Bacillus clausii strains, compositions and methods of use thereof

Abstract

The present invention provides a strain of Bacillus clausii comprising a purified population of microorganisms comprising one or more bacteria having a gyrB having at least 97% identity to SEQ ID NO: 1; and / or the population of microorganisms comprises one or more bacteria having a 16S rRNA having at least 97% identity to SEQ ID NO: 2. Optionally, the Bacillus clausii strain has at least 97% identity to SEQ ID NO: 3. Compositions and methods using Bacillus clausii are also part of the invention.

Description

Technical Field

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

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

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

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

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

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

[0007] Description of Related Technology

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

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

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

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

[0012] Although long considered to be soil microorganisms, species of the genus Bacillus have been used for more than 50 years in the form of fermentation products or spore-based supplements (Cutting et al., 2011). Bacilli are ubiquitous in nature and are continuously introduced into the gastrointestinal tract and respiratory tract of healthy humans through food, water, and air (Benno & Mitsuoka, 1986). They are isolated from the intestine and can reach up to 107 CFU / g, and are therefore considered to be one of the main components of the normal intestinal microbiota (Lakshmi et al., 2017). Recently, strains of Bacillus clausii have been isolated to provide more specific functions and their safety has been evaluated. Bacillus clausii has been previously used in patients with diarrhea (Sudha et al., 2013, Horosheva et al., 2014) and children with recurrent respiratory tract infections (Marseglia et al., 2007) with no adverse events reported. Although the countries and strains are not specified, Bacillus clausii has been commercialized in 55 countries around the world (Nista et al., 2004; Gabrielli et al., 2009). A literature review of Bacillus clausii showed no adverse events associated with probiotics, and the presence of the bacteria in different countries around the world supplements the narrative of its safety for human consumption.

[0013] In general, the probiotic-competent Bacillus strain should be able to adhere to other suitable cells, such as the cell line Caco-2 cells. In addition, it is also desirable that the probiotic-competent Bacillus strain show inhibitory activity against other bacterial species in vitro, produce acid and / or produce hydrogen peroxide after growth in liquid culture. Summary of the invention

[0014] The object of the present invention is to provide strains and compositions as described in the present application, such as pharmaceutical preparations or absorption products of suitable probiotic strains (such as Bacillus strains) having desired properties. In an embodiment, the present invention relates to Bacillus clausii strain CSI08 alone or with other Bacillus strains such as Bacillus megaterium strain MIT411 (disclosed in corresponding PCT application PCT / US2022 / xxxxx, and claiming priority to Irish patent application No. 2021 / 0211, the contents of which are incorporated herein in their entirety) and Bacillus coagulans strain CGI314 (disclosed in corresponding PCT application PCT / US2022 / xxxxx, and claiming priority to Irish patent application No. 2021 / 0210, the contents of which are incorporated herein in their entirety). In embodiments, these strains have similar or substantially identical advantageous properties, such as the ability to colonize by adhering to mucous membranes / surfaces, and thus they are suitable for treating or preventing infections or diseases of the vagina, urinary tract, gastrointestinal tract, sinuses, pharynx, esophagus, oral cavity, and / or other areas of the body having mucous membranes, and for treating or preventing infections or diseases of the skin and / or other areas of the body having epithelium; immune health, protection from oxidative stress, cleansing and detoxification, metabolic health, and cardiovascular health, such as providing antimicrobial activity, anti-inflammatory activity, inhibition of pro-inflammatory responses, activation and and / or stimulate an immune response, etc., such as providing immune protection by stimulating macrophages, aiding digestion and / or fermentation, for example in the intestine, producing branched-chain amino acids, essential amino acids and B vitamins, maintaining a healthy intestine and / or skin, protecting mucosa and other epithelial tissues from toxic agents, reducing the incidence of loose stools, improving the gut-brain axis, and treating and / or preventing dysbiosis and its effects, such as periodontal disease, inflammatory bowel disease, chronic fatigue syndrome, metabolic disorders, heart disease, respiratory tract infections, urinary tract infections, GI infections and diarrhea; and restoring normal and / or healthy flora. In an embodiment, the present invention allows for the use of Bacillus clausii strain CSI08 and compositions for fecal transplantation.

[0015] Gastrointestinal disorders include, but are not limited to, treating gastrointestinal irregularity in a subject wherein the subject has at least one 24 hour episode per month of bowel movements measured as 1 or 2 on the Bristol stool scale (i.e., treating constipation); or wherein the subject has at least one 24 hour episode per month of bowel movements measured as 6 to 7 on the Bristol stool scale (tending toward diarrhea), wherein the subject's frequency of 24 hour episodes per month of bowel movements measured as 1 or 2 (or 6 or 7) on the Bristol stool scale is reduced.

[0016] Also included are methods of restoring gastrointestinal regularity in an individual wherein the individual has at least one 24 hour episode per month of bowel movements measured as 1 or 2; or 6 to 7 on the Bristol stool scale, wherein the individual has an increased frequency of 24 hour cycles of bowel movements measured as 3 to 5 on the Bristol stool scale.

[0017] The present invention also includes maintaining a healthy intestinal microflora with one or more compositions containing bacillus. The one or more compositions containing bacillus can be used as a probiotic supplement to the gastrointestinal microflora and can compete with or otherwise inhibit pathogenic bacteria in the intestine, such as Escherichia coli, Listeria monocytogenes, and Salmonella species.

[0018] Another object of the present invention is to provide a pharmaceutical preparation having an increased colonization ability by adhering to the mucosa using a mucoadhesive excipient.

[0019] Yet another object of the present invention is to provide vaginal preparations having an enhanced ability to inhibit the growth of Candida albicans and Gram-negative pathogens.

[0020] Another object of the present invention is to provide compositions, such as dairy products, nutritional products and functional foods, comprising Bacillus clausii strain CSI08 alone or in combination with other Bacillus strains such as Bacillus megaterium strain MIT411 and / or Bacillus coagulans strain CGI314, in embodiments, the compositions have substantially the same properties, in embodiments, have the ability to colonize on mucous membranes, and are therefore suitable for treating or preventing vaginal infections, urinary tract infections and gastrointestinal diseases. The compositions of the present invention can be administered for 1 dose, 1 day, 1 day to 1 week, 1 day to 1 month, 1 month to 45 days, 45 days to 2 months, 3 months, 6 months, 1 year or longer, including any time period determined and / or falling within these ranges.

[0021] Attached photos

[0022] In the attached picture:

[0023] Figure 1 Genome analysis of Bacillus clausii CSI08 is shown.

[0024] Figure 2 Phylogenetic tree showing Bacillus species arranged by clade (16S).

[0025] Figure 3 Phylogenetic tree showing Bacillus species arranged by clade (gyrB).

[0026] Figure 4 The stability of MuniSpore in PBS during pasteurization is shown.

[0027] Figure 5 Shown is the pH viability of B. clausii CSI08 spores at 0-, 3-, and 24-hour time points.

[0028] Figure 6 Shown is the viability of Bacillus clausii CSI08 spores in bile salts at 0-, 3-, and 24-hour time points.

[0029] Figure 7 Shown is the survival of Bacillus clausii CSI08 spores and Lactobacillus rhamnosus (L. rhamnosus) GG under in vitro simulated gastric and small intestinal digestion conditions.

[0030] Figure 8 and 9 The antimicrobial activity of Munispore (Bacillus clausii CSI08) in liquid culture is shown.

[0031] Fig.10 The total antioxidant capacity of Bacillus clausii CSI08 and Lactobacillus rhamnosus GG is shown.

[0032] Fig.11 The cytoprotective effect of vegetative B. clausii CSI08 on epithelia exposed to H2O2 is shown.

[0033] Fig.12 Shown with 10 8 and 10 9 Survival of C. elegans N2 fed with CFU / ml Bacillus clausii CSI08 and then induced to oxidative stress by H2O2. NGM-control fed nematodes; Vitamin C-positive control.

[0034] Fig.13 Shown is the effect of propagating somatic cells Bacillus clausii CSI08 on HT-29 cell viability. Bacillus clausii CSI08 compared to untreated cells (medium).

[0035] Fig.14 Shown is the adhesion of vegetative somatic cells and spores of Bacillus clausii CSI08 to HT-29-MTX. Comparison of Bacillus clausii CSI08 and L. fermentum.

[0036] Fig.15A and 15BShown is the modulation of LPS-induced pro-inflammatory responses by Bacillus clausii CSI08 in the HT-29 cell line and the gene expression pattern after co-incubation of HT-29 cells with Bacillus clausii CSI08 and its CFS under unstimulated conditions (LPS-).

