Bacillus megaterium strains, compositions thereof and methods of use

Abstract

The present invention provides a Bacillus megaterium 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 comprising one or more bacteria having a 16S rRNA that shares at least 97% identity with SEQ ID NO:2. Optionally, the Bacillus megaterium strain shares at least 97% identity with SEQ ID NO:3. This strain may be used in the compositions and methods.

Description

[Technical field]

[0001] The present invention relates to novel Bacillus megaterium 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 and personal care products, comprising the Bacillus megaterium strains alone or in combination, and to the use of the strains for the prevention or treatment of gastrointestinal, urinary tract, vaginal and other 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 can reach CFU / g and is therefore considered to be one of the dominant components of the normal gut microbiota ( Lakshmi et al., 2017 ).

[0012] 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 & Sikora, 2007; Pelletier & Sygusch, 1990; Vary et al., 2007; Von Tersch & 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 were ancillary experiments that isolated Bacillus megaterium in fish (Sumathi et al., 2017). Afrilasari et al., 2015 also succeeded in isolating Bacillus megaterium from the digestive tract of catfish and identified the strain 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 has been confirmed through genomic analysis to be nearly identical (>99%) to Bacillus megaterium MIT411, which is claimed in the present invention. Health Canada has declared that this organism is not harmful to human health or the environment; and that the 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).

[0013] 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

[0014] 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 as discussed above. In one embodiment, the present invention relates to Bacillus megaterium MIT411 alone or in combination with other strains, such as Bacilli strains such as Bacillus coagulans strain CGI314 (disclosed and claimed in corresponding PCT application PCT / US2022 / xxxxx, which claims priority from Irish Patent Application No. 2021 / 0210, 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, such as the ability to colonize by adhesion to mucous membranes / surfaces, thus allowing the treatment or prevention of infections or diseases of the vagina, urethra, gastrointestinal, naso-sinal, pharyngeal, esophageal, oral cavity and / or other areas of the body having mucous membranes, as well as the treatment or prevention of infections or diseases of other areas of the body having skin and / or epithelia; immune health, protection against oxidative stress, cleansing and detoxification, metabolic health and cardiovascular health, inter alia providing antimicrobial activity, anti-inflammatory activity, suppression of pro-inflammatory responses, for example by stimulating macrophages, The present invention is suitable for activating and / or inducing an immune response, providing immune defense, for example aiding in digestion and / or fermentation in the gut, producing branched amino acids, essential amino acids and group B vitamins, maintaining a healthy gut and / or skin, 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 consequences, 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, etc. In one embodiment, the present invention enables the use of Bacillus megaterium strain MIT411 and compositions for use in fecal transplants.

[0015] 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 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 6-7 on the Bristol Stool Scale (tendency towards diarrhea), and the frequency of the individual's 24-hour episodes per month of bowel movements that are 1 or 2 (or 6-7) on the Bristol Stool Scale is reduced.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] It is yet another object of the present invention to provide compositions, such as dairy products, nutraceutical products and functional foods, comprising Bacillus megaterium MIT411 strains alone or in combination with other Bacilli strains, such as Bacillus coagulans and Bacillus clausii strains, having essentially the same properties of having the ability to colonize mucous membranes and thus being 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. [Brief description of the drawings]

[0021] In the drawings [Figure 1] FIG. 1 illustrates the genome analysis of Bacillus megaterium MIT411. [Diagram 2] FIG. 2 illustrates a phylogenetic tree of Bacillus spp. arranged in clades (16S). [Diagram 3] FIG. 3 illustrates a phylogenetic tree of Bacillus spp. arranged in a clade (gyrB). [Figure 4] Figure 4 shows the stability of Bacillus megaterium in phosphate buffer during the pasteurization process. Results are shown as mean concentrations ± standard deviation. [Diagram 5] FIG. 5 shows the antimicrobial activity of Renuspore against opportunistic pathogens of the intestine, skin and urinary tract in solid medium (TSA). [Figure 6]FIG. 6 shows the antimicrobial activity of B. megaterium MIT411 in liquid TSB medium against opportunistic pathogens of the intestine, skin and urinary tract: E. coli (*p<0.05), Salmonella enteritidis (****p<0.0001), Pseudomonas aeruginosa (****p<0.0001) and S. aureus. [Figure 7] FIG. 7 shows the total antioxidant capacity of PBS and B. megaterium. [Figure 8A] FIG. 8A shows the bioaccumulation of heavy metals by Renuspore in TSB medium supplemented with 1 ppm lead. [Figure 8B] FIG. 8B shows heavy metal bioaccumulation by Renuspore in TSB medium supplemented with 1 ppm mercury. [Figure 9] FIG. 9 shows the iron concentration in the extracellular fraction of Renuspore. [Figure 10] FIG. 10 shows the calcium concentration in the extracellular fraction of Renuspore. [Figure 11] FIG. 11 shows the magnesium concentration in the extracellular fraction of Renuspore. [Figure 12] FIG. 12 shows that B. megaterium does not affect bisphenol A concentrations in TST or MM medium. [Figure 13] FIG. 13 shows the nitrite concentration in the extracellular matrix of Renuspore. [Figure 14A] FIG. 14A shows the decomposition of ammonia by Renuspore. [Figure 14B] FIG. 14B shows the concentration of ammonia remaining in TSB+1 mM ammonia after incubation with Renuspore versus control at 37° C. for 24 hours. [Figure 15]FIG. 15 shows adhesion of B. megaterium MIT411 spores and vegetative cells to HT-29 and HT-29MTX cells at 37° C. [Figure 16] FIG. 16 shows the caseinolytic activity of Bacillus megaterium MIT411 (positive) versus B. coagulans (negative) as detected by the conventional method using skim milk agar at 24 hours. [Figure 17] FIG. 17 shows that Renuspore demonstrated protease activity using a quantitative extracellular protease assay with the EnzCheck Kit. [Figure 18] FIG. 18 shows that FAA was increased in Renuspore UHT fermented milk samples. [Figure 19] FIG. 19 shows that FAA was increased in Renuspore UHT fermented milk samples. [Figure 20] FIG. 20 shows that FAA was increased in Renuspore UHT fermented milk samples. [Figure 21] FIG. 21 shows that FAA was increased in Renuspore UHT fermented milk samples. [Figure 22] FIG. 22 shows that SCFAs were increased in Renuspore UHT fermented milk samples. [Figure 23] FIG. 23 shows that Fibersol® did not significantly increase the concentration of Renuspore (CFU / mL) in minimal medium after 24 hours of incubation compared to the control. [Figure 24] Figures 24 and 25 show that Renuspore increased cytokine expression in a human macrophage cell culture model. [Diagram 25] Figures 24 and 25 show that Renuspore increased cytokine expression in a human macrophage cell culture model. [Figure 26]FIG. 26 shows that Renuspore did not improve C. elegans survival after exposure to H2O2. [Figure 27] FIG. 27 shows the adhesion ability of Bacillus megaterium MIT411 vegetative cells and spores to intestinal epithelial cell lines HT-29 and HT-29-MTX at 37° C. [Figure 28] FIG. 28 shows a schematic flow chart of the study design. [Figure 29] FIG. 29 shows that the probiotic cocktail administered during the study significantly reduced the occurrence of loose stools throughout the study course compared to the placebo control. [Diagram 30] FIG. 30 shows that there was no effect of any of the treatments administered during the study on the percentage of hard stools compared to the placebo control. [Diagram 31] Figure 31 is a box plot showing the distribution of Chao1 values ​​in each experimental group on days 1 and 45 of the study. Dotted lines connect paired samples. Group distributions were compared using paired Wilcoxon tests. [Diagram 32] Figure 32 is a box plot showing the distribution of Chao1 values ​​in each experimental group on study days 1 and 45. The distributions of each experimental group were compared against placebo using the Wilcoxon test. [Diagram 33] FIG. 33 illustrates PCoA clustering performed on a Bray-Curtis dissimilarity matrix. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Genotyping The applicants collaborated with Cornell University (Ithaca NY, USA) for genome sequencing and identification.

