BACILLUS COAGULANS STRAINS, COMPOSITIONS THEREOF AND METHODS OF USE

Abstract

The present invention is directed to a new Bacillus coagulans strain, which may be used alone or in combination with other Bacilli strains, as a probiotic or with prebiotics and synbiotics. The present invention also relates to compositions, such as pharmaceutical compositions, dairy products, functional foods, nutraceuticals, dietary supplements and personal care products, comprising the new Bacillus coagulans strain, alone or in combination with other strains, and to the use of the strain for the prevention or treatment of gastrointestinal infections and diseases, and other uses.

Description

[Technical field]

[0001] The present invention relates to novel Bacillus coagulans strains, which may be used alone or in combination with other Bacilli strains as probiotics or with prebiotics and synbiotics. The present invention also relates to compositions, such as pharmaceutical compositions, dairy products, functional foods, nutraceuticals, dietary supplements and personal care products, comprising the novel Bacillus coagulans strains, alone or in combination with other strains, and to the use of the strains for the prevention or treatment of gastrointestinal infections and diseases and other uses. [Background technology]

[0002] Probiotics are live microorganisms or mixtures of microorganisms administered to improve the microbial balance of a patient, especially the environment of the respiratory and gastrointestinal tract. Bacillus strains have been used to treat respiratory infections, prevent diarrhea, and treat immune-related diseases (Elshaghabee et al., 2017).

[0003] Normal gut flora is dominated by various bacterial species, which produce substances that help regulate the growth of pathogens. Dysbiosis is a condition characterized by a reduction in certain bacterial species and an increased growth of pathogenic bacteria. Dysbiosis has been associated with the development of periodontal disease, inflammatory bowel disease and chronic fatigue syndrome. Some studies suggest that patients with dysbiosis may have an increased risk of developing metabolic and cardiac disorders (Chan et al., 2013).

[0004] By administering probiotic Bacilli, it is possible to regenerate the intestinal flora in men and women with frequent 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 the maintenance of the gut microbial ecosystem. Bacilli have been shown to possess inhibitory activity against the growth of pathogenic bacteria such as Listeria monocytogenes, Escherichia coli, Salmonella spp. and others (Yilmaz et al., 2005). This inhibition could be due to the production of inhibitory compounds such as organic acids, hydrogen peroxide, bacteriocins or reuterin or competitive adhesion to the epithelium (Abriouel et al., 2010).

[0006] Bacilli are also being investigated as a treatment for respiratory tract infections (Marseglia et al., 2007). For example, the introduction of Bacilli and stimulation of the indigenous flora has been used to prevent recurrent urinary tract infections (Marseglia et al., 2007). The role of Bacilli in preventing intestinal infections is also being investigated.

[0007] 2. Description of Related Art The importance of Bacilli as probiotics has been documented in the literature.

[0008] Hyronimus et al., 2000 disclose the screening of a number of Bacilli strains for probiotic activity by in vitro techniques and the evaluation of the colonization ability of 13 selected strains in humans. The strains were examined for resistance to pH 2.5 and 0.3% Oxgall adhesion to Caco-2 cells and antimicrobial activity against enteropathogenic bacteria (Khochamit et al., 2015). In addition to possessing good adhesion and biotherapeutic properties, Bacilli have been shown to possess the basic requirements for GIT stress resistance (Thakur et al., 2016).

[0009] Pharmaceutical compositions of Bacilli known in the art are not efficient enough in recolonizing in vivo, i.e., mammalian microbial ecosystems, and therefore it is necessary to find Bacilli that have the inherent ability to recolonize upon administration of Bacilli 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 upon administration due to their inherent survival ability in the human microbial ecosystem. Identifying Bacilli strains with enhanced ability to colonize upon administration is often a cumbersome process, and therefore it is important to select the best test system to predict in vivo colonization ability.

[0010] There appears to be a large variability in the reported in vitro adhesion properties of probiotic strains in the literature. This variability indeed reflects biological differences between strains, but certainly also depends on the experimental conditions. Furthermore, there appears to be variability regarding the methods for measuring adhesion. It can be argued that in vitro experiments serve only as a means to estimate the in vivo colonization potential by adhesion to epithelial cells.

[0011] Although long considered as soil microorganisms, Bacillus spp. have been used for more than 50 years in the form of supplements based on fermentation products or spores (Cutting et al., 2011). Ubiquitous in nature, Bacilli constantly invade the gastrointestinal and respiratory tracts of healthy humans through food, water and air (Benno & Mitsuoka, 1986). They have been isolated from the intestine and are present in up to 10 7 It is considered to be one of the dominant components of normal gut microbiota, as it can reach CFU / g (Lakshmi et al., 2017). More recently, strains of Bacillus clausii have been isolated to serve more specific functions and their safety evaluated. Bacillus clausii has been used in the past without reports of adverse events in patients with diarrhea (Sudha et al., 2013; Horosheva et al., 2014) and children with recurrent respiratory infections (Marseglia et al., 2007). Although the country and strain were not specified, Bacillus clausii is commercially available in 55 countries worldwide (Nista et al., 2004; Gabrielli et al., 2009). Review literature for Bacillus clausii shows no adverse events associated with the probiotic, and the global presence of the bacterium in different countries complements the narrative of its safety for human consumption.

[0012] Bacillus coagulans has a long history of use in various foods. There are many strains of Bacillus coagulans that have been widely consumed worldwide for decades (Endres et al., 2009). The presence of this bacterium can be found in foods such as yogurt, milk, sauerkraut, kimchi and other dairy products, all of which are found in abundance at 5x10 9 CFU / g (Sudha et al., 2016)~9.38x10 10(Endres et al., 2011) contain Bacillus coagulans levels. Several Bacillus species have been reported to be common 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). The origin of this bacterial species for use in probiotics originates from India, where many manufacturers produce Bacillus coagulans as a food ingredient for export and relabeling in Europe and the United States (Cutting, 2011). To assess the safety of Bacillus coagulans, the genome was examined and it was found to contain no harmful genes (Salvetti et al., 2016). Due to the harmless nature of Bacillus coagulans, the bacterium has been reported as safe by the US Food and Drug Administration (FDA) and the European Union Food Safety Authority (EFSA) and is listed on the Generally Recognized as Safe (GRAS) and Qualified Presumption of Safety (QPS) lists (European Food Safety Authority, 2017).Among the 100 known Bacillus spp., several non-pathogenic strains, including Bacillus coagulans and Bacillus subtilis, have been declared safe for human consumption for all ages (Nithya & Halami, 2012). Review literature on Bacillus coagulans showed no adverse events associated with probiotics, reinforcing the finding that it is well tolerated and safe. Therefore, Bacillus coagulans can be considered a safe probiotic whose consumption benefits the host.

[0013] Bacillus megaterium has been found in a variety of habitats, from soil to marine water, sediments, rice fields, honey, fish, milk and dry foods (Alfoldi, 1957; Alippi & Reynaldi, 2006; Padgham and Sikora, 2007; Pelletier & Sygusch, 1990; Vary et al., 2007; Von Tersch and Carlton, 1983; Scholle et al., 2003, Kotb, 2014). Further qualitative analysis of the microorganisms isolated from honey revealed that one of the most frequent species of Bacillus was Bacillus megaterium (Alippi, 1995; Alippi et al., 2004; Snowdon & Cliver, 1996; Tysset, Durand, & Taliergio, 1970). There have been ancillary experiments in which Bacillus megaterium was isolated in fish (Sumathi et al., 2017). Afrilasari et al., 2015 also successfully isolated Bacillus megaterium from the digestive tract of catfish and identified it as PTB 1.4. The harmless nature of Bacillus megaterium has led to the bacterium being included in the Qualified Presumption of Safety (QPS) list (European Food Safety Authority, 2017). Bacillus megaterium strain ATCC14581 was confirmed to be nearly identical (>99%) to Bacillus megaterium MIT411 through genomic analysis. Health Canada has declared that the organism poses no hazard to human health or the environment; exposure to the environment and Canadians is moderate.Therefore, it can be concluded that Bacillus megaterium strain ATCC14581 poses no hazard to human health or to the environment (Health Canada, 2018).

[0014] In summary, Bacilli strains with probiotic potential should be able to adhere to other suitable cells, such as the cell line Caco-2 cells. Furthermore, it is also desirable that Bacilli strains with probiotic potential show in vitro inhibitory activity against other bacterial species, produce acid and / or produce hydrogen peroxide after growth in liquid culture. Summary of the Invention

[0015] It is an object of the present invention to provide suitable probiotic Bacilli strains and compositions as described throughout this application, such as pharmaceutical formulations or absorbent products, that have the desired properties. In one embodiment, the present invention relates to Bacillus coagulans strain CGI314, alone or in combination with other strains, such as Bacilli strains such as Bacillus megaterium strain MIT411 (disclosed and claimed in corresponding PCT application PCT / US2022 / xxxxx, which claims priority from Irish Patent Application No. 2021 / 0211, the contents of which are incorporated herein in their entirety) and Bacillus clausii strain CSI08 (disclosed and claimed in corresponding PCT application PCT / US2022 / xxxxx, which claims priority from Irish Patent Application No. 2021 / 0209, the contents of which are incorporated herein in their entirety).In one embodiment, these strains have similar or essentially the same advantageous properties, e.g. colonization ability due to adhesion to mucous membranes, thus treatment or prevention of infections or diseases, e.g. vaginal, urinary tract, gastrointestinal, naso-sinal, pharyngeal, esophageal, oral and / or other areas of the body with e.g. mucous membranes, and treatment or prevention of infections or diseases, e.g. of the vagina, urinary tract, gastrointestinal, naso-sinal, pharyngeal, esophageal, oral and / or other areas of the body with skin and / or epithelium; immune health, protection against oxidative stress, cleansing and detoxification, metabolic health and cardiovascular health, inter alia antimicrobial activity, including e.g. lactic acid, benzoic acid and succinic acid, anti-inflammatory activity, providing suppression of pro-inflammatory responses, e.g. stimulating macrophages. The Bacillus clausii strain CSI08 is suitable for activating and / or eliciting an immune response by inhibiting the production of lactic acid, benzoic acid and succinic acid, providing immune protection, for example aiding in digestion and / or fermentation in the gut, producing branched amino acids, essential amino acids and group B vitamins, maintaining healthy gut and / or skin with lactic acid, benzoic acid and succinic acid in support of skin health, protecting mucous membranes and other epithelial tissues from toxic substances, reducing the occurrence of loose stools, improving the gut-brain axis, and treating and / or preventing dysbiosis and its effects such as periodontal disease, inflammatory bowel disease, chronic fatigue syndrome, metabolic disorders, cardiac disorders, respiratory tract infections, urinary tract infections, GI infections and diarrhea; and restoring normal and / or healthy bacterial flora. In one embodiment, the present invention enables the use of Bacillus clausii strain CSI08 and compositions for use in fecal transplants.

