.2% propyl gallate, about 6.4% sodium caseinate, and about 0.6% sodium citrate; about 17.5% sucrose, about 0.9% sorbitol, about 8.5% sodium glutamate and about 0.6% sodium citrate; about 15% trehalose, about 8.5% sodium glutamate, and about 0.1% cysteine; or about 10% trehalose, about 8.5% sodium glutamate, about 0.2% ascorbic acid and about 10% maltodextrin.

In some aspects, the compositions comprise from about 1 × E3 to about 1 × E11 Colony Forming Units (CFU) of SCFA-producing bacteria and mixtures thereof. In some aspects, the bacteria and/or mixtures thereof produce at least about 1000 micromolar SCFA in about 24 hours. In some aspects, the SCFA are acetate, propionate, or butyrate, or a combination thereof. In some aspects, the SCFA are butyrate.

In some aspects, the bacteria in the composition survive less than about 1 log unit CFU reduction, less than about 2 log unit CFU reduction, less than about 3 log unit CFU reduction, less than about 4 log unit CFU reduction, less than about 5 log unit CFU reduction, or less than about 6 log unit CFU reduction of viable bacteria within about 12 months after lyophilization in the cryoprotectant.

In some aspects, the composition further comprises a prebiotic and/or a resistant starch.

In some aspects, the composition further comprises at least one additional bacterium that degrades resistant starch. Such additional bacteria include, but are not limited to, Bifidobacterium adolescentis (Bifidobacterium adolescentis), Ruminococcus branchii (Ruminococcus braunii), Bacteroides thetaiotaomicron (Bacterodes thetaiotaomicron), Bacteroides ovatus (Bacteroides ovatus), Bifidobacterium breve (Bifidobacterium breve), or Roseburia enteralis (Roseburia intestinalis).

In some aspects, the compositions of the present disclosure are probiotic compositions.

The present disclosure also provides a method of increasing SCFA in the gastrointestinal tract of a subject, comprising administering to the subject an effective amount of any of the compositions disclosed herein. In some aspects, the SCFA are acetate, propionate, or butyrate, or a combination thereof. In some aspects, the SCFA are butyrate. In some aspects, the subject has or is at risk of developing a disorder. In some aspects, the disorder is an intestinal disorder, a metabolic disorder, an inflammatory disorder, or an immune disorder. In some aspects, the disorder is insulin resistance, insulin sensitivity, prediabetes, diabetes or type 2 diabetes (T2DM), irritable bowel syndrome, metabolic irregularity, obesity-related disorders, hypertension, stress-related disorders, irregular drug metabolism, gastrointestinal infection, Inflammatory Bowel Disease (IBD), or Crohn's Disease.

The present disclosure also provides a method for reducing or maintaining glucose levels and/or body weight in a subject in need thereof, comprising administering to the subject an effective amount of any of the compositions disclosed herein. In some aspects, the subject has diabetes or pre-diabetes.

The present disclosure also provides a method for treating, reducing, or preventing a disorder in a subject having or at risk of having the disorder, comprising administering to the subject an effective amount of any of the compositions disclosed herein. In some aspects, the disorder is an intestinal disorder, a metabolic disorder, an inflammatory disorder, or an immune disorder. In some aspects, the disorder is insulin resistance, insulin sensitivity, prediabetes, diabetes or T2DM, irritable bowel syndrome, metabolic irregularity, obesity-related disorders, hypertension, stress-related disorders, drug metabolism, gastrointestinal infections, IBD, or Crohn's Disease.

The present disclosure provides the use of a composition as disclosed herein in the treatment of, or in the manufacture of a medicament or nutritional product for the treatment of, a metabolic, immune, intestinal or inflammatory disorder.

Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the disclosed subject matter, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

Drawings

The compositions, methods, and uses disclosed herein may be more fully understood by reference to the accompanying drawings. These drawings are merely schematic representations based on convenience in demonstrating the present disclosure, and are, therefore, not intended to limit or restrict the scope of the exemplary embodiments.

FIG. 1 shows a protocol for isolating, growing and screening bacteria that produce Short Chain Fatty Acids (SCFA)/butyrate.

FIG. 2 shows the results of a screen for bacterial strains that grow and butyrate production on YCFA or RCB. At least about 1700 bacterial isolates were screened, many of which showed no growth or very low butyrate production. Growth (OD600nm) was on the x-axis and butyrate production was on the y-axis. The identified bacteria show high butyrate production and diversity in their classification. Dots of the same color represent different isolates.

FIGS. 3A-B show the growth curves and survival after lyophilization for Ralstonia faecalis (RC 1-78; i.e., SEQ ID NO: 1). Fig. 3A shows the growth curve before lyophilization. Fig. 3B shows the post-lyophilization survival of four cryoprotectants for each of the pre-lyophilization growth periods in fig. 3A.

FIGS. 4A-B show the growth curves and survival after lyophilization of coprococcus chaperone (RC 1-148; i.e., SEQ ID NO: 10). Fig. 4A shows the growth curve before lyophilization. Fig. 4B shows the post-lyophilization survival of four cryoprotectants for each of the pre-lyophilization growth periods in fig. 4A.

FIGS. 5A-B show the growth curve and survival after lyophilization for Anaerosties hadrus (MS-5.1; i.e., SEQ ID NO: 9). Fig. 5A shows the growth curve before lyophilization. Fig. 5B shows the post-lyophilization survival of four cryoprotectants for each of the pre-lyophilization growth periods in fig. 5A.

FIGS. 6A-B show the growth curves and survival after lyophilization for Agathobacter recalis (RC 2-6; i.e., SEQ ID NO: 11). Fig. 6A shows the growth curve before lyophilization. Fig. 6B shows the post-lyophilization survival of four cryoprotectants for each of the pre-lyophilization growth periods in fig. 6A.

FIGS. 7A-B show the growth curve and survival after lyophilization of butylricococcus faecalis (ODS-26; i.e., SEQ ID NO: 13). Fig. 7A shows the growth curve before lyophilization. Fig. 7B shows the post-lyophilization survival of four cryoprotectants for each of the pre-lyophilization growth periods in fig. 7A.

FIGS. 8A-B show the growth curves and survival after lyophilization of anaerobic fecal coryneform bacteria (FLM9 MS-25; SEQ ID NO: 15). Fig. 8A shows the growth curve before lyophilization. Fig. 8B shows the post-lyophilization survival of four cryoprotectants for each of the pre-lyophilization growth periods in fig. 8A.

FIGS. 9A-B show the growth curves and survival after lyophilization for L.hominis (RC 1-32; i.e., SEQ ID NO: 21). Fig. 9A shows the growth curve before lyophilization. Fig. 9B shows the post-lyophilization survival of four cryoprotectants for each of the pre-lyophilization growth periods in fig. 9A.

FIGS. 10A-B show the growth curves and survival after lyophilization for L.gluconeophaga (YC 1-156; i.e., SEQ ID NO: 22). Fig. 10A shows the growth curve before lyophilization. Fig. 10B shows the post-lyophilization survival of four cryoprotectants for each of the pre-lyophilization growth periods in fig. 10A.

FIGS. 11A-B show the growth curve and survival after lyophilization of Ralstonia enterobacteria (ODS-29; i.e. SEQ ID NO: 2). Fig. 11A shows the growth curve before lyophilization. Fig. 11B shows the post-lyophilization survival of four cryoprotectants for each of the pre-lyophilization growth periods in fig. 11A.

FIGS. 12A-B show the growth curves and survival after lyophilization for Flavonifactor platutii (FLM9 MS-20; i.e., SEQ ID NO: 19). Fig. 12A shows the growth curve before lyophilization. Fig. 12B shows the post-lyophilization survival of four cryoprotectants for each of the pre-lyophilization growth periods in fig. 12A.

FIG. 13 shows a comparison of bacterial growth under static conditions in various types of media known in the art (1mL) (e.g., RCB, TSB, YCFA + RS, PYG, GM-17, BHI, and MTGE) in a deep-well plate in an anaerobic chamber at 37 ℃. (+++++ ═ OD600> 2;, OD 1.5-2;, OD 1-1.5;, OD 0.5-1; and OD 0-0.5. ANG.). This data indicates a medium that can achieve optimal growth at small scale.

Fig. 14 shows the time of peak CFU production during the growth phase and bacterial survival at peak CFU after lyophilization for cells harvested immediately after lyophilization and 30 days after lyophilization using 10 different bacteria in four different cryoprotectants.

Figure 15 shows the oxygen sensitivity of various bacterial isolates. For O₂Sens¹Bacteria were spotted on RCA plates with 1:100 dilutions of the strain in an anaerobic chamber, then air was removed before being placed back into the anaerobic chamber at various time points. Data are expressed as the longest exposure time that resulted in visible growth on the plate. For micro-aerobic growth plates²The plates were spotted in an anoxic chamber (oxygen set to 0, no oxygen scavenger in the chamber) with a 1:100 dilution of the strain on RCA plates to maintain oxygen levels between about 40ppm and 160 ppm. Growth was measured by visualization of colonies after about 32 hours.

Detailed Description

The present disclosure provides probiotic bacterial strains or strains that produce butyrate and related compositions comprising the same. The disclosed bacterial strains are suitable for delivery to the colon where Short Chain Fatty Acids (SCFAs) may be endogenously produced at their site of action, thereby providing health benefits. The health benefits may alleviate or mitigate a variety of disorders, including metabolic disorders, immune disorders, intestinal disorders, and inflammatory disorders. In certain embodiments, the bacterial strains or compositions are used in the preparation of foods, supplements, pharmaceutical compositions, and other consumer products to provide health benefits, including therapeutic applications, for a variety of disorders, including metabolic disorders, immune disorders, intestinal disorders, and inflammatory disorders.