[0037] Fig.16 Shown is the regulation of LPS-induced IL8 gene expression by live and heat-killed Bacillus clausii CSI08.

[0038] Fig.17 Shown are NF-κB levels in nuclear fractions of control HT-29s, cells exposed to LPS, and cells treated with B. clausii CSI08 prior to the addition of LPS.

[0039] Fig.18A and 18B Modulation of PolyI·C-triggered pro-inflammatory response by Bacillus clausii CSI08 in HT-29 cell line is shown. qPCR analysis of IL-8, TNF-α, IL-17C and CXCL10 gene expression was performed after 4 hours of exposure to PolyI·C in HT-29 cells pre-incubated with Bacillus clausii CSI08 or its cell-free supernatant (CFS).

[0040] Fig.19 The immunostimulatory effect of CSI08 is shown. Cytokine levels in the cell culture supernatant of U937-derived macrophages challenged with propagated somatic B. clausii CSI08 or LPS for 5 hours.

[0041] Fig. 20 Shown are the lifespan of C. elegans wild-type N2 strain under control conditions (NGM) and the lifespan of C. elegans wild-type N2 fed with Bacillus clausii CSI08.

[0042] Fig.21 Shown are the lifespan of C. elegans daf-16 mutant worms under control conditions (NGM) and the lifespan of C. elegans daf-16 mutants fed with Bacillus clausii CSI08.

[0043] Fig. 22 It was shown that B. clausii CSI08 was slightly caseinolytic using the streak method, and that B. megaterium and B. clausii CSI08 had caseinolytic activity.

[0044] Fig.23 Shown by Proteolytic activity measured by the kit assay.

[0045] Fig.24 Shown are the concentrations of essential amino acids and vitamins in overnight cultures of Bacillus clausii CSI08 determined by RP-HPLC-FLD and RP-HPLC-MS.

[0046] Fig.25 A graphical flow chart showing the study design.

[0047] Fig.26 Administration of the probiotic cocktail during the study was shown to significantly reduce the incidence of loose stools during the study compared to the placebo control.

[0048] Fig. 27 It was shown that any of the treatments administered during the study had no effect on the percentage of hard stools compared to the placebo control.

[0049] Fig.28 is a box plot showing the distribution of Chao1 values ​​for each experimental group on study day 1 and day 45. Dashed lines connect paired samples. Paired Wilcoxon test was used to compare the distributions of the groups.

[0050] Fig.29 is a box plot showing the distribution of Chao1 values ​​for each experimental group on study day 1 and day 45. The Wilcoxon test was used to compare the distribution of each experimental group with placebo.

[0051] Fig.30 Shows PCoA clustering performed on the Bray-Curtis dissimilarity matrix. DETAILED DESCRIPTION

[0052] Genotype identification

[0053] The applicant collaborated with Cornell University (Ithaca NY, USA) to perform genome sequencing and characterization.

[0054] WGS DNA composition

[0055] Whole genome sequencing (WGS) was performed by Cornell University.

[0056] The whole genome sequence of the B. clausii isolate was obtained, assembled, and annotated at Cornell University. Bioinformatics analysis was performed at Cornell University and Deerland Probiotics and Enzymes (Kennesaw GA, USA). The DNA nucleotide content, base pair length, and marker sequence of the B. clausii CSI08 genome are shown below (Table 1). The genome size (4.2 Mbp) and GC% (44.6%) of B. clausii CSI08 are consistent with those of previously sequenced B. clausii strains (4.3 Mbp and 44.6%, respectively) ( https: / / www.ncbi.nlm.nih.gov / genome / ?term=txid66692[Organism:noexp ]).

[0057] Table 1

[0058] CSI08 Whole Genome Sequencing Indicators

[0059]

[0060]

[0061] gyrB gene

[0062] The applicant has identified the polymorphism of the gyrB gene. The gyrB gene encodes DNA gyrase subunit B. DNA gyrase negatively supercoils closed circular double-stranded DNA in an ATP-dependent manner to maintain the underwound state of chromosomes.

[0063] Gene sequencing analysis using gyrB gene polymorphism, a well-established method for species identification of prokaryotes (Bavykin et al., 2004; Wang et al., 2007), showed that B. clausii CSI08 was most closely related to the B. clausii group (>99%) (Table 2).

[0064] Table 2

[0065] gyrB gene distance matrix

[0066] Bacillus clausii subsp. B106 UBBC-07 CSI08 99.983 98.952

[0067] Representative genomes were previously reviewed and curated by NCBI and coordinated with the UniProt Consortium (NCBI, 2016; UniProt, 2016). The R package SequinR combined with UniProt Consortium analysis was used to compare the base pair length and GC content of the whole genome sequence (WGS) and GyrB sequence of B. clausii CSI08 and two reference sequences (Table 3). Independent whole genome sequence (WGS) analysis performed by DiLan Probiotics and Enzymes Ltd. identified CSI08 as having the most similar homology to B. clausii B106.

[0068] Table 3

[0069] Whole genome sequence comparison

[0070]

[0071] 16S rRNA

[0072] Whole genome sequencing and 16S rRNA analysis of the presently claimed strain (Bacillus clausii CSI08) compared to the two reference strains showed an average nucleotide identity (ANI) score of >99% for 16S rRNA when compared to Bacillus clausii strain B106 (the genome sequence of Bacillus clausii B106 has been deposited in GenBank under accession number NFZO00000000). The genome size (4.2 MBP) and GC content (44.6%) of CSI08 are comparable to those of the two reference strains.

[0073] Further deposits and accession numbers

[0074] The complete genome sequence of Bacillus clausii UBBC07 has been deposited in DDBJ / ENA / GenBank under accession number LATY00000000.

[0075] The genome sequence data of Bacillus clausii strain CSI08 (Munispore) was deposited into the NCBI GenBank database, and the genome sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The genome is publicly available, the strain's GenBank accession number is JABBNL000000000.1, and can be obtained through the following link: Alkalihalobacillus clausii strain CSI08, whole genome shotgun sequenci-Nucleotide-NCBI (nih.gov) .

[0076] The genome sequence data of Bacillus megaterium strain MIT411 (Renuspore) was deposited into the NCBI GenBank database, and the genome sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The genome is publicly available, the strain's GenBank accession number is JABBNK000000000.1, and can be obtained through the following link: Priestia megaterium strain MIT411, whole genome shotgun sequencing pro-Nucleotide-NCBI (nih.gov).

[0077] The genome sequence data of Bacillus coagulans strain CGI314 (Fortispore) was deposited into the NCBI GenBank database and the genome sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The genome is publicly available, the strain's GenBank accession number is JABBFU000000000.1, and can be obtained through the following link: https: / / www.ncbi.nlm.nih.gov / nuccore / JABBFU000000000.1.

[0078] Phylogenetic Positioning DiLan Probiotics and Enzymes Ltd.

[0079] Genome-to-genome distance calculation (GGDC) is a digital gold standard that is as reliable as DNA-DNA hybridization (DDH) (Auch et al., 2010). GGDC has a stronger discriminatory power in subspecies delineation and was subsequently used for confirmation of multiple alignments and phylogenetic analyses. GGDC generated three computationally based models that further confirmed that B. clausii CSI08 is a close relative of B. clausii B106 (Table 3).

[0080] Although the conserved 16S rRNA sequence is a well-established method for comparing and studying bacterial phylogeny, the high percentage of sequence similarity between closely related species limits its usefulness (Wang et al., 2007). The high rate of 16S rRNA sequence similarity among closely related bacterial species is due to the slower rate of molecular evolution. Past studies (Bavykin et al., 2004; Wang et al., 2007) support the validity of using gyrB sequences as taxonomic biomarkers because of their base substitution rates and their significant and reliable correlation with DNA-DNA hybridization analysis (Dauga et al., 2002; Kasai et al., 1998; Wang et al., 2007). gyrB encodes DNA gyrase B and a type II topoisomerase that plays an important role in DNA replication. The gyrase B subunit is encoded by the gyrB gene.

[0081] Phylogenetic analysis using the neighbor-joining (NJ) method (Saitou & Nei, 1987) placed Bacillus clausii CSI08 and Bacillus clausii B106 in the same evolutionary branch ( Figure 2 ). This confirms all previous genome identity determinations. B. clausii B106 has been assigned to the B. clausii group.

[0082] definition

[0083] "Excipient" refers to any inactive ingredient added to form part of the final formulation.

[0084] "Probiotics" refers to live microbial supplements that have a beneficial effect on a subject by acting in the intestinal tract, urinary tract, vagina, and / or other areas of the subject's body.