[0016] Gastrointestinal disorders include, but are not limited to, treating gastrointestinal disorders in an individual, wherein the individual has at least one 24 hour episode per month of bowel movements that are a 1 or 2 on the Bristol Stool Scale (i.e., treating constipation); or the individual has at least one 24 hour episode per month of bowel movements that are a 6-7 on the Bristol Stool Scale (tendency towards diarrhea), wherein the frequency of the individual's 24 hour episodes per month of bowel movements that are a 1 or 2 (or 6-7) on the Bristol Stool Scale is reduced.

[0017] Also included is a method of restoring gastrointestinal regularity in an individual, wherein the individual has at least one 24-hour episode per month of bowel movements that are 1 or 2 on the Bristol Stool Scale; or 6-7, and the frequency of a 24-hour period in which the individual has bowel movements that are 3-5 on the Bristol Stool Scale is increased.

[0018] The present invention further includes maintaining a healthy gut microbiome using Bacillus-containing compositions, which may be used as probiotic supplements to the gastrointestinal microbiome and may compete with or otherwise interfere with pathogenic bacteria in the gut, such as Escherichia coli, Listeria monocytogenes, Salmonella spp.

[0019] Another object of the present invention is to provide pharmaceutical formulations that have an increased ability to colonize by adhesion to mucosa through the use of mucosal adhesive excipients.

[0020] It is a further object of the present invention to provide a vaginal formulation that has an increased ability to inhibit the growth of Candida albicans and gram-negative pathogenic bacteria.

[0021] It is yet another object of the present invention to provide compositions, such as dairy products, nutraceutical products and functional foods, comprising Bacillus coagulans strain CGI314 alone or in combination with other Bacilli strains, such as Bacillus megaterium and / or Bacillus clausii strains, having essentially the same properties of having the ability to colonize mucous membranes and thus indicated for the treatment or prevention of vaginal infections, urinary tract infections and gastrointestinal diseases. The compositions of the present invention may be administered in one dose, one day, one day to one week, one day to one month, one month to 45 days, 45 days to two months, three months, six months, one year or more, including any time frame specified and / or falling within these ranges.

[0022] The present invention includes methods of treatment and / or prevention and various other methods, which may include providing a Bacillus coagulans strain or a composition of the present invention to a subject (e.g., a mammalian subject, a human, including a human patient, etc.) and administering the strain or composition to the subject. In one embodiment, the present invention is directed to a composition, e.g., the use of the composition, for treating a disease or infection or other condition. In one embodiment, the present invention is directed to the use of a Bacillus coagulans strain or a composition thereof, as described throughout this application (including the claims), in the manufacture of a medicament for treating vaginal infections, urinary tract infections, gastrointestinal diseases, improving immune health, defense against oxidative stress, cleansing and detoxification, metabolic health, cardiovascular health and / or skin health, and / or other treatments or other reasons for the applications described throughout this application. [Brief description of the drawings]

[0023] In the drawings [Figure 1] FIG. 1 illustrates a phylogenetic tree of Bacillus spp. arranged in clades (16S). [Diagram 2]FIG. 2 illustrates a phylogenetic tree of Bacillus spp. arranged in clades (gyrB). [Diagram 3] Figure 3 shows the antibacterial activity of B. coagulans CGI314 against opportunistic pathogens of the intestine, skin and urinary tract on MRS agar plates overlaid with 0.4% TSA agar (solid medium): AE. E. coli; BS. S. enteritidis and CS. aureus. [Figure 4] Figure 4 shows the antibacterial activity of B. coagulans CGI314 in liquid TSB medium against opportunistic pathogens of the intestine, skin and urinary tract. Pathogens: E. coli, Salmonella, Pseudomonas aeruginosa and S. aureus. (Concentration Log10CFU / ml) [Diagram 5] Figure 5 shows the strongest antibacterial activity observed with B. coagulans CGI314 (Fortispore) against E. coli at 24 hours. (a) B. coagulans MTCC5856 (Lactospore®), (b) B. coagulans CG314 (Fortispore), (c) B. coagulans 6086 (BC30™) inoculated onto MRS agar against E. coli 0.4% TSA agar overlay plates. [Figure 6] Figure 6 shows the strongest antibacterial activity observed with Fortispore against S. enteritidis at 48 hours. (a) Lactospore® (b) Fortispore (c) BC30™ inoculated onto MRS agar against S. enteritidis 0.4% TSA agar overlay plates. [Figure 7]Figure 7 shows that no antibacterial activity was detected over Bacillus coagulans against P. aeruginosa. (a) B. coagulans MTCC5856 (Lactospore®), (b) B. coagulans CG314, (c) B. coagulans 6086 (BC30™) inoculated onto TSA agar against P. aeruginosa 0.4% TSA agar overlay plates at 24 hours. [Figure 8] Figure 8 shows the limited antibacterial activity detected across B. coagulans against S. aureus using MRS agar at 24 hours: (a) B. coagulans inoculated onto TSA agar versus S. aureus 0.4% TSA agar overlay plate; (b) B. coagulans inoculated onto MRS agar versus S. aureus 0.4% TSA agar overlay plate. [Figure 9] FIG. 9 shows the total antioxidant capacity of B. coagulans (Fortispore) and L. rhamnosus. [Figure 10] FIG. 10 shows the total antioxidant capacity of DE111, B. coagulans (Fortispore), B. clausii CSI08 (Munispore), B. megaterium MIT411 (Renuspore) and L. rhamnosus. [Figure 11] FIG. 11 shows that adhesion of Fortispore to the HT-29 cell line, measured as the percentage of adherent bacteria, was negligible. [Figure 12] FIG. 12 shows that adhesion of Fortispore to the HT-29-MTX cell line, measured as the percentage of adherent bacteria, is negligible. [Figure 13] FIG. 13 shows a study of the adhesion of B. coagulans strains to the HT-29 cell line. [Figure 14] FIG. 14 shows a study of the adhesion of B. coagulans strains to the HT-29-MTX cell line. [Figure 15] FIG. 15 shows the lack of caseinolytic activity in B. coagulans CGI314 using both the streak method at 24 or 48 hours and the overnight TSB broth method. [Figure 16] FIG. 16 shows that the proteolytic activity of Fortispore on casein derivatives is lower than the positive control, Proteinase K, using the EnzCheck® kit after 24 hours of incubation at 37° C. [Figure 17] FIG. 17 shows the lack of caseinolytic activity across (left to right) B. coagulans CGI314, B. coagulans 6086 (BC30™) and B. coagulans MTCC5856 (Lactospore®) using both the streak and overnight TSB broth methods at 24 or 48 hours. [Figure 18] FIG. 18 shows that Fortispore B. coagulans exhibits low protease activity against casein. [Figure 19] FIG. 19 shows a quantitative analysis of caseinolytic activity across B. coagulans strains as determined by the EnzCheck® kit after 24 hours of incubation at 37° C. [Figure 20] Figure 20 shows the FAA profile of Fortispore UTH fermented milk, showing the relative concentrations of methionine, alanine, proline, tryptophan, lysine and cis-aconitic acid (white bar (left) corresponds to the control). [Figure 21]Figure 21 shows the FAA profile of Fortispore UTH fermented milk. The relative concentrations of succinic acid, lactic acid, benzoic acid and isocitrate are shown (white bar (left) corresponds to the control). [Figure 22] FIG. 22 compares Lactospore and BC30 with Fortispore in the production of lactate, succinate and benzoate (from left to right: control, BC30, Lactospore, Fortispore). [Figure 23] FIG. 23 compares Lactospore and BC30 with Fortispore in the production of amino acids (from left to right: control, BC30, Lactospore, Fortispore; also shows the relative concentrations of methionine, proline, tryptophan, lysine). [Figure 24] FIG. 24 shows that Fibersol® (F) significantly increased the concentration of Fortispore (CFU / mL) by 1 log10 in minimal medium after 24 hours of incubation compared to the control. [Diagram 25] FIG. 25 shows that Fibersol® (F) significantly increased the concentration (CFU / mL) of Fortispore in minimal medium, while no significance in the growth of BC30 and Lactospore was seen. [Figure 26] FIG. 26 shows that Fibersol® (F) did not show a significant increase in Fortispore concentration (CFU / mL) in TSB medium compared to the control. [Figure 27] FIG. 27 shows that Fibersol® (F) showed no significant increase in the concentrations (CFU / mL) of DE111, Fortispore, BC30, E. coli, and Salmonella enteritidis in TSB medium compared to the control. [Figure 28] FIG. 28 shows that Fibersol® (F) did not significantly increase the concentration of Fortispore (CFU / mL) in 50% TSB medium compared to the control. [Figure 29] FIG. 29 shows that Fibersol® (F) significantly increased the yield (CFU / mL) of DE111 by 1 log10 after 24 hours in 50% TSB medium. [Diagram 30] FIG. 30 shows that Fibersol® (F) did not show a significant increase in the concentration of Fortispore (CFU / mL) compared to the control in BHI medium. [Diagram 31] FIG. 31 shows that Fibersol® (F) did not show a significant increase in the concentration (CFU / mL) of DE111, Fortispore and BC30 compared to the control in BHI medium. [Diagram 32] FIG. 32 shows that Fibersol® (F) showed no significant increase in Fortispore concentration (CFU / mL) compared to the control in 50% BHI medium. [Diagram 33] FIG. 33 shows that Fibersol® (F) showed no significant increase in the concentration (CFU / mL) of DE111, Fortispore and BC30 compared to the control in 50% BHI medium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Genotyping The applicants collaborated with Cornell University (Ithaca NY, USA) for genome sequencing and identification.