The terms "microorganism" and "microorganism" are used interchangeably herein and refer to a bacterium. The terms "population", "microbiota" and "microbiota" are used interchangeably herein and may refer to a microbial ecological community that lives on or within the body of a subject. The flora may be present on or in many, if not most, parts of the subject. Some non-limiting examples of habitats of flora may include: body surfaces, body cavities, body fluids, intestinal tract, colon, skin surfaces and pores, vaginal cavity, umbilical region, conjunctival region, intestinal region, stomach, nasal and nasal passages, gastrointestinal tract, urogenital tract, saliva, mucus, and feces.

Bacteria are classified and identified using criteria of interest to microbiologists or other scientists to distinguish organisms from each other and to group similar organisms. The bacterial species classes define organisms based on genetic similarity, biochemical and phenotypic criteria. Within a species, strains and subgroups may differ in the host response they elicit, their environmental habitat, and many other characteristics. Strain names typically reflect progeny of a single organism and are typically classified based on unique characteristics characterized by serotype, enzyme type, protein/nucleic acid/plasmid, and defined for important functional characteristics of probiotic strains including, but not limited to, undesirable bacterial inhibition, gastric acid tolerance, adhesion/colonization, hydrophobicity, and immunoregulatory cytokine production. DNA correlation was used to group strains based on overall genetic similarity.

As used herein, the term "genome" may refer to the entire genetic information of an organism encoded in its primary DNA sequence. The genome includes both genetic and non-coding sequences. For example, in some aspects, the genome represents a microbial genome. The genetic content of the microbiome may include: genomic DNA, RNA and ribosomal RNA, epigenome, plasmid, and all other types of genetic information present in the microorganisms comprising the microbiome.

In bacteria, the gene that has proven to be the most informative for studying evolutionary relatedness is the 16S ribosomal RNA (16S-rRNA) coding gene sequence, a DNA sequence that encodes the RNA component of the smaller subunit of the bacterial ribosome. The 16S-rRNA coding gene sequence is highly conserved evolutionarily among microbial species. The 16S-rRNA-encoding gene sequence is present in all bacteria, and the relevant forms are present in all cells, including eukaryotic cells. Thus, sequencing of 16S-rRNA subunits can be used to identify and/or compare microorganisms (e.g., bacteria) present in a sample (e.g., microbiome). 16S-rRNA gene sequencing is a well-established method for studying the phylogenetic and taxonomic classification of samples from complex microorganisms or environments that are difficult to study. In various aspects, the bacteria are identified by their 16S-rRNA sequence or 16S-rRNA encoding gene sequence.

As used herein, "nucleic acid sequence" and "nucleotide sequence" refer to oligonucleotides or polynucleotides and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and represent the sense or antisense strand. The nucleic acid sequence may be composed of adenine, guanine, cytosine, thymine and uracil (A, T, C, G and U) as well as modified forms (e.g., N6-methyladenosine, 5-methylcytosine, etc.). The term "sequencing" as used herein refers to a sequencing method for determining the order of nucleotide bases-A, T, C, G and U in a nucleic acid molecule (e.g., a DNA or RNA nucleic acid molecule). In some aspects of the disclosure, bacteria that produce the highest levels of SCFA and exhibit lyophilization tolerance are isolated and 16S-rRNA encoding gene sequences from each of these bacteria are sequenced. The 16S-rRNA-encoding gene sequences (SEQ ID NOS: 1-23) of each of these bacteria are provided in Table 1.

TABLE 1-16S-rRNA coding gene sequences of probiotics of the present disclosure

R ═ a or G; y ═ C or T; k ═ G or T; m ═ a or C; s ═ G or C; and W ═ a or T, as shown in the standard ambiguity code

Table 2-whole genome DNA sequences of probiotics of the present disclosure

Since the size of these whole genome sequences of the bacteria of the present disclosure is very large, these sequences are provided only in the sequence listing that is part of the present disclosure.

In some aspects, the bacteria comprise a nucleic acid sequence having a particular degree of homology or identity to other bacteria. As used herein, the terms "identity", "homology" and "homology" refer to the degree to which other nucleotide sequences are complementary or share similarity. There may be partial homology or complete homology (i.e., identical sequences). A nucleotide sequence that is partially complementary (i.e., "substantially homologous" or "substantially identical") to a nucleic acid sequence is a sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid sequence. In some aspects, bacteria of the disclosure comprise 16S-rRNA encoding gene sequences having at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, or any one of the nucleotide sequences of SEQ ID NOs 1-23, At least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity (100% homology). In some aspects, a bacterium of the present disclosure comprises a DNA sequence that has at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or at least about 95% of the nucleotide sequence of SEQ ID NOs 24-35, At least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity (100% homology). In some aspects, the whole genome sequence of the bacteria of the present disclosure is present in any one of the nucleotide sequences of SEQ ID NOs: 24-35.

In some aspects, the bacterium comprising the nucleotide sequence of any one of SEQ ID NOs 1-35 is a probiotic. As used herein, the term "probiotic" means a microbial cell preparation or microbial cell component that has a beneficial effect on the health of the host. (Salminen et al, Trends Food Sci.Technol.10107-10,1999). As used herein, the term "probiotic" may mean one or more microorganisms (e.g., bacteria) that, when properly administered, can confer a health benefit to a subject.

Some non-limiting examples of probiotic bacteria of the present disclosure include, but are not limited to, bacteria such as Agrobacterium retrievalis (previously known as Eubacterium retrievale), Corynebacterium thermoacidophilum (Akkermansia), Corynebacterium faecalis, Escherichia hadrus, Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium infantis (Bifidobacterium infantis), Bifidobacterium longum (Bifidobacterium longum), Clostridium butyricum, Clostridium cellulolyticum (Clostridium butyricum), Clostridium acetobutylicum (Clostridium acetobacter), Clostridium aminophilum (Clostridium butyricum), Clostridium beijerinckii (Clostridium bifidum), Clostridium acetobutylicum (Clostridium butyricum), Clostridium butyricum (Clostridium faecalicum), Clostridium nitrobacter asiaticum (Clostridium), Clostridium faecalis (Clostridium), Clostridium bifidum), Clostridium butyricum (Clostridium), Clostridium butyricum (Clostridium), Clostridium faecalis (Clostridium), Clostridium faecalis (Clostridium), Clostridium (Clostridium), Clostridium faecalis), Clostridium (Clostridium), Clostridium (Clostridium faecalis), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium faecalis), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium, Clostridium praerussitum, filamentous succinic acid-producing bacteria (Fibrobacter succinogenes), Clostridium praerussitum (Flavonifactor platutii), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus casei (Lactobacillus casei), Lactobacillus caucasicus (Lactobacillus caucasicus), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus lactis (Lactobacillus lactis), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus casei (Lactobacillus plantarum), Lactobacillus plantarum (Lactobacillus), Lactobacillus sanfrancisei), Lactobacillus casei (Lactobacillus), Lactobacillus sanfrancisei), Lactobacillus plantarum (Lactobacillus sanfrancisella), Lactobacillus sanfranciscii), Lactobacillus rhamnosus (Lactobacillus sanfranciscensis), Lactobacillus sanctici (Lactobacillus sana), Lactobacillus sanctici), Lactobacillus sancus (Lactobacillus sanfranciscensis), Lactobacillus sanctici (rumen), Lactobacillus sanctici (Lactobacillus sana), Lactobacillus sancus (Lactobacillus sancus), Lactobacillus sancus (Lactobacillus), Lactobacillus sancus (Lactobacillus sancus), Lactobacillus sancus (Lactobacillus sancus), Lactobacillus (Lactobacillus sancus), Lactobacillus (Lactobacillus sancus), Lactobacillus (Lactobacillus sancus), Lactobacillus (Lactobacillus sancus), Lactobacillus (Lactobacillus sancus), Lactobacillus (Lactobacillus sancus), Lactobacillus (Lactobacillus sancus), Lactobacillus (ilecus), Lactobacillus sancus), Lactobacillus (Lactobacillus sancus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus sancus), Lactobacillus (Lactobacillus sancus), Lactobacillus (Lactobacillus sancus), Lactobacillus sancus), Lactobacillus (Lactobacillus sancus), Lactobacillus (Lactobacillus sancus), Lactobacillus (Lactobacillus sancus), Lactobacillus (Lactobacillus sancus), Lactobacillus (ilex (Lactobacillus (ilecus), Lactobacillus (Lactobacillus sancus), Lactobacillus (Rotunica, Luciosus), and (Rotunica), and (Rotunica (Roxicus), or (Rotunica (Roxiella, Streptococcus casei (Streptococcus cremoris), Streptococcus faecalis (Streptococcus faecalis), Streptococcus infantis (Streptococcus infantis), Streptococcus mutans (Streptococcus mutans), Streptococcus thermophilus (Streptococcus thermophilus), human anaerobacterium faecalis (Anaerococcus thermophilus), Anaerococcus hadens, human colonic anaerobium (Anaerococcus colis), Clostridium sporogenes (Clostridium sporogenes), Clostridium tetani (Clostridium tei), enterococcus faecalis (Coprococcus), Coprococcus chaperones (Coprococcus comatus), enterococcus regularis (Coprococcus), Eubacterium columniformis (Eubacterium cylindracea), Eubacterium chrysosporium (Clostridium cylindracea), Eubacterium elongatum (Eubacterium hum), Eubacterium rhodobacter ventris (Eubacterium), Eubacterium ventris (Streptococcus faecalis), Eubacterium faecalis (Roscoenospora), and any combination thereof.