[0023] 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.

[0024] The gyrB gene encodes DNA gyrase subunit B. DNA gyrase negatively supercoils closed circular double-stranded DNA in an ATP-dependent manner to maintain chromosomes in an underwound state. Gene sequencing analysis used gyrB gene polymorphism, a well-established method for species-level identification of prokaryotes (Bavykin et al., 2004; 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 A, B, and C below).

[0025] Genotype, gyrB and 16S rRNA identification of Bacillus megaterium MIT411

[0026] The MIT411 isolate and genome was considered a success.

[0027] The genome size (5.4 MBp) and GC content (37.8%) of the isolated strain were comparable to those of a Bacillus megaterium strain. [Table 1] [Table 2] [Table 3]

[0028] 16S rRNA Compared to one reference strain, whole genome sequencing (WGS) and 16S rRNA analysis of MIT-411 showed an average nucleotide identity (ANI) score for 16S rRNA of >99% when compared to Bacillus megaterium ATCC-14581. Genome size (5.4 Mbp) and GC content (37.8%) for Bacillus megaterium MIT-411 were comparable to the reference strain.

[0029] Further deposit and accession numbers 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).

[0030] 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 seuenci-Nucleotide-NCBI (nih.gov).

[0031] The genome sequence data of Bacillus coagulans strain CGI314 (Fortispore) 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, JABBFU000000000.1, and is available, for example, at the link: https: / / www.ncbi.nlm.nih.gov / nuccore / JABBFU000000000.1.

[0032] Phylogenetic placement was performed by Deerland Probiotics and Enzymes, Inc.

[0033] 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 confirmation for multiple alignments and phylogenetic analyses. GGDC verified the close relationship of Bacillus megaterium MIT411 to ATCC 14581.

[0034] 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.

[0035] Phylogenetic analysis using the Neighbor-Joining (NJ) method (Saitou & Nei, 1987) placed Bacillus megaterium MIT411 in the clade with Bacillus megaterium ATCC 15481 (Figure 2), confirming all previous genome identity determinations. Bacillus megaterium MIT411 is placed in the Bacillus megaterium group.

[0036] definition "Excipient" means any inactive ingredient that is added to form part of the final formulation.

[0037] "Probiotics" refers to viable microbial supplements that have beneficial effects on a subject through their effects on the intestinal tract, urinary tract, vaginal tract, skin and / or other areas of the subject's body. The term 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 gut flora may promote healthy gut homeostasis.

[0038] "Prebiotics" are used herein as substrates that have a beneficial effect on probiotics and thus on the individual subject that ingests (e.g., is administered) the probiotic. Suitable prebiotics may be selected from inulin, oligosaccharides and / or vitamins.

[0039] "Subject," as used herein, includes those suffering from any clinical condition related to microbial imbalance, as well as those using a bacterial preparation prophylactically, for health, or for any other purpose, including, for example, to benefit from administration of a Bacillus megaterium strain of the invention (e.g., MIT411). Optionally, the subject is a human, patient, and / or mammal.

[0040] "Synbiotic product" means a combination of a probiotic and a prebiotic, which is synergistic and has a beneficial effect on the patient.

[0041] "Hardy growth" means that the bacteria exhibits excellent proliferation.

[0042] The abbreviation "CFU" stands for colony forming unit.

[0043] 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.

[0044] According to a first aspect, the present invention includes Bacillus megaterium MIT411 alone or in combination with other probiotic Bacilli strains having essentially the same properties. Such other probiotic Bacilli strains include, but are not limited to, Bacillus clausii and Bacillus coagulans strains. Such other Bacilli strains further include Bacillus clausii and Bacillus coagulans strains, each of which is filed today under their respective headings, the contents of which are incorporated herein in their entirety.

[0045] SEQ ID NO:1 as recited in the claims appended hereto comprises gyrB of Bacillus megaterium MIT411.

[0046] SEQ ID NO:2 as recited in the claims appended hereto comprises the 16S rRNA of Bacillus megaterium MIT411.

[0047] SEQ ID NO:3 as recited in the claims appended hereto contains the assembled complete genome sequence of Bacillus megaterium MIT411.

[0048] 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:

[0049] Bacillus megaterium MIT411:

[0050] This strain exhibits bile stability.

[0051] This strain exhibits acid stability.

[0052] This strain exhibits heat tolerance.

[0053] This strain produces a natural antibiotic in the form of a bacteriocin.

[0054] 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.

[0055] This strain has been shown to pose little or no antibiotic resistance and safety concerns.

[0056] This strain was found to be acid and bile stable.

[0057] 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.

[0058] In another preferred embodiment, a composition, such as a pharmaceutical composition, is provided comprising Bacillus megaterium MIT144, alone or in combination with other probiotic Bacilli strains having similar and / or essentially the same properties, together with a pharma- ceutically acceptable carrier and / or diluent. Such other probiotic Bacilli strains include, but are not limited to, Bacillus clausii and Bacillus coagulans strains. These bacterial strains are formulated into compositions, such as pharmaceutical formulations, to allow easy administration of the probiotic strains and by means known to those skilled in the art.

[0059] 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 apoptosis induction 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.

[0060] 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 produce 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).

[0061] 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).

[0062] Preferably, the probiotic bacteria used in the pharmaceutical preparation according to the invention are from 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 ~10 10 In terms of CFU, or for example, about 10 6 , about 10 7 , about 10 8 , about 109 , 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 of amounts 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 megaterium MIT411 (e.g., about 10 9 CFU). In one embodiment, the composition of the present invention is orally administered in capsule form. In one embodiment, the Bacillus megaterium MIT411 is in spore form or is not in spore form.

[0063] In certain embodiments, the composition comprising Bacillus megaterium MIT411 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 megaterium MIT411 in a weight percentage of about 1% to about 95% by weight of the composition.

[0064] In certain embodiments, the composition comprising Bacillus megaterium MIT411 may include one or more liquid or gel-based carriers selected from the group consisting of water and physiological salt solutions, 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 megaterium MIT411 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 megaterium MIT411 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 megaterium MIT411 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 megaterium MIT411 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 megaterium MIT411 at a weight / volume percentage of about 1% weight / volume of the composition.

[0065] 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 oral cavity or 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 oral cavity or strips that dissolve in the oral cavity may be used. Other useful delivery systems include oral or nasal sprays or inhalants, and the like.

[0066] 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.

[0067] 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 especially tablets, filled capsules, powders and pellets, all for oral use; liquids and especially 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] Compositions suitable for topical administration in the mouth include, but are not limited to, lozenges containing the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles containing the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes containing the active ingredient in a suitable liquid carrier.

[0075] 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.

[0076] The pharmaceutical preparation is preferably in unit dosage form.In such form, the preparation is subdivided into unit doses containing appropriate amounts of active ingredient.The unit dosage form may be a packaged preparation, the package containing discrete 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.

[0077] 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.

[0078] 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).

[0079] In certain embodiments, compositions of the present invention, including compositions 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. 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 auxiliaries. 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.

[0080] In certain embodiments, nutraceutical compositions, including nutraceutical compositions administered according to the disclosed methods, 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.

[0081] Route of Administration The compounds may be administered by any route, including but not limited to oral, sublingual, buccal, ocular, pulmonary, rectal 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 present 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.