[0085] "Prebiotics" are used herein as substrates which have a beneficial effect on probiotics and thus on a subject taking (eg administering) the probiotics. Suitable prebiotics may be selected from inulin, oligosaccharides and / or vitamins.

[0086] As used herein, "subject" includes humans suffering from any clinical condition associated with microbial imbalance, as well as humans who use bacterial preparations prophylactically for health or any other purpose, including, for example, benefiting from the administration of the Bacillus clausii strain of the present invention (e.g., CSI08). Optionally, the subject is a human, a patient, and / or a mammal.

[0087] A “synbiotic product” is a combination of probiotics and prebiotics that work synergistically to have a beneficial effect on patients.

[0088] "Hardy growth" means that the bacteria show very good growth.

[0089] The abbreviation "CFU" means colony forming units.

[0090] As will become apparent during the course of the following detailed description, the present invention relates to probiotic Bacillus strains capable of regenerating the microbiota in a subject.

[0091] According to a first aspect, the present invention includes Bacillus clausii strain CSI08 alone or in combination with other probiotic strains having substantially the same properties. Such other probiotic bacillus strains include, but are not limited to, Bacillus coagulans strains and Bacillus megaterium strains. Such other bacillus strains also include Bacillus coagulans strains and Bacillus megaterium strains, each of which is filed on the same day under its own title, the contents of which are incorporated herein in their entirety.

[0092] As described in the appended claims, SEQ ID NO: 1 comprises gyrB of Bacillus clausii CSI08.

[0093] As described in the appended claims, SEQ ID NO: 2 comprises the 16S rRNA of Bacillus clausii CSI08.

[0094] As described in the appended claims, SEQ ID NO: 3 comprises the assembled complete genome sequence of Bacillus clausii CSI08.

[0095] The Bacillus clausii CSI08 strain claimed herein has at least 97% identity to SEQ ID NO: 1 and / or 2; or has at least 97% identity to SEQ ID NO: 3, and has the following properties:

[0096] In order to determine the genus and species of the strains disclosed herein, the whole genome was sequenced. The number and composition of the strains were identified and determined.

[0097] The strain was shown to have little or no antibiotic resistance and presented no safety concerns.

[0098] The strain was found to be stable to acid and bile and exhibited heat resistance. The strain produced natural antimicrobial substances in the form of bacteriocins.

[0099] According to a second aspect, the Bacillus strains of the invention are suitable for medical use in preventing or treating vaginal infections, urinary tract infections and gastrointestinal diseases, as well as improving immune health, protection from oxidative stress, cleansing and detoxification, metabolic health and cardiovascular health.

[0100] In another preferred embodiment, a composition such as a pharmaceutical composition is provided, which includes Bacillus clausii CSI08 alone or in combination with other probiotic strains having similar and / or substantially the same properties, and a pharmaceutically acceptable carrier and / or diluent. Such other probiotic bacillus strains include, but are not limited to, Bacillus coagulans strains and Bacillus megaterium strains. The bacterial strains are formulated into a composition, such as a pharmaceutical preparation, by methods known to those skilled in the art to facilitate administration of the probiotic strains.

[0101] Bacillus coagulans has been shown to alleviate symptoms of irritable bowel syndrome (Sudha et al., 2018), improve muscle integrin and cytokine responses (Gepner et al., 2017; Jager et al., 2018), modulate the gut microbiome and immune response (Kimmel et al., 2010), reduce functional intestinal gas symptoms (Kalman et al., 2009), reduce the occurrence and duration of diarrhea (Dolin et al., 2009), improve symptoms of functional abdominal pain and bloating (Hun et al., 2009), protect against acetaminophen-induced acute liver injury (Neag et al., 2020), enhance butyrogenesis (Sasaki et al., 2020), reduce the severity of bacterial vaginosis (Sudha et al., 2012), and reduce cholesterol (Sudha et al., 2012), all in vivo. Bacillus coagulans has also been shown to induce immune responses and anti-inflammatory effects (Jensen et al., 2017), improve plant protein digestion (Keller et al., 2017), adhere to Caco-2 cells (Sharma & Kanwar, 2017), improve the colonic microenvironment in patients with ulcerative colitis (Sasaki et al., 2020), reduce adhesion, cytotoxicity, and induction of cell apoptosis caused by S. typhimurium in HT-29 cells (Kawarizadeh et al., 2019), hydrolyze lactose from whey protein (Liu et al., 2019), and enhance T-cell responses (Baron, 2009), all in vitro.

[0102] Bacillus clausii has been shown to be effective in preventing recurrent respiratory tract infections (Marseglia et al., 2007) and reducing the duration and severity of diarrhea (Sudha et al., 2019), all in vivo. Bacillus clausii has also been shown to produce protein hydrolysates with antimicrobial and antioxidant properties (Rochinmedina et al., 2017), protect against acetaminophen-induced acute liver injury (Neag et al., 2020), and inhibit the cytotoxic effects induced by Clostridium difficile and Bacillus cereus toxins (Ripert et al., 2016), all in vitro.

[0103] Bacillus megaterium has been shown to protect against oxidative stress both in vitro and in vivo (Mazzoli et al., 2019). Bacillus megaterium has also been shown to be able to adapt and survive acidic stress conditions and to chelate heavy metals, all in vitro (Ferreira et al., 2019).

[0104] In an embodiment, the probiotics used in the present invention are 10 6 -10 13 The bacterial concentration of CFU (colony forming units) is used, for example, as a daily dose, including any amount or range contained in the range. In an embodiment, the amount of bacteria used is 10 7 -10 12 CFU, or 10 8 -10 11 CFU, or 10 9 -10 10 CFU, or for example about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , about 10 12 and / or about 10 13 CFU, and any amount or range including or between said amounts. In an embodiment, the composition of the present invention comprises about 10 6 - Approx. 10 13 CFU, such as about 10 9 Bacillus clausii CSI08, basically composed of about 10 6 - Approx. 10 13 CFU, such as about 10 9 Bacillus clausii CSI08, consisting of about 10 6 - Approx. 10 13 CFU, such as about 10 9 Bacillus clausii CSI08 and / or about 10 6 - Approx. 10 13 CFU, such as about 10 9 In one embodiment, the composition of the present invention comprises Bacillus clausii CSI08 (e.g., about 10 9 In one embodiment, the composition of the present invention is orally administered in the form of a capsule. In one embodiment, Bacillus clausii CSI08 is in spore form, or is not in spore form.

[0105] In certain embodiments, the composition comprising Bacillus clausii CSI08 may include one or more dry carriers selected from the group consisting of trehalose, maltodextrin, rice flour, microcrystalline cellulose, magnesium stearate, inositol, oligofructose, oligogalactose, dextrose, dry dairy products, etc. In certain embodiments, the dry carrier may be added to the composition comprising Bacillus clausii CSI08 at a weight percentage of about 1% to about 95% by weight of the composition.

[0106] In certain embodiments, the composition comprising Bacillus clausii CSI08 may include one or more liquid or gel-based carriers selected from the group consisting of water and physiological salt solutions, urea, alcohols and their derivatives (e.g., methanol, ethanol, propanol, butanol), glycols (e.g., ethylene glycol, propylene glycol), etc.; natural or synthetic flavoring agents and food-grade colorants, all compatible with organisms; thickeners selected from the group consisting of corn starch, guar gum, xanthan gum, etc.; one or more spore germination inhibitors selected from the group consisting of high salt carriers, methyl parabens, guar gum, polysorbates, preservatives, etc. In certain embodiments, one or more liquid or gel-based carriers may be added to the composition comprising Bacillus clausii CSI08 at a weight / volume percentage of about 0.6% to about 95% weight / volume of the composition. In certain embodiments, one or more natural or synthetic flavoring agents may be added to the composition comprising Bacillus clausii CSI08 at a weight / volume percentage of about 3.0% to about 10.0% weight / volume of the composition. In certain embodiments, one or more colorants may be added to a composition including Bacillus clausii CSI08 at a weight / volume percentage of about 1.0% to about 10.0% weight / volume of the composition. In certain embodiments, one or more thickeners may be added to a composition including Bacillus clausii CSI08 at a weight / volume percentage of about 2% weight / volume of the composition. In certain embodiments, one or more spore germination inhibitors may be added to a composition including Bacillus clausii CSI08 at a weight / volume percentage of about 1% weight / volume of the composition.