In particular aspects, the probiotic is a bacterium, including but not limited to: agrobacterium recalis, anaerobic Corynebacterium faecalis (Anaerobiosis caccae), Anaerobiosis hadrus, Butyricoccus faecalis, Clostridium butyricum (Clostridium butyricum), Clostridium curosum (Clostridium cochleariae), Clostridium innocuum (Clostridium innoculum), Coprococcus (Coprococcus comatus), Flavonociceptor platii, Ralstonia faecalis (Roseburia faecia), Rourella mansonii (Roseburia hominis), Rourella intestinalis (Roseburia intestinalis), and Gluconobacter carnea (Roseburia inivorans), and any combination or mixture thereof.

As described above, the bacteria of the present disclosure are of interest for their ability to produce Short Chain Fatty Acids (SCFAs). In some aspects, the disclosed bacteria produce at least about 0.5 millimoles (mmol) of SCFA, at least about 0.6mmol of SCFA, at least about 0.7mmol of SCFA, at least about 0.8mmol of SCFA, at least about 0.9mmol of SCFA, at least about 1.0mmol of SCFA, at least about 1.2mmol of SCFA, at least about 1.4mmol of SCFA, at least about 1.6mmol of SCFA, at least about 1.8mmol of SCFA, at least about 2.0mmol of SCFA, at least about 2.2mmol of SCFA, at least about 2.4mmol of SCFA, at least about 2.6mmol of SCFA, at least about 2.8mmol of SCFA, at least about 3.0mmol of SCFA, at least about 3.2mmol of SCFA, at least about 3.4mmol of SCFA, at least about 3.6mmol of SCFA, at least about 3.8mmol of SCFA, at least about 4.4mmol of SCFA, at least about 4.5 mmol of SCFA, at least about 4.4mmol of SCFA, at least about 4mmol of SCFA, at least about 4.6mmol of SCFA, at least about 4.4mmol of SCFA, at least about 4.5 mmol of SCFA, at least about 4, At least about 6.5mmol of SCFA, at least about 7.0mmol of SCFA, at least about 7.5mmol of SCFA, at least about 8.0mmol of SCFA, at least about 8.5mmol of SCFA, at least about 9.0mmol of SCFA, at least about 9.5mmol of SCFA, at least about 10mmol of SCFA, at least about 11mmol of SCFA, at least about 12mmol of SCFA, at least about 13mmol of SCFA, at least about 14mmol of SCFA, at least about 15mmol of SCFA, at least about 16mmol of SCFA, at least about 17mmol of SCFA, at least about 18mmol of SCFA, at least about 19mmol of SCFA, at least about 20mmol of SCFA, at least about 25mmol of SCFA, at least about 30mmol of SCFA, at least about 35mmol of SCFA, at least about 40mmol of SCFA, at least about 45mmol of SCFA, at least about 50mmol of SCFA, at least about 55mmol of SCFA, at least about 60mmol of SCFA, at least about 65mmol of SCFA, at least about 70mmol of SCFA, at least about 85mmol of SCFA, at least about 95mmol of SCFA, at least about 80mmol of SCFA, At least about 100mmol of SCFA, at least about 110mmol of SCFA, at least about 120mmol of SCFA, at least about 130mmol of SCFA, at least about 140mmol of SCFA, at least about 150mmol of SCFA, at least about 160mmol of SCFA, at least about 170mmol of SCFA, at least about 180mmol of SCFA, at least about 190mmol of SCFA, at least about 200mmol of SCFA, at least about 250mmol of SCFA, or at least about 300mmol of SCFA.

In some aspects, SCFA production ranges from about 1 millimole over about 24 hours to about 300 millimoles over about 24 hours. In some aspects, SCFA production ranges from about 1 millimole over about 24 hours to about 100 millimoles over about 24 hours. In some aspects, SCFA production ranges from about 1 millimole over about 24 hours to about 50 millimoles over about 24 hours. In some aspects, SCFA production ranges from about 4 millimoles over about 24 hours to about 40 millimoles over about 24 hours.

In some aspects, such amounts or concentrations of SCFA are measured over a time period varying from about 24 hours. For example, in some aspects, SCFA production is measured over a period of about 12 hours, over a period of about 36 hours, over a period of about 2 days, over a period of about 3 days, over a period of about 4 days, over a period of about 5 days, over a period of about 6 days, over a period of about one week, and the like. Thus, in those aspects, the SCFA production will be greater than or less than the SCFA production disclosed herein over a period of about 24 hours. In a particular aspect, SCFA are measured over a period of about 24 hours.

In various aspects, the SCFA are acetate, butyrate, or propionate, or a combination thereof. In a particular aspect, the SCFA is butyrate. Thus, in some more particular aspects, butyrate production is measured over a period of about 24 hours.

As described herein, SCFA are known to be important for intestinal health and metabolic function. Health benefits of SCFA, particularly butyrate, include, but are not limited to, anti-inflammatory activity, maintenance of the intestinal epithelial barrier, pathogen inhibition by lowering local pH, and increased mucus production. In various aspects, SCFAs produced by bacteria include, but are not limited to, acetic acid, propionic acid, butyric acid (butyric/butyroic acid), isobutyric acid (isobutric/isobutanoic acid), valeric acid (valeric/pentanoic acid), or isovaleric acid (isovaleric/isovaleroic acid), or combinations thereof, typically expressed as their conjugate bases, such as acetate, propionate, butyrate (butyrate/butanoate), isobutyrate (isobutryate/isobutanoate), valerate/pentanoate, isovalerate (isovaletate/isopentanoate), or combinations thereof. In some aspects, the SCFA produced by the bacteria is butyrate.

Accordingly, the present disclosure includes bacteria that modulate or increase butyrate production. In the colon, dietary fiber can be processed by butyrate producing microorganisms to produce butyrate (butyrate/butyrate). In turn, butyrate may trigger G protein-coupled receptor (GPCR) signaling, leading to glucagon-like peptide-1 (GLP-1) secretion, which may lead to increased insulin sensitivity and/or decreased appetite. By altering the butyrate-producing microbiome in a subject, the pathway responsible for insulin sensitivity can be stimulated. In some subjects, the microbial composition can be used to increase insulin sensitivity and/or restore insulin sensitivity to pre-diabetic levels.

In some aspects, the bacteria of the present disclosure are provided in a composition (i.e., a probiotic composition). In some aspects, the various types of bacteria of the present disclosure are combined in a composition. In particular aspects, the compositions comprise at least one human isolate, or mixtures thereof, of a SCFA-producing bacterium comprising a 16S-rRNA encoding gene sequence having at least about 80% identity to any of the nucleotide sequences of SEQ ID NOs 1-23, or a whole genome DNA sequence having at least about 80% identity to any of the nucleotide sequences of SEQ ID NOs 24-35, and an excipient, carrier, and/or diluent. In various aspects, the mixture is any two or more bacteria. Thus, a mixture can comprise three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, and so forth. The composition may be in the form of a nutritional supplement or a pharmaceutical composition. In some aspects, one or more of the bacterial species or strains described herein are used to produce a formulation comprising an effective amount of a composition for treating a subject. In some aspects, an effective amount is an amount effective to provide a health benefit to a subject. In some aspects, the effective amount is a therapeutically effective amount. The composition may be any formulation known in the art. Some non-limiting examples may include topical dosage forms, capsules, pills or tablets, lozenges, sachets, enemas, gels, liquids, bulk powders for reconstitution or beverages prepared from bulk powders, and the like.

In various aspects, the compositions comprise one or more strains or species having metabolic activity as described herein, i.e., live and/or lyophilized or non-live, heat-inactivated, irradiated or lysed probiotic bacteria, and a physiologically, pharmaceutically or nutritionally acceptable excipient, carrier or diluent. In some aspects, one or more of the strains or strains disclosed herein can be included in a food or beverage product, a cosmetic product, or a nutritional supplement.

Nutritionally acceptable excipients, carriers or diluents include, but are not limited to, those suitable for human or animal consumption, and those standard for the food or supplement industry. Typical nutritionally acceptable excipients, carriers or diluents are familiar to those skilled in the art.

In some aspects, examples of such suitable Excipients for use in the various compositions described herein can be found in "Handbook of Pharmaceutical Excipients" edited by a Wade and P J Weller (2 nd edition, 1994). In some aspects, acceptable carriers or diluents are described, for example, in "Remington's Pharmaceutical Sciences" (Mack Publishing co., a.r. gennaro editors, 1985). Such suitable carriers include, but are not limited to, lactose, starch, glucose, cellulose, microcrystalline cellulose, methylcellulose, magnesium stearate, mannitol, sorbitol, and the like. Such suitable diluents include, but are not limited to, water, ethanol, propylene glycol, and glycerol.

In some aspects, suitable excipients are stabilizing excipients or cryoprotectants, which maintain the viability of the bacteria. In various aspects, the terms "stabilizing excipient" and "cryoprotectant" are used interchangeably herein. In some aspects, such cryoprotectants maintain the viability of the bacteria after they are lyophilized. In some aspects, suitable cryoprotectants include inositol, sorbitol, mannitol, trehalose, glucose, sucrose, corn syrup, DMSO, all types of starch and/or modified starch, PVP, maltose or other mono-and disaccharides. In some aspects, an effective amount of cryoprotectant is used to minimize cell damage upon freezing.