[0082] The pharmaceutical compositions of the present invention may be in a form suitable for oral, rectal, bronchial, nasal, pulmonal, topical (including buccal and sublingual), transdermal, vaginal 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 present invention, which matrices may be in the form of shaped articles, such as films or microcapsules.

[0083] 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.

[0084] Working Example Example 1 Further characterization of Bacillus megaterium MIT411 (hereinafter also referred to as Renuspore)

[0085] ·Temperature stability: B. megaterium MIT411 in PBS-pH 7.51 remains stable in PBS for 30 seconds to 3 minutes at 45°C, 75°C, and 90°C (Figure 4).

[0086] Renuspore is stable during pasteurization processes and other manufacturing processes in food and beverage and other applications.

[0087] Figure 4 shows the stability of Bacillus megaterium in phosphate buffer during the pasteurization process; results show the mean concentration ± standard deviation.

[0088] Antimicrobial activity against intestinal and skin pathogens in soil environment: B. megaterium MIT411 (Renuspore) had weak antimicrobial activity against E. coli, Salmonella enteritidis, and S. aureus on TSA overlaid with 0.4% TSA agar, with cloudy zones of inhibition observed (Figure 5 and Table 1). No antimicrobial activity was observed against P. aeruginosa on solid medium. [Table 4]

[0089] Figure 5 shows Renuspore antimicrobial activity against opportunistic pathogens of the intestine, skin and urinary tract on solid medium (TSA). A cloudy inhibition zone is observed around the B. megaterium MIT411 growth. Antimicrobial activity is shown as inhibition zone (mm) standard deviation. AE. E. coli and BS. enteritidis and CS. aureus.

[0090] Renuspore demonstrated a broad antimicrobial profile that was active against the enteric pathogen Salmonella enteritidis and the opportunistic enteric and urinary tract pathogen E. coli in solid media. Renuspore was also active against the opportunistic skin pathogen S. aureus.

[0091] Renuspores have the potential to keep out bacterial pathogens and maintain a healthy gut and skin flora.

[0092] Antimicrobial activity against intestinal and skin pathogens in a liquid environment: Figure 6 shows the antimicrobial activity of B. megaterium MIT411 in liquid TSB medium against the intestinal, skin and urinary tract opportunistic pathogens: E. coli, Salmonella enteritidis, Pseudomonas aeruginosa and S. aureus. Controls show the growth of the pathogens individually; treatments show the growth of the pathogens in the presence of B. megaterium MIT411. * p<0.05 and **** p<0.0001.

[0093] Renuspore inhibited the growth of the enteric pathogen Salmonella enteritidis and the opportunistic enteric and urinary tract pathogen E. coli in liquid medium. Renuspore was active against the opportunistic skin and urinary tract pathogen P. aeruginosa in liquid medium conditions.

[0094] Renuspores have the potential to keep out bacterial pathogens and maintain a healthy gut and skin flora.

[0095] Antimicrobial activity against intestinal and skin pathogens in a liquid environment: B. megaterium MIT411 (Renuspore) had significant antimicrobial activity against Salmonella enteritidis and P. aeruginosa in liquid TSB medium (Figure 6 and Table 2). Weak antimicrobial activity was detected against E. coli, and no antimicrobial activity was observed against S. aureus under these conditions. [Table 5]

[0096] Table 2. Antimicrobial activity of B. megaterium MIT411 against intestinal, skin and urinary tract opportunistic pathogens. Antimicrobial activity detected (+), no antimicrobial activity observed (-).

[0097] Renuspores have the potential to keep out bacterial pathogens and maintain a healthy gut and skin flora.

[0098] Antioxidant activity: The total antioxidant activity of Renuspore B. megaterium was compared with that of L. rhamnosus. Figure 7 shows the total antioxidant capacity of PBS and B. megaterium. Results show the mean concentration of Trolox equivalents in nmoles / g (n=3) ± standard error. Renuspore has higher antioxidant levels compared to the potential probiotic L. rhamnosus (not shown).

[0099] Renuspore showed significant antioxidant levels, not significantly different from Fortispore (Bacillus coagulans CGI314) according to Tukey's multiple comparison test.

[0100] Renuspores bioaccumulate lead and eliminate it from the environment: Renuspore can eliminate 37.97% of bioavailable lead (Figure 8A). Renuspore has also proven effective in bioaccumulating heavy metals.

[0101] FIG. 8A shows heavy metal bioaccumulation by Renuspore in TSB medium supplemented with 1 ppm lead. Results show the mean concentration in ppm (n=5)±standard error. A significant reduction was observed between Renuspore and the control, as shown: *** p=0.001.

[0102] Renuspore has the ability to bioaccumulate lead, the most commonly occurring heavy metal in our environment. These data demonstrate the potential of Renuspore to bioaccumulate environmental contaminants, such as heavy metals, present in the environment and prevent their deleterious effects.

[0103] Renuspore may act as a potential probiotic for the biological removal of heavy metals, thereby mitigating the effects of heavy metals in the human body.

[0104] ·Heavy metal bioaccumulation - mercury: Renuspore can effectively bioaccumulate mercury, reducing the bioavailable free mercury by 85.80% (Figure 8B).

[0105] FIG. 8B shows heavy metal bioaccumulation by Renuspore in TSB medium supplemented with 1 ppm mercury. Results show the mean concentration in ppm (n=5)±standard error. The significant reduction observed between Renuspore and the control is shown: **** p<0.0001.

[0106] Renuspore has the ability to bioaccumulate lead and mercury, the two most commonly occurring heavy metals in our environment. Collectively, these data indicate the potential of Renuspore to bioaccumulate environmental contaminants, such as heavy metals, present in the environment and prevent their harmful effects.

[0107] Renuspore may act as a potential probiotic for the biological removal of heavy metals, thereby mitigating the effects of heavy metals in the human body.

[0108] Iron bioaccumulation: Renuspore was grown in TSB medium in the presence of iron and the supernatant was assayed. TSB medium + iron was used as a control. The results revealed that Renuspore does not bioaccumulate iron in TSB medium, as the iron concentration in the extracellular fraction remained unchanged (Figure 9).

[0109] Figure 9 shows the iron concentration in the extracellular fraction of Renuspore. Results show the mean concentration (n=3) ± standard error of the total iron concentration in nmole / ml.

[0110] Renuspore may act as a potential probiotic for the biological removal of toxic heavy metals without compromising the body's natural absorption of essential minerals such as iron.

[0111] Renuspore does not bioaccumulate calcium: Renuspore was grown in TSB medium in the presence of calcium and the supernatant was assayed. TSB medium + calcium was set as the control. Renuspore does not bioaccumulate calcium in TSB medium, as the calcium concentration in the supernatant remained unchanged (Figure 10).

[0112] Figure 10 shows calcium concentration in the extracellular fraction of Renuspore. Results show the mean (n=3) ± standard error of calcium concentration in μM using Dunnett's test.

[0113] Renuspore may act as a potential probiotic for the biological removal of toxic heavy metals without impairing the body's natural absorption of essential minerals such as calcium.

[0114] Renuspore does not bioaccumulate magnesium: Renuspore was grown in TSB medium in the presence of magnesium and the supernatant was assayed. TSB medium + magnesium was set as the control. Using Dunnett's test, the results revealed that Renuspore was not significantly different compared to the control (TSB medium). This indicates that Renuspore does not bioaccumulate magnesium from the environment (Figure 11).

[0115] Figure 11 shows magnesium concentration in the extracellular fraction of Renuspore. Results show the mean concentration (n=3) ± standard error of magnesium concentration in mmol / L using Dunnett's test.