[0107] Delivery System

[0108] Suitable dosage forms include tablets, capsules, solutions, suspensions, powders, gels and candies. Sublingual delivery systems include, but are not limited to, sublingual and supralingual dissolvable tablets, drops and beverages. Edible films, hydrophilic polymers, orally dissolvable films or orally dissolvable strips can be used. Other useful delivery systems include oral or nasal sprays or inhalers, etc. Suitable dosage forms include tablets, capsules, solutions, suspensions, powders, gels and candies. Sublingual delivery systems include, but are not limited to, sublingual and supralingual dissolvable tablets, drops and beverages. Edible films, hydrophilic polymers, orally dissolvable films or orally dissolvable strips can be used. Other useful delivery systems include oral or nasal sprays or inhalers, etc.

[0109] For oral administration, probiotics can be further combined with one or more solid inactive ingredients for the preparation of tablets, capsules, pills, powders, granules or other suitable dosage forms. For example, the active agent can be combined with at least one excipient selected from the group consisting of fillers, binders, humectants, disintegrants, dissolution retardants, absorption promoters, wetting agents, absorbents and lubricants. Other useful excipients include but are not limited to magnesium stearate, calcium stearate, mannitol, xylitol, sweeteners, starch, carboxymethyl cellulose, microcrystalline cellulose, silica, gelatin, silicon dioxide, etc.

[0110] In certain embodiments, the components of the composition administered according to the methods of the present disclosure, together with one or more conventional adjuvants, carriers or diluents, may therefore be placed in the form of pharmaceutical compositions and unit dosage forms thereof. These forms include: solid, particularly tablets, filled capsules, powders and pill forms; liquid, particularly aqueous or non-aqueous solutions, suspensions, emulsions, elixirs; and capsules filled with the composition; all forms for oral administration, suppositories for rectal administration, and sterile injection solutions for parenteral administration. Such pharmaceutical compositions and unit dosage forms thereof may include conventional ingredients in conventional proportions, with or without additional active compounds or ingredients, and such unit dosage forms may include any suitable effective amount of active ingredients commensurate with the intended daily dosage range used.

[0111] The components of the compositions administered according to the methods of the present disclosure can be administered in a variety of oral and parenteral dosage forms. It will be apparent to those skilled in the art that, in certain embodiments, the following dosage forms may include a compound of the present disclosure or a pharmaceutically acceptable salt of a compound of the present disclosure as an active component.

[0112] For the preparation of pharmaceutical compositions for administration according to the methods of the present disclosure, pharmaceutically acceptable carriers may be solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier may be one or more substances which may also be used as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials.

[0113] In powders, the carrier is a finely divided solid which is mixed with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.

[0114] In certain embodiments, powders and tablets administered according to the methods of the present disclosure may preferably contain from 5 or 10 percent to about 70 percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, and the like. The term "preparation" means a preparation comprising an active compound and an encapsulating material as a carrier to provide a capsule in which the active component is present, and the active component (with or without additional carriers) is surrounded by the carrier, thereby binding to the carrier. Similarly, cachets and lozenges are also included. Tablets, powders, capsules, pills, cachets, and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.

[0115] Liquid preparations include but are not limited to solutions, suspensions and emulsions, such as water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in polyethylene glycol aqueous solutions. In certain embodiments, the compound used according to the method of the present disclosure can be formulated for parenteral administration (for example, by injection, for example, bolus injection or continuous infusion), and can be present in unit doses, for use in ampoule, prefilled syringe, small volume infusion or multiple dose containers with added preservatives. The composition can take the form of suspensions, solutions or emulsions in oily or aqueous carriers, and can contain formulations such as suspending agents, stabilizers and / or dispersants. Alternatively, active ingredient can be in powder form, obtained by aseptic separation of sterile solids or by freeze drying from solution, for example, prepared with suitable carriers (for example, sterile, pyrogen-free water) before use.

[0116] Aqueous solutions suitable for oral administration can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavoring agents, stabilizers and thickeners as desired. Aqueous suspensions suitable for oral use can be prepared by dispersing the finely divided active ingredient in water containing a viscous substance, such as a natural or synthetic gum, resin, methylcellulose, sodium carboxymethylcellulose or other well-known suspending agents.

[0117] Compositions for topical administration in the mouth include, but are not limited to, lozenges comprising the active agent in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0118] The solution or suspension is applied directly to the nasal cavity by conventional methods, such as with a dropper, pipette or sprayer. The composition can be provided in a single dose or multiple dose form. In compositions (including intranasal compositions) for administration to the respiratory tract, the compound generally has a small particle size, such as on the order of about 5 microns or less. Such particle size can be obtained by methods known in the art, such as by micronization.

[0119] The pharmaceutical formulation is preferably in unit dosage form. In this form, the formulation is subdivided into unit doses containing appropriate quantities of the active ingredient. The unit dosage form can be a packaged formulation, the package containing discrete quantities of the formulation, such as packaged tablets, capsules, and powders in vials or ampoules. Furthermore, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself; or it can be an appropriate number of any of these in packaged form.

[0120] Tablets, capsules and lozenges for oral administration and liquids for oral administration are preferred compositions. Solutions or suspensions for nasal or respiratory tract administration are preferred compositions. Transdermal patches for topical administration to the epidermis are preferred.

[0121] Further details on formulation and administration techniques may be found in the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA).

[0122] In certain embodiments, the composition of the present invention including the composition used according to the method of the present disclosure may also include one or more excipients, most preferably one or more nutrition or pharmaceutical excipients. The composition containing one or more excipients and incorporating one or more probiotics can be prepared by methods known in the art. Optionally, the composition may include one or more adjuvants, excipients, carriers, buffers, diluents and / or other conventional pharmaceutical adjuvants. For example, probiotics can be formulated into tablets, capsules, powders, suspensions, solutions for oral administration, solutions for parenteral administration (including intravenous, intradermal, intramuscular and subcutaneous administration), and solutions for application to patches for transdermal administration.

[0123] In certain embodiments, the nutrient composition including the nutrient composition used according to the method of the present disclosure may include a pharmaceutically acceptable carrier and may be co-administered with a pharmaceutically acceptable carrier. In certain embodiments, the active ingredient in this type of preparation may include about 1% by weight to about 99% by weight. In other embodiments, the active ingredient in this type of preparation may include about 0.1% by weight to about 99.9% by weight. "Pharmaceutically acceptable carrier" refers to any carrier, diluent or excipient that is compatible with the other ingredients of the preparation and harmless to the user. Useful excipients include but are not limited to microcrystalline cellulose, magnesium stearate, calcium stearate, any acceptable sugar (e.g., mannitol, xylitol), etc., and for cosmetic purposes, water-based or oil-based or mixtures thereof may be used, including such as emulsions.

[0124] Route of administration

[0125] The strain Bacillus clausii CSI08 or a composition comprising the strain of the invention can be administered by any route, including but not limited to oral, sublingual, oral, ocular, pulmonary, rectal, vaginal, urethral, ​​ureteral and parenteral administration, or as an oral or nasal spray (e.g., inhalation of atomized vapor, droplets or solid particles). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, intravaginal, intravesical (e.g., to the bladder), intradermal, transdermal, topical or subcutaneous administration. It is also contemplated within the scope of the present invention that the pharmaceutical composition is instilled into the patient in the form of a controlled agent, and systemic or local release of the drug occurs at a later time. For example, the drug can be located in a reservoir for controlled release into the circulation, or for release to a local location.

[0126] The pharmaceutical composition of the present invention can be suitable for and formulated for any of the above-mentioned routes, including, for example, oral, rectal, bronchial, nasal, pulmonary, topical (including buccal and sublingual), transdermal, vaginal, urethral, ​​ureteral or parenteral (including cutaneous, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intracerebral, intraocular injection or infusion) administration, or a form suitable for administration by inhalation or insufflation, including powder and liquid aerosol administration, or by sustained release system administration. Suitable examples of sustained release systems include semipermeable matrices of solid hydrophobic polymers containing the compounds of the present invention, which may be in the form of shaped articles, such as films or microcapsules.

[0127] The above embodiments may be further understood in conjunction with the following examples. In addition, the following non-limiting examples are provided to illustrate the present invention. However, it will be appreciated by those skilled in the art that, for any given embodiment of the present invention, it may be necessary to change the process, such as changing the order or step.

[0128] Example

[0129] Example 1

[0130] Study on the characteristics of Bacillus clausii CSI08 strain

[0131] Temperature stability:

[0132] The spores of Bacillus clausii CSI08 were stable in PBS (pH 7.72) at 45°C and at 75°C for 30 seconds to 3 minutes, and the number of spores decreased after treatment at 90°C for 3 minutes.