In some aspects, the cryoprotectant maintains the bacteria viable for an extended period of time. In some aspects, the bacteria survive less than about 1 log unit CFU reduction, less than about 2 log unit CFU reduction, less than about 3 log unit CFU reduction, less than about 4 log unit CFU reduction, less than about 5 log unit CFU reduction, or less than about 6 log unit CFU reduction of viable bacteria within about 12 months after lyophilization in a cryoprotectant.

In various aspects, the cryoprotectant comprises a carbohydrate. In various aspects, the carbohydrate is a sugar. In some aspects, the sugar does not crystallize. In some aspects, the sugar is a monosaccharide or disaccharide, including but not limited to sucrose, trehalose, glucose, galactose, lactose, maltose, mannose; or a sugar alcohol including, but not limited to, ethylene glycol, glycerol, erythritol, threitol, arabitol, sorbitol, mannitol, xylitol, ribitol, galactitol, fucitol, iditol, inositol, heptatol, isomalt, maltitol, lactitol, maltotriose, maltotetratol, polyglucitol or allose, or a polysaccharide such as maltodextrin and inulin, or any combination or mixture thereof. In some aspects, the sugar or sugar alcohol is present from about 0.1% to about 25%. In some aspects, the sugar is present at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25%. In some aspects, the sugar is sucrose. In some aspects, sucrose is present at about 15% to about 20%, or at about 18%, or at about 17.8%. In some aspects, the saccharide is trehalose. In some aspects, trehalose is present at about 5% to about 20%, or at about 10%. In some aspects, the sugar alcohol is sorbitol. In some aspects, sorbitol is present in the cryoprotectant from about 0.1% to about 5%. In some aspects, sorbitol is present from about 0.5% to about 1.5%.

In various aspects, the cryoprotectant comprises sodium citrate. In some aspects, the cryoprotectant comprises a sugar or sugar alcohol and sodium citrate. In some aspects, the sodium citrate is present in the cryoprotectant at about 0.1% to about 5.0%. In some aspects, the sodium citrate is present in the cryoprotectant at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%.

In some aspects, the cryoprotectant comprises propyl gallate. In some aspects, the cryoprotectant comprises a sugar and/or sugar alcohol, sodium citrate, and propyl gallate. In some aspects, the propyl gallate is present in the cryoprotectant at about 0.05% to about 1.0%. In particular aspects, the propyl gallate is present in the cryoprotectant at about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 1.0%.

In some aspects, the cryoprotectant comprises sodium caseinate. In some aspects, sodium caseinate is present in the cryoprotectant from about 0.5% to about 10%. In some aspects, the sodium caseinate is present in the cryoprotectant at about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.

In some aspects, the cryoprotectant comprises sodium glutamate. In some aspects, sodium glutamate is present from about 1% to about 15%. In some aspects, the sodium glutamate is present at about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%.

In some aspects, the cryoprotectant comprises cysteine. In some aspects, the cysteine is present from about 0.01% to about 2%. In some aspects, the cysteine is present at about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2.0%.

In some aspects, the cryoprotectant comprises ascorbic acid. In some aspects, the ascorbic acid is present from about 0.005% to about 5%. In some aspects, the ascorbic acid is present at about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, or about 5%.

In some aspects, the cryoprotectant comprises maltodextrin. In some aspects, the maltodextrin is present from about 1% to about 20%. In some aspects, the maltodextrin is present at about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%.

In some aspects, the cryoprotectant comprises a combination of any one or more of sucrose, trehalose, sorbitol, propyl gallate, sodium caseinate, sodium citrate, sodium glutamate, cysteine, ascorbic acid, and/or maltodextrin.

In some aspects, the cryoprotectant comprises sucrose, propyl gallate, sodium caseinate, and sodium citrate. In some aspects, the cryoprotectant comprises sucrose, sorbitol, sodium glutamate, and sodium citrate. In some aspects, the cryoprotectant comprises trehalose, sodium glutamate, and cysteine. In some aspects, the cryoprotectant comprises trehalose, sodium glutamate, ascorbic acid, and maltodextrin.

In some aspects, the cryoprotectant comprises about 15% to about 20% sucrose, about 0.05% to about 1.0% propyl gallate, about 4% to about 8% sodium caseinate, and about 0.2% to about 1.0% sodium citrate. In some aspects, the cryoprotectant comprises about 15% to about 20% sucrose, about 0.5% to about 1.5% sorbitol, about 5% to about 12% sodium glutamate, and about 0.1% to about 1.5% sodium citrate. In some aspects, the cryoprotectant comprises about 5% to about 20% trehalose, about 3% to about 15% sodium glutamate, and about 0.01% to about 1.0% cysteine. In some aspects, the cryoprotectant comprises about 5% to about 20% trehalose, about 3% to about 15% sodium glutamate, about 0.01% to about 2% ascorbic acid, and about 2% to about 18% maltodextrin.

In some aspects, the cryoprotectant further comprises corn syrup, DMSO, all types of starch and/or modified starch, and/or PVP.

The choice of pharmaceutical or stabilizing excipient (cryoprotectant), carrier or diluent is selected based on the intended route of administration and standard pharmaceutical or nutritional practice. In some aspects, such compositions may comprise additional ingredients in addition to the excipient, carrier, or diluent. Such additional ingredients include, but are not limited to, any suitable binders, lubricants, suspending agents, coating agents, solubilizing agents, preservatives, dyes, flavoring agents, antioxidants, and/or suspending agents.

Examples of suitable binders include, but are not limited to, starch, gelatin, and natural sugars. Such natural sugars include, but are not limited to, glucose, anhydrous lactose, free-flowing lactose, beta-lactose, corn sweeteners, and natural and/or synthetic gums such as gum arabic, tragacanth or sodium alginate, carboxymethylcellulose, and polyethylene glycol. Examples of suitable lubricants include, but are not limited to, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. In some aspects, preservatives, stabilizers, dyes, and flavoring agents are also provided in the compositions. Examples of preservatives include, but are not limited to, sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. In some aspects, antioxidants and suspending agents are also present in the composition.

In some aspects, the composition comprises one or more active ingredients and a probiotic as described herein. The active ingredient may be selected from: antibiotics, prebiotics, probiotics, glycans (e.g., baits that limit the binding of specific bacteria/viruses to the intestinal wall), bacteriophages, microorganisms, and the like. As used herein, the term "prebiotic" may be a general term referring to a chemical and/or ingredient that may affect the growth and/or activity of a microorganism in a subject or host (e.g., may allow for a particular change in the composition and/or activity of the flora) and may impart a health benefit to the subject. Prebiotics include, but are not limited to, complex carbohydrates, complex sugars, resistant dextrins, resistant starches, amino acids, peptides, nutritional compounds, biotin, polydextrose, Fructooligosaccharides (FOS), Galactooligosaccharides (GOS), inulin, lignin, psyllium, chitin, chitosan, gums (e.g., guar gum), high amylose corn starch (HAS), cellulose, beta-glucan, hemicellulose, lactulose, Mannooligosaccharides (MOS), fructooligosaccharide-rich inulin, fructooligosaccharides, dextrose oligosaccharides, tagatose, transgalactooligosaccharides, pectin, and Xylooligosaccharides (XOS). In some aspects, an antioxidant ingredient such as, for example, vitamin C is included as a prebiotic substrate to act as an oxygen scavenger. Prebiotic substrates such as these improve colonization and survival of bacteria in the body. In some aspects, the prebiotic is selectively fermented, for example, in the colon.

In some aspects, the prebiotic is present in an amount of about 1 weight percent to about 50 weight percent, relative to the total weight of the composition. In some aspects, the prebiotic is present in an amount of about 2% to about 40% by weight. In some aspects, the prebiotic is present in an amount of about 3% to about 30% by weight. In some aspects, the prebiotic is present in an amount of about 4% to about 25% by weight. In some aspects, the prebiotic is present in an amount of about 5% to about 20% by weight. In some aspects, the prebiotic is present in an amount of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, or 50%.

In some aspects, the prebiotic is present in a greater amount than the probiotic. In some aspects, the prebiotic to probiotic ratio is about 5,000:1, about 4,500:1, about 4,000:1, about 3,500:1, about 3,000:1, about 2,500:1, about 2,000:1, about 1,500:1, about 1,000:1, about 900:1, about 800:1, about 700:1, about 600:1, about 500:1, about 400:1, about 300:1, about 200:1, about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1: 1.

In various aspects, prebiotics are present in food products (e.g., gum arabic, guar seeds, brown rice, rice bran, barley hulls, chicory roots, jerusalem artichoke, dandelion leaves, garlic, leek, onion, asparagus, wheat bran, oat bran, baked beans, whole wheat flour, bananas) and breast milk. In some aspects, the prebiotic is administered in other forms (e.g., a capsule or dietary supplement).

In some aspects, the compositions of the present disclosure further comprise at least one additional bacterium that degrades resistant starch. In some aspects, the bacteria that degrade resistant starch is bifidobacterium adolescentis, ruminococcus brucei, bacteroides thetaiotaomicron, bacteroides ovatus, bifidobacterium breve, or ralstonia enterica.

In some aspects, the disclosure includes a composition, wherein the bacteria survive less than about 1 log unit CFU reduction, less than about 2 log unit CFU reduction, less than about 3 log unit CFU reduction, less than about 4 log unit CFU reduction, less than about 5 log unit CFU reduction, or less than about 6 log unit CFU reduction of viable bacteria within about 12 months after lyophilization in a cryoprotectant.