[0116] This study demonstrated how Renuspore does not bioaccumulate magnesium and does not compete with the intestine for absorption of this essential mineral.

[0117] Renuspore may act as a potential probiotic for the biological removal of toxic heavy metals without impairing the body's natural absorption of essential minerals such as magnesium, iron and calcium.

[0118] · Renuspore does not utilize or break down Bisphenol A (BPA): Bacillus megaterium MIT411 (also known as Renuspore) was unable to utilize BPA as a sole carbon source as no growth was observed in minimal medium (MM) agar and in broth across all BPA concentrations analyzed (5 mg / L to 100 mg / L). B. megaterium cell growth decreased with increasing BPA concentrations (Table 3). B. megaterium did not tolerate 5 mg / L BPA overnight in both MM and TSB broth (Figure 12).

[0119] Figure 12 shows that B. megaterium does not affect bisphenol A concentrations in TST or MM medium (leftmost vertical bar: control; T = 24, 48, 72, 96, 120 hours, vertical bars from left to right). [Table 6]

[0120] Renuspore does not have the ability to break down BPA present in the environment: Renuspore does not utilize or break down DEET (N,N-diethyl-m-toluamide): No gene for DEET hydrolase was detected in the Renuspore genome. Renuspore cannot utilize DEET as an energy source using minimal media. Also, increasing concentrations of this synthetic chemical in rich media has a toxic effect on Renuspore growth (Tables 4, 5 and 6). Thus, Renuspore cannot use DEET as a food source and cannot break it down into less toxic products. [Table 7] [Table 8] [Table 9]

[0121] Renuspore does not have the ability to break down DEET present in the environment.

[0122] Renuspore can detoxify nitrites from the environment: Renuspore was grown in TSB medium in the presence of nitrite and their supernatants were assayed. TSB medium + nitrite was set as the control. Renuspore completely removed nitrite from the environment and started converting it to nitrate or nitric oxide (Figure 13). Renuspore can either reduce nitrite to nitric oxide using nitrite reductase or oxidize nitrite to nitrate using oxidoreductase, both enzymes were found in its genome.

[0123] Figure 13 shows the nitrite concentration in the extracellular matrix of Renuspore. Results show the mean concentration of nitrite in nmole / ml (n=3) ± standard error. A significant reduction is observed between control and Renuspore, as shown: **** p<0.0001.

[0124] Renuspore can scavenge environmental nitrites and play a significant role in reducing the toxic levels of nitrite in the human body.

[0125] Taken together, Renuspore may act as a potential probiotic for the biological removal of toxic nitrites, oxidizing them to less harmful products such as nitrates.

[0126] Renuspore does not biodegrade ammonia: Minimal salts medium containing ammonium chloride as the sole nitrogen source was used to assess whether Renuspore can use ammonia as a nitrogen source. In the presence of glucose, magnesium sulfate and calcium chloride, Renuspore can grow in minimal medium, thereby using 30% of the ammonia from the medium (Figure 14A). In TSB medium, Renuspore can synthesize ammonia, presumably from peptide sources present in TSB medium, since there is a 47.9% increase in ammonia concentration observed compared to the control (Figure 14B).

[0127] FIG. 14A shows the degradation of ammonia by Renuspore. Results show the mean concentration of ammonia in μmol / L (n=3) ± standard error and Tukey's multiple comparison test. Note: ** indicates significance between Renuspore and control (P<0.05).

[0128] FIG. 14B shows the concentration of ammonia remaining in TSB+1 mM ammonia after incubation of Renuspore versus controls for 24 hours at 37° C. ** indicates significance between Renuspore and control (P<0.05).

[0129] Renuspore can utilize ammonia as a nutrient source.

[0130] Renuspores attach to epithelial intestinal cells: FIG. 15 shows adhesion of B. megaterium MIT411 spores and vegetative cells to HT-29 and HT-29MTX cells at 37° C.

[0131] B. megaterium MIT411 vegetative cells do not adhere to HT-29 and HT-29-MTX intestinal cell lines. B. megaterium MIT411 spores adhere to HT-29 and mucus-producing HT-29-MTX cell lines; therefore, it can adhere to and bud into intestinal cells.

[0132] Vegetative cells are able to bioaccumulate toxic environmental contaminants and eliminate them from the human body without attaching to the intestine.

[0133] Renuspore exhibits high protease activity: Renuspore showed caseinolytic activity on skim milk agar plates (see FIG. 16). Quantitative analysis of Renuspore caseinolytic activity was assessed by using a commercial kit with fluorescently tagged casein derivatives. Renuspore showed extracellular protease activity. Genome analysis of Renuspore revealed the presence of multiple genes (prtP) encoding caseinolytic proteases CEP, which explains this high protease activity of Renuspore.

[0134] Figure 16 shows the caseinolytic activity of Bacillus megaterium MIT411 (positive) versus B. coagulans (negative) as detected by conventional methods using skim milk agar at 24 hours. The clearing zone gives an indication of the degree of caseinolysis. The plate on the left shows a streaked plate and the plate on the right shows an inoculation from strain MIT411 overnight in TSB.

[0135] FIG. 17 shows that Renuspore demonstrated protease activity using a quantitative extracellular protease assay with the EnzCheck Kit.

[0136] Both in silico and in vitro analyses suggest the ability of Renuspore to hydrolyze milk proteins, specifically casein.

[0137] Renuspore has a diverse carbohydrate profile: Renuspore metabolizes a variety of monosaccharides, sugar alcohols, amine sugars and glycosides: Renuspore tested positive for 11 carbohydrates out of 49 tested using the commercially available API 50 CH. Most of these carbohydrates were monosaccharides such as D-ribose, L-arabinose, D-xylose, D-glucose, D-fructose and D-saccharose. Genome analysis of Renuspore reveals the presence of transporters and enzymes involved in the metabolism of most of these sugars. Furthermore, genes involved in the metabolism of polysaccharides, amylase A involved in starch metabolism were also identified in the Renuspore genome. [Table 10]

[0138] Both in silico and in vitro analyses suggest a diverse ability of Renuspore to ferment different carbohydrates. Renuspore has enzymatic activity towards esters, proteins and carbohydrates: Renuspore is positive for esterase, alpha-chymotrypsin, alkaline phosphatase (ALP) and galactosidase activity using the API ZYM kit, which means that: ·High potential of Renuspore to generate free fatty acids from the action of esterases in the presence of an appropriate lipid source. · α-chymotrypsin activity of Renuspore, the ability to hydrolyze amide bonds where the amino acid N-terminal to the bond is tryptophan, tyrosine, phenylalanine or leucine. This activity should enhance Renuspore's proteolytic ability. Galactosidases enhance the carbohydrate catabolic potential of Renuspore since they are active towards various oligosaccharides, lactosylceramide, lactose and many glycoproteins.

[0139] Indeed, in silico analysis identified genes encoding esterases, ALPs, proteases and galactosidases. [Table 11]

[0140] This study confirms Renuspore's ability to hydrolyze proteins and oligosaccharides and indicates its potential to break down fats.

[0141] These data suggest that Renuspore may aid in the digestion of these molecules in the intestine.

[0142] Renuspore produces a broad range of amino acids from milk protein hydrolysates: The proteolytic ability of Renuspore was analyzed using the UHT milk model. In silico analysis of Renuspore revealed the presence of various proteases, peptide transporters and peptidases, suggesting the presence of a robust proteolytic system in Renuspore. GC-MS analysis identified a total of 38 free amino acid (FAA) compounds, 28 of which were found to be statistically significant in Renuspore. The results from this analysis support the presence of a highly active proteolytic system in Renuspore that can completely degrade milk proteins to release FAA. Furthermore, the proteolytic system in Renuspore shows the potential to further catabolize these amino acids to generate aromatic carboxylic acids (4-methyl-2-oxopentanoic acid, benzoic acid, oxopentanoic acid and proponoic acid). [Table 12]

[0143] Both in silico and in vitro analyses suggest that Renuspore possesses a strong and active proteolytic system, since high release of amino acids and their downstream products was obtained in Renuspore fermented UHT milk.

