Bacillus coagulans strains, compositions and methods of use thereof

Abstract

The present invention relates to a novel Bacillus coagulans strain, which can be used as a probiotic, alone or in combination with other Bacillus strains, or in combination with prebiotics and synbiotics. The present invention also relates to a composition, such as a pharmaceutical composition, a dairy product, a functional food, a nutraceuticals, a dietary supplement and a product for personal care, comprising a novel Bacillus coagulans strain, alone or in combination with other one or more strains, and to the use and other uses of said strains for the prevention or treatment of gastrointestinal infections and diseases.

Description

Technical Field

[0001] The present invention relates to a novel strain of Bacillus coagulans, which can be used as a probiotic alone or in combination with other Bacillus strains or used with prebiotics and synbiotics. The present invention also relates to a composition, such as a pharmaceutical composition, a dairy product, a functional food, a nutraceutical, a dietary supplement and a product for personal care, comprising the novel strain of Bacillus coagulans alone or in combination with one or more other strains, and the use of the strain for preventing or treating gastrointestinal infections and diseases and other uses. Background Art

[0002] Probiotics are live microorganisms or mixtures of microorganisms for administration to improve the patient's microbial balance, particularly the respiratory and gastrointestinal environments. Bacillus (Bacillus) strains have been used to treat respiratory infections, prevent diarrhea, and treat immune-related diseases (Elshaghabee et al., 2017).

[0003] Normal intestinal flora is dominated by various bacterial species, which produce substances that help control the growth of pathogens. Dysbiosis is a condition characterized by a decrease in certain bacterial species and an increase in the growth of pathogens. Dysbiosis has been associated with the development of periodontal disease, inflammatory bowel disease, and chronic fatigue syndrome. Some studies have shown that patients with dysbiosis may be at increased risk for metabolic and cardiac diseases (Chan et al., 2013).

[0004] By administering probiotic Bacillus, it was possible to regenerate the intestinal flora in men and women with recurrent episodes of dysbiosis. Dysbiosis is a common gastrointestinal problem. Dysbiosis caused by Escherichia coli is also a common problem (Chan et al., 2013).

[0005] The presence of bacilli is important for maintaining the intestinal microbial ecosystem. Bacilli have been shown to have inhibitory activity against the growth of pathogens such as Listeria monocytogenes, Escherichia coli, and Salmonella spp (Yilmaz et al., 2005). This inhibitory effect may be due to the production of inhibitory compounds (such as organic acids, hydrogen peroxide, bacteriocins or reuterins) or competitive adhesion to the epithelium (Abriouel et al., 2010).

[0006] Bacilli have also been examined for the treatment of respiratory tract infections (Marseglia et al., 2007). For example, assembly of bacilli and stimulation of indigenous organisms have been used to prevent recurrence of urinary tract infections (Marseglia et al., 2007). Bacilli have also been studied for their role in preventing intestinal infections.

[0007] Description of Related Technology

[0008] The importance of Bacillus species as probiotics has been described in the literature.

[0009] Hyronimus et al., 2000 disclosed the screening of many Bacillus strains for probiotic activity by in vitro techniques and evaluated the colonization ability of thirteen selected strains in humans. The strains adhering to Caco-2 cells were tested for resistance to pH 2.5 and 0.3% ox bile, as well as antimicrobial activity against enteric pathogens (Khochamit et al., 2015). In addition to having good adhesion and biotherapeutic properties, Bacillus also showed the first condition of having GIT stress tolerance (Thakur et al., 2016).

[0010] Pharmaceutical compositions of bacilli known in the art are not sufficiently effective in recolonizing the body (i.e., the mammalian microbial ecosystem), and therefore, there is a need to discover bacilli that have inherent recolonization capabilities when administered in the form of pharmaceutical compositions, nutraceuticals, dairy products, functional foods, or absorbent products. Bacilli isolated from soil may have the ability to recolonize in vivo after administration because they have an inherent ability to survive in the human microbial ecosystem. Identifying bacilli strains that have enhanced colonization capabilities after administration is often a cumbersome process, and therefore it is important to select the best test system to predict their in vivo colonization capabilities.

[0011] In the literature, there appears to be a great deal of variability in the reported in vitro adhesion of probiotic strains. This variability does reflect biological differences between strains, but of course also depends on the experimental conditions. Furthermore, there also appears to be variability in how adhesion is measured. It could be argued that in vitro experiments serve only as a means of assessing in vivo colonization capacity via adhesion to epithelial cells.

[0012] Although long considered soil microorganisms, species of the genus Bacillus have been used for more than 50 years in the form of fermentation products or spore-based supplements (Cutting et al., 2011). Bacillus are ubiquitous in nature and are continuously introduced into the gastrointestinal tract and respiratory tract of healthy humans through food, water, and air (Benno & Mitsuoka, 1986). They are isolated from the intestine and can reach up to 10 7CFU / g and is therefore considered one of the major components of the normal intestinal microflora (microbiota) (Lakshmi et al., 2017). Recently, strains of Bacillus clausii have been isolated to provide more specific functions and their safety has been evaluated. Bacillus clausii has been used previously in patients with diarrhea (Sudha et al., 2013, Horosheva et al., 2014) and children with recurrent respiratory tract infections (Marseglia et al., 2007) with no adverse events reported. Although the country and strain were not specified, Bacillus clausii has been commercialized in 55 countries around the world (Nista et al., 2004; Gabrielli et al., 2009). A literature review of Bacillus clausii showed no adverse events associated with probiotics and the presence of the bacterium in different countries around the world complements the narrative of its safety for human consumption.

[0013] Bacillus coagulans has a long history of use in various foods. For decades, many strains of Bacillus coagulans have been widely consumed around the world (Endres et al., 2009). The bacteria can be found in foods such as yogurt, milk, sauerkraut, kimchi, and other dairy products, all of which contain levels ranging from 5x10 9 CFU / g (Sudha et al., 2016) to 9.38x10 10(Endres et al., 2011) varies. Several Bacillus species have been reported to be commonly found in honey, including Bacillus megaterium, Bacillus coagulans, Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus (Alippi, 1995; Alippi et al., 2004; Gilliam, 1979; Gilliam & Valentine, 1976; Snowdon & Cliver, 1996). This bacterial species used for probiotics originated in India, where many manufacturers produce Bacillus coagulans as a food ingredient for export and relabeling in Europe and the United States (Cutting, 2011). In order to evaluate the safety of Bacillus coagulans, its genome was studied and found to contain no harmful genes (Salvetti et al., 2016). Due to the harmless nature of Bacillus coagulans, the U.S. Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA) have reported the bacteria as safe and included it in the Generally Recognized as Safe (GRAS) and Qualified Presumption of Safety (QPS) lists (EFSA, 2017). Some non-pathogenic strains of the 100 known species of the genus Bacillus, including Bacillus coagulans and Bacillus subtilis, are considered safe for consumption by people of all ages (Nithya & Halami, 2012). A review of the literature on Bacillus coagulans showed no adverse events associated with probiotics, which consolidates the findings of good tolerability and safety. Therefore, Bacillus coagulans can be considered a safe probiotic, and consumption can be beneficial to the host.

[0014] Bacillus megaterium has been found in a variety of habitats, from soil to seawater, sediments, rice fields, honey, fish, milk, and dried foods (Alfoldi, 1957; Alippi & Reynaldi, 2006; Padgham & Sikora, 2007; Pelletier & Sygusch, 1990; Vary et al., 2007; VonTersch & Carlton, 1983; Scholle et al., 2003; Kotb, 2014). Further qualitative analysis of microorganisms isolated from honey showed that one of the most common Bacillus species was Bacillus megaterium (Alippi, 1995; Alippi et al., 2004; Snowdon & Cliver, 1996; Tysset, Durand & Taliergio, 1970). Additional studies have isolated Bacillus megaterium in fish (Sumathi et al., 2017). Afrilasari et al., 2015 also successfully isolated B. megaterium from the digestive tract of catfish and identified the strain as PTB 1.4. The harmless nature of B. megaterium has led to the inclusion of the bacterium in the Qualified Presumption of Safety (QPS) list (EFSA, 2017). It has been confirmed through genomic analysis that B. megaterium strain ATCC 14581 is almost identical (>99%) to B. megaterium MIT411. Health Canada has stated that the organism is not harmful to human health or the environment; exposure to the environment and Canadians is moderate. Therefore, it can be concluded that B. megaterium strain ATCC14581 is not harmful to human health or the environment (Health Canada, 2018).

[0015] In general, the probiotic-competent Bacillus strain should be able to adhere to other suitable cells, such as the cell line Caco-2 cells. In addition, it is also desirable that the probiotic-competent Bacillus strain show inhibitory activity against other bacterial species in vitro, produce acid and / or produce hydrogen peroxide after growth in liquid culture. Summary of the invention

[0016] The object of the present invention is to provide strains and compositions as described in the present application, such as pharmaceutical preparations or absorption products of suitable probiotic Bacillus strains having the desired properties. In an embodiment, the present invention relates to Bacillus coagulans strain CGI314 alone or with other strains such as Bacillus strains such as Bacillus megaterium strain MIT411 (disclosed in corresponding PCT application PCT / US2022 / xxxxx and claiming priority to Irish Patent Application No. 2021 / 0211, the contents of which are incorporated herein in their entirety) and Bacillus clausii strain CSI08 (disclosed in corresponding PCT application PCT / US2022 / xxxxx and claiming priority to Irish Patent Application No. 2021 / 0209, the contents of which are incorporated herein in their entirety). In embodiments, these strains have similar or substantially identical advantageous properties, such as the ability to colonize by adhering to mucous membranes, and thus they are suitable for treating or preventing infections or diseases of, for example, the vagina, urinary tract, gastrointestinal tract, sinuses, pharynx, esophagus, oral cavity and / or other areas of the body having, for example, mucous membranes, and treating or preventing infections or diseases of the skin and / or other areas of the body having epithelium; immune health, protection from oxidative stress, cleansing and detoxification, metabolic health and cardiovascular health, etc., such as providing antimicrobial activity (including lactic acid, benzoic acid and succinic acid), anti-inflammatory activity, inhibition of pro-inflammatory responses, activation and / or Stimulate immune response, for example, by stimulating macrophages to provide immune protection, aid digestion and / or fermentation, for example in the intestine, produce branched-chain amino acids, essential amino acids and B vitamins, maintain healthy intestine and / or skin, including lactic acid, benzoic acid and succinic acid to support skin health, protect mucosa and other epithelial tissues from toxic agents, reduce the incidence of loose stools, improve the gut-brain axis, and treat and / or prevent dysbiosis and its effects, such as periodontal disease, inflammatory bowel disease, chronic fatigue syndrome, metabolic disorders, heart disease, respiratory tract infections, urinary tract infections, GI infections and diarrhea; and restore normal and / or healthy flora. In an embodiment, the present invention allows for the use of Bacillus clausii strain CSI08 and compositions for fecal transplantation.