[0133] Figure 4 The stability of MuniSpore in PBS during pasteurization is shown. Data represent mean ± SEM. Spore counts compared to 0 min. *p < 0.0001.

[0134] Bacillus clausii CSI08 spores are stable during pasteurization and in other production methods for food and beverages.

[0135] The strain showed bile stability, and the strain showed acid stability. Survival of Bacillus clausii CSI08 spores in nutrient broth medium under acidic and bile salt conditions for 24 hours. After 3 hours of contact with concentrated nutrient broth under acidic conditions or bile salt conditions, the total spore counts of Bacillus clausii CSI08 did not show a significant decrease in viability or concentration. Under the most extreme conditions (pH 1.2 and 0.45% bile salt concentration), a significant decrease in counts was observed after 24 hours. Both pH and bile salts at these concentrations and exposures greatly exceed the conditions of the stomach and intestines.

[0136] Figure 5 Shown is the pH viability of B. clausii CSI08 spores at 0-, 3-, and 24-hour time points.

[0137] Figure 6 Shown is the viability of Bacillus clausii CSI08 spores in bile salts at 0-, 3-, and 24-hour time points.

[0138] Bacillus clausii CSI08 spores are insensitive to acidic conditions above 1.2 for 24 h and are resistant to bile salt concentrations up to 0.30% for 24 h. Therefore, Bacillus clausii CSI08 spores can survive low pH foods and beverages (e.g., juices) as well as gastric and intestinal conditions.

[0139] The strain showed heat resistance. Spores of Bacillus clausii CSI08 were stable in PBS (pH 7.72) at 45°C and at 75°C for 30 seconds to 3 minutes, and the number of spores decreased after treatment at 90°C for 3 minutes.

[0140] Bacillus clausii CSI08 spores are stable during pasteurization and in other production methods of food and beverages.

[0141] The survival of Bacillus clausii CSI08 spores during simulated in vitro digestion (gastric phase: 0.3% pepsin, pH=3, for 2 h; small intestinal phase: 0.1% pancreatin, 0.3% bile salts, pH7.5, for 2 h) was studied. No reduction in the spore count of Bacillus clausii CSI08 was detected after exposure to simplified gastric and small intestinal conditions. The number of Lactobacillus rhamnosus decreased during the small intestinal phase.

[0142] The results show that B. clausii CSI08 spores are able to effectively survive during passage through the upper gastrointestinal tract.

[0143] Figure 7 Shown is the survival of Bacillus clausii CSI08 spores and Lactobacillus rhamnosus GG under simulated gastric and small intestinal digestion conditions in vitro. Data represent mean ± SEM. *P < 0.0001 for survival rate compared to 0 h.

[0144] This strain produces natural antimicrobial substances in the form of bacteriocins.

[0145] The propagule of Bacillus clausii CSI08 is active against common enteric pathogens Escherichia coli and Salmonella spp., and has the potential to protect and restore the intestinal microbiota.

[0146] Propagative strain B. clausii CSI08 was also shown to have antimicrobial activity against the well-known skin and urinary tract pathogen Pseudomonas aeruginosa in liquid culture.

[0147] Table 4

[0148] Antimicrobial activity

[0149]

[0150] Table 4 Bacillus clausii CSI08 spores show antimicrobial activity against common pathogens. Antimicrobial activity was detected (+), and no antimicrobial activity was observed (-).

[0151] The vegetative form of Bacillus clausii CSI08 has antimicrobial activity against Escherichia coli, Salmonella, Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa in liquid culture medium.

[0152] Figure 8 and 9Shown is the antimicrobial activity of Munispore (Bacillus clausii CSI08) in liquid culture. Values ​​represent the average concentration lof 10 CFU / ml±standard deviation. *p<0.05, ****p<0.0001.

[0153] Therefore, the vegetative strain B. clausii CSI08 has the potential to crowd out bacterial pathogens and maintain a healthy gut and skin microbiome.

[0154] Antioxidant activity: The total antioxidant activity of Lactobacillus rhamnosus GG (DI_AS_030) and vegetative Bacillus clausii CSI08 was compared.

[0155] Compared with Lactobacillus rhamnosus GG, Munispore showed higher antioxidant activity.

[0156] Fig.10 The total antioxidant capacity of Bacillus clausii CSI08 and Lactobacillus rhamnosus GG is shown. The results show the average concentration of Trolox equivalents in nanomoles (nmole) / g (n=3) ± standard error.

[0157] The increased Trolox equivalent concentration levels in B. clausii CSI08 spores can neutralize the effects of reactive oxidative species. The antioxidant properties in B. clausii CSI08 spores enable this strain to act as a producer of antioxidant enzymes and molecules, thereby mitigating oxidative damage.

[0158] Therefore, Bacillus clausii CSI08 spores are potential probiotics with strong antioxidant properties.

[0159] The antioxidant activity of B. clausii CSI08 was confirmed using cell culture models and an in vivo Caenorhabditis elegans model of oxidative stress.

[0160] Propagative strain Bacillus clausii CSI08 attenuated the hydrogen peroxide-induced decrease in cell viability in epithelial cells (HT-29 cell line).

[0161] Propagative strains of Bacillus clausii CSI08 attenuated the hydrogen peroxide-induced decrease in survival of Caenorhabditis elegans N2 after oxidative stress.

[0162] Fig.11 Shown is the cytoprotective effect of vegetative Bacillus clausii CSI08 on epithelia exposed to H2O2. Values ​​are mean ± SEM. *-P < 0.0001 vs H2O2

[0163] Fig.12 Shown with 10 8and 10 9 Survival of C. elegans N2 fed with CFU / ml Bacillus clausii CSI08 and then induced to oxidative stress by H2O2. NGM-control fed nematodes; Vitamin C-positive control. Values ​​are the mean of two independent experiments (n=100 / condition). *p<0.05 vs NGM (+H2O2).

[0164] Bacillus clausii CSI08 spores exhibit reversal of oxidative stress in mammalian cell lines and host organisms.

[0165] Adhesion ability and cytotoxic effect: Propagated Bacillus clausii CSI08 did not affect the viability of intestinal epithelial cells (HT-29 model).

[0166] The propagules and spores of Bacillus clausii CSI08 have a significant adhesion ability to the mucus-producing cell line HT-29-MTX.

[0167] The adhesion of propagules and spores of B. clausii CSI08 to a non-mucus producing intestinal epithelial cell line (HT-29) was negligible and is not shown.

[0168] Fig.13 Shown is the effect of propagule B. clausii CSI08 on HT-29 cell viability. B. clausii CSI08 compared to untreated cells (medium). Values ​​are mean ± SEM.

[0169] Fig.14 Figure 2 shows the adhesion of vegetative cells and spores of Bacillus clausii CSI08 to HT-29-MTX. Bacillus clausii CSI08 compared to Lactobacillus fermentum (L.fermentum). Values ​​are mean ± SEM

[0170] Bacillus clausii CSI08 does not negatively affect mammalian cell viability and can adhere to the intestinal wall.

[0171] Anti-inflammatory activity: The ability of vegetative bodies of B. clausii CSI08 and its cell-free supernatant to attenuate the pro-inflammatory response triggered by LPS was investigated in an in vitro model of the intestinal epithelium (HT-29 cell line).

[0172] HT-29 cells pretreated with propagules of B. clausii CSI08 but not its supernatant (CFS) showed a strong inhibition of the expression of genes associated with pro-inflammatory responses after exposure to LPS.

[0173] Fig.15A and 15BModulation of LPS-induced pro-inflammatory response by Bacillus clausii CSI08 in HT-29 cell line is shown. Also shown are gene expression patterns after co-incubation of HT-29 cells with Bacillus clausii CSI08 and its CFS under unstimulated conditions (LPS-). $p<0.05 vs LPS, #p<0.001 vs LPS, *p<0.0001 vs. LPS.

[0174] Propagates of B. clausii CSI08, but not its cell-free supernatant, can reduce the pro-inflammatory response triggered by LPS in HT-29 cells.

[0175] After heat inactivation, B. clausii CSI08 partially retained the ability to inhibit the pro-inflammatory response triggered by LPS in HT-29 cells.

[0176] Fig.16 Shown is the regulation of LPS-induced IL8 gene expression by live and heat-killed Bacillus clausii CSI08. *p<0.0001.

[0177] Bacillus clausii CSI08 can downregulate LPS-induced NF-κB activation in HT-29 cell line.

[0178] Fig.17 Shown are NF-κB levels in nuclear fractions of control HT-29s, cells exposed to LPS, and cells treated with B. clausii CSI08 prior to addition of LPS. *p<0.001, LPS vs. LPS+B. clausii by t-test.