[0025] WGS DNA composition Whole genome sequencing (WGS), including assembly and annotation, was performed by Cornell University. Bioinformatics analysis was completed at Cornell University and at Deerland Probiotics and Enzymes (Kennesaw GA, USA). Identification of the gyrB gene polymorphism was performed by the applicant.

[0026] 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 chromosome in an underwound state. Gene sequencing analysis used gyrB gene polymorphism, a well-established method for species-level identification of prokaryotes (Bavykin et al., 2014; Wang et al., 2007). Representative genomes were reviewed and curated by NCBI and coordinated by the UniProt Consortium (NCBI, 2016; UniProt, 2016). Using the R package SequinR in conjunction with UniProt Consortium analysis, the whole genome sequence (WGS) and GyrB sequence of the Bacillus coagulans strain CGI314 claimed in the present invention were compared with other reference strains (Tables 1-3 below).

[0027] Identification of genotype, gyrB and 16S rRNA of Bacillus coagulans CGI314 The isolation and genome of CGI314 was deemed a success.

[0028] The genome size of CGI314 (3.0 Mbp) was shorter than that of a previously sequenced B. coagulans strain (3.4 Mbp) (Upadrasta et al., 2016). The %GC (47.3%) was consistent with that of a previously sequenced B. coagulans strain (46.5%) (Upadrasta et al., 2016). [Table 1] [Table 2] [Table 3]

[0029] 16S rRNA Whole genome sequencing (WGS) and 16SrRNA analysis of CGI314 compared to four reference sequences showed an average nucleotide identity (ANI) score for 16SrRNA of 99.9% when compared to B. coagulans strain ATCC 7050. The genome size (3.0 Mbp) and GC content (47.30%) for CGI314 were comparable to the four reference strains.

[0030] Further deposit and accession numbers The genome sequence data of Bacillus coagulans strain CGI314 (Fortispore) has been deposited in the NCBI GenBank database and the genome sequence was annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The genome is publicly available under the GenBank accession number for the strain, JABBFU000000000.1, and is available, for example, at the following link:https: / / www.ncbi.nlm.nih.gov / nuccore / JABBFU000000000.1.

[0031] The genome sequence data of Bacillus clausii strain CSI08 (Munispore) have been deposited in the NCBI GenBank database and the genome sequence was annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The genome is publicly available under the GenBank accession number for the strain, JABBNL000000000.1, and is available at, for example, the link: Alkalihalobacillus clausii strain CSI08, whole genome shotgun sequence-Nucleotide-NCBI (nih.gov).

[0032] The genome sequence data of Bacillus megaterium strain MIT411 (Renuspore) have been deposited in the NCBI GenBank database and the genome sequence was annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The genome is publicly available under the GenBank accession number for the strain, JABBNK000000000.1, and is available at, for example, the link: Priestia megaterium strain MIT411, whole genome shotgun sequencing pro - Nucleotide - NCBI (nih.gov).

[0033] Phylogenetic placement was performed by Deerland Probiotics and Enzymes, Inc.

[0034] Genome-to-genome distance calculation (GGDC), the digital gold standard, is as reliable as DNA-DNA hybridization (DDH) (Auch et al., 2010). GGDC holds a higher discriminatory power for the evaluation of subspecies and was subsequently used as a confirmation of multiple alignments and phylogenetic analyses. GGDC yielded a computational model that further validated that Bacillus coagulans CGI314 is closely related to ATCC7050.

[0035] Conserved 16S rRNA sequences are a well-established method for comparing and testing phylogeny in bacteria, but the high rate of sequence similarity between closely related species limits its usefulness (Wang et al., 2007). The high rate of 16S rRNA sequence similarity in closely related bacterial species is due to the slower rate of molecular evolution. Previous studies (Bavtlin et al., 2004; Wang et al., 2007) support the validity of using gyrB sequences as taxonomic biomarkers due to their rate of base substitution and 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 type II topoisomerase, which play an important role in DNA replication. The gyrase B subunit is encoded by the gyrB gene.

[0036] Phylogenetic analysis using the Neighbor-Joining (NJ) method (Saitou & Nei, 1987) placed Bacillus coagulans CGI314 in the clade with Bacillus coagulans ATCC 7050 (Figure 2), confirming all previous genome identity determinations. Bacillus coagulans CGI314 is placed in the Bacillus coagulans group.

[0037] definition "Excipient" means any inactive ingredient that is added to form part of the final formulation.

[0038] "Probiotics" refers to viable microbial supplements that have a beneficial effect on the patient through their effects in the intestinal, urinary or vaginal tracts. The term "probiotics" may refer to live microorganisms that, when administered in adequate amounts, confer a health benefit to the host. Foods and food additives that contain probiotics may help restore a healthy balance of the gut microflora. Furthermore, probiotic supplementation of the gut microflora may promote healthy gut homeostasis.

[0039] "Prebiotics" are used herein as substrates, which have a beneficial effect on probiotics and thus on the individual patient taking the probiotics. Suitable prebiotics may be selected from inulin, oligosaccharides and / or vitamins.

[0040] As used herein, a "subject" includes those suffering from any clinical condition related to microbial imbalance as well as those using a bacterial preparation prophylactically. Optionally, the subject is a human.

[0041] "Synbiotic product" means a combination of a probiotic and a prebiotic, which is synergistic and has a beneficial effect on the patient.

[0042] "Hardy growth" means that the bacteria exhibits excellent proliferation.

[0043] The abbreviation "CFU" stands for colony forming unit.

[0044] The present invention, which relates to probiotic Bacilli strains capable of regenerating the in vivo bacterial flora in a subject, will become apparent as the following detailed description proceeds.

[0045] According to a first aspect, the present invention comprises Bacillus coagulans strain CGI314 alone or in combination with other probiotic Bacilli strains having essentially the same properties. Such other probiotic Bacilli strains may include, but are not limited to, Bacillus clausii and Bacillus megaterium strains. Such other Bacilli strains may further include Bacillus clausii and Bacillus megaterium strains, each of which is filed today under their respective headings, the contents of which are incorporated herein in their entirety.

[0046] SEQ ID NO:1 as recited in the claims appended hereto comprises gyrB of Bacillus coagulans CGI314.

[0047] SEQ ID NO:2 as recited in the claims appended hereto comprises the 16S rRNA of Bacillus coagulans CGI314.

[0048] SEQ ID NO:3, as recited in the claims appended hereto, contains the assembled entire genome sequence of Bacillus coagulans CGI314.

[0049] Bacillus strains claimed herein with at least 97% identity to SEQ ID NO: 1 and / or 2; or with at least 97% identity to SEQ ID NO: 3 have the following properties:

[0050] Bacillus coagulans CGI314

[0051] This strain exhibits bile stability.

[0052] This strain exhibits acid stability.

[0053] This strain exhibits heat tolerance.

[0054] This strain produces a natural antibiotic in the form of a bacteriocin.

[0055] The entire genome was sequenced to determine the genus and species of the strain disclosed herein. The abundance and composition of this strain were identified and determined.

[0056] This strain has been shown to pose little or no antibiotic resistance and safety concerns.

[0057] This strain was found to be acid and bile stable.

[0058] According to a second aspect, the Bacilli strains of the invention are suitable for medical use in preventing or treating vaginal infections, urinary tract infections and gastrointestinal diseases (including gastrointestinal infections), as well as in improving immune health, protection against oxidative stress, cleansing and detoxification, metabolic health and cardiovascular health.

[0059] In another preferred embodiment, a pharmaceutical composition is provided comprising Bacillus coagulans CGI314, alone or in combination with other probiotic Bacilli strains having essentially the same properties, together with pharma- ceutically acceptable carriers and / or diluents. Such other probiotic Bacilli strains include, but are not limited to, Bacillus clausii and Bacillus megaterium strains. These bacterial strains are formulated into pharmaceutical formulations to allow easy administration of the probiotic strains and by means known to those skilled in the art.

[0060] Bacillus coagulans has been shown to alleviate symptoms of irritable bowel syndrome (Sudha et al., 2018), improve muscle integrity 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 instances and duration of diarrhea (Dolin et al., 2009), improve symptoms of functional abdominal pain and bloating (Hun et al., 2009), prevent acetaminophen-induced acute liver injury (Neag et al., 2020), promote butyrogenesis (Sasaki et al., 2020), and reduce the severity of bacterial vaginosis (Sudha et al., 2018), 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 colony microenvironment in patients with ulcerative colitis (Sasaki et al., 2020), reduce adhesion, cytotoxicity and induction of 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.

[0061] Bacillus clausii has been proven effective in preventing recurrent respiratory infections in vivo (Marseglia et al., 2007) and reducing the duration and severity of diarrhea (Sudha et al., 2019). Bacillus clausii has also been shown to be able to generate protein hydrolysates with antibacterial and antioxidant capacity in vitro (Rochin-Medina et al., 2017), prevent acute liver injury induced by acetaminophen (Neag et al., 2020), and inhibit the cytotoxic effects induced by Clostridium difficile and Bacillus cereus toxins (Ripert et al., 2016).

[0062] Bacillus megaterium has been shown to exert a protective effect against oxidative stress both in vitro and in vivo (Mazzoli et al., 2019). It has also been shown to adapt and survive acid stress conditions and to be able to chelate heavy metals in vitro (Ferreira et al., 2019).