[0017] Gastrointestinal disorders include, but are not limited to, treating gastrointestinal irregularity in a subject wherein the subject has at least one 24 hour episode per month of bowel movements measured as 1 or 2 on the Bristol stool scale (i.e., treating constipation); or wherein the subject has at least one 24 hour episode per month of bowel movements measured as 6 to 7 on the Bristol stool scale (tending toward diarrhea), wherein the subject's frequency of 24 hour episodes per month of bowel movements measured as 1 or 2 (or 6 or 7) on the Bristol stool scale is reduced.

[0018] Also included are methods of restoring gastrointestinal regularity in an individual wherein the individual has at least one 24 hour episode per month of bowel movements measured as 1 or 2; or 6 to 7 on the Bristol stool scale, wherein the individual has an increased frequency of 24 hour cycles of bowel movements measured as 3 to 5 on the Bristol stool scale.

[0019] The present invention also includes maintaining a healthy intestinal microflora with one or more compositions containing bacillus. The one or more compositions containing bacillus can be used as a probiotic supplement to the gastrointestinal microflora and can compete with or otherwise inhibit pathogenic bacteria in the intestine, such as Escherichia coli, Listeria monocytogenes, and Salmonella species.

[0020] Another object of the present invention is to provide a pharmaceutical preparation having an increased colonization ability by adhering to the mucosa using a mucoadhesive excipient.

[0021] Yet another object of the present invention is to provide vaginal preparations having an enhanced ability to inhibit the growth of Candida albicans and Gram-negative pathogens.

[0022] Another object of the present invention is to provide compositions, such as dairy products, nutritional products and functional foods, comprising Bacillus coagulans strain CGI314 alone or in combination with other Bacillus strains such as Bacillus megaterium strains and / or Bacillus clausii strains, said compositions having substantially the same properties, having the ability to colonize on mucous membranes, and therefore being suitable for treating or preventing vaginal infections, urinary tract infections and gastrointestinal diseases. The compositions of the present invention can be administered for 1 dose, 1 day, 1 day to 1 week, 1 day to 1 month, 1 month to 45 days, 45 days to 2 months, 3 months, 6 months, 1 year or longer, including any time period determined and / or falling within these ranges.

[0023] The present invention includes methods of treatment and / or prevention and various other methods, which may include providing a subject (e.g., a mammalian subject, a person including a human patient, etc.) with a Bacillus coagulans strain or composition of the present invention, and the step of administering the strain or composition to the subject. In embodiments, the present invention relates to compositions, such as the use of a composition for treating a disease or infection or other condition. In embodiments, the present invention relates to the use of a Bacillus coagulans strain or composition thereof as described throughout this application (including its claims) in the preparation of a medicament for treating vaginal infections, urinary tract infections, gastrointestinal diseases, improving immune health, protecting from oxidative stress, cleansing and detoxification, metabolic health, cardiovascular health and / or skin health, and / or other therapeutic or other application reasons described in this application.

[0024] Attached photos

[0025] In the attached picture:

[0026] Figure 1 Phylogenetic tree showing Bacillus species arranged by clade (16S).

[0027] Figure 2 Phylogenetic tree showing Bacillus species arranged by clade (gyrB).

[0028] Figure 3 The antimicrobial activity of B. coagulans CGI314 against intestinal, skin and urinary tract opportunistic pathogens is shown on MRS agar plates with 0.4% TSA agar overlay (solid medium). A - E. coli; B - S. enteritidis and C - S. aureus.

[0029] Figure 4 The antimicrobial activity of Bacillus coagulans CGI314 against opportunistic pathogens of the intestinal tract, skin and urinary tract in liquid TSB medium is shown. Pathogens: Escherichia coli, Salmonella, Pseudomonas aeruginosa and Staphylococcus aureus. (Concentration Log 10 CFU / ml)

[0030] Figure 5 It is shown that the strongest antimicrobial activity of Bacillus coagulans CGI314 (Fortispore) against E. coli was observed at 24 hr. (a) Bacillus coagulans MTCC5856 (b) Bacillus coagulans CG314 (Fortispore) (c) Bacillus coagulans 6086 (BC30 TM) were plated on MRS agar against E. coli and 0.4% TSA agar was used to cover the plates.

[0031] Figure 6 It is shown that the strongest antimicrobial activity of Fortispore against Salmonella Enteritidis was observed at 48 hr. (a) (b) Fortispore (c) BC30 TM Plated on MRS agar against S. Enteritidis, 0.4% TSA agar overlay plates.

[0032] Figure 7 It is shown that no antimicrobial activity of Bacillus coagulans against Pseudomonas aeruginosa was observed at 24 hr. (a) Bacillus coagulans MTCC5856 (b) Bacillus coagulans CG314 (c) Bacillus coagulans 6086 (BC30 TM ) were plated on TSA agar against Pseudomonas aeruginosa, and 0.4% TSA agar was overlaid on the plates.

[0033] Figure 8 Showing limited antimicrobial detection in Bacillus coagulans against S. aureus using MRS agar at 24 hr. (a) Bacillus coagulans seeded on TSA agar against S. aureus, 0.4% TSA agar overlay plate. (b) Bacillus coagulans seeded on MRS agar against S. aureus, 0.4% TSA agar overlay plate.

[0034] Fig. 9 The total antioxidant capacity of Bacillus coagulans (Fortispore) and Lactobacillus rhamnosus (L. rhamnosus) is shown.

[0035] Fig.10 The total antioxidant capacity of DE111, Bacillus coagulans (Fortispore), Bacillus clausii CSI08 (Munispore), Bacillus megaterium MIT411 (Renuspore) and Lactobacillus rhamnosus is shown.

[0036] Fig.11 The adhesion of Fortispore to the HT-29 cell line, measured as the percentage of adherent bacteria, was shown to be negligible.

[0037] Fig.12 The adhesion of Fortispore to the HT-29-MTX cell line, measured as the percentage of adherent bacteria, was shown to be negligible.

[0038] Fig.13 Shown is a study of the adhesion of Bacillus coagulans strains to the HT-29 cell line.

[0039] Fig.14 Shown is a study of the adhesion of Bacillus coagulans strains to the HT-29-MTX cell line.

[0040] Fig.15 It was shown that there was no caseinolytic activity in Bacillus coagulans CGI314 at 24h or 48h using both the streak method and the overnight TSB broth method.

[0041] Fig.16 After incubation at 37°C for 24 h, Kit, Fortispore, had lower proteolytic activity against casein derivatives than the positive control proteinase K.

[0042] Fig.17 The results showed that the streak method and overnight TSB broth method showed that the expression of Bacillus coagulans CGI314, Bacillus coagulans 6086 (BC30 TM ) and Bacillus coagulans MTCC5856 (from left to right) No caseinolytic activity is present.

[0043] Fig.18 Bacillus coagulans Fortispore was shown to exhibit low protease activity towards casein.

[0044] Fig.19 After incubation at 37°C for 24 h, Quantitative analysis of caseinolytic activity of Bacillus coagulans strains determined by the kit.

[0045] Fig. 20 The FAA profile of Fortispore UTH fermented milk is shown. The relative concentrations of methionine, alanine, proline, tryptophan, lysine, and cis-aconitic acid are shown (the white bar (left) represents the control).

[0046] Fig.21 The FAA profile of Fortispore UTH fermented milk is shown. The relative concentrations of succinic acid, lactic acid, benzoic acid, and isocitric acid are shown (the white bar (left) represents the control).

[0047] Fig. 22 The effects of Fortispore were compared with those of Lactospore and BC30 in the production of lactate, succinate and benzoate (from left to right: control, BC30, Lactospore, Fortispore).

[0048] Fig.23The effects of Fortispore on amino acid production were compared with those of Lactospore and BC30 (from left to right: control, BC30, Lactospore, Fortispore; relative concentrations of methionine, proline, tryptophan, lysine are also shown).

[0049] Fig.24 Shown are the results of 24 hours post-incubation in minimal medium compared to the control. (F) The concentration of Fortispore (CFU / mL) increased significantly by 1 log 10 .

[0050] Fig.25 Show (F) The concentration of Fortispore (CFU / mL) in minimal medium was significantly increased, while no significance was observed in the growth of BC30 and Lactospore.

[0051] Fig.26 Compared with the control, (F) No significant increase in the concentration of Fortispore (CFU / mL) in TSB medium was shown.

[0052] Fig. 27 Compared with the control, (F) No significant increase in the concentrations (CFU / mL) of DE111, Fortispore, BC30, Escherichia coli, and S. Enteritidis in TSB medium was shown.

[0053] Fig.28 Compared with the control, (F) There was no significant increase in the concentration (CFU / mL) of Fortispore in 50% TSB medium.

[0054] Fig.29 After 24 hours in 50% TSB medium, (F) The production of DE111 (CFU / mL) increased significantly by 1 log 10 .

[0055] Fig.30 Compared with the control in BHI medium, (F) No significant increase in Fortispore concentration (CFU / mL) was shown.

[0056] Fig.31 Compared with the control in BHI medium, (F) No significant increase in the concentrations (CFU / mL) of DE111, Fortispore, and BC30 was shown.

[0057] Fig.32 Compared with the control in 50% BHI medium, (F) No significant increase in Fortispore concentration (CFU / mL) was shown.

[0058] Fig.33 Compared with the control in 50% BHI medium, (F) No significant increase in the concentrations (CFU / mL) of DE111, Fortispore, and BC30 was shown. DETAILED DESCRIPTION

[0059] Genotype identification

[0060] The applicant collaborated with Cornell University (Ithaca NY, USA) to conduct genome sequencing and characterization.

[0061] WGS DNA composition

[0062] Whole genome sequencing (WGS) was performed by Cornell University, including assembly and annotation. Bioinformatics analysis was performed at Cornell University and Deerland Probiotics and Enzymes (Kennesaw GA, USA). The applicant identified the polymorphism of the gyrB gene.

[0063] The gyrB gene encodes DNA gyrase subunit B. DNA gyrase negatively supercoils closed circular double-stranded DNA in an ATP-dependent manner to maintain the underwound state of chromosomes. Gene sequencing analysis was performed using gyrB gene polymorphism, which is a well-established method for prokaryotic species identification (Bavykin et al., 2014; Wang et al., 2007). Representative genomes were reviewed and curated by NCBI and coordinated with the UniProtein Consortium (NCBI, 2016; Uniprot, 2016). SequinR, an R software package, combined with UniProt Consortium analysis, was used to compare the whole genome sequence (WGS) and GyrB sequence of the currently claimed Bacillus coagulans CGI314 with other reference strains (Tables 1 to 3 below).

[0064] Identification of genotype, gyrB, and 16S rRNA of Bacillus coagulans CGI314

[0065] CGI314 was isolated and the genome was considered successful.

[0066] The genome size of CGI314 (3.0 Mbp) is shorter than that of previously sequenced Bacillus coagulans strains (3.4 Mbp) (Upadrasta et al., 2016). The GC% (47.3%) is consistent with the GC% (46.5%) of previously sequenced Bacillus coagulans strains (Upadrasta et al., 2016).