[0179] The immunomodulatory effect of Bacillus clausii CSI08 is partially mediated through the Nf-κB pathway.

[0180] The ability of vegetative B. clausii CSI08 and its cell-free supernatant to attenuate PolyI·C-triggered proinflammatory responses (a viral mimetic) was investigated in an in vitro model of the intestinal epithelium (HT-29 cell line).

[0181] HT-29 cells pretreated with CSI08 and its supernatant (CFS) showed significant suppression of the expression of genes associated with pro-inflammatory responses after exposure to PolyI·C.

[0182] Fig.18A and 18BModulation of PolyI·C-triggered pro-inflammatory responses by Bacillus clausii CSI08 in HT-29 cell line is shown. qPCR analysis of IL-8, TNF-α, IL-17C and CXCL10 gene expression was performed 4 hours after exposure to PolyI·C in HT-29 cells pre-incubated with Bacillus clausii CSI08 or its cell-free supernatant (CFS). $p<0.05, &p<0.01; #p<0.001, *p<0.0001.

[0183] The propagules of Bacillus clausii CSI08 and its cell-free supernatant can reduce the pro-inflammatory response triggered by the viral mimic PolyI·C in HT-29 cells.

[0184] Immunity: Bacillus clausii CSI08 can induce a strong immune response in U937-derived macrophages

[0185] The effects of the propagule of Bacillus clausii CSI08 on the innate immune system were investigated using a cellular model of U937-derived macrophages.

[0186] The propagules of Bacillus clausii CSI08 stimulated a strong immune response, resulting in the secretion of high levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-18), regulatory factors (G-CSF, GM-CSF, IL-6), and anti-inflammatory factors (IL-10, IL-1RA, EGF) by macrophages.

[0187] Fig.19 The immunostimulatory effect of CSI08 is shown. Cytokine levels in the cell culture supernatant of U937-derived macrophages challenged with propagated Bacillus clausii CSI08 or LPS for 5 hours. Values ​​are mean ± SEM. *p < 0.05, &p < 0.01, %p < 0.001; $p < 0.0001 LPS or CSI08 vs NC (negative control). ++p < 0.05; +p < 0.0001 CSI08 vs LPS.

[0188] Bacillus clausii CS108 extends the lifespan of Caenorhabditis elegans N2 worms compared to control conditions - NGM medium. This effect is DAF-16 dependent.

[0189] Fig. 20 Shown are the lifespan of C. elegans wild-type N2 strain under control conditions (NGM) and the lifespan of C. elegans wild-type N2 fed with Bacillus clausii CSI08. Mean values ​​of two independent analyses (n=100 / replicate / condition).

[0190] The transcription factor DAF-16, an ortholog of FOXO, mediates the effects of Bacillus clausii CSI08 on lifespan in Caenorhabditis elegans.

[0191] The data suggest a role for B. clausii CSI08 as a potential anti-inflammatory effector or immune activator.

[0192] Fig.21 Shown are the lifespan of C. elegans daf-16 mutant worms under control conditions (NGM) and the lifespan of C. elegans daf-16 mutants fed with Bacillus clausii CSI08. Mean values ​​of two independent analyses (n=100 / replicate / condition).

[0193] Bacillus clausii CSI08 exhibits strong immune protection in host organisms.

[0194] Bacillus clausii CSI08 showed moderate caseinolytic / protease activity.

[0195] The casein degradation / caseinolytic activity of Bacillus clausii CSI08 on skim milk agar plates was weakly positive.

[0196] Quantitative analysis of the caseinolytic activity of B. clausii CSI08 was assessed using a commercial kit employing a fluorescently labeled casein derivative.

[0197] Bacillus clausii CSI08 showed moderate protease activity (Lactobacillus rhamnosus served as negative control).

[0198] Fig. 22 It is shown that Bacillus clausii CSI08 has slight caseinolysis using the streak method. The figure also shows that Bacillus megaterium and Bacillus clausii CSI08 have caseinolytic activity.

[0199] Fig.23 Shown by Proteolytic activity measured by the kit assay. Data represent mean ± SEM.

[0200] Bacillus clausii CSI08 showed moderate caseinolytic / protease activity

[0201] Diverse Carbohydrate Assimilation Profile of Bacillus Clausii CSI08: Using the commercial API 50CH test strips, Bacillus Clausii CSI08 was positive for 20 out of 49 carbohydrates.

[0202] These carbohydrates are mainly monosaccharides such as D-ribose, D-glucose, D-fructose, D-mannose and disaccharides such as trehalose, sucrose, cellobiose, etc. In addition, Bacillus Clausii CSI08 can also metabolize compounds belonging to cyanogenic glycosides (amygladin), amine sugars (glucosamine) and coumarin glucosides (aesculin). Bacillus Clausii CSI08 can also metabolize the polysaccharide glycogen, but the ability to metabolize Amidon (starch) is weak.

[0203] Table 5

[0204] Carbohydrates fermented by Bacillus clausii CSI08

[0205]

[0206] Table 5. List of carbohydrates that are efficiently fermented using the API 50Ch bar.

[0207] These data suggest that B. clausii CSI08 can aid in the digestion of these compounds in the gut.

[0208] Bacillus clausii CSI08 is an esterase and has phosphatase and β-galactosidase activities: Using the API ZYM kit, Bacillus clausii CSI08 tested positive for esterase, phosphohydrolase, and β-galactosidase activities, indicating that

[0209] Bacillus clausii CSI08 can produce free fatty acids through the action of esterase in the presence of an appropriate lipid source.

[0210] β-Galactosidase – an enzyme that catalyzes the hydrolysis of lactose into glucose and galactose and, importantly, is responsible for the formation of galacto-oligosaccharides (GOS), which promote the growth of Bifidobacterium and Lactobacillus species

[0211] Phosphohydrolase catalyzes the hydrolysis of phosphate

[0212] No harmful enzyme activities (β-glucuronidase, α-chymotrypsin and β-glucosaminidase) were detected.

[0213] Table 6

[0214] Enzymatic properties of Bacillus clausii CSI08

[0215]

[0216] Table 6 Enzymatic properties of Bacillus clausii CSI08 using API ZYM kit

[0217] This indicates that B. clausii CSI08 has the potential to break down lactose and fat. These data suggest that B. clausii CSI08 can help digest these molecules in the intestine.

[0218] Proteomic analysis of Bacillus clausii CSI08 identified proteins with potential probiotic benefits:

[0219] The extracellular secretions of Bacillus clausii CSI08 grown in TSB broth for 24 h were sent to a mass spectrometer to identify proteins released by the probiotic strain. A total of 29 proteins were detected, of which 5 had potential probiotic benefits.

[0220] Table 7

[0221] Proteomic Study of Bacillus Clausii CSI08

[0222]

[0223] These data confirm previous in vitro results showing how B. clausii CSI08 can aid in the digestion of proteins and carbohydrates and can aid in the production of branched-chain amino acids.

[0224] Bacillus clausii CSI08 can produce essential amino acids and B vitamins: The ability of Bacillus clausii CSI08 to synthesize essential amino acids and vitamins during cultivation in liquid medium (TSB) was evaluated. The results showed that several amino acids (alanine, glutamine and glutamic acid, histidine, methionine, proline, tyrosine and threonine) and two B vitamins (pantothenic acid / B5 and cyanocobalamin / B12) were produced by Bacillus clausii CSI08.

[0225] Fig.24 The concentrations of essential amino acids and vitamins in overnight cultures of Bacillus clausii CSI08 determined by RP-HPLC-FLD and RP-HPLC-MS, respectively, are shown. Values ​​are mean ± SEM. *p < 0.05, &p < 0.01, $p < 0.0001.

[0226] Bacillus Clausii CSI08 can produce essential amino acids and B vitamins

[0227] Example 2

[0228] Assessing adhesion capacity on an in vitro model of intestinal epithelium

[0229] Cell lines: Human colorectal adenocarcinoma cell line HT-29 and mucus secreting cell line HT-29-MTX were propagated in low glucose DMEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin and 2 μg / ml amphotericin B at 37°C in a 5% CO2 atmosphere.

[0230] The cells were plated at 5x10 5 Cells were seeded into 24-well plates at a density of 100 cells / well and cultured for 21-28 days to complete maturation. The culture medium was changed every 2-3 days.

[0231] Prior to the experiment, wash the cells twice with 0.5 ml DPBS. After the second round of washing, completely aspirate the DPBS from the wells.