[0063] Preferably, the probiotic bacteria used in the medicament according to the invention are from 10 6 ~10 13 In one embodiment, the probiotic bacteria used in the present invention are at a bacterial concentration of 10 6 ~10 13 The bacteria concentration is in CFU (colony forming units), e.g., as a daily dose including any amount or range included in the ranges above. In one embodiment, the bacteria is present in a concentration of 10 7 ~10 12 CFU or 10 8 ~10 11 CFU or 10 9 ~1010 In terms of 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 In one embodiment, the compositions of the present invention are used in amounts of about 10 CFU and any amount or range including or between the amounts recited above. 6 ~about 10 13 CFU, e.g., about 10 9 In one embodiment, the composition of the invention comprises, consists essentially of, consists of, and / or is characterized by Bacillus coagulans CGI314 (e.g., about 10 9 In one embodiment, the composition of the present invention is orally administered in capsule form. In one 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.

[0064] 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, fructooligosaccharides, galactooligosaccharides, dextrose, dried dairy products, etc. In certain embodiments, the dry carriers may be added to the composition comprising Bacillus coagulans CGI314 in a weight percentage of about 1% to about 95% by weight of the composition.

[0065] 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 saline solution, urea, alcohol and its derivatives (e.g., methanol, ethanol, propanol, butanol), glycols (e.g., ethylene glycol, propylene glycol); natural or synthetic flavors and food-quality colorants, all biocompatible; thickeners selected from the group consisting of cornstarch, guar gum, xanthan gum, etc.; one or more spore germination inhibitors selected from the group consisting of hypersaline carriers, methylparaben, 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 in a weight / volume percentage of about 0.6% to about 95% weight / volume of the composition. In certain embodiments, a natural or synthetic flavoring agent may be added to the composition comprising Bacillus coagulans CGI314 at a weight / volume percentage of about 3.0% to about 10.0% weight / volume of the composition. In certain embodiments, a coloring agent may be added to the composition comprising Bacillus coagulans CGI314 at a weight / volume percentage of about 1.0% to about 10.0% weight / volume of the composition. In certain embodiments, a thickening agent may be added to the composition comprising 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 the composition comprising Bacillus coagulans CGI314 at a weight / volume percentage of about 1% weight / volume of the composition.

[0066] delivery system Suitable dosage forms include tablets, capsules, solutions, suspensions, powders, gums and confectionery. Sublingual delivery systems include, but are not limited to, tablets, liquid drops and beverages that dissolve under and on the tongue. Edible films, hydrophilic polymers, films that dissolve in the mouth or oral strips that dissolve may be used. Other useful delivery systems include oral or nasal sprays or inhalants, and the like. Suitable dosage forms include tablets, capsules, solutions, suspensions, powders, gums and confectionery. Sublingual delivery systems include, but are not limited to, tablets, liquid drops and beverages that dissolve under and on the tongue. Edible films, hydrophilic polymers, films that dissolve in the mouth or oral strips that dissolve may be used. Other useful delivery systems include oral or nasal sprays or inhalants, and the like.

[0067] For oral administration, probiotics may be further combined with one or more solid inactive ingredients to prepare tablets, capsules, pills, powders, granules or other suitable dosage forms.For example, active substances may be combined with at least one excipient selected from the group consisting of fillers, binders, humectants, disintegrants, dissolution retarders, absorption enhancers, wetting agents, absorbents and lubricants.Other useful excipients include, but are not limited to, magnesium stearate, calcium stearate, mannitol, xylitol, sweeteners, starch, carboxymethylcellulose, microcrystalline cellulose, silica, gelatin, silicon dioxide, etc.

[0068] In certain embodiments, the components of the composition administered according to the method of the present disclosure together with one or more conventional adjuvants, carriers or diluents can be put into the form of pharmaceutical compositions and their unit dosages.Such forms include solids and in particular tablets, filled capsules, powders and pellets, all for oral use; liquids and in particular aqueous or non-aqueous solutions, suspensions, emulsions, elixirs; and capsules filled with them; suppositories for rectal administration and sterile injectable solutions for parenteral use.Such pharmaceutical compositions and their unit dosages can contain conventional ingredients in conventional proportions, with or without additional active compounds or active ingredients, and such unit dosages can contain any suitable effective amount of active ingredients that is commensurate with the intended daily dosage range used.

[0069] The components of the compositions administered according to the methods of the present disclosure can be administered in a wide variety of oral and parenteral dosage forms. It will be apparent to those skilled in the art that the following dosage forms may, in certain embodiments, contain, as the active ingredient, either the chemical compounds of the present disclosure or pharma- ceutically acceptable salts of the chemical compounds of the present disclosure.

[0070] The pharma- ceutically acceptable carrier for preparing the pharmaceutical composition to be administered according to the method of the present disclosure can be either solid or liquid.Solid form preparations include powder, tablet, pill, capsule, cachet, suppository and dispersible granule.Solid carrier can be one or more substances that can also act as diluents, flavorings, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents or encapsulating materials.

[0071] In powders, the carrier is a finely divided solid, which is in a mixture 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.

[0072] In certain embodiments, powders and tablets administered according to the disclosed method may preferably contain 5 or 10 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" is intended to include formulation of the active compound with an encapsulating material as a carrier to provide a capsule in which the active ingredient is surrounded by, and thus combined with, a carrier, with or without additional carrier. Similarly, cachets and lozenges are 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.

[0073] 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 aqueous polyethylene glycol solutions. In certain embodiments, the chemical compounds administered according to the methods of the present disclosure can thus be formulated for parenteral administration (e.g., by injection, such as bolus injection or continuous infusion), and can be provided in ampoules, pre-filled syringes, unit doses for administration in low-volume injections, or in multi-dose containers with added preservatives. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be in powder form, obtained by aseptic isolation of sterile solids or by lyophilization from solution, for constitution with a suitable vehicle, such as sterile, pyrogen-free water, before use.

[0074] Aqueous solutions suitable for oral use can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavors, stabilizers and thickeners as required. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active ingredient in water with viscous substances such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose or other well-known suspending agents.

[0075] Compositions suitable for topical administration in the mouth include, but are not limited to, lozenges which contain the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles which contain the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes which contain the active ingredient in a suitable liquid carrier.

[0076] The solution or suspension is applied directly to the nasal cavity by conventional means, for example, by using a dropper, pipette or spray.The composition can be provided in single or multiple dose form.In compositions intended for administration to the respiratory tract, including intranasal compositions, the compound generally has a small particle size, for example, on the order of 5 microns or less.Such a particle size can be obtained by means known in the art, for example, by micronization.

[0077] Pharmaceutical preparations are preferably in unit dosage form.In such form, the preparation is further divided into unit doses containing appropriate amounts of active ingredient.The unit dosage form may be a packaged preparation, the package containing individual amounts of the preparation, such as packaged tablets, capsules, and powders in vials or ampoules.Also, the unit dosage form may be a capsule, tablet, cachet, or lozenge itself; or it may be the appropriate number of any of these in packaged form.

[0078] Tablets, capsules and lozenges for oral administration and liquids for oral use are preferred compositions. Solutions or suspensions for application to the nasal cavity or to the respiratory tract are preferred compositions. Transdermal patches for topical administration to the epidermis are preferred.

[0079] Further details on techniques for formulation and administration may be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., Easton, PA).

[0080] In certain embodiments, the compositions of the present invention, including those administered according to the methods of the present disclosure, may also include one or more excipients, most preferably one or more nutritional supplement excipients or pharmaceutical excipients. The compositions containing one or more excipients and incorporating one or more probiotics may be prepared by procedures known in the art. Optionally, the compositions may include one or more adjuvants, excipients, carriers, buffers, diluents and / or other conventional pharmaceutical auxiliary agents. For example, probiotics may 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 on patches for transdermal application with common and conventional barriers, binders, diluents and excipients.

[0081] In certain embodiments, nutraceutical compositions, including nutraceutical compositions administered according to the methods of the present disclosure, may include and be administered in combination with a pharma- ceutral carrier. In certain embodiments, the active ingredient in such formulations may comprise from about 1% to about 99% by weight. In other embodiments, the active ingredient in such formulations may comprise from about 0.1% 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 formulation and is not harmful to the user. Useful excipients include, but are not limited to, microcrystalline cellulose, magnesium stearate, calcium stearate, any acceptable sugar (e.g., mannitol, xylitol), and the like, and for cosmetic use, water or oil bases may be used, or mixtures thereof, including emulsions, and the like.

[0082] Route of Administration The strain Bacillus coagulans CGI314 or compositions comprising the strains of the invention may be administered by any route, including but not limited to oral, sublingual, buccal, ocular, pulmonary, rectal, vaginal, urethral, ​​ureteral and parenteral administration, or as an oral or nasal spray (e.g., inhalation of aerosol vapor, liquid droplets or solid particles). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, intravaginal, intravesical (e.g., into the bladder), intradermal, transdermal, topical or subcutaneous administration. Also contemplated within the scope of the invention is the instillation of a pharmaceutical composition in a patient's body in a controlled formulation, where systemic or local release of the drug occurs at a later time. For example, the drug may be localized in a depot for controlled release into the circulation or for release to a local site.

[0083] The pharmaceutical compositions of the invention may be suitable and formulated for any of the above-specified routes, including, for example, oral, rectal, bronchial, nasal, pulmonal, 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 may be in a form suitable for administration by inhalation or insufflation, including powder and liquid aerosol administration, or by sustained release systems.Suitable examples of sustained release systems include semipermeable matrices of solid hydrophobic polymers containing the compounds of the invention, which matrices may be in the form of shaped articles, such as films or microcapsules.

[0084] The above embodiments may be further understood in connection with the following examples. Furthermore, the following non-limiting examples are provided to illustrate the present invention. However, one skilled in the art will recognize that it may be necessary to modify the procedure, for example, to change the order or steps, for any given embodiment of the present invention.

[0085] Example 1 Characterization of Bacillus coagulans CGI314 (hereinafter referred to as Fortispore) B. coagulans CGI314 (Fortispore) had significant antimicrobial activity against E. coli, Salmonella, and S. aureus on solid MRS agar overlaid with 0.4% TSA agar.