[0067] Table 1

[0068] CGI314 whole genome sequencing indicators

[0069]

[0070] Table 2

[0071] gyrB gene distance matrix

[0072]

[0073] Table 3

[0074] Whole genome sequence comparison

[0075] Bacillus coagulans subsp. Accession Number %GC Sequence length CGI314 JABBFU000000000.1 47.30％ 3,028,595 ATCC7050 NZ_CP009709.1 46.90％ 3,366,995 BC-30 JPSK01000000 46.39％ 3,458,616 IS-2 JZDH01000000 46.41％ 3,446,692 MTCC 5856 NZ_CP011939.1 46.23％ 3,694,837

[0076] 16S rRNA

[0077] Whole genome sequencing (WGS) and 16S rRNA analysis of CGI314 compared with the four reference strains showed an average nucleotide identity (ANI) score of 99.9% for 16S rRNA compared with Bacillus coagulans strain ATCC 7050. The genome size (3.0 Mbp) and GC content (47.30%) of CGI314 were comparable to those of the four reference strains.

[0078] Further deposits and accession numbers

[0079] The genome sequence data of Bacillus coagulans strain CGI314 (Fortispore) was deposited into the NCBI GenBank database and the genome sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The genome is publicly available, the strain's GenBank accession number is JABBFU000000000.1, and can be obtained through the following link: https: / / www.ncbi.nlm.nih.gov / nuccore / JABBFU00000000.1.

[0080] The genome sequence data of Bacillus clausii strain CSI08 (Munispore) was deposited into the NCBI GenBank database and the genome sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The genome is publicly available, the strain's GenBank accession number is JABBNL000000000.1, and can be obtained through the following link: Alkalihalobacillus clausii strain CSI08, whole genome shotgun sequence -Nucleotide-NCBI(nih.gov) .

[0081] The genome sequence data of Bacillus megaterium strain MIT411 (Renuspore) was deposited into the NCBI GenBank database and the genome sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The genome is publicly available, the strain's GenBank accession number is JABBNK000000000.1, and can be obtained through the following link: Priestia megaterium strain MIT411- whole genome shotgun sequencing pro-Nucleotide-NCBI(nih.gov) .

[0082] Phylogenetic positioning was performed by DiLan Probiotics and Enzymes Ltd.

[0083] Genome-to-genome distance calculation (GGDC) is a digital gold standard that is as reliable as DNA-DNA hybridization (DDH) (Auch et al., 2010). GGDC has a stronger discriminatory power in subspecies delineation and was subsequently used for confirmation of multiple alignments and phylogenetic analyses. GGDC generated a computationally based model that further confirmed that Bacillus coagulans CGI314 is a close relative of ATCC7050.

[0084] Although the conserved 16S rRNA sequence is a well-established method for comparing and studying bacterial phylogeny, the high ratio of sequence similarity between closely related species limits its usefulness (Wang et al., 2007). The high ratio of 16S rRNA sequence similarity in closely related bacterial species is due to the slower rate of molecular evolution. Past studies (Bavtlin et al., 2004; Wang et al., 2007) support the validity of using gyrB sequences as taxonomic biomarkers because of their base substitution rates and their significant and reliable correlation with DNA-DNA hybridization analysis (Dauga et al., 2002; Kasai et al., 1998; Wang et al., 2007). gyrB encodes DNA gyrase B and a type II topoisomerase that plays an important role in DNA replication. The gyrase B subunit is encoded by the gyrB gene.

[0085] Phylogenetic analysis using the neighbor-joining (NJ) method (Saitou & Nei, 1987) placed Bacillus coagulans CGI314 and Bacillus coagulans ATCC 7050 in the same evolutionary branch ( Figure 2 ). This confirms all previous genome identity determinations. Bacillus coagulans CGI314 has been assigned to the Bacillus coagulans group.

[0086] definition

[0087] "Excipient" refers to any inactive ingredient added to form part of the final formulation.

[0088] "Probiotics" refers to live microbial supplements that have a beneficial effect on a patient by acting in the intestinal tract, urinary tract, or vagina. The term "one or more probiotics" may refer to live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Foods and food additives containing probiotics can help restore a healthy balance of the intestinal microflora. In addition, probiotics that supplement the intestinal flora can promote a healthy intestinal balance.

[0089] "Prebiotics" are used herein as substrates which have a beneficial effect on the probiotics and on the individual patient taking the probiotics. Suitable prebiotics may be selected from inulin, oligosaccharides and / or vitamins.

[0090] As used herein, "subject" includes humans suffering from any clinical symptoms associated with microbial imbalance, as well as humans using bacterial preparations prophylactically. Optionally, the subject is a human.

[0091] A “synbiotic product” is a combination of probiotics and prebiotics that work synergistically to have a beneficial effect on patients.

[0092] "Hardy growth" means that the bacteria show very good growth.

[0093] The abbreviation "CFU" means colony forming units.

[0094] As will become apparent during the course of the following detailed description, the present invention relates to probiotic Bacillus strains capable of regenerating the microbiota in a subject.

[0095] According to a first aspect, the present invention includes Bacillus coagulans strain CGI314 alone or in combination with other probiotic strains having substantially the same properties. Such other probiotic bacillus strains may include, but are not limited to, Bacillus clausii strains and Bacillus megaterium strains. Such other bacillus strains may also include Bacillus clausii strains and Bacillus megaterium strains, each of which is filed on the same day under its own title, the contents of which are incorporated herein in their entirety.

[0096] As described in the appended claims, SEQ ID NO: 1 comprises gyrB of Bacillus coagulans CGI314.

[0097] As described in the appended claims, SEQ ID NO: 2 comprises the 16S rRNA of Bacillus coagulans CGI314.

[0098] As set forth in the appended claims, SEQ ID NO: 3 comprises the assembled complete genome sequence of Bacillus coagulans CGI314.

[0099] The spore-coagulating strain claimed herein has at least 97% identity to SEQ ID NO: 1 and / or 2; or has at least 97% identity to SEQ ID NO: 3, and has the following properties:

[0100] Bacillus coagulans CGI314

[0101] This strain showed bile stability.

[0102] This strain showed acid stability.

[0103] This strain showed heat tolerance.

[0104] This strain produces natural antimicrobial substances in the form of bacteriocins.

[0105] In order to determine the genus and species of the strains disclosed herein, the whole genome was sequenced. The number and composition of the strains were identified and determined.

[0106] The strain was shown to have little or no antibiotic resistance and presented no safety concerns.

[0107] The strain was found to exhibit stability to acid and bile.

[0108] According to a second aspect, the Bacillus strains of the invention are suitable for the prevention or treatment of vaginal infections, urinary tract infections and gastrointestinal diseases (including gastrointestinal infections), as well as medical uses for improving immune health, protection from oxidative stress, cleansing and detoxification, metabolic health and cardiovascular health.

[0109] In another preferred embodiment, a pharmaceutical composition is provided, which comprises Bacillus coagulans CGI314 alone or in combination with other probiotic strains having substantially the same properties, and a pharmaceutically acceptable carrier and / or diluent. Such other probiotic bacillus strains include, but are not limited to, Bacillus clausii strains and Bacillus megaterium strains. The bacterial strains are formulated into pharmaceutical preparations by methods known to those skilled in the art to facilitate administration of the probiotic strains.

[0110] Bacillus coagulans has been shown to alleviate symptoms of irritable bowel syndrome (Sudha et al., 2018), improve muscle integrin and cytokine responses (Gepner et al., 2017; Jager et al., 2018), modulate the gut microbiome and immune response (Kimmel et al., 2010), reduce functional intestinal gas symptoms (Kalman et al., 2009), reduce the occurrence and duration of diarrhea (Dolin et al., 2009), improve symptoms of functional abdominal pain and bloating (Hun et al., 2009), protect against acetaminophen-induced acute liver injury (Neag et al., 2020), enhance butyrogenesis (Sasaki et al., 2020), reduce the severity of bacterial vaginosis (Sudha et al., 2012), and reduce cholesterol (Sudha et al., 2012), all in vivo. Bacillus coagulans has also been shown to induce immune responses and anti-inflammatory effects (Jensen et al., 2017), improve plant protein digestion (Keller et al., 2017), adhere to Caco-2 cells (Sharma & Kanwar, 2017), improve the colonic microenvironment in patients with ulcerative colitis (Sasaki et al., 2020), reduce adhesion, cytotoxicity, and induction of cell apoptosis caused by S. typhimurium in HT-29 cells (Kawarizadeh et al., 2019), hydrolyze lactose from whey protein (Liu et al., 2019), and enhance T-cell responses (Baron, 2009), all in vitro.

[0111] Bacillus clausii has been shown to be effective in preventing recurrent respiratory tract infections (Marseglia et al., 2007) and reducing the duration and severity of diarrhea (Sudha et al., 2019), all in vivo. Bacillus clausii has also been shown to produce protein hydrolysates with antimicrobial and antioxidant properties (Rochinmedina et al., 2017), protect against acetaminophen-induced acute liver injury (Neag et al., 2020), and inhibit the cytotoxic effects induced by Clostridium difficile and Bacillus cereus toxins (Ripert et al., 2016), all in vitro.

[0112] Bacillus megaterium has been shown to protect against oxidative stress both in vitro and in vivo (Mazzoli et al., 2019). Bacillus megaterium has also been shown to be able to adapt and survive acidic stress conditions and to chelate heavy metals, all in vitro (Ferreira et al., 2019).

[0113] Preferably, according to the present invention, the bacterial concentration of the probiotics used in the medicine is 10 6 -10 13 In an embodiment, the probiotics used in the present invention are 10 6 -10 13 The bacterial concentration of CFU (colony forming units) is used, for example, as a daily dose, including any amount or range contained in the range. In an embodiment, the amount of bacteria used is 10 7 -10 12 CFU, or 10 8 -10 11 CFU, or 10 9 -10 10 CFU, or for example about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , about 10 12 and / or about 10 13 CFU, and any amount or range including or between said amounts. In an embodiment, the composition of the present invention comprises about 10 6 - Approx. 10 13 CFU, such as about 10 9 Bacillus coagulans CGI314, consisting essentially of about 10 6 - Approx. 10 13 CFU, such as about 109 Bacillus coagulans CSI08, consisting of about 10 6 - Approx. 10 13 CFU, such as about 10 9 Bacillus coagulans CSI08 and / or about 10 6 - Approx. 10 13 CFU, such as about 10 9 In one embodiment, the composition of the present invention comprises Bacillus coagulans CGI314 (e.g., about 10 9 In an embodiment, the composition of the present invention is orally administered in the form of a capsule. In an embodiment, in the composition and / or use or method of the present invention, Bacillus coagulans CGI314 is in spore form, or is not in spore form.

[0114] In certain embodiments, the composition comprising Bacillus coagulans CGI314 may include one or more dry carriers selected from the group consisting of trehalose, maltodextrin, rice flour, microcrystalline cellulose, magnesium stearate, inositol, oligofructose, oligogalactose, dextrose, dry dairy products, etc. In certain embodiments, the dry carrier may be added to the composition comprising Bacillus coagulans CGI314 at a weight percentage of about 1% to about 95% by weight of the composition.