[0232] Preparation of spores: Ten milligrams of spore powders of Bacillus clausii CSI08, Bacillus megaterium MIT411 and Bacillus coagulans CGI314 were weighed into a 15 ml Falcon tube and resuspended in 10 ml of Not included The suspension was aliquoted and stored at -20 °C until use. The suspension was used within 2 weeks of preparation.

[0233] Adhesion test: 500 μl of spore suspension (1.3x10 7 -9.2x10 7 CFU / ml) were added to HT-29 and HT-29-MTX cells, mixed by gentle vortexing, and incubated in a CO2 incubator at 37°C for 2.5 h. Not included Control wells of mammalian cells were incubated in parallel in the same manner (0.5 ml of spore suspension).

[0234] After incubation, HT-29 and HT-29-MTX cells were washed 4 times with 0.5 ml PBS. Afterwards, 50 μl of trypsin / EDTA solution and 50 μl PBS were added to the wells and incubated for 10 min at 37° C. with gentle shaking (~100 rpm). Fifty microliters of trypsin / EDTA solution were added to the control wells.

[0235] Therefore, 450 μL of PBS was added to the wells with spores, the contents of the wells were transferred to Eppendorf tubes by scraping and subjected to three rounds of vigorous shaking for 30 seconds each. The contents of the control wells were transferred to Eppendorf tubes and subjected to one round of shaking.

[0236] Serial dilutions (plus dilutions from control wells) were prepared in PBS and plated on BC agar (Bacillus coagulans CGI314) or PetriFilm TM(Bacillus clausii CSI08, Bacillus megaterium MIT411). Plates were incubated at 37°C for 48 h before counting, and PetriFilm was incubated at 37°C for 24 h before counting.

[0237] Experiments were performed in duplicate or triplicate with three technical replicates per experiment. Results are expressed as mean ± SEM.

[0238] Table 8

[0239] Adhesion of Bacillus clausii CSI08, Bacillus megaterium MIT411 and Bacillus coagulans CGI314 spores to HT-29-MTX cell line

[0240]

[0241] Table 9

[0242] Adhesion of Bacillus clausii CSI08, Bacillus megaterium MIT411 and Bacillus coagulans CGI314 spores to HT-29 cell line

[0243]

[0244] in conclusion

[0245] 1. The above results indicate that the ability of spores to adhere to the mucus-secreting cell line HT-29-MTX is higher than that of non-mucus-secreting cells, which may be due to the physical properties of the spores.

[0246] 2. Bacillus megaterium MIT411 and Bacillus coagulans CGI314 spores had a higher (but overall lower) ability to adhere to the non-mucoid-producing cell line HT-29 compared to Bacillus clausii CSI08 spores.

[0247] Example 3

[0248] Evaluation of the safety, tolerability, and gastrointestinal health of Bacillus clausii CSI08, Bacillus megaterium MIT411, and a mixture of Bacillus spores in healthy adults: a randomized, double-blind, placebo-controlled trial.

[0249] The efficacy of 1X 10 9 CFU Bacillus clausii CSI08, 1X 10 9 Safety, tolerability, and effects of CFU Bacillus megaterium MIT411 and a probiotic mixture comprising 0.5 x 10 9 CFU Bacillus subitilis 0.5x 10 9CFU Bacillus megaterium MIT411, 0.5x10 9 CFU Bacillus coagulans CGI314, 0.5x10 9 CFU Bacillus clausii CSI08 (Bacillus subtilis The total counts of Bacillus megaterium MIT411, Bacillus coagulans CGI314, and Bacillus clausii CSI08 were 2.0x10 9 CFU). A total of 98 study participants received daily doses for 45 days, followed by a 2-week washout period. Throughout the 45 days, a questionnaire was maintained daily to record the incidence and duration of upper respiratory, urinary and / or gastrointestinal complaints, and a diary was maintained daily to record the regularity and consistency of stool to record compliance. At the beginning and end of the treatment period, stool and blood samples were collected for microbiological and hematological analysis. Throughout the study, the probiotic mixture significantly reduced the incidence of loose stools. Recorded respiratory, urinary and gastrointestinal symptoms, bowel movement frequency and other stool consistency were not affected. During and after administration, there were no clinically relevant changes in blood indicators such as liver and kidney function, and no serious adverse events occurred. At baseline and at the end of the treatment period, there were no changes in symptoms including sadness, irritability, energy, appetite, tension, stress, sleep, cardiovascular events, pain and dizziness, as determined by mood questionnaires administered to participants. Similarly, measured inflammatory cytokines, antioxidant levels, cholesterol, triglycerides, free amino acids or minerals were not affected. There were no negative changes in the alpha or beta diversity of the microbiota in any of the treatment groups. These promising data suggest that these treatments are safe and well tolerated, and further work in larger cohorts is justified to determine the efficacy of these potential probiotics in selected demographic groups.

[0250] Probiotics are live microorganisms that reside in the human intestine, have low or no pathogenicity, and exhibit beneficial effects on the host. Common products containing probiotic bacteria include dietary supplements and foods such as fermented dairy products, sauerkraut, and salami. Probiotic supplements have shown positive effects in alleviating various conditions such as: antibiotic-associated diarrhea, constipation, allergies, and diabetes. Probiotics also exhibit protective properties.

[0251] Probiotic supplements can contain one or more different strains that have different effects on the human gut. Common probiotic strains are lactic acid producers such as Lactobacillus, Bifidobacterium, and Streptococcus due to their tolerance to gastric acid, bile salts, and pancreatic enzymes. Studies have shown that lactic acid bacteria are effective inhibitors of colonization by pathogenic Gram-negative bacteria (e.g., Salmonella typhimurium, Clostridium difficile, and Escherichia coli) in vitro.

[0252] However, not all probiotic supplements produce lactic acid. Bacillus subtilis spores have been used as probiotics, competitive excluders, and preventive agents for human and animal administration. All four Bacillus strains are Gram-positive, spore-forming, rod-shaped bacteria. Under nutrient-limiting conditions, Bacillus species can form dormant endospores that are resistant to environmental stress and nutrient deficiency, making these bacteria a viable option for probiotic supplementation.

[0253] DE111, CSI08, CGI314 and MIT411 are unique strains of probiotics. As probiotic Bacillus strains, they are able to withstand the harsh digestive environment and colonize the intestines, thereby supporting a healthy GI (gastrointestinal) tract. To date, DE111 is sold in the United States and Canada as a probiotic food ingredient and probiotic capsules for adults. The other three Bacillus probiotics used in this trial, CSI08, CGI314 and MIT411, are not currently on the market and are claimed here.

[0254] The trial was designed to determine the safety of three novel probiotic strains and to assess their efficacy in reducing the incidence and / or duration of gastrointestinal problems and infections and respiratory tract infections in healthy adults.

[0255] Materials and methods

[0256] Subjects

[0257] Healthy adult volunteers aged 18-65 years were recruited from February to July 2021 through flyers, posters, and physicians. Inclusion criteria included willingness to provide informed consent and good overall health. Exclusion criteria included the presence of any pre-existing adverse events (e.g., gastric ulcer, Crohn's disease, UC, diabetes, kidney disease, HIV / AIDS, hepatitis, cancer, and organ transplant recipients), taking medications for digestive discomfort (constipation, bloating, or diarrhea), antibiotic use within four weeks before randomization, unwillingness to discontinue any probiotic supplements other than those provided in this study, known immunodeficiency or use of immunosuppressive drugs, pregnancy, 6 months postpartum or lactation, women of childbearing age planning to become pregnant during the study, participation in another study, and use of mood medications (e.g., antidepressants, antianxiety drugs, antipsychotics).

[0258] This study was approved by the Nutritional Research Ethics Committee of the University of Ljubljana, Biotechnical Faculty, Slovenia, and was conducted according to the guidelines established by the Declaration of Helsinki. All participants were informed of the aims, requirements, and risks of the study and were informed that they could withdraw from the study at any time. Participants provided their written consent indicating that they fully understood the study protocol.

[0259] Experimental design

[0260] This study was a double-blind, placebo-controlled, randomized, parallel trial. The study was conducted through the University Clinical Centre Maribor, Slovenia, and coordinated by the Slovenian CRO Vizera doo. Participants were randomly assigned to one of three treatment groups or to take a placebo daily. The treatment groups were administered 1x 10 9 CFU / dose of Bacillus clausii CSI08, 1x 10 9 CFU / dose Bacillus megaterium MIT411 and Bacillus subtilis A probiotic mixture of Bacillus megaterium MIT411, Bacillus coagulans CGI314 and Bacillus clausii CSI08, the total count of the probiotic mixture being 2.0 x 10 9 CFU / dose. Placebo was rice maltodextrin.