[0086] However, no antimicrobial activity was observed against P. aeruginosa in solid medium. Fortispore has antimicrobial activity against enteric, urinary and skin pathogens in solid environments. [Table 4]

[0087] Antimicrobial activity is expressed as zone of inhibition (mm) ± standard deviation.

[0088] Figure 3 shows the antimicrobial activity of B. coagulans CGI314 against opportunistic pathogens of the intestine, skin and urinary tract on MRS agar plates overlaid with 0.4% TSA agar (solid medium): AE. E. coli; BS. enteritidis and CS. aureus.

[0089] Fortispore had significant antimicrobial activity against E. coli, Salmonella, and P. aeruginosa in liquid TSB medium, but no antimicrobial activity was observed against S. aureus in liquid TSB medium. [Table 5]

[0090] Figure 4 shows the antimicrobial activity of B. coagulans CGI314 in liquid TSB medium against opportunistic pathogens of the intestine, skin and urinary tract. Pathogens: E. coli, Salmonella, Pseudomonas aeruginosa and S. aureus. Controls represent the growth of pathogens individually; treatments represent the growth of pathogens in the presence of B. coagulans CGI314. ** p<0.01, *** p<0.001, **** p<0.0001.

[0091] Fortispore has the potential to control the presence of opportunistic pathogens in the intestine and urinary tract where semi-liquid to liquid conditions may be prevalent. Additionally, Fortispore has the potential to prevent the spread of opportunistic pathogens in drier environments such as human skin.

[0092] Fortispore has higher antimicrobial activity than competitor B. coagulans probiotics. The antimicrobial activity of B. coagulans CGI1314 (Fortispore) was compared to that of B. coagulans MTCC5856 (Lactospore®) and B. coagulans 6086 (BC30™) using the agar diffusion method against the following human skin and intestinal opportunistic and zoonotic pathogens: Escherichia coli 25922, Salmonella enteritidis 13076, Staphylococcus aureus RF122 and Pseudomonas aeruginosa DSM3227.

[0093] B. coagulans CGI314 (Fortispore) showed stronger antimicrobial activity against E. coli at 24 hours and against S. enteritidis at 48 hours than BC30™ and Lactospore®. Fortispore may have a higher potential to control the presence of opportunistic and zoonotic pathogens that may be present in the human gut or on the skin: High antimicrobial activity of Fortispore against E. coli. High antimicrobial activity of Fortispore against S. enteritidis at 48 hours. No antimicrobial activity was detected across Bacillus coagulans against P. aeruginosa. Limited antimicrobial activity of Bacillus coagulans against S. aureus using TSA agar.

[0094] Antimicrobial activity - High antimicrobial activity against E. coli: B. coagulans CGI314 (Fortispore) had the strongest antimicrobial activity against E. coli tested using MRS agar. B. coagulans MTCC5856 (Lactospore®) and B. coagulans 6086 (BC30™) had limited antimicrobial activity. [Table 6]

[0095] Figure 5 shows the strongest antimicrobial activity observed with B. coagulans CGI314 (Fortispore) against E. coli at 24 hours. (a) B. coagulans MTCC5856 (Lactospore®), (b) B. coagulans CG314 (Fortispore), (c) B. coagulans 6086 (BC30™) inoculated onto MRS agar against E. coli 0.4% TSA agar overlay plates.

[0096] Fortispore inhibited the growth of the opportunistic intestinal and urinary tract pathogen E. coli.

[0097] Antimicrobial activity - High antimicrobial activity against S. enteritidis at 48 hours: At 24 hours, B. coagulans 6086 (BC30™) had the strongest antimicrobial activity against S. enteritidis, but at 48 hours, B. coagulans CGI314 (Fortispore) had the strongest antimicrobial activity against this pathogen. [Table 7]

[0098] Figure 6 shows the strongest antimicrobial activity observed with Fortispore against S. enteritidis at 48 hours. (a) Lactospore® (b) Fortispore (c) BC30™ inoculated onto MRS agar against S. enteritidis 0.4% TSA agar overlay plates.

[0099] Fortispore inhibited the growth of Salmonella, a zoonotic pathogen known to affect the intestinal tract.

[0100] Antimicrobial Activity - No antimicrobial activity was detected against P. aeruginosa and Bacillus coagulans:

[0101] Using the agar diffusion method, no antimicrobial activity was observed over B. coagulans against P. aeruginosa.

[0102] Figure 7 shows that no antimicrobial activity was detected over Bacillus coagulans against P. aeruginosa. (a) B. coagulans MTCC5856 (Lactospore®), (b) B. coagulans CG314, and (c) B. coagulans 6086 (BC30™) inoculated onto TSA agar against P. aeruginosa 0.4% TSA agar overlay plates at 24 hours.

[0103] Fortispore does not release antimicrobial agents against the skin pathogen P. aeruginosa, however, Fortispore does show potential activity against P. aeruginosa in liquid media (see Table 5 and Figure 7).

[0104] Antimicrobial Activity-Limited antimicrobial activity of Bacillus coagulans against S. aureus using MRS agar:

[0105] There was no evidence of antimicrobial activity by B. coagulans strains against S. aureus using TSA agar, however B. coagulans 6086 (BC30™) and B. coagulans CGI1314 (Fortispore) showed little activity against this pathogen using MRS agar. [Table 8]

[0106] Figure 8 shows the limited antimicrobial detection across B. coagulans versus S. aureus using MRS agar at 24 hours: (a) S. aureus 0.4% TSA agar overlay plate versus B. coagulans inoculated onto TSA agar (left to right: BC30, CGI314, Lactospore). (b) S. aureus 0.4% TSA agar overlay plate versus B. coagulans inoculated onto MRS agar (left to right: CGI314, BC30, Lactospore).

[0107] Fortispore can release limited amounts of antimicrobial agents on dry surfaces such as skin against the skin pathogen S. aureus.

[0108] Fortispore is a potential antioxidant probiotic: The total oxidative activity of Fortispore B. coagulans was compared with that of L. rhamnosus. Fortispore has higher antioxidant activity than L. rhamnosus.

[0109] Figure 9 shows the total antioxidant capacity of B. coagulans and L. rhamnosus. Results show the mean concentration of Trolox equivalents in nmole / ml (n=3) ± standard error. ** P-value=0.0014.

[0110] The elevated levels of Trolox equivalent concentration in Fortispore can neutralize and scavenge free radicals and prevent oxidative damage to cells.

[0111] Fortispore antioxidant activity is the second highest amount of antioxidant activity among all Deerland spores.

[0112] The total antioxidant activity of Fortispore B. coagulans was compared with B. megaterium MIT411, B. clausii CSI08, DE111 B. subtilis and L. rhamnosus. Fortispore has high antioxidant activity, higher than L. rhamnosus.

[0113] Figure 10 shows the total antioxidant capacity of DE111, B. coagulans of the present invention, B. clausii CSI08, B. megaterium MIT411 and L. rhamnosus. Results show the mean concentration of Trolox equivalents in nmole / ml (n=3) ± standard error.

[0114] The elevated levels of Trolox equivalent concentration in Fortispore can neutralize and scavenge free radicals and prevent oxidative damage to cells.

[0115] Fortispore (trophozoite, non-sporulating form) did not adhere to intestinal epithelial cell lines.

[0116] The adhesion ability of Bacillus coagulans strain CGI314 to an in vitro model of intestinal epithelium was evaluated. The adhesion of B. coagulans strain CGI314 to the HT-29 cell line was negligible at 37°C. The adhesion ability of B. coagulans strain CGI314 to the mucus-producing cell line HT-29-MTX was negligible at 37°C.

[0117] FIG. 11 shows that adhesion of Fortispore to the HT-29 cell line was negligible.

[0118] FIG. 12 shows that Fortispore adhesion to the HT-29-MTX cell line is negligible.

[0119] The Fortispore strain does not adhere to intestinal mucus or epithelial cells.

[0120] The adhesion capacity of various Bacillus coagulans strains (CGI314, BC30™, Lactospore®) to an in vitro model of the intestinal epithelium was evaluated. The adhesion of B. coagulans strains CGI314, BC30™, Lactospore® to the HT-29 cell line is negligible at 37°C. The adhesion capacity of B. coagulans BC30™ to the mucus-producing cell line HT-29-MTX is higher than B. coagulans CGI314 and Lactospore® at 37°C.

[0121] FIG. 13 shows a study of the adhesion of B. coagulans strains to the HT-29 cell line.

[0122] FIG. 14 shows a study of the adhesion of B. coagulans strains to the HT-29-MTX cell line.

[0123] B. coagulans strains (liquid culture, non-spore forming form) do not adhere to intestinal mucus or epithelial cells.

[0124] Fortispore exhibits low caseinolytic activity.

[0125] Fortispore was negative for caseinolytic activity on skim milk agar plates. Quantitative analysis of Fortispore caseinolytic activity was assessed by using a commercial kit with a fluorescently tagged casein derivative. Fortispore showed low extracellular protease activity.

[0126] FIG. 15 shows a test for the absence of caseinolytic activity in B. coagulans CGI1314 using both the streak method and the overnight TSB broth method at 24 or 48 hours.

[0127] Figure 16 shows that the proteolytic activity of Fortispore on casein derivatives is lower than the positive control Proteinase K using the EnzCheck® kit after 24 hours of incubation at 37°C. Error bars indicate standard error (n=3).

[0128] Fortispore exhibits low protease activity towards casein.

[0129] Among the Deerland strains, Fortispore exhibits low extracellular protease activity against casein derivatives.

[0130] FIG. 17 shows the lack of caseinolytic activity across B. coagulans CGI1314, B. coagulans 6086 (BC30™) and B. coagulans MTCC5856 (Lactospore®) using both the streak and overnight TSB broth methods at 24 or 48 hours.

[0131] FIG. 18 shows that among the Deerland strains, Fortispore B. coagulans exhibits the lowest protease activity against casein.