[0115] In certain embodiments, the composition comprising Bacillus coagulans CGI314 may include one or more liquid or gel-based carriers selected from the group consisting of: water and physiological salt solutions, urea, alcohols and their derivatives (e.g., methanol, ethanol, propanol, butanol), glycols (e.g., ethylene glycol, propylene glycol), etc.; natural or synthetic flavoring agents and food-grade colorants, all compatible with organisms; thickeners selected from the group consisting of corn starch, guar gum, xanthan gum, etc.; one or more spore germination inhibitors selected from the group consisting of high salt carriers, methyl parabens, guar gum, polysorbates, preservatives, etc. In certain embodiments, one or more liquid or gel-based carriers may be added to the composition comprising Bacillus coagulans CGI314 at a weight / volume percentage of about 0.6% to about 95% weight / volume of the composition. In certain embodiments, one or more natural or synthetic flavoring agents may be added to a composition including Bacillus coagulans CGI314 at a weight / volume percentage of about 3.0% to about 10.0% weight / volume of the composition. In certain embodiments, one or more coloring agents may be added to a composition including Bacillus coagulans CGI314 at a weight / volume percentage of about 1.0% to about 10.0% weight / volume of the composition. In certain embodiments, one or more thickening agents may be added to a composition including Bacillus coagulans CGI314 at a weight / volume percentage of about 2% weight / volume of the composition. In certain embodiments, one or more spore germination inhibitors may be added to a composition including Bacillus coagulans CGI314 at a weight / volume percentage of about 1% weight / volume of the composition.

[0116] Delivery System

[0117] Suitable dosage forms include tablets, capsules, solutions, suspensions, powders, gels and candies. Sublingual delivery systems include, but are not limited to, sublingual and supralingual dissolvable tablets, drops and beverages. Edible films, hydrophilic polymers, orally dissolvable films or orally dissolvable strips can be used. Other useful delivery systems include oral or nasal sprays or inhalers, etc. Suitable dosage forms include tablets, capsules, solutions, suspensions, powders, gels and candies. Sublingual delivery systems include, but are not limited to, sublingual and supralingual dissolvable tablets, drops and beverages. Edible films, hydrophilic polymers, orally dissolvable films or orally dissolvable strips can be used. Other useful delivery systems include oral or nasal sprays or inhalers, etc.

[0118] For oral administration, probiotics can be further combined with one or more solid inactive ingredients for the preparation of tablets, capsules, pills, powders, granules or other suitable dosage forms. For example, the active agent can be combined with at least one excipient selected from the group consisting of fillers, binders, humectants, disintegrants, dissolution retardants, absorption promoters, wetting agents, absorbents and lubricants. Other useful excipients include but are not limited to magnesium stearate, calcium stearate, mannitol, xylitol, sweeteners, starch, carboxymethyl cellulose, microcrystalline cellulose, silica, gelatin, silicon dioxide, etc.

[0119] In certain embodiments, the components of the composition administered according to the methods of the present disclosure, together with one or more conventional adjuvants, carriers or diluents, may therefore be placed in the form of pharmaceutical compositions and unit dosage forms thereof. These forms include: solid, particularly tablets, filled capsules, powders and pill forms; liquid, particularly aqueous or non-aqueous solutions, suspensions, emulsions, elixirs; and capsules filled with the composition; all forms for oral administration, suppositories for rectal administration, and sterile injection solutions for parenteral administration. Such pharmaceutical compositions and unit dosage forms thereof may include conventional ingredients in conventional proportions, with or without additional active compounds or ingredients, and such unit dosage forms may include any suitable effective amount of active ingredients commensurate with the intended daily dosage range used.

[0120] The components of the compositions administered according to the methods of the present disclosure can be administered in a variety of oral and parenteral dosage forms. It will be apparent to those skilled in the art that, in certain embodiments, the following dosage forms may include a compound of the present disclosure or a pharmaceutically acceptable salt of a compound of the present disclosure as an active component.

[0121] For the preparation of pharmaceutical compositions for administration according to the methods of the present disclosure, pharmaceutically acceptable carriers may be solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier may be one or more substances which may also be used as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials.

[0122] In powders, the carrier is a finely divided solid which is mixed with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.

[0123] In certain embodiments, powders and tablets administered according to the methods of the present disclosure may preferably contain from 5 or 10 percent to about 70 percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, and the like. The term "preparation" means a preparation comprising an active compound and an encapsulating material as a carrier to provide a capsule in which the active component is present, and the active component (with or without additional carriers) is surrounded by the carrier, thereby binding to the carrier. Similarly, cachets and lozenges are also included. Tablets, powders, capsules, pills, cachets, and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.

[0124] Liquid preparations include but are not limited to solutions, suspensions and emulsions, such as water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in polyethylene glycol aqueous solutions. In certain embodiments, the compound used according to the method of the present disclosure can be formulated for parenteral administration (for example, by injection, for example, bolus injection or continuous infusion), and can be present in unit doses, for use in ampoule, prefilled syringe, small volume infusion or multidose containers with added preservatives. The composition can take the form of suspensions, solutions or emulsions in oily or aqueous carriers, and can contain formulations such as suspending agents, stabilizers and / or dispersants. Alternatively, active ingredient can be in powder form, obtained by aseptic separation of sterile solids or by freeze drying from solution, for example, prepared with suitable carriers (for example, sterile, pyrogen-free water) before use.

[0125] Aqueous solutions suitable for oral use can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavoring agents, stabilizers and thickeners as desired. Aqueous suspensions suitable for oral use can be prepared by dispersing the finely divided active ingredient in water containing a viscous substance, such as a natural or synthetic gum, resin, methylcellulose, sodium carboxymethylcellulose or other well-known suspending agents.

[0126] Compositions for topical administration in the mouth include, but are not limited to, lozenges comprising the active agent in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0127] The solution or suspension is applied directly to the nasal cavity by conventional methods, such as with a dropper, pipette or sprayer. The composition can be provided in a single dose or multiple dose form. In compositions (including intranasal compositions) for administration to the respiratory tract, the compound generally has a small particle size, such as on the order of about 5 microns or less. Such particle size can be obtained by methods known in the art, such as by micronization.

[0128] The pharmaceutical formulation is preferably in unit dosage form. In this form, the formulation is subdivided into unit doses containing appropriate quantities of the active ingredient. The unit dosage form can be a packaged formulation, the package containing discrete quantities of the formulation, such as packaged tablets, capsules, and powders in vials or ampoules. Furthermore, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself; or it can be an appropriate number of any of these in packaged form.

[0129] Tablets, capsules and lozenges for oral administration and liquids for oral administration are preferred compositions. Solutions or suspensions for nasal or respiratory administration are preferred compositions. Transdermal patches for topical administration to the epidermis are preferred.

[0130] Further details on formulation and administration techniques may be found in the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA).

[0131] In certain embodiments, the composition of the present invention including the composition used according to the method of the present disclosure may also include one or more excipients, most preferably one or more nutrition or pharmaceutical excipients. The composition containing one or more excipients and incorporating one or more probiotics can be prepared by methods known in the art. Optionally, the composition may include one or more adjuvants, excipients, carriers, buffers, diluents and / or other conventional pharmaceutical adjuvants. For example, probiotics can be formulated into tablets, capsules, powders, suspensions, solutions for oral administration, solutions for parenteral administration (including intravenous, intradermal, intramuscular and subcutaneous administration), and solutions for application to patches for transdermal administration.

[0132] In certain embodiments, the nutrient composition including the nutrient composition used according to the method of the present disclosure may include a pharmaceutically acceptable carrier and may be co-administered with a pharmaceutically acceptable carrier. In certain embodiments, the active ingredient in this type of preparation may include about 1% by weight to about 99% by weight. In other embodiments, the active ingredient in this type of preparation may include about 0.1% by weight to about 99.9% by weight. "Pharmaceutically acceptable carrier" refers to any carrier, diluent or excipient that is compatible with the other ingredients of the preparation and harmless to the user. Useful excipients include but are not limited to microcrystalline cellulose, magnesium stearate, calcium stearate, any acceptable sugar (e.g., mannitol, xylitol), etc., and for cosmetic purposes, water-based or oil-based or mixtures thereof may be used, including such as emulsions.

[0133] Route of administration

[0134] The strain Bacillus coagulans CGI314 or a composition comprising the strain of the invention can be administered by any route, including but not limited to oral, sublingual, oral, ocular, pulmonary, rectal, vaginal, urethral, ​​ureteral and parenteral administration, or as an oral or nasal spray (e.g., inhalation of atomized vapor, droplets or solid particles). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, intravaginal, intravesical (e.g., to the bladder), intradermal, transdermal, topical or subcutaneous administration. It is also contemplated within the scope of the present invention that the pharmaceutical composition is instilled into the patient in the form of a controlled agent, and systemic or local release of the drug occurs at a later time. For example, the drug can be located in a reservoir for controlled release into the circulation, or for release to a local location.

[0135] The pharmaceutical composition of the present invention can be suitable for and formulated for any of the above-mentioned routes, including, for example, oral, rectal, bronchial, nasal, pulmonary, topical (including buccal and sublingual), transdermal, vaginal, urethral, ​​ureteral or parenteral (including cutaneous, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intracerebral, intraocular injection or infusion) administration, or a form suitable for administration by inhalation or insufflation, including powder and liquid aerosol administration, or by sustained release system administration. Suitable examples of sustained release systems include semipermeable matrices of solid hydrophobic polymers containing the compounds of the present invention, which may be in the form of shaped articles, such as films or microcapsules.

[0136] The above embodiments may be further understood in conjunction with the following examples. In addition, the following non-limiting examples are provided to illustrate the present invention. However, it will be appreciated by those skilled in the art that, for any given embodiment of the present invention, it may be necessary to change the process, such as changing the order or step.

[0137] Example 1

[0138] Characterization of Bacillus coagulans CGI314 (hereinafter referred to as Fortispore)

[0139] Bacillus coagulans CGI314 (Fortispore) has significant antimicrobial activity against Escherichia coli, Salmonella and Staphylococcus aureus in solid MRS agar overlaid with 0.4% TSA agar.

[0140] However, no antimicrobial activity was observed against Pseudomonas aeruginosa in solid media. Fortispore exhibits antimicrobial activity against enteric, urinary, and skin pathogens in solid environments.

[0141] Table 4

[0142] Summary of the antimicrobial activity of Bacillus coagulans CGI314 against opportunistic pathogens of the intestinal tract, skin and urinary tract (solid culture medium)

[0143]

[0144] The antimicrobial activity was expressed as inhibition zone (mm) ± standard deviation.

[0145] Figure 3 The antimicrobial activity of Bacillus coagulans CGI314 against intestinal, skin and urinary tract opportunistic pathogens is shown on MRS agar plates with 0.4% TSA agar overlay (solid medium). A - Escherichia coli (E. coli); B - Salmonella enteritidis (S. enteritidis) and C - Staphylococcus aureus (S. aureus).

[0146] Fortispore exhibited significant antimicrobial activity against Escherichia coli, Salmonella, and Pseudomonas aeruginosa in liquid TSB medium. However, no antimicrobial activity was observed against Staphylococcus aureus in liquid TSB medium.

[0147] Table 5

[0148] Summary of the antimicrobial activity of Fortispore against intestinal, skin and urinary tract opportunistic pathogens (in liquid culture)

[0149]

[0150] Antimicrobial activity was detected (+), and no antimicrobial activity was observed (-).