[0144] Renuspore produces a broad range of amino acids from milk protein hydrolysates: Renuspore FAA analysis shown in bar graph (mean + SEM):

[0145] Figure 18 shows that FAA was increased in Renuspore UHT fermented milk samples. Statistical analysis was performed using a multiple T-test - unpaired parametric, with a two-stage setting (Benjamini, Krieger and Yekutieli and P-value ≤ 0.01 = * White bars represent controls.

[0146] Figure 19 shows that FAA was increased in Renuspore UHT fermented milk samples. Statistical analysis was performed using a multiple T-test - unpaired parametric, with a two-stage setting (Benjamini, Krieger and Yekutieli and P value ≤ 0.01 = * White bars represent controls.

[0147] Figure 20 shows that FAA was increased in Renuspore UHT fermented milk samples. Statistical analysis was performed using a multiple T-test - unpaired parametric, with a two-stage setting (Benjamini, Krieger and Yekutieli and P-value ≤ 0.01 = * White bars represent controls.

[0148] Figure 21 shows that FAA was increased in Renuspore UHT fermented milk samples. Statistical analysis was performed using a multiple T-test - unpaired parametric, with a two-stage setting (Benjamini, Krieger and Yekutieli and P value ≤ 0.01 = * White bars represent controls.

[0149] · Renuspore exhibits weak lipolytic activity: Limited short chain fatty acids (SCFAs) produced by Renuspore fermentation of UHT milk: Although Renuspore exhibits esterolytic activity and possesses genes encoding esterase A and lipase, only two SCFAs were significantly increased in Renuspore-fermented UHT milk samples.

[0150] Figure 22 shows that SCFAs were increased in Renuspore UHT fermented milk samples. Statistical analysis was performed using a multiple T-test - unpaired parametric, with a two-stage setting (Benjamini, Krieger and Yekutieli and P value ≤ 0.01 = * White bars represent controls.

[0151] The only two SCFAs associated with Renuspore are propionate and 2-methyl-propionate, which are normally associated with amino acid metabolism, specifically alanine and valine, respectively. Collectively, these data suggest a narrow lipolytic activity of Renuspore.

[0152] Renuspore proteomic analysis identifies proteins with potential probiotic benefit: Proteomic Study - Renuspore Secretome: Extracellular secretions of Renuspore grown in TSB broth for 24 hours were sent to mass spectrometry to identify proteins released by the probiotic strains. A total of 23 proteins were detected, of which 4 had potential probiotic benefits (Table 9A): [Table 13]

[0153] These data support previous in vitro results showing how Renuspores can aid in the digestion of proteins and carbohydrates, detoxify harmful compounds, and have antimicrobial properties against pathogens.

[0154] Renuspore in the presence of Fibersol® (F) in minimal medium: FIG. 23 shows that Fibersol® did not significantly increase the concentration of Renuspore (CFU / mL) in minimal medium after 24 hours of incubation compared to the control.

[0155] ·Immunomodulatory potential of Renuspore in an in vitro human macrophage model: Figures 24 and 25 show that Renuspore increased the expression of cytokines in a human macrophage cell culture model. Unlike the LPS positive control, Renuspore increased the expression of all cytokines tested (TNF-α, IL-1β, IL-18, IL-6, GM-CSF, IL-10, IL-1RA and EGF). Renuspore was more effective than LPS in inducing the expression of TNF-α, GM-CSF and EGF. Thus, Renuspore can be considered as a strong stimulator of the innate immune system. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 significantly higher than negative control; + p<0.05, ++ p<0.01, ++++ p<0.0001, significantly higher than the positive control.

[0156] Antioxidant potential of Renuspore in a C. elegans model of H2O2 oxidative stress: Figure 26 shows that Renuspore did not improve C. elegans survival after exposure to H2O2. Vitamin C was used as a positive control for the assay. * p<0.05 significantly higher than control.

[0157] 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.

[0158] Cells, 5x10 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.

[0159] Before the experiment, the cells were washed twice with 0.5 ml DPBS, which was completely aspirated from the wells after the second wash.

[0160] 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.

[0161] Adhesion assay: 500 μl of spore suspension (1.3 x 10 7 ~9.2x10 7 CFU / 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).

[0162] 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.

[0163] 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.

[0164] 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.

[0165] Experiments were performed two or three times with three technical replicates per experiment. Results are expressed as mean±SEM.

[0166] Figure 27 shows the adhesion ability of Bacillus megaterium MIT411 vegetative cells and spores on intestinal epithelial cell lines HT-29 and HT-29-MTX at 37°C (DI_EK_03). (Left graph) See Figure 27 showing the percentage of adherent bacteria on HT-29 with B. megaterium MIT411 spores (left bar) and vegetative B. megaterium MIT411 (right bar). Also see Figure 27 showing (right graph) the percentage of adherent bacteria on HT-29-MTX with B. megaterium MIT411 spores (left bar) and vegetative B. megaterium MIT411 (right bar).

[0167] In comparative studies, the adhesion of B. clausii CSI08, B. megaterium MIT411 and B. coagulans CGI314 spores to the HT-29-MTX cell line was as follows (Table D): [Table 14]

[0168] Adhesion of B. clausii CSI08, B. megaterium MIT411 and B. coagulans CGI314 spores to the HT-29 cell line (Table E): [Table 15]

[0169] 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.

[0170] Example 3 Evaluation of Bacillus clausii CSI08, Bacillus megaterium MIT411 and Bacillus cocktail on safety, tolerability and gastrointestinal health: a randomized, double-blind, placebo-controlled study in healthy adults

[0171] Administered daily, 1X10 9 CFU Bacillus clausii CSI08, 1X10 9 CFU Bacillus megaterium MIT411 and 0.5x10 9 CFU of Bacillus subitilis DE111®, 0.5x10 9 CFU of Bacillus megaterium MIT411, 0.5x10 9 CFU of Bacillus coagulans CGI314, 0.5x10 9 Probiotic cocktail containing CFU Bacillus clausii CSI08 (i.e., total number of 2.0x10 9The safety, tolerability and effects of CFUs of Bacillus subitilis DE111®, Bacillus megaterium MIT411, Bacillus coagulans CGI314 and Bacillus clausii CSI08 were evaluated compared to a placebo control containing maltodextrin. A total of 98 study participants received a daily dose for 45 days, followed by a 2-week washout period. Compliance was recorded throughout the 45 days with questionnaires to record the occurrence and duration of upper respiratory, urinary and / or gastrointestinal complaints and daily diaries to record bowel movements and stool consistency. Fecal and blood samples were collected for microbiological and hematological analysis at the beginning and end of the treatment period. The probiotic cocktail significantly reduced the occurrence of loose stools throughout the study. Recorded respiratory, urinary and gastrointestinal symptoms, stool frequency and other stool consistency were not affected. No clinically significant changes in blood parameters such as liver and kidney function and no serious adverse events were noted during and after dosing. There were no changes in symptoms, including sadness, agitation, energy, appetite, tension, stress, sleep, cardiovascular events, aches and pains and dizziness, as determined by mood questionnaires administered to participants at baseline and at the end of the treatment period. Similarly, measured inflammatory cytokines, antioxidant levels, cholesterol, triglycerides, free amino acids or minerals remained unaffected. There were no negative changes in alpha or beta diversity of the microbiota in any of the treatment groups. These encouraging data suggest that these treatments were safe and well tolerated, and further studies in larger cohorts are justified to determine the efficacy of these potential probiotics in selected populations.