[0132] Figure 19 shows a quantitative analysis of caseinolytic activity across B. coagulans strains as determined by the EnzCheck® kit after 24 hours of incubation at 37°C. Significance is indicated by a, b and / or c, with multiple letters indicating that the results are not significantly different from more than one group (P<0.05). Error bars indicate standard deviation.

[0133] In comparison to comparators (Lactospore® and BC30™), Fortispore shows superior protease activity against casein.

[0134] Diverse carbohydrate profile of Fortispore Fortispore can metabolise a range of monosaccharides, disaccharides, sugar alcohols, amine sugars and polysaccharides.

[0135] Fortispore was positive for the metabolism of 22 carbohydrates out of 49 tested using the commercial API 50 CH strip. Most of these carbohydrates were monosaccharides such as D-ribose, D-glucose, D-galactose, D-fructose, D-mannose, and disaccharides such as trehalose, maltose, and cellobiose. In addition, there were compounds belonging to the glycosylated hydroquinones (arbutin), cyanogenic glycosides (amigladin), and alcoholic β-glucosides (Salicin) that Fortispore could metabolize. [Table 9]

[0136] These data suggest that Fortispore may help digest these compounds in the intestine.

[0137] Fortispore was strongly positive for D-sorbitol and starch amidon, whereas the other two competitor strains (BC30™ and Lactospore®) were negative or weakly positive for these. [Table 10]

[0138] These data suggest that Fortispore may help digest these compounds in the intestine.

[0139] Fortispore has esterase, peptidase, phosphatase and glucosidase activities:

[0140] Fortispore was positive for esterase, peptidase, phosphatase and glucosidase activity using the API ZYM kit, which means: High potential of Fortispore to generate free fatty acids from the action of esterases in the presence of suitable lipid sources. Galctosidase enhances the carbohydrate catabolic potential of Fortispore, as it has activity against a variety of oligosaccharides, lactosylceramide, lactose and numerous glycoproteins. An aminopeptidase that catalyzes the hydrolysis of leucine, valine, and cysteine ​​substrates.

[0141] Indeed, in silico analysis identified genes encoding esterases, peptidases and galactosidases. [Table 11]

[0142] This study confirms the hydrolytic ability of Fortispore towards oligosaccharides and demonstrates its potential to break down fats and peptides to release free amino acids.

[0143] These data suggest that Fortispore may help digest these molecules in the intestine.

[0144] B. coagulans enzyme profile shows peptidase and esterolytic activity, confirming oligosaccharide degradation:

[0145] All B. coagulans were positive for esterase, peptidase, phosphatase and galactosidase activity using the API ZYM kit, which means: High potential of B. coagulans to generate free fatty acids from the action of esterases in the presence of an appropriate lipid source. Galactosidases enhance the carbohydrate catabolic potential of B. coagulans since they are active against a variety of oligosaccharides, lactosylceramide, lactose and numerous glycoproteins. An aminopeptidase that catalyzes the hydrolysis of leucine, valine, and cysteine ​​substrates. [Table 12]

[0146] This study shows that there are no differences in the hydrolytic capacity of B. coagulans strains towards several enzymes using the commercial API ZYM kit.

[0147] Fortispore releases free amino acids from glycolysis and protease metabolism.

[0148] Metabolomic analysis reveals the fermentative and proteolytic capabilities of Fortispore towards milk carbohydrates and proteins to produce various amino acids.

[0149] The carbohydrate fermentation and protein degradation capabilities of Fortispore were analyzed using the UHT milk model. GC-MS analysis identified a total of 38 free amino acid (FAA) compounds, of which 10 were found to be statistically significant in Fortispore. Several of the identified carboxylic acids are related to carbohydrate metabolic pathways, confirming that the lactose / galactose / glucose uptake and the presence of enzyme metabolic systems are active in Fortispore. Furthermore, there is also evidence of an active protein degradation system in Fortispore, as there are several amino acids associated with peptidase activity and the release of benzoic acid associated with further catabolism of phenylalanine. [Table 13]

[0150] Overall, the enzymatic and metabolomic 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). Furthermore, there is evidence of potentially active aminopeptidases, lyases and decarboxylases that generate carboxylic acids and release amino acids from the UHT milk fermented by Fortispore.

[0151] Fortispore releases free amino acids from glycolysis and protease metabolism. Free amino acid analysis shown as bar graph (mean + SEM) FIG. 20 shows the FAA profile of Fortispore UTH fermented milk. P-value≦0.005= ** and P-value ≤ 0.01 = * White bars represent controls.

[0152] FIG. 21 shows the FAA profile of Fortispore UTH fermented milk. P-value≦0.005= ** and P-value ≤ 0.01 = * White bars represent controls.

[0153] Fortispore is slightly more effective at producing compounds beneficial to skin health compared to competing B. coagulans strains.

[0154] Ganeden Biotech markets B. coagulans 6086 (BC30™) as an anti-aging probiotic because it produces the highest amounts of naturally occurring L+ lactic acid, bacteriocins, hydrogen peroxide, enzymes and other metabolic products. Lactic acid is associated with anti-aging and fighting acne due to its antimicrobial activity and hydration. Succinic acid is also associated with anti-inflammatory, antimicrobial and hydration. Benzoic acid is associated with antimicrobial activity.

[0155] Figure 22 compares Fortispore with Lactospore and BC30 in lactate, succinate and benzoate production. Significance is indicated by a, b and / or c, with multiple letters indicating results are not significantly different from multiple groups (P<0.05). Error bars indicate standard deviation.

[0156] Our results show that Fortispore is slightly more efficient than BC30™ for lactate production and superior for succinate and benzoate production.

[0157] Fortispore may produce amino acids beneficial to skin health in comparison to competing B. coagulans strains.

[0158] Fortispore is compared to Lactospore® and BC30. Our results show that Fortispore is superior to BC30™ and Lactospore® in the production of methionine, proline, lysine and tryptophan.

[0159] Methionine is a sulfur-containing amino acid that improves skin tone and elasticity, promotes healthy hair, and strengthens nails. Proline helps reduce inflammation, which boosts a healthy immune system. It also helps trigger a cascade of anti-inflammatory compounds and genes that aid in recovery. Lysine is an essential amino acid with many benefits, ranging from preventing cold sores to reducing anxiety and promoting wound healing. Tryptophan is associated with the relief of depression and anxiety.

[0160] Figure 23 compares Fortispore with Lactospore and BC30 in the production of amino acids. Significance is indicated by a, b and / or c, with multiple letters indicating that the results are not significantly different from multiple groups (P<0.05). Error bars indicate standard deviation.

[0161] Fortispore proteomic analysis identifies proteins with potential probiotic benefit.

[0162] The extracellular secretions of Fortispore were subjected to mass spectrometry to identify proteins released by the probiotic strains. A total of 28 proteins were detected, 6 of which had potential probiotic benefits. [Table 14]

[0163] These data support previous in vitro results showing how Fortispore may aid in the digestion of proteins and carbohydrates and has antimicrobial properties against pathogens.

[0164] Fortispore growth was increased in the presence of Fibersol® in minimal medium.

[0165] FIG. 24 shows that Fibersol® significantly increased the concentration of Fortispore (CFU / mL) by 1 log10 in minimal medium after 24 hours of incubation compared to the control. * p<0.05

[0166] There is a significant increase in growth of Fortispore in the presence of Fibersol® in minimal medium compared to competitor B. coagulan strains.

[0167] Figure 25 shows that Fibersol® significantly increased the concentration (CFU / mL) of Fortispore in minimal medium, while no significant growth of BC30 and Lactospore was observed. Statistical analysis was performed using one-way ANOVA using Tukey's method. ** p-value=<0.01.

[0168] There was no increase in the growth of Fortispore in the presence of Fibersol® in TSB medium.

[0169] Figure 26 shows that Fibersol® did not show a significant increase in the concentration of Fortispore (CFU / mL) in TSB medium compared to the control. Unlike minimal medium, Fibersol® did not increase the growth of Fortispore in rich medium, likely because Fortispore reaches maximum growth due to the nutrients present in TSB medium.

[0170] The growth of Fortispore, BC30, DE111 and the pathogen was compared in the presence of Fibersol® in TSB medium.

[0171] 27 shows that Fibersol® did not significantly increase the concentration (CFU / mL) of DE111, Fortispore, BC30, E. coli, and Salmonella enteritidis in TSB medium compared to the control. Unlike minimal medium, Fibersol® did not improve the growth of DE111, Fortispore, BC30, E. coli, and S. enteritidis in rich medium, likely because S. enteritidis reaches maximum growth due to the nutrients present in TSB medium.

[0172] There was no increase in the growth of Fortispore in the presence of Fibersol® in 50% TSB medium.

[0173] FIG. 28 shows that Fibersol® did not significantly increase the concentration of Fortispore (CFU / mL) in 50% TSB medium compared to the control.

[0174] The growth of Fortispore, BC 30 and DE111 was compared in the presence of Fibersol® in 50% TSB medium.

[0175] FIG. 29 shows that Fibersol® significantly increased the yield (CFU / mL) of DE111 by 1 log10 after 24 hours in 50% TSB medium. **** p<0.0001. Fibersol® did not show a significant increase in the concentrations (CFU / mL) of Fortispore and BC30 in 50% TSB medium compared to the control.

[0176] No significant growth of Fortispore was observed in the presence of BHI medium supplemented with Fibersol®.

[0177] Figure 30 shows that Fibersol® did not show a significant increase in the concentration of Fortispore (CFU / mL) compared to the control in BHI medium. Unlike minimal medium, Fibersol® did not increase the growth of Fortispore in rich medium, likely because Fortispore reaches maximum growth due to the nutrients present in BHI medium.

[0178] The growth of Fortispore, BC30 and DE111 was compared in the presence of Fibersol® in BHI medium.