[0151] Figure 4The antimicrobial activity of Bacillus coagulans CGI314 against opportunistic pathogens of the intestinal tract, skin and urinary tract in liquid TSB medium is shown. Pathogens: Escherichia coli, Salmonella, Pseudomonas aeruginosa and Staphylococcus aureus. Controls represent the growth of pathogens respectively; treatments represent the growth of pathogens in the presence of Bacillus coagulans CGI314. **p<0.01, ***p<0.001, ****p<0.0001.

[0152] Fortispore has the potential to control the presence of opportunistic pathogens in the intestinal and urinary tracts, where semi-liquid to liquid conditions would be common. In addition, Fortispore has the potential to prevent the spread of opportunistic pathogens in dry environments, such as human skin.

[0153] Fortispore has higher antimicrobial activity than competitor Bacillus coagulans probiotics.

[0154] The agar diffusion method was used to compare the activity of Bacillus coagulans CGI1314 (Fortispore) and Bacillus coagulans MTCC5856. and Bacillus coagulans 6086 (BC30 TM ) against the following opportunistic and zoonotic pathogens of human skin and intestine:

[0155] Escherichia coli 25922,

[0156] Salmonella Enteritidis 13076,

[0157] Staphylococcus aureus RF122, and

[0158] Pseudomonas aeruginosa DSM3227

[0159] It has been proven that BC30 TM and In comparison, Bacillus coagulans CGI314 (Fortispore) showed stronger antimicrobial activity against Escherichia coli at 24h and against Salmonella enteritidis at 48h. Fortispore may have a higher potential to control the presence of opportunistic pathogens and zoonotic pathogens that may be present in the human intestine or on the skin:

[0160] Fortispore has high antimicrobial activity against Escherichia coli.

[0161] Fortispore has high antimicrobial activity against Salmonella Enteritidis at 48hr.

[0162] No antimicrobial activity of Bacillus coagulans against Pseudomonas aeruginosa was detected.

[0163] Limited antimicrobial activity of Bacillus coagulans against Staphylococcus aureus using TSA agar.

[0164] Antimicrobial Activity High antimicrobial activity against Escherichia coli:

[0165] Using the MRS agar test, Bacillus coagulans CGI314 (Fortispore) had the strongest antimicrobial activity against Escherichia coli. Bacillus coagulans MTCC5856 and Bacillus coagulans 6086 (BC30 TM ) have limited antimicrobial activity.

[0166] Table 6

[0167] The inhibition zone of Bacillus coagulans CGI314 (Fortispore) strain against E. coli was larger.

[0168]

[0169] Values ​​represent mean inhibition ± standard deviation (n=2).

[0170] Figure 5 It is shown that the strongest antimicrobial activity of Bacillus coagulans CGI314 (Fortispore) against E. coli was observed at 24 hr. (a) Bacillus coagulans MTCC5856 (b) Bacillus coagulans CG314 (Fortispore) (c) Bacillus coagulans 6086 (BC30 TM ) were plated on MRS agar against E. coli and 0.4% TSA agar was used to cover the plates.

[0171] Fortispore inhibits the growth of Escherichia coli, an opportunistic pathogen of the intestinal and urinary tract.

[0172] Antimicrobial Activity - High antimicrobial activity against Salmonella Enteritidis at 48hr:

[0173] At 24h, Bacillus coagulans 6086 (BC30 TM ) showed the strongest antimicrobial activity against Salmonella enteritidis. However, at 48 h, Bacillus coagulans CGI314 (Fortispore) showed the strongest antimicrobial activity against this pathogen.

[0174] Table 7

[0175] At 48 hours, Bacillus coagulans strain CGI314 (Fortispore) obtained a larger inhibition zone against Salmonella Enteritidis.

[0176]

[0177] Values ​​represent mean inhibition ± standard deviation (n=2).

[0178] Figure 6 It is shown that the strongest antimicrobial activity of Fortispore against Salmonella Enteritidis was observed at 48 hr. (a) (b) Fortispore (c) BC30 TM Plated on MRS agar against S. Enteritidis, 0.4% TSA agar overlay plates.

[0179] Fortispore inhibits the growth of Salmonella, an animal-borne pathogen known to affect the intestinal tract.

[0180] Antimicrobial activity - No antimicrobial activity was detected against Pseudomonas aeruginosa by Bacillus coagulans:

[0181] Using the agar diffusion method, no antimicrobial activity of Bacillus coagulans against Pseudomonas aeruginosa was observed.

[0182] Figure 7 It was shown that no antimicrobial activity of Bacillus coagulans against Pseudomonas aeruginosa was observed at 24 hr. (a) Bacillus coagulans MTCC5856 (b) Bacillus coagulans CG314 (c) Bacillus coagulans 6086 (BC30 TM ) were plated on TSA agar against Pseudomonas aeruginosa, and 0.4% TSA agar was overlaid on the plates.

[0183] Fortispore did not release antimicrobial agents against the skin pathogen Pseudomonas aeruginosa. However, in liquid culture medium, Fortispore showed potential activity against Pseudomonas aeruginosa (see Tables 5 and Figure 7 ).

[0184] Antimicrobial Activity - Limited antimicrobial activity of Bacillus coagulans against Staphylococcus aureus using MRS agar:

[0185] Using TSA agar, there was no evidence that B. coagulans strains had antimicrobial activity against S. aureus. However, using MRS agar, B. coagulans 6086 (BC30 TM ) and Bacillus coagulans CGI1314 (Fortispore) did show slight activity against this pathogen.

[0186] Table 8

[0187] Narrow inhibition zones of the three Bacillus coagulans strains against S. aureus were detected at 24 h using MRS agar.

[0188]

[0189] Values ​​represent mean inhibition ± standard deviation (n=2).

[0190] Figure 8 Showing limited antimicrobial detection in Bacillus coagulans against S. aureus using MRS agar at 24 hr. (a) Bacillus coagulans seeded on TSA agar against S. aureus, 0.4% TSA agar overlay plate. (From left to right: BC30, CGI314, Lactospore). (b) Bacillus coagulans seeded on MRS agar against S. aureus, 0.4% TSA agar overlay plate. (From left to right: CGI314, BC30, Lactospore).

[0191] Fortispore can release limited amounts of antimicrobial agents on dry surfaces such as skin against skin pathogens such as Staphylococcus aureus.

[0192] Fortispore is a potential antioxidant probiotic:

[0193] The antioxidant activities of Bacillus coagulans and Lactobacillus rhamnosus were compared. Fortispore had higher antioxidant activity than Lactobacillus rhamnosus.

[0194] Fig. 9 The total antioxidant capacity of Bacillus coagulans and Lactobacillus rhamnosus is shown. The results show the average concentration of Trolox equivalents in nanomoles (nmole) / ml (n=3) ± standard error. **p value = 0.0014.

[0195] The increased Trolox equivalent concentration levels in Fortispore neutralize and scavenge free radicals and prevent oxidative damage to cells.

[0196] Among all Deerland spores, Fortispore had the second highest amount of antioxidant activity.

[0197] The total antioxidant activity of Fortispore Bacillus coagulans was compared with Bacillus megaterium MIT411, Bacillus clausii CSI08, Bacillus subtilis DE111 and Lactobacillus rhamnosus. Fortispore has high antioxidant activity, which is higher than that of Lactobacillus rhamnosus.

[0198] Fig.10The total antioxidant capacity of DE111, Bacillus coagulans of the present invention, Bacillus clausii CSI08, Bacillus megaterium MIT411 and Lactobacillus rhamnosus is shown. The results show the average concentration of Trolox equivalents in nanomoles / g (n=3) ± standard error.

[0199] The increased Trolox equivalent concentration levels in Fortispore neutralize and scavenge free radicals and prevent oxidative damage to cells.

[0200] Fortispore (vegetative, non-spore form) does not adhere to intestinal epithelial cell lines.

[0201] The adhesion ability of Bacillus coagulans strain (CGI314) to an in vitro intestinal epithelial model was evaluated. At 37°C, the adhesion of Bacillus coagulans strain CGI314 to the HT-29 cell line was negligible. At 37°C, the adhesion ability of Bacillus coagulans strain CGI314 to the mucus-producing cell line HT-29-MTX was negligible.

[0202] Fig.11 Fortispore showed negligible adhesion to the HT-29 cell line.

[0203] Fig.12 Fortispore showed negligible adhesion to the HT-29-MTX cell line.

[0204] Fortispore strains do not adhere to intestinal mucus or epithelial cells.

[0205] Various Bacillus coagulans strains (CGI314, BC30 TM , ) to the in vitro intestinal epithelial model. At 37°C, Bacillus coagulans strains CGI314, BC30 TM , Adhesion to HT-29 cell line was negligible. At 37°C, Bacillus coagulans BC30 TM The adhesion ability to the mucus-producing cell line HT-29-MTX was higher than that of Bacillus coagulans CGI314 and

[0206] Fig.13 Shown is a study of the adhesion of Bacillus coagulans strains to the HT-29 cell line.

[0207] Fig.14 Shown is a study of the adhesion of Bacillus coagulans strains to the HT-29-MTX cell line.

[0208] Bacillus coagulans strains (liquid cultures, non-spore form) do not adhere to intestinal mucus or epithelial cells.

[0209] Fortispore exhibits low caseinolytic activity

[0210] Fortispore was negative for caseinolytic activity on skim milk agar plates. Quantitative analysis of Fortispore caseinolytic activity was assessed using a commercial kit that employs fluorescently labeled casein derivatives. Fortispore exhibited low extracellular protease activity.

[0211] Fig.15 Studies using the streak method and the overnight TSB broth method show the absence of caseinolytic activity in Bacillus coagulans CGI1314 at 24h or 48h.

[0212] Fig.16 After incubation at 37°C for 24 h, The proteolytic activity of the kit, Fortispore, against casein derivatives was lower than that of the positive control proteinase K. Error bars represent standard deviations (n=3).

[0213] Fortispore exhibits low protease activity towards casein.

[0214] In the Deerland strain, Fortispore exhibited low extracellular protease activity against casein derivatives.

[0215] Fig.17 The results showed that the streak method and overnight TSB broth method showed that the expression of Bacillus coagulans CGI1314, Bacillus coagulans 6086 (BC30 TM ) and Bacillus coagulans MTCC5856 There is no caseinolytic activity in the

[0216] Fig.18 It is shown that among the Deerland strains, Fortispore Bacillus coagulans exhibits low protease activity towards casein.

[0217] Fig.19 After incubation at 37°C for 24 h, Quantitative analysis of caseinolytic activity of Bacillus coagulans strains determined by the kit. Significance is indicated by a, b and / or c, and multiple letters indicate that the result is not significantly different from more than one group (P<0.05). Error bars indicate standard deviation.

[0218] Compared with the comparison and BC30 TM ), Fortispore showed high protease activity against casein.

[0219] Fortispore's diverse carbohydrate profile

[0220] Fortispore can metabolize a range of monosaccharides, disaccharides, sugar alcohols, amine sugars and polysaccharides.