[0172] Probiotics are live microorganisms that are present in the human intestine and have low or no pathogenicity, and have beneficial effects on the host.Common products that contain probiotic bacteria include dietary supplements and foods, such as fermented dairy products, sauerkraut and salami.Probiotic supplementation has shown good results in the relief of various disorders, such as antibiotic-associated diarrhea, constipation, allergies and diabetes.Probiotics have also shown protective properties.

[0173] Probiotic supplements may contain one or more different bacterial strains that exert different effects on the human gut. Common probiotic strains are lactic acid producers such as Lactobacillus, Bifidobacterium and Streptococcus due to their resistance to gastric acid, bile salts and pancreatic enzymes. Studies have shown that lactic acid bacteria are effective inhibitors of pathogenic Gram-negative bacterial colonization in vitro (e.g. Salmonella typhimurium, Clostridium difficile and Escherichia coli).

[0174] However, not all probiotic supplements are lactic acid producers. Bacillus subtilis spores have been used as probiotics, competitive excluders and prophylactics for human and animal consumption. All four Bacilli strains are Gram-positive, spore-forming, rod-shaped bacteria. Under nutrient-limited conditions, Bacillus sp. can form dormant endospores that are resistant to environmental stressors and nutrient deprivation, making these bacteria a viable option for probiotic supplements.

[0175] DE111, CSI08, CGI314 and MIT411 are unique strains of probiotics. They are probiotic Bacillus strains that can withstand the harsh digestive environment and colonize the intestine, thus supporting a healthy GI tract. To date, DE111 has been sold in both the United States and Canada as a probiotic food ingredient for adults and as a probiotic capsule. The other three Bacillus probiotics used in this trial, CSI08, CGI314 and MIT411, are not currently on the market and are claimed herein.

[0176] The study was to determine the safety of three new probiotic strains and to evaluate their effectiveness in reducing the onset and / or duration of gastrointestinal problems and infections, and respiratory infections in healthy adults.

[0177] Materials and Methods subject Healthy adult volunteers aged 18-65 years were recruited using flyers, posters and from their physicians from February to July 2021. Inclusion criteria included willingness to provide informed consent and good overall health. Exclusion criteria included: presence of any pre-existing adverse event condition (e.g. gastric ulcer, Crohn's disease, UC, diabetes, kidney disease, HIV / AIDS, hepatitis, cancer and organ transplant recipients), taking medication for digestive disorders (constipation, bloating or diarrhea), use of antibiotics within the past 4 weeks before randomization, unwillingness to discontinue any probiotic supplement other than that provided by this study, known immunodeficiency or use of immunosuppressant medications, pregnancy, 6 months postpartum or breastfeeding, women of childbearing age planning pregnancy during the course of the study, participation in another study and use of medications for mood (e.g. antidepressants, anxiolytics, antipsychotics).

[0178] The study was approved by the Nutritional Research Ethics Committee, Biotechnical Faculty, University of Ljubljana, Slovenia, and was conducted in accordance with the guidelines established by the Declaration of Helsinki. All participants were informed of the purpose, requirements, and risks of the study, as well as being informed that they could withdraw from the study at any time. Participants provided their written consent indicating that they were fully aware of the study protocol.

[0179] Experimental design The study was double-blind, placebo-controlled, randomized, parallel design. The study was conducted through the University Clinical Centre Maribor, Slovenia and coordinated by CRO Vizera doo, Slovenia. Participants were randomized to one of three treatment arms or placebo, administered daily. Treatment arms consisted of 1x10 9 CFU / dose of Bacillus clausii CSI08, 1x10 9 CFU / dose of Bacillus megaterium MIT411 and total 2.0x10 9 The probiotic cocktail contained CFU / dose of Bacillus subtilis DE111®, Bacillus megaterium MIT411, Bacillus coagulans CGI314, and Bacillus clausii CSI08. The placebo was rice maltodextrin.

[0180] The randomization scheme was performed by CRO Vizera doo, Slovenia, with the allocation sequence concealed from the study personnel and participants until the day of randomization in a sealed opaque envelope. After the assessment of baseline characteristics (age, sex, height, weight by digital scale) and the collection of the first stool sample, the envelope was opened and participants were assigned to the intervention. The physician received an individually closed envelope containing the link between the randomization number and the treatment group for a particular participant. The closed envelope could only be opened in case of emergency. The sponsor was informed immediately if the participant's treatment was unblinded during the course of the study. Information regarding unblinding had to be recorded in the data source documentation and in the participant's case report form (CRF). Participants were then instructed to take one capsule per day after a meal.

[0181] Participants visited the study center three times and spoke with a designated physician by phone twice: Visit 0 for screening purposes (screening visit), two visits during the treatment period, Visit 1 was the baseline visit, where randomization and product distribution took place, and Visit 2 was the last treatment visit. In addition, patients spoke with a physician by phone 21 days after product intake (In between visits call) and after a 2-week follow-up after the second visit (follow-up call). A schematic flow chart of the study is shown in Figure 28.

[0182] After screening, consenting and randomization, participants provided blood and stool samples before any treatment. At the end of the 45-day intervention period, study participants provided a second stool sample and again a blood sample.

[0183] FIG. 28 shows a schematic flow chart of the study design.

[0184] Probiotics Administration Protocol Deerland Probiotics and Enzymes (Kennesaw, Georgia, US) provided the investigational product as identical, rectangular 300 mg capsules and the placebo indistinguishable by appearance. The investigational capsules were provided in bottles labeled with the treatment code by a study collaborator who had no contact with the study personnel or participants.

[0185] Test Protocol Participants completed a questionnaire daily to monitor bowel movement times and type of stool samples based on the Bristol Fecal Chart Index and whether they had any symptoms, including gastrointestinal upset, respiratory upset, urinary symptoms, head, ear, nose and throat, behavior, vomiting, loss of appetite, fever and superficial skin. If there were any visits to the participant's GP or any medication prescriptions during the study, this was also recorded and reported. A mood questionnaire was administered to participants at baseline and at the end of the treatment period to assess their experience over the previous month. This questionnaire consisted of 14 recorded symptoms, including sadness, irritability, energy, appetite, tension, stress, sleep, cardiovascular events, pain and aches and dizziness, on a scale of 1 (no noticeable symptoms) to 3 (severe). Any adverse events were reported to study staff.

[0186] Blood sample collection and preparation For blood safety, 3 mL red cap serum clot activator tubes were used for blood collection (Greiner Bio-One, 454029). For biochemistry blood panel high and low density lipoprotein, total cholesterol and triglyceride determination, 3.5 mL SST II Advanced / gel yellow cap vials (Greiner Bio-One, 454029) were used. For antioxidant and cytokine measurements, whole blood was collected in 4 mL lithium-heparin-containing tubes (Greiner Bio-One, 454029). Plasma samples were prepared by centrifugation at 2000G for 15 min. The supernatant was aliquoted and stored at -80°C for later analysis.

[0187] Determination of LDL, HDL, total cholesterol and triglycerides Hematology and biochemistry evaluations were performed at the University Clinical Centre Maribor, Slovenia. Blood safety was performed using a Sysmex EN-1000, while biochemistry assays for LDL, HDL, total cholesterol and triglycerides were assayed according to the manufacturer's instructions and analyzed by an Abbott Allinity C.