[0179] 31 shows that Fibersol® did not significantly increase the concentration (CFU / mL) of DE111, Fortispore, and BC30 compared to the control in BHI medium. Unlike minimal medium, Fibersol® did not increase the growth of DE111, Fortispore, and BC30 in rich medium, likely because they achieve maximum growth using nutrients present in BHI medium.

[0180] No increase in Fortispore growth was observed in the presence of Fibersol® in 50% BHI medium.

[0181] Figure 32 shows that Fibersol® did not significantly increase the concentration of Fortispore (CFU / mL) compared to the control in 50% BHI medium. Unlike minimal medium, Fibersol® did not improve the growth of Fortispore in rich medium, likely because Fortispore achieves maximum growth using nutrients present in BHI medium.

[0182] No increase in growth of Fortispore, BC30 and DE111 was observed in the presence of Fibersol® in 50% BHI medium.

[0183] 33 shows that Fibersol® did not significantly increase the concentration (CFU / mL) of DE111, Fortispore, and BC30 compared to the control in 50% BHI medium. Unlike minimal medium, Fibersol® did not improve the growth of DE111, Fortispore, and BC30 in rich medium, likely because they achieve maximum growth using nutrients present in BHI medium.

[0184] Example 2 Assessment of adhesive ability to an in vitro model of intestinal epithelium Cell lines: The human colorectal adenocarcinoma cell line HT-29 and the mucus-secreting cell line HT-29-MTX were grown using low glucose DMEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin and 2 μg / ml amphotericin B at 37°C in a 5% CO2 atmosphere.

[0185] Cells, 5x10 cells 5 The cells were seeded onto 24-well plates at a density of 10 cells / well and cultured for 21–28 days until full maturation. The medium was changed every 2–3 days.

[0186] Before the experiment, the cells were washed twice with 0.5 ml DPBS, which was completely aspirated from the wells after the second wash.

[0187] Spore preparation: 10 milligrams of B. clausii CSI08, B. megaterium MIT411 and B. coagulans CGI314 spore powder were weighed into a 15 ml Falcon tube and resuspended in 10 ml of antibiotic-free complete culture medium. The suspension was aliquoted and stored at -20°C until use. The suspension was used within 2 weeks of preparation.

[0188] Adhesion assay: 500 μl of spore suspension (1.3 x 10 7 ~9.2x10 7CFU / ml) were added to HT-29 and HT-29-MTX cells, mixed by gentle swirling, and incubated for 2.5 hours at 37° C. in a CO2 incubator. Control wells containing no mammalian cells were prepared and incubated in parallel in the same way (0.5 ml of spore suspension).

[0189] At the time of incubation, HT-29 and HT-29-MTX cells were washed 4 times with 0.5 ml PBS. Then, 50 μL of trypsin / EDTA solution and 50 μL of PBS were added to the wells and incubated for 10 minutes at 37° C. with gentle shaking (~100 rpm). 50 microliters of trypsin / EDTA solution was added to the control wells.

[0190] Consequently, 450 μL of PBS was added to the wells containing spores, and the contents of the wells were transferred, with scraping, to Eppendorf tubes and subjected to three bouts of vigorous shaking for 30 seconds each, whereas the contents of the control wells were transferred to Eppendorf tubes and subjected to one bout of shaking.

[0191] Serial dilutions (plus dilutions of control wells) were prepared in PBS and plated onto BC agar (B. coagulans CGI314) or PetriFilm™ (B. clausii CSI08, B. megaterium MIT411). Plates were incubated at 37° C. for 48 hours before counting, and PetriFilms were incubated at 37° C. for 24 hours before counting.

[0192] Experiments were performed two or three times with three technical replicates per experiment. Results are expressed as mean±SEM. [Table 15] [Table 16]

[0193] Conclusion: 1. The results presented above indicate a higher ability of spores to adhere to the mucus-secreting cell line HT-29-MTX compared to non-mucus-secreting cells, probably due to the physical properties of the spores. 2. B. megaterium MIT411 and B. coagulans CGI314 spores have a higher (but overall lower) ability to adhere to the non-mucus producing cell line HT-29 compared to B. clausii CSI08 spores.

[0194] Example 3 Evaluation of Bacillus coagulans CGI314 for safety, tolerability and gastrointestinal health: a randomized, double-blind, placebo-controlled study in healthy adults.

[0195] Study intervention 1x10 administered daily 9 Probiotic 1 containing CFU of Bacillus coagulans CGI314 and Bacillus subtilis DE111®, Approx. 0.5x10 9 CFU (and 0.5x10 9 CFU of Bacillus subtilis DE111®, approximately 0.5x10 9 CFU of Bacillus megaterium MIT411, approximately 0.5x10 9 CFU (and 0.5x10 9 Bacillus coagulans CGI314 (less than CFU), approximately 0.5x10 9 A probiotic combination containing CFU of Bacillus clausii CSI08; a total of 2x10 administered daily 9 Bacillus spores, Placebo: rice maltodextrin administered daily.

[0196] Indications tested Gastrointestinal homeostasis and the immune system

[0197] Exam description Healthy adults aged 18-65 years were recruited and screened for participation in this study. Eligible participants were randomized 1:1:1 to one of two experimental or control groups for 45 days of treatment and 2 weeks of follow-up to evaluate the safety and efficacy of a novel probiotic strain in healthy adults.

[0198] For this, the investigational product was packaged in individual capsules corresponding to the daily dose. Each capsule contained 300 mg of the ingredients: Probiotic formulation 1: Bacillus coagulans CGI314 and Bacillus subtilis DE111® (total 1x10 9 CFU), ~4.17mg; Low Moisture Rice Maltodextrin, 292.23mg; Medium Chain Triglycerides, 3.6mg. Probiotic Cocktail: Bacillus subtilis DE111 (Registered Trademark) (approximately 0.5 x 10 9 CFU (and 0.5x10 9 CFU), 2.1mg; Bacillus megaterium MIT411 (approximately 0.5x10 9 CFU), 2.1mg; Bacillus clausii CSI08 (approximately 0.5x10 9 CFU), 2.1mg; Bacillus coagulans CGI314 (approximately 0.5x10 9 CFU (and 0.5x10 9 Contains less than CFU), 2.1mg; Low Moisture Rice Maltodextrin, 288mg; Medium Chain Triglycerides, 3.6mg, 2x10 9 CFU of Bacillus spores. The placebo consisted of all the same ingredients as the investigational product, with the exception of the probiotic organisms. The placebo was in individual capsules corresponding to the daily dose. Each capsule contained 300 mg of the following ingredients: Placebo: Low-moisture rice maltodextrin, 296.4 mg; Medium-chain triglycerides, 3.6 mg.

[0199] Table 17 summarizes the change in gut-brain axis questionnaire scores from baseline to the end of the treatment period for the four probiotic groups and the placebo group. Mean changes with 95% confidence intervals are shown. ANOVA omnibus test (p * The results of the mean mean (p-value) and one-sample T-test (p-value) are also shown. A test of normality for the changes in gut-brain axis scores showed that the data do not follow a normal distribution, which may affect the results, which are borderline significant (p-values ​​between 0.05 and 0.10). This affects two items: loss of energy and changes in appetite. An alternative non-parametric Kruskal-Wallis test was applied to these items; p-values ​​of 0.111 (loss of energy) and 0.123 (changes in appetite) were observed.

[0200] In general, the mean change in gut-brain axis questionnaire score was negative for all symptoms tested, meaning that the symptoms were less intense (participants were less bothered by these symptoms) at the end of the treatment period. The results of the one-sample T-test show that in one third of the studies (out of 70), a statistically significant change in gut-brain axis questionnaire score was observed. However, this could be observed for all treatment groups, including the placebo group. As a result, the results of the ANOVA test show that no significant differences in gut-brain axis score change were detected between the treatment groups, but borderline significance was observed for the items loss of energy and change in appetite. Participants in the Bacillus megaterium group experienced the greatest change for these two items. [Table 17] TIFF2024544211000019.tif169159

[0201] Mean differences with 95% confidence intervals and p-values ​​for pairwise comparisons between each probiotic and placebo group are shown in Table 17. Compared to placebo, none of the probiotic-containing test products demonstrated statistically significant differences in gut-brain axis score change from baseline to the end of the treatment period.

[0202] The mean differences with 95% confidence intervals and p-values ​​for pairwise comparisons between each probiotic and placebo group are shown in Table 18. Compared to placebo, none of the probiotic-containing test products demonstrated statistically significant differences in the total gut-brain axis item scores at baseline or the end of the treatment period. [Table 18]

[0203] Mean differences with 95% confidence intervals and p-values ​​for pairwise comparisons between each probiotic and placebo group are shown in Table 19. Compared to placebo, none of the probiotic-containing test products demonstrated statistically significant differences in HDL, LDL, TC, or TG changes from baseline to the end of the treatment period. [Table 19]

[0204] Mean differences with 95% confidence intervals and p-values ​​for pairwise comparisons between each probiotic group and the placebo group are presented in Table 20. Compared to placebo, none of the probiotic-containing test products demonstrated statistically significant differences in the relative change in HDL, LDL, TC, or TG from baseline to the end of the treatment period. [Table 20]

[0205] Table 21 shows the proportion of participants who reported at least one day of clinically significant infection in Diary 1. Table 22 shows the descriptive statistics and Kruskal-Wallis test (p * -values) and the results of the Mann-Whitney U test with Holm correction (p-values) are shown. [Table 21] [Table 22]

[0206] The Kruskal-Wallis test showed no significant differences in the number of days with clinically significant infections between the treatment groups. However, borderline statistically significant results were observed for clinically significant gastrointestinal infections. This is likely due to the fact that none of the four probiotic treatment groups experienced clinically significant gastrointestinal infections, whereas a total of two days of such infections were observed in the probiotic group, which may have occurred by chance.

[0207] Nevertheless, compared to placebo, none of the test products containing probiotics showed statistically significant differences.