[0221] Using commercial API 50 CH test strips, Fortispore was positive for the metabolism of 22 out of 49 carbohydrates. These carbohydrates are mainly monosaccharides such as D-ribose, D-glucose, D-galactose, D-fructose, D-mannose and disaccharides such as trehalose, maltose and cellobiose. In addition, Fortispore can also metabolize compounds belonging to glycosylated hydroquinones (arbutin), cyanogenic glycosides (amygladin) and alcohol β-glucosides (salicin).

[0222] Table 9

[0223] API 50 Ch strips were used, which were made from Bacillus coagulans (Fortispore, BC30TM, ) List of effectively fermentable carbohydrates

[0224]

[0225] These data suggest that Fortispore may aid digestion of these compounds in the gut.

[0226] Fortispore was strongly positive for D-sorbitol and Amidon, while the other two competitor strains (BC30TM and ) were negative or weakly positive for these.

[0227] Table 10

[0228] API 50 Ch strips were used, which were prepared from Bacillus coagulans (Fortispore, BC30 TM , ) List of effectively fermentable carbohydrates

[0229]

[0230] These data suggest that Fortispore may aid digestion of these compounds in the gut.

[0231] Fortispore has esterase, peptidase, phosphatase and glucosidase activities:

[0232] Using the API ZYM kit, Fortispore tested positive for esterase, peptidase, phosphatase, and galactosidase activities, indicating that:

[0233] In the presence of an appropriate lipid source, Fortispore has a high probability of producing free fatty acids through the action of esterases.

[0234] Galactosidase enzymes increase the carbohydrate catabolic potential of Fortispore as they are active against various oligosaccharides, lactosylceramide, lactose and many glycoproteins.

[0235] Aminopeptidases that catalyze the hydrolysis of leucine, valine, and cysteine ​​substrates.

[0236] In fact, in silico analysis has identified genes encoding esterases, peptidases and galactosidases.

[0237] Table 11

[0238] Zymogram of Fortispore using API ZYM kit

[0239]

[0240] The study confirmed Fortispore's ability to hydrolyze oligosaccharides and showed its potential to break down fats and peptides to release free amino acids.

[0241] These data suggest that Fortispore can aid digestion of these molecules in the intestine.

[0242] The zymogram of Bacillus coagulans showed peptidase and ester hydrolysis capabilities and confirmed oligosaccharide degradation ability:

[0243] All Bacillus coagulans tested positive for esterase, peptidase, phosphatase and galactosidase activities using the API ZYM kit, indicating that:

[0244] In the presence of an appropriate lipid source, Bacillus coagulans has a high probability of producing free fatty acids through the action of esterases.

[0245] Galactosidase increases the carbohydrate catabolic potential of B. coagulans as they are active against various oligosaccharides, lactosylceramide, lactose, and many glycoproteins.

[0246] Aminopeptidases that catalyze the hydrolysis of leucine, valine, and cysteine ​​substrates.

[0247] Table 12

[0248] Zymogram of Fortispore using API ZYM kit

[0249]

[0250] This study showed that there were no differences in the hydrolysis abilities of Bacillus coagulans strains to several enzymes using the commercial API ZYM kit.

[0251] Fortispore releases free amino acids through glycolysis and protease metabolism

[0252] Metabolomic analysis revealed the fermentation and proteolytic capabilities of Fortispore towards milk carbohydrates and proteins, generating a range of amino acids.

[0253] The carbohydrate fermentation and proteolytic capabilities of Fortispore were analyzed using the UHT milk model. In Fortispore, a total of 38 free amino acid (FAA) compounds were identified by GC-MS analysis, of which 10 were statistically significant. Some of the carboxylic acids identified were related to carbohydrate metabolic pathways, confirming the presence of an active lactose / galactose / glucose uptake and enzyme metabolism system in Fortispore. In addition, there was also evidence for the presence of an active proteolytic system in Fortispore, as some amino acids were released, which were associated with peptidase activity and benzoic acid, which was associated with further catabolism of phenylalanine.

[0254] Table 13

[0255] Statistically significant compounds analyzed by GC-MS using the FAA method are listed together with potential precursors

[0256]

[0257] Overall, the enzymology and metabolomics data suggest that Fortispore has active carbohydrate metabolizing enzymes, as most of the compounds produced are from the glycolytic pathway (conversion of carbohydrates in milk). In addition, there is evidence that UHT milk from Fortispore fermentations contains potentially active aminopeptidases, lyases, and decarboxylases that generate carboxylic acids and release amino acids.

[0258] Fortispore releases free amino acids through glycolysis and protease metabolism

[0259] The bar graph shows free amino acid analysis (mean + SEM)

[0260] Fig. 20 The FAA profile of Fortispore UTH fermented milk is shown. P-value ≤ 0.005 = ** and P-value ≤ 0.01 = *. White bars represent controls.

[0261] Fig.21The FAA profile of Fortispore UTH fermented milk is shown. P-value ≤ 0.005 = ** and P-value ≤ 0.01 = *. White bars represent controls.

[0262] Compared to a competitor strain of Bacillus coagulans, Fortispore was slightly more efficient at producing compounds that benefit skin health.

[0263] Ganeden Biotech will be coagulant Bacillus 6086 (BC30 TM ) is marketed as an anti-aging probiotic because it produces the largest amount of naturally derived L+ lactic acid, bacteriocins, hydrogen peroxide, enzymes, and other metabolites. Lactic acid has been associated with anti-aging, anti-acne effects due to its antimicrobial activity and hydration. Succinic acid has also been associated with anti-inflammatory, antimicrobial, and hydration effects. Benzoic acid has been associated with antimicrobial activity.

[0264] Fig. 22 The effects of Fortispore on lactic acid, succinic acid and benzoic acid production were compared with those of Lactospore and BC30. Significance is indicated by a, b and / or c, and multiple letters indicate that the results are not significantly different from more than one group (P < 0.05). Error bars indicate standard deviation.

[0265] Our results showed that Fortispore was slightly superior to BC30 in lactate production. TM , and is superior in the production of succinic acid and benzoic acid.

[0266] Compared to competitor Bacillus coagulans strains, Fortispore produces amino acids that benefit skin health.

[0267] Fortispore and Our results showed that Fortispore was superior to BC30 in producing methionine, proline, lysine and tryptophan. TM and

[0268] Methionine is a sulfur-containing amino acid that improves skin tone and elasticity, promotes healthy hair, and strengthens nails. Proline helps lower inflammation, which promotes a healthy immune system. It also helps trigger a cascade of anti-inflammatory compounds and genes that aid recovery. Lysine is an essential amino acid that has a variety of benefits, including preventing cold sores, reducing anxiety, and promoting wound healing. Tryptophan has been linked to relief from depression and anxiety.

[0269] Fig.23The effects of Fortispore on amino acid production were compared with those of Lactospore and BC30. Significance is indicated by a, b and / or c, and multiple letters indicate that the results are not significantly different from more than one group (P < 0.05). Error bars indicate standard deviation.

[0270] Fortispore proteomic analysis identifies proteins with potential probiotic benefits.

[0271] The extracellular secretions of the Fortispore were sent to a mass spectrometer to identify proteins released by the probiotic strains. A total of 28 proteins were detected, of which 6 had potential probiotic benefits.

[0272] Table 14

[0273] Fortispore proteomics identifies proteins with potential probiotic benefits

[0274]

[0275] These data confirm previous in vitro findings showing that Fortispore can aid in the digestion of proteins and carbohydrates and has antimicrobial properties against pathogens.

[0276] In the basic medium In this case, the growth of Fortispore increased.

[0277] Fig.24 Shown are the results of 24 hours post-incubation in minimal medium compared to the control. The concentration of Fortispore (CFU / mL) increased significantly by 1 log 10 *p<0.05

[0278] Compared with competitor Bacillus coagulans strains, the presence of Under these conditions, the growth of Fortispore increased significantly.

[0279] Fig.25 Show The concentration of Fortispore in minimal medium (CFU / mL) was significantly increased, while no significance was observed in the growth of BC30 and Lactospore. Statistical analysis was performed using one-way ANOVA with Tukey's method. **p-value = <0.01.

[0280] In TSB medium In the absence of , there was no increase in the growth of Fortispore.

[0281] Fig.26Compared with the control, There was no significant increase in the Fortispore concentration (CFU / mL) in TSB medium. The inability to increase the growth of Fortispore in rich medium may be because they reach maximal growth in the presence of nutrients in TSB medium.

[0282] In TSB medium Under these conditions, the growth of Fortispore, BC 30, DE111 and pathogens was compared.

[0283] Fig. 27 Compared with the control, There was no significant increase in the concentrations (CFU / mL) of DE111, Fortispore, BC30, Escherichia coli and Salmonella enteritidis in TSB medium. The inability to increase the growth of DE111, Fortispore, BC30, E. coli, and S. Enteritidis in rich medium is likely because they reach maximum growth in the presence of nutrients in TSB medium.

[0284] In 50% TSB medium In the absence of , there was no increase in the growth of Fortispore.

[0285] Fig.28 Compared with the control, There was no significant increase in the concentration of Fortispore (CFU / mL) in 50% TSB medium.

[0286] In 50% TSB medium Under the condition of , the growth of Fortispore, BC 30 and DE111 was compared.

[0287] Fig.29 After 24 hours in 50% TSB medium, Significantly increased the production of DE111 (CFU / mL) by 1 log 10 ****p<0.0001. Compared with the control, There was no indication of a significant increase in the concentrations (CFU / mL) of Fortispore and BC30 in 50% TSB medium.

[0288] When added to BHI medium No significant growth of Fortispore was observed.

[0289] Fig.30 Compared with the control in BHI medium, There was no significant increase in the concentration of Fortispore (CFU / mL). There was no increase in the growth of Fortispore in rich medium, most likely because they reach maximal growth in the presence of nutrients in BHI medium.

[0290] In BHI medium Under these conditions, the growth of Fortispore, BC 30 and DE111 was compared.

[0291] Fig.31 Compared with the control in BHI medium, There was no significant increase in the concentrations (CFU / mL) of DE111, Fortispore, and BC30. There was no increase in the growth of DE111, Fortispore, and BC30 in rich medium, most likely because they reach maximal growth in the presence of nutrients in BHI medium.

[0292] In 50% BHI medium No increase in the growth of Fortispore was observed under these conditions.

[0293] Fig.32 Compared with the control in 50% BHI medium, No significant increase in Fortispore concentration (CFU / mL) was shown. There was no increase in the growth of Fortispore in rich medium, most likely because they reach maximal growth in the presence of nutrients in BHI medium.

[0294] In 50% BHI medium No growth enhancement was observed for Fortispore, BC 30 and DE111.

[0295] Fig.33 Compared with the control in 50% BHI medium, There was no significant increase in the concentrations (CFU / mL) of DE111, Fortispore, and BC30. There was no increase in the growth of DE111, Fortispore, and BC30 in rich medium, most likely because they reach maximal growth in the presence of nutrients in BHI medium.