[0188] Cytokine quantification The concentrations of IL-8 and TNF-alpha in serum samples were determined by sandwich ELISA: Human IL-8 (CXCL8) ELISA kit (ELH-IL8-1, RayBiotech) and Human TNF-alpha ELISA kit (ELH-TNFa-1, RayBiotech) according to the manufacturer's instructions. Prior to ELISA, serum samples were diluted 1:2 using the dilution buffer provided with the kit.

[0189] Determination of antioxidant activity Total antioxidant activity was assessed using a Total Antioxidant Capacity Assay Kit (Sigma, Ireland) according to the manufacturer's instructions and absorbance was measured at 340 nm.

[0190] Fecal collection Feces were collected using Zymokit DNA / RNA Shield™ Fecal Collection Tubes (ZymoResearch, California, US) at the pre-treatment baseline visit and again at the final visit on day 45. Participants were instructed to place the collection system containing the sample on ice immediately after defecation and to deliver the sample to the study personnel at the clinic visit.

[0191] DNA extraction and 16S rRNA sequencing Total fecal DNA from approximately 200 mg of sample was extracted using a ZymoBIOMICS DNA Miniprep kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's instructions. Briefly, fecal samples were placed in ZR BashingBead™ Lysis tubes containing 750 μl ZymoBIOMICS™ Lysis Solution and processed with a BeadBug™ 6 homogenizer (Benchmark Scientific, China): 5x1 min beating at 4350 rpm with a 1 min pause between each beating cycle. The lysis tube was then centrifuged at 10,000 g for 1 min. 400 microliters of the supernatant was transferred to a Zymo-Spin™ III-F filter in a collection tube and further centrifuged at 8,000 g for 1 min. The filtrate was mixed with 1,200 μL of ZymoBIOMICS™ DNA Binding Buffer, transferred to a Zymo-Spin™ II CR column in a collection tube, and centrifuged at 10,000 g for 1 min. After three washes, the DNA was eluted in 100 μL of ZymoBIOMICS™ DNase / RNase-free water and further purified using a Zymo-Spin™ III-HRC filter according to the protocol. DNA concentration was determined using the Qubit dsDNA BR Assay Kit (ThermoFisher Scientific).

[0192] Data Generation Library preparation was performed following the Illumina guidelines for 16S Metagenomic Sequencing Library Preparation (https: / / support.illumina.com / documents / documentation / chemistry_documentation / 16s / 16s-metagenomic-library-prep-guide-15044223-b.pdf). Briefly, 16S degenerate primers are used to amplify targets from each sample. At the same time, Illumina adapters and barcodes are included to allow for library creation. Sequencing was performed on a Novaseq 6000 instrument generating paired-end 250bp reads. Quality control of the sequencing data was performed using the software QIIME2. On average, 670000 read pairs were generated per sample. Taxonomic classification of ASVs (also called OTUs) was performed using QIIME2 / DADA2 and Silva132 databases.

[0193] statistical analysis Twenty-five participants per arm were determined to be sufficient to assess the occurrence and nature of possible adverse events, including the occurrence and duration of urinary, gastrointestinal, and upper respiratory complaints. Descriptive statistics were used to evaluate these outcomes in this study. The Kruskal-Wallis test was used to confirm that there were no statistically significant differences in the occurrence of any of these individual symptoms between the four treatment groups at the start of the study or in the occurrence and duration of gastrointestinal, upper respiratory, or urinary complaints over the study period. In addition, nonparametric Mann-Whitney U tests with Holm correction were used for pairwise comparisons between each of the three probiotic product groups compared with the placebo group.

[0194] For the Gastrointestinal Health Questionnaire and blood analyses, differences in changes in individual symptom scores from baseline to the end of the treatment period were compared among treatment groups using analysis of variance (one-way ANOVA test) with post-hoc tests evaluating pairwise comparisons between each of the three treatment groups compared to the placebo group.

[0195] For sequencing data, multiple alpha diversity indices were calculated, including Observed, Chao1, ACE, Shannon, and Simpson indices. Alpha diversity was then compared between experimental groups and against placebo to detect treatment- or within-treatment differences from baseline to post-treatment time points.

[0196] To quantify the compositional differences between the various samples, Bray-Curtis dissimilarity was calculated and used to generate multiple clustering plots. This method collapses information from multiple dimensions for ease of visualization and interpretation. Group distributions were compared using paired Wilcoxon tests.

[0197] Abundance variation analysis was performed to detect significant differences in genera abundance across different treatments and time points. Day 1 samples from all treatments were compared to the placebo group on day 1 to determine whether there were any resting differences at baseline. Pairwise comparisons were performed for each group on day 45 versus day 1. Bifactorial analysis was also performed using the placebo group as a reference to detect whether there were significant differences in the response of treatment on day 45 relative to day 1 compared to the response of the placebo group on day 45 relative to day 1.

[0198] result participants Ninety-eight participants completed the 45-day intervention (Figure 28). After screening, one participant declined to participate and another withdrew due to pregnancy. A total of 12 adverse events were reported in the study. These included gastroesophageal reflux (3 AEs), rash (2 AEs) and dizziness (2 AEs). One case of rash was reported as a fungal rash (Tinea corporis) and one case of dizziness was due to the use of an approved co-medication. All other AEs reported, namely: vaginal irritation, fecal worms (possibly related to international travel), right wrist spin, metal fuss, lower back pain, sebaceous gland inflammation, granuloma, dark brown stool and acne, occurred only once.

[0199] Causality assessment revealed no relationship between the reported AEs and the study product.

[0200] No serious adverse events were reported throughout this study.

[0201] Participant demographics: [Table 16]

[0202] Gastrointestinal health status at screening visit: [Table 17] TIFF2024546089000019.tif18155

[0203] There were no significant differences between groups for any of the individual readouts.

[0204] Stool consistency and regularity The mean bowel movement frequency (regularity) ranged from 0.33 to 2.16 bowel movements / day in study participants. It was concluded that the various time periods and intervention group comparisons were not comparable. Bowel movement frequency was not significantly different when comparing the means with the placebo treatment group or washout period (Table 12). [Table 18]

[0205] Stool consistency is reported as the percentage of participants with loose stools and the percentage of participants with hard stools during the entire treatment period. The baseline questionnaire reported no differences in the occurrence of loose or hard stools / constipation in the study group compared to the control (Table 2). Participants were asked to report on the frequency of loose or hard stools / constipation over the past month. The scale was as follows: 0=none, 1=monthly, 2=weekly, 3=daily.

[0206] FIG. 29 shows that the probiotic cocktail significantly reduced the incidence of loose stools across the study series when compared to the placebo control.

[0207] Over the first 6 weeks of the study, the probiotic cocktail significantly reduced the incidence of loose stools as an overall effect when compared to the control as determined by repeated measures one-way ANOVA (Figure 29) (Treatment: F (2.615、13.08) =20.07, P<0.0001; time (F (5、15) = 2.803, p = 0.055). Sixteen of 25 study participants in the probiotic group reported no loose stools over the course of the study, compared with only eight in the placebo group, 10 in the B. clausii group, and 10 in the B. megaterium group.

[0208] FIG. 30 shows no effect of either treatment on the percentage of hard stools when compared to the placebo control.

[0209] There was no significant effect of any of the treatment groups on the percentage of hard stools over the course of this study (Figure 30) (F (1.829、9.146) =2.831, P=0.113; time (F (5、15)= 1.121, p=0.391).

[0210] Onset and duration of gastrointestinal symptoms [Table 19]

[0211] The Kruskal-Wallis test showed no significant differences in the number of days with gastrointestinal symptoms between the treatment groups.Compared to placebo, none of the test products containing probiotics showed a statistically significant difference in the number of days with gastrointestinal symptoms.