[0208] Table 23 shows the proportion of participants who reported at least one day with individual symptoms of gastrointestinal infection in Diary 2. In Table 24, descriptive statistics and Kruskal-Wallis tests (p * -values) and the results of the Mann-Whitney U test with Holm correction (p-values) are shown. [Table 23] [Table 24]

[0209] A significant difference between the groups was only detected in the number of days with constipation (p* = 0.013), likely due to the fact that only three participants in the placebo group reported this symptom in participant diary 2, whereas none of the participants in the other four treatment groups reported this symptom. However, further analyses comparing the number of days with constipation between the individual probiotic and placebo groups (Mann-Whitney U test with Holm correction) did not show significant differences, likely due to small sample sizes.

[0210] Efficacy outcomes: treatment duration No significant differences in participants' gastrointestinal health at baseline between treatment groups were detected. Participants were randomly assigned to five treatment groups, so such results were expected.

[0211] The scores of the items in the gut-brain axis questionnaire were lower at the end of the treatment period compared to the baseline scores, meaning that participants were less bothered by these symptoms at the end of the treatment period. This could be observed for all treatment groups, including the placebo group.

[0212] No significant differences in the change in gut-brain axis item scores (at the end of the treatment period minus baseline) between treatment groups were detected. None of the test products containing probiotics showed a statistically significant difference in the change in gut-brain axis scores compared to the placebo group.

[0213] No significant differences in the improvement of gut-brain axis item score changes between treatment groups were detected. Compared to the placebo group, none of the test products containing probiotics showed a statistically significant difference in the improvement of gut-brain axis scores.

[0214] No significant differences in the sum of all items scores on the gut-brain axis questionnaire between treatment groups were detected. Compared to placebo, none of the probiotic-containing test products demonstrated statistically significant differences in the sum of gut-brain axis item scores at baseline or at the end of the treatment period.

[0215] No significant differences in cholesterol and triglyceride changes (absolute and relative change from baseline to end of treatment period) were detected between treatment groups. Compared to placebo, none of the test products containing probiotics showed statistically significant differences in HDL, LDL, TC, or TG changes (absolute or relative) from baseline to end of treatment period. The same findings were observed in the PP population.

[0216] Cytokine (TNFα, IFNα, IFNβ, IFNγ, and IL6) levels at the end of the treatment period were below the limit of quantification (LOQ) in all treatment groups. Although some values ​​above the LOQ were reported for the cytokine IL13, no statistically significant differences were observed between treatment groups.

[0217] No significant changes in heavy metal (cadmium, lead, mercury, copper, nickel, zinc, and arsenic) levels were detected in the Bacillus megaterium group at the end of the treatment period compared to baseline values. The same findings were observed in the PP cohort.

[0218] Compared with placebo, none of the test products containing probiotics showed a statistically significant difference in the number of days with symptoms of gastrointestinal infection.However, results of borderline statistical significance were observed for clinically significant gastrointestinal infection.This is probably due to the fact that none of the participants in the four probiotic treatment groups experienced clinically significant gastrointestinal infection, while in the probiotic group, a total of 2 days of such infection were observed, which may have occurred by chance.

[0219] Compared to placebo, none of the probiotic-containing test products showed a statistically significant difference in the number of days with symptoms of respiratory tract infection. However, a statistically significant difference between the treatment groups was detected in the number of days with nasal mucus (p=0.018), likely due to the fact that only three participants in the probiotic cocktail group reported this symptom, whereas none of the participants in the other four treatment groups reported this symptom.

[0220] However, further post-hoc analyses (pairwise comparisons vs. placebo) did not show any significant differences.

[0221] Compared to placebo, none of the probiotic-containing test products demonstrated a statistically significant difference in the number of days with urinary tract infection symptoms.

[0222] No statistically significant differences were detected between treatment groups in bowel regularity.

[0223] Significant differences between groups were detected for stool consistency, i.e., the percentage of loose stools during the total treatment period and during weeks 6 and 7 of the treatment period. However, further post-hoc analyses (pairwise comparisons vs. placebo) did not show any significant differences, likely due to small sample sizes. Participants in the probiotic cocktail group had the smallest percentage of loose stools per total bowel movement.

[0224] Efficacy outcomes: follow-up period A significant difference between groups was only detected in the number of days with constipation (p=0.013), but further post-hoc analyses (pairwise comparisons vs placebo) did not show any significant differences, probably due to the small sample size.

[0225] None of the probiotic-containing test products demonstrated a statistically significant difference in the number of days with respiratory tract infection symptoms.

[0226] None of the probiotic-containing test products demonstrated a statistically significant difference in the number of days with urinary tract infection symptoms.

[0227] No significant differences were detected between treatment groups in bowel regularity and stool consistency.

[0228] Safety outcomes A total of 17 AEs were reported. Between one and five ADEs were reported for each study product, the most common being gastroesophageal reflux (3 AEs), rash (2 AEs), and dizziness (2 AEs). No SAEs were reported.

[0229] Causality assessment revealed no relationship between the reported AEs and the study product.

[0230] In summary, this study addressed the safety and efficacy of novel probiotics, namely Bacillus coagulans, Bacillus clausii, Bacillus megaterium, and a probiotic cocktail containing Bacillus subtilis, Bacillus megaterium, Bacillus clausii, and Bacillus coagulans.

[0231] The gastrointestinal health of participants at baseline between treatment groups did not differ between study arms, which was expected due to randomization.

[0232] A total of 17 AEs and no SAEs were reported, thus achieving the primary outcome of this study (safety). Causality assessment did not indicate a relationship between the reported AEs and the study product.

[0233] None of the efficacy-related outcomes showed any statistically significant differences, which is not surprising given the small sample size per study group. Furthermore, some trends in favor of the active product were observed, especially in gut-brain axis score and rate of loose stool.

[0234] In conclusion, the probiotic product was shown to be safe for use in adults and showed some beneficial trends with regard to the gut-brain axis and stool consistency, however further trials with larger sample sizes, a run-in period to determine baseline stool consistency for each participant and the use of validated questionnaires to determine gut-brain axis domains are needed to scientifically prove the efficacy of the tested product.

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[0236] The present invention is not limited to the embodiments described herein, which may be modified or varied without departing from the scope of the invention.

[0237] The use of "a", "an", "the" and similar referents in the context of describing the present invention (particularly in the context of the claims) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. The recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of referring individually to each individual value falling within the range, unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually recited herein. The use of the term "about" is intended to describe values ​​either above or below the stated value in a range of approximately ±10%; in other embodiments, values ​​may range values ​​either above or below the stated value in a range of approximately ±5%; in other embodiments, values ​​may range values ​​above or below the stated value in a range of approximately ±2%; in other embodiments, values ​​may range values ​​above or below the stated value in a range of approximately ±1%. The foregoing ranges are intended to be made clear by the context, and no further limitations are implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or representative language (e.g., "etc.") provided herein is merely to better clarify the invention and does not pose a limitation on the scope of the invention unless otherwise specified. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0238] While the invention has been described in the foregoing specification with reference to specific embodiments thereof, and numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to further embodiments and that the specific details described herein may be modified considerably without departing from the underlying principles of the invention.

[0239] All references cited herein are incorporated by reference in their entirety. The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof, and therefore, reference should be made to the appended claims, rather than the foregoing specification, as indicating the scope of the invention.

Claims

1. A Bacillus coagulans strain comprising a purified microbial population comprising one or more bacteria having a gyrB that shares at least 97% identity with SEQ ID NO:1; and / or one or more bacteria having a 16S rRNA that shares at least 97% identity with SEQ ID NO:

2.

2. 2. The Bacillus coagulans strain of claim 1, which shares at least 97% identity with SEQ ID NO:

3.

3. 2. The Bacillus coagulans strain of claim 1, wherein the purified microbial population comprises bacteria having a 16S nucleic acid sequence comprising SEQ ID NO:

2.

4. 2. The Bacillus coagulans strain of claim 1, wherein the purified microbial population comprises bacteria having a gyrB nucleic acid sequence comprising SEQ ID NO:

1.

5. 2. The Bacillus coagulans strain of claim 1, wherein the purified microbial population comprises bacteria having a 16S nucleic acid sequence comprising SEQ ID NO:2 and a gyrB nucleic acid sequence comprising SEQ ID NO:

1.

6. A microbial composition comprising a Bacillus coagulans strain according to any one of claims 1 to 5 together with a edible acceptable carrier and / or diluent.

7. The unit dose of the composition is 10 ６ ~10 １３ 7. The microbial composition of claim 6, comprising CFU of a Bacillus coagulans strain.

8. The microbial composition of claim 6 further comprising a mucilaginous adhesive excipient.

9. 7. The microbial composition of claim 6, further comprising at least one additional probiotic Bacillus strain.

10. 7. The microbial composition of claim 6, formulated as a tablet, pill, capsule, powder, solution, suspension, or emulsion.

11. The microbial composition of claim 6 formulated as a food product.

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

13. 10. A composition for use in a method for preventing or treating vaginal infections, urinary tract infections, gastrointestinal infections and / or gastrointestinal diseases, or for improving immune health, protection against oxidative stress, cleansing and detoxification, metabolic health and / or cardiovascular health, said composition comprising the Bacillus clausii strain of any one of claims 1 to 5, said method comprising administering said composition.

14. 10. The microbial composition of claim 6 for use in preventing or treating vaginal infections, urinary tract infections, gastrointestinal infections, and / or gastrointestinal diseases, or improving immune health, protection against oxidative stress, cleansing and detoxification, metabolic health, and / or cardiovascular health.

15. 7. The microbial composition of claim 6, for use in a method for preventing or treating vaginal infections, urinary tract infections, gastrointestinal infections, and / or gastrointestinal diseases, or for improving immune health, protection against oxidative stress, cleansing and detoxification, metabolic health, and / or cardiovascular health, wherein the method comprises administering the microbial composition.

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

17. 6. A Bacillus coagulans strain according to any one of claims 1 to 5 for use as a probiotic, optionally wherein the strain is associated with an acceptable carrier or delivery vehicle and optionally an adjuvant component in a single composition, or wherein separate compositions comprise a mixture of the different strains.

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

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