[0296] Example 2

[0297] Assessing adhesion capacity on an in vitro model of intestinal epithelium

[0298] Cell lines: Human colorectal adenocarcinoma cell line HT-29 and mucus secreting cell line HT-29-MTX were propagated in low glucose DMEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U / ml penicillin, 100 μg / l streptomycin and 2 μg / ml amphotericin B at 37°C in a 5% CO2 atmosphere.

[0299] The cells were plated at 5x10 5 Cells were seeded into 24-well plates at a density of 100 cells / well and cultured for 21-28 days to complete maturation. The culture medium was changed every 2-3 days.

[0300] Prior to the experiment, wash the cells twice with 0.5 ml DPBS. After the second round of washing, completely aspirate the DPBS from the wells.

[0301] Preparation of spores: Ten milligrams of spore powders of Bacillus clausii CSI08, Bacillus megaterium MIT411 and Bacillus coagulans CGI314 were weighed into a 15 ml Falcon tube and resuspended in 10 ml of Not included The suspension was aliquoted and stored at -20 °C until use. The suspension was used within 2 weeks of preparation.

[0302] Adhesion test: 500 μl of spore suspension (1.3x10 7 -9.2x10 7 CFU / ml) were added to HT-29 and HT-29-MTX cells, mixed by gentle vortexing, and incubated in a CO2 incubator at 37°C for 2.5 h. No Control wells containing mammalian cells were incubated in parallel in the same manner (0.5 ml of spore suspension).

[0303] After incubation, HT-29 and HT-29-MTX cells were washed 4 times with 0.5 ml PBS. Afterwards, 50 μl of trypsin / EDTA solution and 50 μl of PBS were added to the wells and incubated for 10 min at 37° C. with gentle shaking (~100 rpm). Fifty microliters of trypsin / EDTA solution were added to the control wells.

[0304] Therefore, 450 μL of PBS was added to the wells with spores, the contents of the wells were transferred to Eppendorf tubes by scraping and subjected to three rounds of vigorous shaking for 30 seconds each. The contents of the control wells were transferred to Eppendorf tubes and subjected to one round of shaking.

[0305] Serial dilutions (plus dilutions from control wells) were prepared in PBS and plated on BC agar (Bacillus coagulans CGI314) or PetriFilm TM (Bacillus clausii CSI08, Bacillus megaterium MIT411). Plates were incubated at 37°C for 48 h before counting, and PetriFilm was incubated at 37°C for 24 h before counting.

[0306] Experiments were performed in duplicate or triplicate with three technical replicates per experiment. Results are expressed as mean ± SEM.

[0307] Table 15

[0308] Adhesion of Bacillus clausii CSI08, Bacillus megaterium MIT411 and Bacillus coagulans CGI314 spores to HT-29-MTX cell line

[0309]

[0310] Table 16

[0311] Adhesion of Bacillus clausii CSI08, Bacillus megaterium MIT411 and Bacillus coagulans CGI314 spores to HT-29 cell line

[0312]

[0313] in conclusion

[0314] 1. The above results show that compared with non-mucus secreting cells, spore The ability to adhere to the mucus-secreting cell line HT-29-MTX was higher, which may be due to the physical properties of the spores.

[0315] 2. Bacillus megaterium MIT411 and Bacillus coagulans CGI314 spores had a higher (but overall lower) ability to adhere to the non-mucoid-producing cell line HT-29 compared to Bacillus clausii CSI08 spores.

[0316] Example 3

[0317] Evaluation of the safety, tolerability, and gastrointestinal health of Bacillus coagulans CGI314 in healthy adults: a randomized, double-blind, placebo-controlled trial:

[0318] Study Intervention

[0319] Daily administration of 1x10 9 CFU of Bacillus coagulans CGI314 and Bacillus subtilis Probiotics 1,

[0320] A probiotic combination comprising about (and greater than) 0.5 x 109 CFU of Bacillus subtilis About 0.5x10 9 CFU of Bacillus megaterium MIT411, about (and less than) 0.5x10 9 CFU of Bacillus coagulans CGI314, about 0.5x10 9 CFU of Bacillus clausii CSI08; total daily application amount 2x10 9 Bacillus spores

[0321] Placebo: Daily administration of rice maltodextrin

[0322] Indications studied

[0323] Gastrointestinal homeostasis and the immune system.

[0324] Study Description

[0325] Healthy adults aged 18-65 years were recruited and screened to participate in this study. Eligible participants were randomly assigned to one of the two experimental groups or the control group in a 1:1:1 ratio and received 45 days of treatment and 2 weeks of follow-up to evaluate the safety and efficacy of the novel probiotic strain in healthy adults.

[0326] For the purposes of this study, the study product was packaged in individual capsules consistent with the daily dose. Each capsule contained 300 mg of the following ingredients:

[0327] Probiotic Formula 1: Bacillus coagulans CGI314 and Bacillus subtilis (1x10 9 Total CFU), ~4.17mg; low moisture rice maltodextrin, 292.23mg; medium chain triglycerides, 3.6mg.

[0328] Probiotic cocktail: 2 x 10 9 CFU Bacillus spores, including Bacillus subtilis (approximately (and greater than) 0.5x10 9 CFU), 2.1 mg; Bacillus megaterium MIT411 (0.5x10 9 CFU), 2.1 mg; Bacillus clausii CSI08 (about 0.5x10 9 CFU), 2.1 mg; Bacillus coagulans CGI314 (approximately (and less than) 0.5x10 9 CFU), 2.1mg; low moisture rice maltodextrin, 288mg; medium chain triglycerides, 3.6mg.

[0329] Placebo consisted of all the same ingredients as the study product, but without the probiotics. Placebo was a single capsule, equivalent to the daily dose. Each capsule contained 300 mg of ingredients: Placebo: Low-moisture rice maltodextrin, 296.4 mg; Medium-chain triglycerides, 3.6 mg.

[0330] Table 17 summarizes the changes in gut-brain axis questionnaire scores from baseline to the end of the treatment period for the four probiotic groups and the placebo group. The changes in the mean values ​​with 95% confidence intervals are shown. The results of the ANOVA comprehensive test (p*-value) and the one-sample T test (p-value) are also provided. The normality test of the changes in the gut-brain axis scores showed that the data did not follow a normal distribution, which may affect the results with borderline significance (p-values ​​between 0.05 and 0.10). This affects two items: loss of energy and changes in appetite. A non-parametric Kruskal Wallis test was performed on these items; the observed p-values ​​were 0.111 (fatigue) and 0.123 (changes in appetite).

[0331] In short, the mean values ​​of the changes in the gut-brain axis questionnaire scores for all tested symptoms were negative, which means that at the end of the treatment period, the intensity of the symptoms decreased (participants were less bothered by these symptoms). The results of the one-sample T test showed that statistically significant changes in the gut-brain axis questionnaire scores were observed in one-third of the tests (a total of 70 times). However, this was observed in all treatment groups, including the placebo group. Therefore, the results of the ANOVA test showed that no significant differences in the changes in the gut-brain axis scores were detected between the treatment groups, but borderline significance was observed for the fatigue and appetite change items. Participants in the Bacillus megaterium group experienced the greatest changes in these two items.

[0332] Table 17

[0333] Changes in the Gut-Brain Axis Questionnaire Item Scores from Baseline to the End of the Treatment Period: Pairwise Comparisons between the Probiotic and Placebo Groups (Post Hoc ANOVA: Dunnet t-Test)

[0334]

[0335]

[0336] N* = number of participants included in the ITT population, *p-value from Dunnett's T-test (pairwise comparison)

[0337] Mean differences with 95% confidence intervals and p-values ​​for pairwise comparisons between individual probiotic groups and placebo are shown in Table 17. None of the investigational products containing probiotics demonstrated statistically significant differences in the change in gut-brain axis scores from baseline to the end of the treatment period compared to placebo.

[0338] Mean differences with 95% confidence intervals and p-values ​​for pairwise comparisons between individual probiotic groups and placebo are shown in Table 18. None of the sum of gut-brain axis item scores for the probiotic-containing investigational products showed statistically significant differences compared to placebo at either baseline or at the end of the treatment period.

[0339] Table 18

[0340] Sum of the Gut-Brain Axis Questionnaire item scores at baseline and at the end of the treatment period: pairwise comparisons between the probiotic and placebo groups (post hoc ANOVA: Dunnett's t-test)

[0341]

[0342]

[0343] N* = number of participants included in the ITT population, *p-value from Dunnett's T-test (pairwise comparison)

[0344] The mean differences with 95% confidence intervals and p-values ​​for pairwise comparisons between the individual probiotic groups and the placebo group are shown in Table 19. None of the investigational products containing probiotics demonstrated statistically significant differences in changes in HDL, LDL, TC, or TG from baseline to the end of the treatment period compared to placebo.

[0345] Table 19

[0346] Changes in cholesterol and triglycerides from baseline to the end of the treatment period in the ITT population: pairwise comparisons of the probiotic group with the placebo group (post hoc ANOVA: Dunnett's t-test)

[0347]

[0348] N* = number of participants included in the ITT population

[0349] The mean differences with 95% confidence intervals and p-values ​​for pairwise comparisons between the individual probiotic groups and the placebo group are shown in Table 20. None of the investigational products containing probiotics demonstrated statistically significant differences in relative changes in HDL, LDL, TC, or TG from baseline to the end of the treatment period compared to placebo.

[0350] Table 20

[0351] Relative changes in cholesterol and triglycerides from baseline to the end of the treatment period in the ITT population: pairwise comparisons of the probiotic group with the placebo group (post hoc ANOVA: Dunnett's t-test)

[0352]

[0353] N* = number of participants included in the ITT population, *p-value from Dunnett's T-test (pairwise comparison)

[0354] Table 21 shows the proportion of participants who reported at least one day of clinically relevant infection in Diary 1. Table 22 shows the descriptive statistics of the number of days with clinically relevant infection and the results of the Kruskal-Wallis test (p*-value) and the Mann-Whitney U test with Holm correction (p-value).

[0355] Table 21

[0356] Proportion of patients with clinically relevant infections reported in participant diary 1 (N=123)

[0357]

[0358]

[0359] N* = number of participants included in the ITT population

[0360] Table 22

[0361] Number of days with clinically relevant infections reported in participant diary 1 (N=123)

[0362]

[0363] N* = number of participants included in the ITT population, NC = not calculable, P* = p-value of Kruskal-Wallis test, P = p-value of Mann-Whitney U test.

[0364] The Kruskal-Wallis test did not reveal any significant differences in the number of days with clinically relevant infections between the treatment groups. However, a borderline statistically significant result was observed for clinically relevant gastrointestinal infections. This may have occurred by chance because no participant in any of the four probiotic treatment groups developed a clinically relevant gastrointestinal infection, whereas a total of 2 days of such infections were observed in the probiotic treatment groups.

[0365] Despite this, none of the study products that included probiotics showed a statistically significant difference compared with placebo.

[0366] Table 23 shows the proportion of participants who reported having individual symptoms of gastrointestinal infection on at least one day in their Diary 2. Table 24 shows the descriptive statistics of the number of days with gastrointestinal infection symptoms and the results of the Kruskal-Wallis test (p*-value) and the Mann-Whitney U test with Holm correction (p-value).