In some aspects, the present disclosure includes products, such as feeds, food products, dietary supplements, nutraceuticals, nutritional formulas, beverages, and/or pharmaceuticals, comprising one or more bacterial species as disclosed herein, as well as their uses.

In some aspects, the compositions of the present disclosure additionally comprise at least one other type of other food grade bacteria. Such suitable food grade bacteria include, but are not limited to, lactic acid bacteria, bifidobacteria, propionibacteria, and/or mixtures thereof.

In one aspect, the food products of the present disclosure comprise one or more of the bacterial species disclosed herein. The term "food product" is intended to cover all consumable products which may be solid, jelly or liquid. Suitable food products include, but are not limited to, functional food products, food compositions, health foods, pet foods, livestock feeds, and the like. In some aspects, the food product is a health food.

As used herein, the term "functional food product" refers to a food product that is capable of not only providing a nutritional effect, but also delivering additional benefits to the consumer. Thus, a functional food is a general food that incorporates components or ingredients (such as those described herein) that impart specific functional benefits, e.g., medical or physiological benefits, in addition to purely nutritional benefits to the food.

Examples of specific food products include, but are not limited to, dairy-based products, ready-to-eat desserts, powders reconstituted with, for example, milk or water, chocolate milk beverages, malt beverages, ready-to-eat dishes, instant dishes and/or beverages for humans or animals, representing a complete or partial diet. In some aspects, the food product is intended for use with humans, pets or livestock. In some aspects, the compositions are intended for animals, including but not limited to dogs, cats, pigs, cattle, horses, goats, sheep, and/or poultry. In a particular aspect, the food product is intended for use in humans. In some aspects, the food product is intended for adults.

In some aspects, a "dairy-based product" is any liquid or semi-solid dairy product or whey-based product having different fat contents. In some aspects, the milk-based product is, for example, cow's milk, goat's milk, sheep's milk, skim milk, whole milk, milk reconstituted from powdered milk and whey without any processing, or a processed product such as yogurt, curd (cured milk/cure), yogurt, sour whole milk, buttermilk, and/or other yogurt products. Dairy-based products include milk beverages such as whey beverages, fermented milks, condensed milks, baby milks (infant/baby milk); flavored milk, ice cream; and milk-containing foods such as confections.

In some aspects, the product is a feed or animal feed comprising a bacterial species described herein.

In some aspects, the compositions described herein are or may be added to a food supplement, i.e., also referred to herein as a dietary or nutritional supplement or food additive. Accordingly, the present disclosure includes a dietary supplement or food additive comprising one or more of the bacterial species described herein.

The bacterial species or strains and compositions described herein are useful for human and/or animal nutrition. In some aspects, the bacterial species and compositions are used in the premature weaning and growing-finishing stages. Probiotics comprising one or more bacteria as described herein are expected to enhance immune function, treat and/or prevent infectious diseases, beneficially alter the microbiota flora of the treated subject, and improve growth and performance. In some aspects, bacterial species and compositions comprising them provide such benefits by increasing feed conversion efficiency.

In some aspects, the composition is formulated as a dietary supplement. Such compositions may be incorporated with vitamin supplements. Such compositions may be formulated in chewable forms, such as probiotic gummies or soft chews. Such compositions may be incorporated into the form of food and/or beverages. For example, non-limiting examples of food and beverages incorporating the compositions include bars, milkshakes, fruit juices, infant formulas, beverages, frozen food products, fermented food products, and cultured dairy products (such as yogurt, yogurt drinks, cheese, lactic acid bacteria drinks, and kefir).

The formulations of the present disclosure may be administered as part of a fecal transplantation procedure. The formulation may be administered to a subject via a tube, such as a nasogastric tube, nasojejunal tube, nasoduodenal tube, oral gastric tube, oral jejunal tube, or oral duodenal tube. The formulation may be administered to the subject by colonoscopy, endoscopy, sigmoidoscopy, and/or enema.

In some aspects, the composition is formulated such that the one or more bacteria present in the composition are replicable once delivered to the target habitat (e.g., intestinal tract). In one non-limiting example, the microbial composition is formulated as a pill, powder, capsule, tablet, enteric-coated dosage form, or package such that the composition has a shelf life of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 months. In some aspects, other components are added to the composition to aid the shelf life of the composition. In some aspects, the one or more bacteria may be formulated in a manner that allows survival in a non-natural environment. For example, bacteria native to the gut may not survive in an oxygen-rich environment. To overcome this limitation, the bacteria may be formulated in a pill or package that reduces or eliminates exposure to oxygen. Other strategies to extend the shelf life of bacteria may include the use of other microorganisms (e.g., if the bacterial consortium comprises a composition whereby one or more strains contribute to the survival of one or more strains).

In some aspects, the compositions of the present disclosure are lyophilized (e.g., freeze-dried) and formulated into a powder, tablet, capsule, enteric-coated dosage form (e.g., for delivery to the ileum/colon), or pill that can be administered to a subject by any suitable route. The lyophilized formulation may be mixed with saline or other solution or solvent prior to administration.

In some aspects, the compositions are formulated for oral administration, e.g., as a powder, tablet, capsule, enteric-coated dosage form, or pill, to deliver the contents of the formulation to the ileum and/or colonic region of a subject.

In some aspects, the composition is formulated for oral administration. In some aspects, the composition is formulated as a powder, tablet, capsule, enteric-coated dosage form, or pill for oral administration. In some aspects, the composition is formulated for delivery of bacteria to the ileal region of a subject. In some aspects, the composition is formulated for delivery of bacteria to a colonic region (e.g., upper colon) of a subject. In some aspects, the compositions are formulated for delivery of bacteria to the ileum and colon regions of a subject.

Enteric coatings protect the contents of an oral formulation (e.g., tablet or capsule) from the acidity of the stomach and provide delivery to the ileum and/or upper colon regions. Non-limiting examples of enteric coatings include pH sensitive polymers (e.g., Eudragit L30D-55, Eudragit S100, or Eudragit FS30D), methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (e.g., hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, shellac, cellulose trimellitate acetate, sodium alginate, zein, other polymers, fatty acids, waxes, shellac, plastics, and vegetable fibers. In some aspects, the enteric coating is formed from a pH-sensitive polymer. In some aspects, the enteric coating is formed from Eudragit L30D-55, Eudragit S100, or Eudragit FS 30D.

In some aspects, the enteric coating may be applied directly to the probiotic or prebiotic to protect the probiotic or prebiotic from the acidity of the stomach and provide delivery to the ileum and/or upper colon regions. For example, the probiotic or prebiotic may be coated with an enteric coating which may include pH sensitive polymers (e.g., Eudragit L30D-55, Eudragit S100, or Eudragit FS30D), methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (e.g., hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, shellac, cellulose trimellitate acetate, sodium alginate, zein, other polymers, fatty acids, waxes, shellac, plastics, and vegetable fibers. In some aspects, the enteric coating is formed from a pH-sensitive polymer. In some aspects, the enteric coating is formed from Eudragit L30D-55, Eudragit S100, or Eudragit FS 30D.

In some aspects, the enteric coating can be designed to dissolve at any suitable pH. In some aspects, the enteric coating is designed to dissolve at a pH greater than about pH 5.0, or at a pH greater than about pH 6.0, or at a pH greater than about pH 7.0. In some aspects, the enteric coating is designed to dissolve at a pH of greater than about pH 5.0 to about pH 7.0. In some aspects, the enteric coating is designed to dissolve at a pH greater than about pH 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, or 7.5.

In some aspects, administration of a composition or formulation of the present disclosure is preceded by, for example, a colon cleansing method, such as colon irrigation/water therapy, enema, administration of laxatives, dietary supplements, dietary fibers, enzymes, and magnesium. In some aspects, administration of a composition or formulation of the present disclosure is preceded by a step of administering one or more antibiotics to reduce existing microbiome, e.g., prior to administration of the composition or formulation.

In some aspects, the bacteria are formulated as a spore population. The spore-containing formulation can be administered by any suitable route described herein. Oral spore-containing preparations survive the low pH conditions of the stomach. The amount of spores employed can be, for example, from about 1% w/w to about 99% w/w of the total formulation.

The formulations provided herein can include the addition of one or more agents to the composition to enhance the stability and/or survival of the microbial preparation. Non-limiting examples of stabilizers include genetic factors, glycerol, ascorbic acid, skim milk, lactose, tween, alginate, xanthan gum, carrageenan, mannitol, palm oil, and poly-L-lysine (POPL).

The terms "subject," "individual," "host," and "patient" are used interchangeably herein and refer to any animal subject, including humans, laboratory animals, livestock, and domestic pets. The subject may host a variety of microorganisms. The subjects may have different microbiomes on their bodies and in various habitats within the bodies. The subject may be diagnosed or suspected of being at high risk for a particular disorder or disease. The subject may have a microbiome status that contributes to disease (dysbiosis). In some aspects, the subject is not necessarily diagnosed or suspected of being at high risk for disease. In some aspects, the subject may have a particular disorder or disease.

In some aspects, the present disclosure provides methods for treating a subject. The terms "treatment" or "treating" are used interchangeably herein. These terms may refer to methods for achieving beneficial or desired results, including but not limited to therapeutic benefits and/or prophylactic benefits. In various aspects, a therapeutic benefit includes eradication or alleviation of the underlying disorder, disease, or condition being treated. Additionally, in some aspects, a therapeutic benefit is achieved by eradicating or alleviating one or more physiological symptoms associated with the underlying disorder, disease, or condition such that an improvement is observed in the subject, even though the subject may still be suffering from the underlying disorder, disease, or condition. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disorder, disease, or condition, delaying or eliminating the onset of symptoms of the disorder, disease, or condition, slowing, stopping, or reversing the progression of the disorder, disease, or condition, or any combination thereof. For prophylactic benefit, a subject at risk for a particular disorder, disease, or condition, or a subject reporting one or more of the physiological symptoms of a disorder, disease, or condition, may be treated even if a diagnosis of the disorder, disease, or condition has not been made.