[0212] Onset and duration of urinary symptoms [Table 20]

[0213] The Kruskal-Wallis test did not show any significant differences in the number of days with urinary tract infection symptoms between treatment groups.Compared to placebo, none of the test products containing probiotics showed a statistically significant difference in the number of days with urinary tract infection symptoms.

[0214] Incidence and duration of upper respiratory tract infection [Table 21]

[0215] The Kruskal-Wallis test did not show any significant differences in the number of days with respiratory tract infection symptoms between the treatment groups.Compared to placebo, none of the test products containing probiotics showed a statistically significant difference in the number of days with symptoms.

[0216] Analysis of daily questionnaires Table 16 summarizes the responses to the mood questionnaire at baseline and end of study for the three treatment groups and placebo. Mean changes with 95% confidence intervals are shown. ANOVA omnibus test (p *The results of the one-sample T-test (p-value) and the one-sample T-test (p-value) are also shown. The test of normality for the changes in gut-brain axis scores shows 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 found. In general, the mean scores were not much higher at the end of the treatment period, including the placebo group (participants were less bothered by these symptoms). As a result, the results of the one-sample T-test show that in one third of the studies (out of 70 performed), a statistically significant change in the gut-brain axis questionnaire scores was observed. However, this can 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 changes were detected between the treatment groups, but borderline significance was observed for loss of energy and change in appetite. Participants in the Bacillus megaterium group experienced the greatest differences for these two items. Nevertheless, no statistically significant differences were observed for pairwise comparisons of the probiotic groups with placebo (Table 16 (bottom)). [Table 22] TIFF2024546089000025.tif153155

[0217] Cholesterol and triglyceride levels Blood samples were collected at the start of the study prior to any treatment and again at the end of the 45-day treatment period. There were no significant effects of treatment within groups or compared to baseline for high-density lipoprotein, low-density lipoprotein, total cholesterol and triglyceride concentrations (Table 17). [Table 23]

[0218] Blood cytokine levels [Table 24]

[0219] Blood samples were collected at the start of the study prior to any treatment and again at the end of the 45-day treatment period. There were no significant effects of treatment within groups or when compared to baseline for IL-8 or TNFα (Table 18).

[0220] Blood antioxidant levels [Table 25]

[0221] Blood samples were collected at the start of the study prior to any treatment and again at the end of the 45-day treatment period. There was no significant effect of treatment within groups on antioxidant levels, nor was there a significant effect of treatment compared to baseline (Table 10).

[0222] Metabolite levels [Table 26] TIFF2024546089000030.tif121153

[0223] Blood samples were collected at the start of the study prior to any treatment and again at the end of the 45-day treatment period. For the amino acids tested, there were no significant effects of treatment within groups, nor when compared to baseline (Table 20). [Table 27]

[0224] Blood samples were collected at the start of the study prior to any treatment and again at the end of the 45-day treatment period. For mineral levels, there was no significant effect of treatment within groups, nor was there a significant effect of treatment compared to baseline (Table 21).

[0225] Changes in the bacterial flora Samples from subjects collected before and after the treatment period were selected for comprehensive microbiome analysis. After removal of short and low quality reads, 202,413 sequences were retained, with an average of 2,736 sequences per sample and an average length of 440 nucleotides. Using the ESPRIT tree and after removal of OTUs containing less than 10 sequences, 1,077 and 1,618 OTUs were retained at the 95 and 98% similarity levels.

[0226] Figure 31 is a box plot showing the distribution of Chao1 values ​​in each experimental group on days 1 and 45. Dotted lines connect paired samples. Group distributions were compared using paired Wilcoxon tests. p-values ​​less than 0.05 should be considered statistically significant.

[0227] Figure 32 is a box plot showing the distribution of Chao1 values ​​in each experimental group on days 1 and 45. The distribution of each experimental group was compared to placebo using the Wilcoxon test. A p-value of less than 0.05 should be considered statistically significant.

[0228] Figure 33 illustrates PCoA clustering performed on a Bray-Curtis dissimilarity matrix. Each treatment is separated in a different tab, while the color and shape are associated with the time point. Samples from the two time points tend to cluster together for all treatments, and the data are not significantly different from each other at the baseline reading on day 1. Samples were not significantly different from each other as a result of treatment, either within or between groups. [Table 28]

[0229] Significant differences between treatment groups were only detected for days with thick nasal discharge (p *= 0.018), likely because only three participants in the probiotic cocktail group reported this symptom, whereas none in the other four treatment groups reported this symptom. However, further analyses comparing the number of days with nasal discharge-thickness between the probiotic cocktail and placebo groups (Mann-Whitney U test with Holm correction) did not show a significant difference, likely due to the small sample size.

[0230] [Table 29]

[0231] Kruskal-Wallis tests showed no significant differences in the number of days in the treatment groups with clinically significant infections. 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.

[0232] Nevertheless, compared to placebo, none of the test products containing probiotics showed statistically significant differences. [Table 30]

[0233] Significant differences between groups were detected in the percentage of loose stools during the total treatment period and during weeks 6 and 7 of the treatment period. However, further analysis (Mann-Whitney U test with Holm correction) did not show significant differences, likely due to small sample sizes. Participants in the probiotic cocktail group had the lowest percentage of loose stools per total stool. [Table 31]

[0234] 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.

[0235] 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.

[0236] The gastrointestinal health of participants at baseline between treatment groups did not differ between study arms, which was expected due to randomization.

[0237] 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.

[0238] 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.

[0239] In conclusion, the probiotic product was shown to be safe for use in adults and provided some useful data regarding the gut-brain axis and stool consistency.

[0240] Consideration The use of Bacillus probiotics in maintaining intestinal health has received much support in the past few years, driving their clinical application. Their beneficial effects have been associated with several properties, such as antimicrobial and immunomodulatory activity, regulation of cell proliferation and differentiation, cell-cell signaling, cell adhesion, signal transcription and transduction, production of vitamins, and intestinal protection against genotoxic agents.

[0241] This study was conducted to evaluate the effect of three probiotic treatments on general health and gastrointestinal symptoms in healthy adults. There were no safety or tolerability concerns or adverse events. In this small study cohort of healthy individuals without gastrointestinal problems, there was no negative impact on bowel regularity and stool consistency, and no negative impact on sadness, restlessness, energy, appetite, tension, stress, sleep, cardiovascular events, aches and pains, and dizziness. Indeed, the inventors report a reduction in the occurrence of loose stools throughout the intervention period due to administration of the probiotic cocktail.

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[0243] 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.

[0244] 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 ​​either above or below the stated value in a range of approximately ±2%; in other embodiments, values ​​may range values ​​either 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.

[0245] 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.

[0246] 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 megaterium 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 megaterium strain of claim 1, which shares at least 97% identity with SEQ ID NO:

3.

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

3.

6. A microbial composition comprising a Bacillus megaterium 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 megaterium 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 megaterium strain according to 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 in improving immune health, protection against oxidative stress, cleansing and detoxification, metabolic health and / or cardiovascular health.

13. Preventing or treating vaginal infections, urinary tract infections, gastrointestinal infections, and / or gastrointestinal disorders, or for immune health, protection against oxidative stress, cleansing and detoxification; 10. A composition for use in a method for improving metabolic 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. Preventing or treating vaginal infections, urinary tract infections, gastrointestinal infections, and / or gastrointestinal disorders, or for immune health, protection against oxidative stress, cleansing and detoxification; 10. The microbial composition of claim 6 for use in a method for improving metabolic and / or cardiovascular health, the method comprising 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 megaterium strain of any one of claims 1 to 5.

17. 6. A Bacillus megaterium 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 the Bacillus megaterium 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 and / or cardiovascular health.

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