[0367] Table 23

[0368] Proportion of participants reporting gastrointestinal infection symptoms in Diary 2 (N=118)

[0369]

[0370] N* = number of participants included in the ITT population

[0371] Table 24

[0372] Number of days with gastrointestinal infection symptoms reported in participant diary 2 (N=118).

[0373]

[0374]

[0375] N* = number of participants included in the ITT population, NC = not calculable, P* = p-value of Kruskal-Wallis test, P = p-value of Mann-Whitney U test.

[0376] A significant difference was detected between the groups only in the number of days with constipation (p*=0.013), which may be due to the fact that only three participants in the placebo group reported this symptom, while no participants in the other four treatment groups reported this symptom in participant diary 2. However, further analysis (Mann-Whitney U test with Holm correction) comparing the number of days with constipation in the individual probiotic groups with the placebo group did not show a significant difference, which may be due to the small sample size.

[0377] Efficacy results: treatment period

[0378] No significant differences were detected in gastrointestinal health among the treatment groups at baseline, a finding that was expected given that participants were randomly assigned to one of five treatment groups.

[0379] Item scores on the gut-brain axis questionnaire were lower at the end of the treatment period compared with baseline scores, meaning that participants were less bothered by these symptoms at the end of the treatment period. This was observed in all treatment groups, including the placebo group.

[0380] No significant differences were detected in the change in gut-brain axis item scores (end of treatment minus baseline) between treatment groups. None of the study products containing probiotics showed a statistically significant difference in the change in gut-brain axis scores compared with the placebo group.

[0381] No significant differences were detected between treatment groups in improvements in gut-brain axis item scores. None of the study products containing probiotics demonstrated statistically significant differences in improvements in gut-brain axis scores compared with placebo.

[0382] No significant differences were detected in the sum of all item scores of the gut-brain axis questionnaire between treatment groups. No sum of gut-brain axis item scores for the study products containing probiotics showed statistically significant differences compared with placebo at baseline or at the end of the treatment period.

[0383] No significant differences were detected between treatment groups in the changes in cholesterol and triglycerides (absolute and relative changes from baseline to the end of the treatment period). The study products containing probiotics did not show statistically significant differences in the changes in HDL, LDL, TC, or TG (absolute and relative) from baseline to the end of the treatment period compared with placebo. The same results were observed in the PP population.

[0384] At the end of the treatment period, the levels of cytokines (TNFα, IFNα, IFNβ, IFNγ and IL6) in all treatment groups were below the limit of quantification (LOQ). For the cytokine IL13, some values ​​above the LOQ were reported, but no statistically significant differences between treatment groups were observed.

[0385] At the end of the treatment period, no significant changes in the levels of heavy metals (cadmium, lead, mercury, copper, nickel, zinc, and arsenic) were detected in the B. megaterium group compared with baseline values. The same results were observed in the PP population.

[0386] None of the study products containing probiotics showed a statistically significant difference in the number of days with symptoms of gastrointestinal infection compared with placebo. However, a borderline statistically significant result was observed for clinically relevant gastrointestinal infection. This may have occurred by chance because no participant in any of the four probiotic treatment groups developed a clinically relevant gastrointestinal infection, whereas a total of 2 days of such infections were observed in the probiotic treatment group.

[0387] None of the study products containing probiotics showed a statistically significant difference in the number of days with respiratory tract infection symptoms compared to placebo. However, there was a statistically significant difference between the treatment groups in the number of days with runny nose (p*=0.018), which may be due to only three participants in the probiotic mixture group reporting this symptom, while no participants in the other four treatment groups reported this symptom.

[0388] However, further post hoc analysis (pairwise comparisons vs placebo) did not show any significant differences.

[0389] The study product containing probiotics did not show a statistically significant difference in the number of days with UTI symptoms compared with placebo.

[0390] No statistically significant differences were detected between treatment groups with regard to stool regularity.

[0391] Significant differences were detected between groups for stool consistency, i.e., the proportion of loose stools, throughout the treatment period and at weeks 6 and 7 of the treatment period. However, further post hoc analyses (paired comparisons vs placebo) did not show any significant differences, which may be due to the small sample size. Participants in the probiotic mixture group had the smallest proportion of all stools that were loose.

[0392] Efficacy results: follow-up period

[0393] Only the number of constipation days was significantly different between the groups (P=0.013).However, further post hoc analysis (pairwise comparisons vs placebo) did not show any significant differences, which may be due to the small sample size.

[0394] The study products containing probiotics did not show a statistically significant difference in the number of days with respiratory tract infection symptoms.

[0395] None of the study products containing probiotics showed a statistically significant difference in the number of days with UTI symptoms.

[0396] No significant differences were detected between treatment groups in stool regularity or stool consistency.

[0397] Safety Results

[0398] A total of 17 AEs (adverse events) were reported. Between 1 and 5 ADEs were reported for each study product, with the most common being gastroesophageal reflux (3 AEs), rash (2 AEs), and dizziness (2 AEs). No SAEs (serious adverse events) were reported.

[0399] Causality assessment showed no relationship between the reported AEs and the study product.

[0400] In conclusion, this study investigated the safety and efficacy of novel probiotics, namely, Bacillus coagulans, Bacillus clausii, Bacillus megaterium, and a probiotic mixture containing Bacillus subtilis, Bacillus megaterium, Bacillus clausii, and Bacillus coagulans.

[0401] There were no differences in gastrointestinal health conditions at baseline between the study groups, as expected from randomization.

[0402] The primary outcome of the study (safety) was achieved as a total of 17 AEs and no SAEs were reported. Causality assessment showed no relationship between the reported AEs and the study product.

[0403] None of the outcomes related to efficacy showed any statistically significant differences, but this is not surprising given the small sample size in each study group. Nevertheless, some trends in favor of the active product were observed, particularly in the gut-brain axis score and the proportion of loose stools.

[0404] In conclusion, probiotic products are safe for use in adults and show some favorable trends in the gut-brain axis and stool consistency, however, to scientifically prove the efficacy of the studied products, further studies with larger sample sizes are needed, a run-in period is needed to determine the baseline stool consistency of each participant, and the use of validated questionnaires to determine the gut-brain axis domain.

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[0458] The invention is not limited to the embodiments described herein and modifications or variations may be made without departing from the scope of the invention.

[0459] The terms "a", "an", "the" and similar references used in the context of describing the invention (especially in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Unless otherwise indicated herein, the recitation of numerical ranges herein is intended merely to serve as a shorthand method of individually referring to each individual value falling within the range, and each individual value is incorporated into the specification as if it were individually recited herein. The use of the term "about" is intended to describe values ​​within a range of about ±10% above or below the stated value; in other embodiments, these values ​​may be within a range of about ±5% above or below the stated value; in other embodiments, these values ​​may be within a range of about ±2% above or below the stated value; in other embodiments, these values ​​may be within a range of about ±1% above or below the stated value. The foregoing ranges are intended to be made clear by the context and are not meant to be further limited. All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Unless otherwise specified, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention rather than to limit the scope of the present invention. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the present invention.

[0460] Although in the foregoing description, the present invention has been described in conjunction with certain embodiments of the present invention and many details have been presented for illustrative purposes, it is obvious to those skilled in the art that the present invention may have additional embodiments and that certain details described herein may be considerably changed without departing from the basic principles of the present invention.

[0461] All references cited herein are incorporated herein by reference in their entirety. The present invention may be implemented in other specific forms without departing from the spirit or essential attributes of the present invention, and therefore, reference should be made to the appended claims rather than the foregoing description to indicate the scope of the present invention.

Claims

1. A Bacillus coagulans strain comprising a purified population of microorganisms comprising one or more bacteria having a gyrB that is at least 97% identical to SEQ ID NO: 1; and / or the population of microorganisms comprising one or more bacteria having a 16S rRNA that is at least 97% identical to SEQ ID NO:

2.

2. The Bacillus coagulans strain according to claim 1, which has at least 97% identity to SEQ ID NO:

3.

3. The Bacillus coagulans strain according to claim 1, wherein The purified microbial population includes bacteria having a 16S nucleic acid sequence including SEQ ID NO:

2.

4. The Bacillus coagulans strain according to claim 1, wherein The purified microbial population includes bacteria having a gyrB nucleic acid sequence including SEQ ID NO:

1.

5. The Bacillus coagulans strain according to claim 1, wherein The purified microbial population includes bacteria having a 16S nucleic acid sequence including SEQ ID NO:2 and a gyrB nucleic acid sequence including SEQ ID NO:

1.

6. A microbial composition comprising the Bacillus coagulans strain according to any one of claims 1 to 5, and an edible carrier and / or diluent.

7. The microbial composition according to claim 6, wherein The unit dose of the composition comprises 10 6 -10 13 CFU of Bacillus coagulans strains.

8. The microbial composition according to claim 6 or 7, further comprising a mucoadhesive excipient.

9. The microbial composition according to any one of claims 6 to 8, further comprising at least one other probiotic Bacillus strain.

10. The microbial composition according to any one of claims 6 to 9, wherein The microbial composition is formulated into tablets, pills, capsules, powders, solutions, suspensions or emulsions.

11. The microbial composition according to any one of claims 6 to 9, wherein The microbial composition is formulated as a food.

12. The Bacillus coagulans strain according to any one of claims 1 to 5, for use in preventing or treating vaginal infections, urinary tract infections, gastrointestinal infections, gastrointestinal diseases, improving immune health, protecting against oxidative stress, cleansing and detoxification, metabolic health and cardiovascular health.

13. A method of preventing or treating vaginal infections, urinary tract infections, gastrointestinal infections, gastrointestinal diseases, improving immune health, protecting against oxidative stress, cleansing and detoxification, metabolic health and cardiovascular health, the method comprising administering the Bacillus coagulans strain according to any one of claims 1 to 5.

14. A microbial composition according to any one of claims 6 to 11 for use in preventing or treating vaginal infections, urinary tract infections, gastrointestinal infections, gastrointestinal diseases, improving immune health, protecting against oxidative stress, cleansing and detoxification, metabolic health and cardiovascular health.

15. A method for preventing or treating vaginal infections, urinary tract infections, gastrointestinal infections, gastrointestinal diseases, improving immune health, protecting against oxidative stress, cleansing and detoxification, metabolic health and cardiovascular health, the method comprising administering a microbial composition according to any one of claims 6 to 11.

16. A method of improving the microbiome in a subject, the method comprising administering to the subject a composition comprising a probiotic, wherein the probiotic comprises the Bacillus coagulans strain according to any one of claims 1 to 5.

17. The Bacillus coagulans strain according to any one of claims 1 to 5, for use as a probiotic, wherein optionally one or more of the bacterial strains are combined with an acceptable carrier or one or more delivery vehicles and optionally one or more adjuvant components in a single composition or separate compositions comprising a mixture of different bacterial strains.

18. Use of the Bacillus coagulans strain according to any one of claims 1 to 5 for the preparation of a medicament for treating vaginal infections, urinary tract infections, gastrointestinal infections, gastrointestinal diseases, improving immune health, protecting against oxidative stress, cleansing and detoxification, metabolic health, cardiovascular health and / or skin health.

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