In some aspects, the present disclosure includes a subject that supplements the present disclosure. In some aspects, the compositions of the present disclosure include supplementing a human diet with a bacterium of the present disclosure and a composition comprising one or more types of the bacterium of the present disclosure.

Altering the composition of the microbiome in a subject may have desirable health consequences. In some aspects, the compositions of the present disclosure are administered as a therapeutic and/or prophylactic agent for the treatment and/or prevention of a disorder, disease, or condition. Treatments designed to alter the host microbiome may alleviate symptoms, prevent disease, and/or treat a disorder, disease, or condition. For example, modifying the gut microbiome may reduce the risk of health disorders such as metabolic disorders.

The present disclosure also provides methods for restoring a microbial habitat of a subject to a healthy state. In some aspects, the methods include microbiome correction and/or adjustment in the subject, including, for example, supplementation with natural microbes, administration of probiotics (including probiotics), removal of pathogenic microbes, administration of prebiotics, and/or administration of growth factors necessary for the survival of the microbiome. In some aspects, the method further comprises administering an antimicrobial agent, such as an antibiotic, to reduce or remove harmful bacteria.

In some aspects, the bacteria of the present disclosure and compositions comprising one or more types of bacteria of the present disclosure are used to alter the composition of microbiome in a subject with a desired health outcome. In some aspects, the compositions of the present disclosure are administered as a therapeutic, prophylactic, or nutraceutical agent. Treatments designed to alter the host microbiome may alleviate a patient's symptoms, prevent a disease, and or treat a disease, disorder, or condition. In various aspects, the terms "disease," "disorder," or "condition" are used interchangeably. Thus, in some aspects, such "disorders" are intestinal disorders, metabolic disorders, inflammatory disorders, or immune disorders. For example, modification of the gut microbiome may reduce the risk of adverse health conditions, such as a disorder being an intestinal disorder, a metabolic disorder, an inflammatory disorder, or an immune disorder. In addition, modification of the gut microbiome may provide beneficial effects for healthy weight, blood glucose management, and/or gastrointestinal health.

In various aspects, such "disorders" include, but are not limited to, insulin resistance, insulin sensitivity, prediabetes, diabetes or type 2 diabetes (T2DM), irritable bowel syndrome, metabolic irregularities, obesity-related conditions, hypertension, stress-related conditions, drug metabolism, gastrointestinal tract infections, Inflammatory Bowel Disease (IBD), crohn's disease. In some aspects, such disorders are low body weight, and the compositions are used to increase the body weight of such subjects. In some aspects, such disorders are high body weight, and the compositions are used to reduce the body weight of such subjects. In another aspect, the composition is used to maintain the current body weight of the subject.

In some aspects, the bacteria of the present disclosure and compositions comprising one or more bacteria of the present disclosure are used to help support healthy weight maintenance, help support healthy weight management, help a subject feel less hungry between meals, help maintain healthy blood glucose levels, help maintain blood glucose levels within a normal range, help support digestive/intestinal health and provide better bacteria.

Butyrate is an anti-inflammatory factor that can affect intestinal permeability. Low levels of butyrate producing bacteria as well as reduced lactate producing bacteria are associated with diabetes, T2DM, obesity and metabolic disorders. Thus, increasing the level of butyrate producing bacteria in a subject is beneficial for preventing and/or treating such diseases, disorders, or conditions.

The present disclosure also provides methods for restoring a microbial habitat of a subject to a healthy state. In some aspects, such methods include microbiome correction and/or adjustment, including, for example, supplementation of native microorganisms, removal of pathogenic microorganisms, administration of prebiotics, and growth factors necessary for the survival of the microbiome. Thus, in some aspects, the method further comprises administering an antimicrobial agent, such as an antibiotic.

Administering a composition comprising a bacterium described herein to achieve a prophylactic and/or therapeutic treatment. In various aspects, administration is by ingestion. In therapeutic applications, the composition is administered to a subject already having a disease, disorder, or condition in an amount effective to cure or at least partially inhibit the symptoms of the disease, disorder, or condition, or to cure, heal, ameliorate, or alleviate the condition. In some aspects, such compositions are administered to reduce the likelihood of suffering from, infecting, or worsening a disorder. The amount effective for this use will vary depending on the severity and course of the disease, disorder or condition, previous therapy, the subject's health, weight and response to the drug, and the judgment of the treating physician.

In some aspects, the composition is administered (e.g., ingested) with another therapeutic agent or active ingredient, and the composition and agent or active ingredient can be administered in any order or simultaneously. As described herein, the composition can be administered before, during, or after the onset or observation of a disease, disorder, or condition, and the time at which the composition is administered can vary. For example, in some aspects, the composition is used as a prophylactic and can be continuously administered to a subject having a predisposition to a disease, disorder or condition, in order to reduce the likelihood of occurrence of the disease, disorder or condition. In some aspects, the composition is administered to the subject during or as soon as possible after the onset of symptoms.

In some aspects, the composition is packaged as a kit. In some aspects, the kit includes written materials or instructions regarding the administration/use of the composition. In some aspects, the written material is a label. In some aspects, the written material provides methods of administration, including methods of consumption.

In some aspects, the composition is administered orally, enterally, or rectally. For example, in some aspects, the composition is an edible composition. By "edible" is meant a material that is approved for human and/or animal consumption. In some aspects, the edible composition is present in a food product or feed product as described herein.

Other suitable oral administrations include, but are not limited to, the use of compressed tablets, pills, gels, drops, capsules, powders, liquids, solutions, and emulsions. In some aspects, it is advantageous to encapsulate the product when the bacteria are anaerobic bacteria. In some aspects, the composition is administered as part of a food or nutritional product (such as milk or a whey-based fermented milk product), or as a pharmaceutical product.

In some aspects, suitable pharmaceutical compositions are in the form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions and/or dusting powders. In some aspects, a suitable composition is administered topically. For example, in some aspects, the active ingredient (e.g., one or more bacterial species of the present disclosure) is incorporated into a cream consisting of an aqueous emulsion of, for example, polyethylene glycol or liquid paraffin, or into an ointment consisting of a white wax or white soft paraffin base, along with such stabilizers and/or preservatives as may be desired.

In some aspects, the compositions are formulated in unit dosage form, i.e., in discrete portions containing a unit dose, or multiple doses, or unit dose subunits.

For example, a typical or common suitable or effective dose of the one or more bacterial species in humans is from about 1 × E3(1 × E3 ═ 1 × 10^3 ═ 1 × (power of 3 of 10)) to about 1 × E13 Colony Forming Units (CFU). In some cases, a suitable or effective dose is about 1 × E6CFU to about 1 × E11 CFU. In particular instances, a suitable or effective dose is about 1 × E7CFU to about 1 × E10 CFU. In some further aspects, a suitable or effective dose of a bacterial species is about 1 × E2CFU, 1 × E3CFU, 1 × E4CFU, 1 × E5CFU, 1 × E6CFU, 1 × E7CFU, 1 × E8CFU, 1 × E9CFU, 1 × E10CFU, 1 × E11CFU, 1 × E12CFU, 1 × E13CFU, 1 × E14CFU, or 1 × E15 CFU.

In some aspects, the compositions of the present disclosure are administered or consumed 1, 2, 3, 4, 5, or more times per day. In some aspects, the dose is daily, every other day, three times a week, twice a week, once every other week, or at other suitable intervals for treating the condition.

Thus, in some aspects, the compositions comprise, as an active ingredient, one or more bacterial species and/or cellular components thereof in an amount of from about 1 × E3 to about 1 × E13 Colony Forming Units (CFU)/gram (g) relative to the weight of the composition. In some aspects, one or more bacterial species and/or cellular components thereof are present in an amount of about 1 × E5CFU/g to about 1 × E11 CFU/g. In some aspects, one or more bacterial species and/or cellular components thereof are present in an amount of about 1 × E6CFU/g to about 1 × E10 CFU/g. In some aspects, one or more bacterial species and/or cellular components thereof are present in the composition in an amount of about 1 × E8CFU/g to about 1 × E10 CFU/g. In some aspects, the compositions comprise one or more bacterial species and/or cellular components thereof present in an amount of about 1 × E1CFU/g, about 1 × E2CFU/g, about 1 × E3CFU/g, about 1 × E4CFU/g, about 1 × E5CFU/g, about 1 × E6CFU/g, about 1 × E7CFU/g, about 1 × E8CFU/g, about 1 × E9CFU/g, about 1 × E10CFU/g, about 1 × E11CFU/g, about 1 × E12CFU/g, about 1 × E13CFU/g, about 1 × E14CFU/g, or about 1 × E15 CFU/g.

Thus, in some aspects, the dose of bacteria in the composition is about 0.1 milligram (mg), about 0.2mg, about 0.3mg, about 0.4mg, about 0.5mg, about 0.6mg, about 0.7mg, about 0.8mg, about 0.9mg, about 1.0mg, about 2.0mg, about 3.0mg, about 4.0mg, about 5.0mg, about 6.0mg, about 7.0mg, about 8.0mg, about 9.0mg, about 10mg, about 15mg, about 20mg, about 25mg, about 30mg, about 35mg, about 40mg, about 45mg, about 50mg, about 55mg, about 60mg, about 65mg, about 70mg, about 75mg, about 80mg, about 85mg, about 90mg, about 95mg, about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 1 gram, or about 1 gram. In some aspects, the dose of bacteria (i.e., probiotics) in the composition is in the range of about 1mg to about 500 mg. In some aspects, the dose of probiotic is in the range of about 2mg to about 300 mg. In some aspects, the dose of probiotic is in the range of about 5mg to about 100 mg.

In the present disclosure, the terms "comprising," including, "" having, "and conjugates thereof mean" including, but not limited to. The term "consisting of … …" means "including and limited to". As used herein, "consisting essentially of … …" means that a composition, method, or structure may include additional ingredients, steps, and/or portions, but only if the additional ingredients, steps, and/or portions do not materially alter the basic and novel characteristics of the bacteria, composition, method, or use as claimed.

The disclosure is further described by the following non-limiting examples. It is understood that the examples, embodiments and aspects described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes to be consistent with the present disclosure.

Examples

Other aspects and details of the present invention will be apparent from the following examples, which are intended to be illustrative and not limiting.

Example 1: isolation of short chain fatty acid producing bacteria

Experiments were performed to isolate SCFA-producing bacteria. Feces were placed under anaerobic conditions shortly after donation and immediately processed. Stool samples from multiple donors were pooled and resuspended 1:10 in M9 minimal medium (Na)₂HPO₄ 6g/L、KH₂PO₄ 3g/L、NaCl 0.5g/L、NH₄Cl 1g/L、CaCl₂ 0.1mM、MgSO₄1mM) and passed through a 100 μm cell sieve to remove larger insoluble material. Samples were washed twice and resuspended in M9 minimal medium to initial concentration. The slurry was then serially diluted and 100 μ l of each sample was plated onto various microbial culture plates. The culture plates are Reinforced Clostridium Agar (RCA) (Hardy Diagnostics C8721), yeast casein peptone fatty acid agar (YCFA) (casein peptone 10g/L, yeast extract 2.5g/L, NaHCO) ₃4g/L, 1g/L, K g cysteine₂HPO₄ 0.45g/L、KH₂PO₄ 0.45g/L、NaCl 0.9g/L、(NH₄)₂SO₄ 0.9g/L、MgSO₄·7H₂O 0.09g/L、CaCl₂0.09g/L, 10mg/L of blood crystal, 1mg/L of resazurin, 10. mu.g/L, Cabalamin 10. mu.g/L of biotin, 30. mu.g/L of p-aminobenzoic acid, 50. mu.g/L of folic acid, 150. mu.g/L of pyridoxine, 50. mu.g/L of thiamine, 50. mu.g/L of riboflavin, 1.9ml/L of acetic acid, 0.7ml/L of propionic acid, 90. mu.l/L of isobutyric acid, n-pentyleneAcid 100. mu.l/L, isovaleric acid 100. mu.l/L), supplemented with 1% unmodified potato starch (Bob's Red Mill Natural Foods), Enriched Trypticase Soy Agar (ETSA) (Anirobe Systems AS-548), Brain Heart Infusion (BHI) (Anirobe Systems AS-6426), MTGE (Anirobe Systems AS-777), M17(Difco BD 218561), supplemented with 1% unmodified potato starch. Under anaerobic conditions (5% H)₂、15％CO₂、80％N₂) Next, the plate was incubated at 37 ℃ for 48 hours.

Morphologically distinct colonies were picked from each plate and placed in RCB medium (AS-606, Anerobe Systems) and modified YCFA (i.e., yeast, casein peptone, fatty acid) broth (casein peptone 10g/L, yeast extract 2.5g/L, NaHCO) in 96 deep-well plates₃4g/L, 1g/L, K g cysteine₂HPO₄ 0.45g/L、KH₂PO₄ 0.45g/L、NaCl 0.9g/L、(NH₄)₂SO₄ 0.9g/L、MgSO₄·7H₂O 0.09g/L、CaCl₂0.09g/L, 10mg/L of hemin, 1mg/L of resazurin, 10. mu.g/L, Cabalamin 10. mu.g/L of biotin, 30. mu.g/L of p-aminobenzoic acid, 50. mu.g/L of folic acid, 150. mu.g/L of pyridoxine, 50. mu.g/L of thiamine, 50. mu.g/L of riboflavin, 1% potato starch). After incubation at 37 ℃ overnight under anaerobic conditions, butyrate, acetate and propionate levels in the supernatants from each well were measured (see analytical methods below).

Organisms positive for butyrate were isolated from the wells and identified by MALDI TOF or 16s-rRNA sequences (accutenix, Charles River Labs, Inc.). Individual bacterial colonies were lysed with 1 μ l of 70% formic acid, dried, and 1 μ l of the matrix was lysed on a MALDI TOF plate, dried and loaded onto the instrument where they were ionized by a laser. The matrix absorbs most of the energy and transfers it to the sample. The ionized sample is broken down into smaller pieces which are then pulled into the mass analyzer region and separated based on their mass-to-charge ratio (m/z). Bacteria were identified by comparing their spectra to spectra of known bacteria in an instrument database.

Example 2: lyophilization of short chain fatty acid producing bacteria

Experiments were performed to determine the extent of bacterial survival after lyophilization. The bacteria were cultured by inoculating 100ml of a suitable bacterial culture medium with a single bacterial isolate, followed by incubation at 37 ℃ under anaerobic conditions. Samples (200 μ Ι) were collected from the cultures at various time points for analysis of OD600, 10 μ Ι were spotted onto solid bacterial media for Colony Forming Unit (CFU) assay, and for aliquoting into four different freeze-dried media. The lyophilized vials were snap frozen in dry ice methanol and stored at-80 ℃ until lyophilized. The four freeze-drying media used were: medium 1 (sucrose 17.8%, propyl gallate 0.2%, sodium caseinate 6.4%, sodium citrate 0.6%); medium 2 (sucrose 17.5%, sorbitol 0.9%, sodium glutamate 8.5%, sodium citrate 0.6%); medium 3 (trehalose 15%, sodium glutamate 8.5%, cysteine 0.1%); and medium 4 (trehalose 10%, sodium glutamate 8.5%, ascorbic acid 0.2%, maltodextrin 10%). The samples were lyophilized in a VirTis Wizard 2.0 advance plus lyophilizer under the conditions set forth in table 3 below.

TABLE 3 Freeze drying conditions

The number of CFUs was determined before and after lyophilization to determine the number of bacteria that survived the lyophilization process. Samples were serially diluted and 10 μ Ι were spotted in triplicate onto square RCB agar plates with grids. After 24-48 hours of incubation, bacterial colonies were visually counted.

Example 3: short chain fatty acid quantification from bacterial isolates

Experiments were performed to quantify short chain fatty acid production in bacterial isolates. The supernatant from the bacterial isolates was diluted 20-fold with acetonitrile and 20. mu.l of each diluted sample was mixed with 500. mu.l of the derivatization mixture (triphenylphosphine; 2,2' -dipyridyl disulfide; hydrazinoquinoline mixed in equal volumes to a final concentration of 2mM, containing the internal standard of work) in a 2ml96 well deep well plate, capped, and heated at 65 ℃ for 1 hour. After incubation, the plates were removed and placed at 4 ℃ for 15 minutes, then vortexed gently and centrifuged at 2000rpm for 1 minute. Samples (150. mu.l in each well) were transferred to a clean 96-well plate containing 300. mu.l acetonitrile per well. The derivatized samples were analyzed by high performance liquid chromatography/MS (AB Sciex triple Quad 4000) using a Waters Xselect HSS C18 SB 2.1 × 50mm × 2.5 μ M column, using 0.1% formic acid in water as mobile phase a, and acetonitrile with 0.1% formic acid as mobile phase B. The internal standards are butyric acid-d 7, propionic acid-d 6 and acetic acid-d 3. The HPLC parameters used are listed in Table 4 below.

TABLE 4 HPLC parameters

The concentrations of butyric acid, propionic acid and acetic acid were determined. For example, the amount of butyrate produced by the bacteria is provided in table 5 below.

TABLE 5 butyrate production

Example 4: determination of preferred media for bacterial growth

Experiments were performed to determine the preferred medium for bacterial growth. Single bacterial isolates were grown in anaerobic chambers at 37 ℃ for 48 hours in various media, such AS RCB (Hardy Diagnostics C8721), RCB (Anserobe Systems (AS-606)), BHI (Anserobe Systems sAS-6426), peptone Yeast glucose agar (PYG) (Anserobe Systems, AS-606), YCFA + 1.0% resistant starch (AS described in example 1 above), TSB (Difco BD 211825), MTGE (Anserob)e Systems AS-777) or GM17 (M17 medium supplemented with 0.5% glucose (Difco BD 218561). In a spectrophotometer (OD)₆₀₀nM) was measured at different time points.

Example 5: selection and sequencing of short chain fatty acid producing bacteria

Bacteria that appeared to produce the highest levels of SCFA and were resistant to lyophilization were isolated and the 16S-rRNA encoding genes from each of these bacteria (see table 1) were sequenced (Charles River' S))。

Example 6: determination of oxygen sensitivity of short chain fatty acid producing bacteria

Because strictly anaerobic bacteria are difficult to grow in large quantities outside anaerobic conditions, the oxygen sensitivity of various bacteria was tested under various microaerobic conditions. Bacteria found to produce the greatest levels of SCFA and resistant to lyophilization were subjected to microaerophilic conditions. The single bacterial isolates were incubated at 37 ℃ for 24h-48h in a microaerobic anaerobic chamber with oxygen controlled between 50ppm-150ppm and serially diluted in phosphate buffered saline. 10 μ L were spotted onto RCB agar plates for Colony Forming Unit (CFU) assay. Immediately after spotting of the plates, one plate was retained in the anaerobic chamber and the other plate was removed from the anaerobic chamber and exposed to oxygen in ambient air. After exposure to oxygen for various times, the plates exposed to oxygen in ambient air were placed back into the anaerobic chamber and incubated for 24h-48h to allow CFU development. Bacterial growth from all plates was determined by CFU formation.

Example 7: determination of the Whole genome sequence of short chain fatty acid producing bacteria

Bacteria found to produce the greatest levels of SCFA and resistant to lyophilization (e.g., one of two isolates per genus and species) were sequenced to determine their whole genome (MOgene, st. The whole genome sequences (SEQ ID NOS: 24-35) of various SCFA-producing bacteria of the present disclosure are provided in Table 6 below, along with their corresponding 16S-rRNA-encoding gene sequences.

Table 6-whole genome sequence of probiotics of the present disclosure

These sequences, i.e., SEQ ID NOs 27, 28 and 31-33, comprise a discontinuous final whole genome of each bacterial species of the present disclosure. The genomic sequence of the asterisked species may be discontinuous due to gaps and/or lack of assembly confidence due to repeated regions, and gaps are identified in the sequences in the sequence listing.

In addition, the presence of potential toxin or antibiotic resistance genes in the bacteria is identified.

Combination of

A. A composition, comprising:

(a) at least one human isolate, or a mixture thereof, of a Short Chain Fatty Acid (SCFA) -producing bacterium, wherein the bacterium comprises a 16S ribosomal RNA (16S-rRNA) encoding gene sequence having at least about 80% identity to any one of the nucleotide sequences of SEQ ID NOS: 1-23, or a DNA sequence having at least about 80% identity to any one of the nucleotide sequences of SEQ ID NOS: 24-35, and

(b) excipients, carriers and/or diluents.

B. The composition of paragraph B, wherein the bacteria is selected from the group consisting of: agrobacterium recalis, anaerobic Corynebacterium faecalis (Anaerobiosis caccae), Anaerobiosis hadrus, Butyricoccus faecalis, Clostridium butyricum (Clostridium butyricum), Clostridium curosum (Clostridium cochleariae), Clostridium innocuum (Clostridium innoculum), Coprococcus (Coprococcus comatus), Flavonociceptor platii, Ralstonia faecalis (Roseburia faecalis), Rourella anthropogonis (Roseburia hominis), Rourella enterica (Roseburia intestinalis), and Gluconobacter acidilans (Roseburia inivorans).

C. The composition of paragraph a or B, wherein the bacteria are lyophilized.

D. The composition of any of paragraphs a-C, wherein the excipient is a cryoprotectant.

E. The composition of paragraph D, wherein the cryoprotectant comprises a sugar or sugar alcohol.

F. The composition of paragraph E, wherein the cryoprotectant further comprises any one or more of propyl gallate, sodium caseinate, sodium citrate, sodium glutamate, cysteine, ascorbic acid, and/or maltodextrin.

G. The composition of any of paragraphs D-F, wherein the cryoprotectant comprises

(a) About 1% to about 25% sucrose;

(b) about 1% to about 25% trehalose;

(c) about 0.1% to about 5% sorbitol;

(d) from about 0.05% to about 1.0% propyl gallate;

(e) from about 0.5% to about 10% sodium caseinate;

(f) from about 0.1% to about 5% sodium citrate;

(g) from about 1% to about 15% sodium glutamate;

(h) about 0.01% to about 2.0% cysteine;

(i) from about 0.005% to about 5.0% ascorbic acid;

(j) about 1% to about 20% maltodextrin; or

(k) Any one or more of (a) - (j) in combination.

H. The composition of any of paragraphs D-G, wherein the cryoprotectant comprises

(a) From about 15% to about 20% sucrose, from about 0.05% to about 1.0% propyl gallate, from about 4% to about 8% sodium caseinate, and from about 0.2% to about 1.0% sodium citrate;

(b) about 15% to about 20% sucrose, about 0.5% to about 1.5% sorbitol, about 5% to about 12% sodium glutamate, and about 0.1% to about 1.5% sodium citrate;

(c) about 5% to about 20% trehalose, about 3% to about 15% sodium glutamate, about 0.01% to about 1.0% cysteine; or

(d) About 5% to about 20% trehalose, about 3% to about 15% sodium glutamate, about 0.01% to about 2% ascorbic acid, and about 2% to about 18% maltodextrin.

I. The composition of any of paragraphs D-H, wherein the cryoprotectant comprises

(a) About 17.8% sucrose, about 0.2% propyl gallate, about 6.4% sodium caseinate, and about 0.6% sodium citrate;

(b) about 17.5% sucrose, about 0.9% sorbitol, about 8.5% sodium glutamate and about 0.6% sodium citrate;

(c) about 15% trehalose, about 8.5% sodium glutamate, and about 0.1% cysteine; or

(d) About 10% trehalose, about 8.5% sodium glutamate, about 0.2% ascorbic acid and about 10% maltodextrin.

J. The composition of any of paragraphs a-I, wherein the bacteria survive less than about 1 log unit CFU reduction, less than about 2 log unit CFU reduction, less than about 3 log unit CFU reduction, less than about 4 log unit CFU reduction, less than about 5 log unit CFU reduction, or less than about 6 log unit CFU reduction of viable bacteria within about 12 months after lyophilization in a cryoprotectant.

K. The composition of any of paragraphs a-J, wherein the composition comprises from about 1 xe 3 to about 1 xe 11 Colony Forming Units (CFU) of SCFA-producing bacteria and mixtures thereof.

L. the composition of any of paragraphs a-K, wherein the bacteria and/or mixtures thereof produce at least about 1000 micromoles of SCFA within about 24 hours.

The composition of any one of paragraphs a-L, wherein the SCFA are acetate, propionate, or butyrate, or a combination thereof.

The composition of any of paragraphs a-M, wherein the SCFA are butyrate.

O. the composition according to any of paragraphs a-N, wherein the composition further comprises a prebiotic and/or a resistant starch.

P. the composition according to any of paragraphs a-O, wherein the composition further comprises at least one additional bacterium that degrades resistant starch.

A composition according to paragraph P, wherein the resistant starch degrading bacteria is Bifidobacterium adolescentis (Bifidobacterium adolescentis), Ruminococcus branchii (Ruminococcus braunii), bacteroides thetaiotaomicron (Bacteriodes thetaiotaomicron), bacteroides ovatus (bacteroides ovatus), Bifidobacterium breve (Bifidobacterium brave) or rosporium entericum (rosebria intestinalis).

R. the composition according to any of paragraphs a-Q, wherein the composition is a probiotic composition.

S. a method of increasing Short Chain Fatty Acids (SCFA) in the gastrointestinal tract of a subject, comprising administering to the subject an effective amount of a composition according to any of paragraphs a-R.

T. the method of paragraph S, wherein the SCFA are acetate, propionate, or butyrate, or a combination thereof.

The method of paragraphs S or T, wherein the SCFA are butyrate.

V. the method of any of paragraphs S-U, wherein the subject has or is at risk of having an obstacle.

The method of any one of paragraphs S-V, wherein the disorder is an intestinal disorder, a metabolic disorder, an inflammatory disorder, or an immune disorder.

X. the method according to paragraph V or W, wherein the disorder is insulin resistance, insulin sensitivity, prediabetes, diabetes or type 2 diabetes (T2DM), irritable bowel syndrome, metabolic irregularity, obesity-related disorders, hypertension, stress-related disorders, drug metabolism irregularity, gastrointestinal infection, Inflammatory Bowel Disease (IBD), or crohn's disease.

A method for reducing or maintaining glucose levels and/or body weight in a subject in need thereof, comprising administering to the subject an effective amount of a composition according to any one of paragraphs a-R.

The method of claim Y, wherein the subject has diabetes or pre-diabetes.

A method for treating, reducing, or preventing a disorder in a subject having or at risk of having the disorder, comprising administering to the subject an effective amount of the composition of any of paragraphs a-R.

BB. the method of paragraph AA, wherein the disorder is an intestinal disorder, a metabolic disorder, an inflammatory disorder, or an immune disorder.

The method according to paragraph AA or BB, wherein the disorder is insulin resistance, insulin sensitivity, prediabetes, diabetes or type 2 diabetes (T2DM), irritable bowel syndrome, metabolic irregularity, obesity-related disorders, hypertension, stress-related disorders, drug metabolism, gastrointestinal infections, Inflammatory Bowel Disease (IBD), or crohn's disease.

DD. use of a composition according to any of paragraphs a-R for the manufacture of a medicament or nutritional product for the treatment of a metabolic, immune, intestinal or inflammatory disorder.

The present disclosure has been described in terms of specific embodiments that find or suggest specific modes for practicing the methods and compositions of the invention described herein. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention. While the present disclosure provides specific embodiments or aspects, it should be understood that the claimed invention should not be unduly limited to such specific embodiments or aspects. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Each document cited herein, including any cross referenced or related patent or patent application and any patent application or patent to which this application claims priority or its benefits, is hereby incorporated by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with any disclosure of the invention or the claims herein or that it alone, or in combination with any one or more of the references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.