[0261] The randomization scheme was performed by Vizera doo, a CRO in Slovenia, and the allocation order was in sealed opaque envelopes before the randomization day, hidden from the investigators and participants. After the assessment of baseline characteristics (age, sex, height, weight (by digital scale)) and the collection of initial stool samples, the envelopes were opened and the participants were assigned to the intervention regimen. The investigators received individually sealed envelopes containing the randomization number and the linkage between the treatment groups for the specific participant. The sealed envelopes should only be opened in emergency situations. If the participant's treatment is unblinded during the study, the sponsor is notified immediately. Information about the unblinding must be recorded in the data source file and the participant's case report form (CRF). Participants were then asked to take one capsule per day at the end of a meal.

[0262] Participants visited the study center three times and had two phone calls with the assigned investigator: visit 0 for screening purposes (screening visit), 2 visits during treatment, of which visit 1 was the baseline visit, where randomization to the product took place, and visit 2 was the end-of-treatment visit. In addition, patients had a phone call with the investigator after taking the product for 21 days (inter-visit call) and after 2 weeks of follow-up at visit 2 (follow-up call). A graphic flow diagram of the study is shown in Fig.25 shown.

[0263] After screening, consent, 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.

[0264] Fig.25 Graphical flow chart showing the study design.

[0265] Probiotic administration regimen

[0266] The study product was provided by DiLan Probiotics and Enzymes, Inc. (Kennesaw, Georgia, US) in the form of identical rectangular 300 mg capsules, and the placebo was indistinguishable in appearance. The study capsules were provided by a study collaborator who had no contact with the investigators or participants and were packaged in bottles labeled with treatment codes.

[0267] Study plan

[0268] Participants completed daily questionnaires to monitor bowel movement timing and stool sample type based on the Bristol Stool Chart Index, as well as any symptoms including: gastrointestinal upset, respiratory distress, urinary symptoms, cephalic, ENT, behaviour, vomiting, loss of appetite, fever and epidermal symptoms. If they visited their GP or were prescribed any medication during the trial, this was also recorded and reported. At baseline and at the end of the treatment period, participants were given a mood questionnaire to assess their experience over the previous month. The questionnaire included 14 symptoms including sadness, irritability, energy, appetite, nervousness, stress, sleep, cardiovascular events, pain and dizziness, with scores ranging from 1 (no significant symptoms) to 3 (severe). Any adverse events were reported to the investigators.

[0269] Blood sample collection and preparation

[0270] For safety blood, 3-mL red-cap serum coagulant tubes (Greiner Bio-One, 454029) were used for blood collection. Biochemical blood samples HDL and LDL, total cholesterol and triglycerides were determined using 3.5mL SST II Advanced / gel yellow-cap bottles (Greiner Bio-One, 454029). For the determination of antioxidants and cytokines, whole blood was collected into 4-mL test tubes containing lithium heparin (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 subsequent analysis.

[0271] LDL, HDL, total cholesterol and triglyceride measurements

[0272] Hematological and biochemical evaluations were performed at the University Clinical Center in Maribor, Slovenia. Safety blood was run with Sysmex EN-1000, while biochemical determinations of LDL, HDL, total cholesterol, and triglycerides were performed according to the manufacturer's instructions and analyzed with Abbott Allinity C.

[0273] Cytokine quantification

[0274] The concentrations of IL-8 and TNF-α in serum samples were determined by sandwich ELISA according to the manufacturer's instructions: human IL-8 (CXCL8) ELISA kit (ELH-IL8-1, RayBiotech) and human TNFα ELISA kit (ELH-TNFa-1, RayBiotech). Before ELISA, serum samples were diluted 1:2 using the dilution buffer provided by the kit.

[0275] Antioxidant activity assay

[0276] Total antioxidant activity was assessed using a total antioxidant capacity assay kit (Sigma, Ireland) according to the manufacturer's instructions, and the absorbance was measured at 340 nm.

[0277] Feces collection

[0278] Using Zymokit DNA / RNA Shield TM Fecal collection tubes (ZymoResearch, California, US) were used to collect stool at the baseline visit before treatment 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 transport the sample to the study staff at the clinical visit.

[0279] DNA extraction and 16S rRNA sequencing

[0280] Total fecal DNA was extracted from approximately 200 mg of sample using the ZymoBIOMICS DNA Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's instructions. Briefly, fecal samples were placed in a 4% paraformaldehyde (pH 7.0) flask containing 750 μl of ZymoBIOMICS DNA Miniprep. TM ZR BashingBead for Lysis Buffer TM Lysis tubes and in BeadBug TM The cells were processed in a Zymo-Spin homogenizer (BenchmarkScientific, China) at 4350 rpm for 5 x 1 min with a 1 min rest step between the shaking cycles. The lysis tubes were then centrifuged at 10,000 g for 1 min. Four hundred microliters of the supernatant were transferred to a collection tube in a Zymo-Spin 6 homogenizer (BenchmarkScientific, China). TM III-F filter and further centrifuged at 8,000 g for 1 min. The filtrate was mixed with 1,200 μl of ZymoBIOMICS TM Mix with DNA Binding Buffer and transfer to the Zymo-Spin in the collection tube TM IICR column and centrifuged at 10,000 g for 1 min. After three rounds of washing, the column was washed with 100 μl of ZymoBIOMICS TM Elute DNA in DNase / RNase-free water and use a Zymo-Spin according to the protocol. TM The DNA concentration was determined using the Qubit dsDNA BR Assay Kit (ThermoFisher Scientific).

[0281] Data Generation

[0282] Library preparation was performed according to the Illumina guide for 16S metagenomic sequencing library preparation (https: / / support.illumina.com / documents / documentation / chemistry_documentation / 16s / 16s-metagenomic-library-prep-guide-15044223-b.pdf). In brief, the target was amplified from each sample using 16S degenerate primers. At the same time, Illumina adapters and barcodes were included to allow library creation. Sequencing was performed on a Novasek 6000 machine that produces paired-end 250bp reads. Sequencing data were quality controlled using QIIME2 software. An average of 670,000 read pairs were generated per sample. Taxonomic classification of ASVs (also known as OTUs) was performed using the QIIME2 / DADA2 and Silva132 databases.

[0283] Statistical analysis

[0284] 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 tract, gastrointestinal tract, and upper respiratory tract discomfort. In this study, descriptive statistics were used to evaluate these results. The Kruskal-Wallis test was used to confirm that there were no statistically significant differences in the incidence of any of these individual symptoms among the four treatment groups at the beginning of the study, or in the incidence and duration of gastrointestinal, upper respiratory, or urinary tract discomfort during the study. In addition, the nonparametric Mann-Whitney U test with Holm's correction was used for pairwise comparisons of the three probiotic product groups with the placebo group.

[0285] For the gastrointestinal health questionnaire and blood analyses, differences in the change in individual symptom scores from baseline to the end of the treatment period were compared between treatment groups using analysis of variance (one-way ANOVA test), with post hoc tests evaluating pairwise comparisons between each of the three treatment groups and the placebo group.

[0286] For sequencing data, multiple alpha diversity indices were calculated, including Observed, Chao1, ACE, Shannon, and Simpson indices. The alpha diversity between experimental groups and between experimental groups and placebo was then compared to detect treatment differences or within-treatment differences from baseline to post-treatment time points.

[0287] To quantify the compositional dissimilarity between samples, the Bray-Curtis dissimilarity index was calculated and used to create multiple clustering plots. This method collapses information from multiple dimensions for easier visualization and interpretation. Paired Wilcoxon tests were used to compare the distributions of the groups.

[0288] Differential abundance analysis was performed to detect significant differences in genus abundance between treatments and time points. Day 1 samples from all treatments were compared to the Day 1 placebo group to determine if there were any resting differences at baseline. Paired comparisons of Day 45 to Day 1 were performed for each group. A two-way analysis was also performed using the placebo group as the reference to detect if there were significant differences in treatment response at Day 45 relative to Day 1 compared to the placebo group response at Day 45 relative to Day 1.

[0289] result

[0290] Participants

[0291] Ninety-eight participants completed the 45-day intervention ( Fig.25 ). After screening, one participant declined to participate and another participant 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 attributed to the use of an approved co-medication. All other reported AEs occurred only once, namely: vaginal inflammation, fecal parasites (possibly related to overseas travel), right wrist spin, metallic taste, lower back pain, sebaceous gland inflammation, granulomas, dark brown stools, and acne.

[0292] Causality assessment showed no relationship between the reported AEs and the study product.

[0293] No serious adverse events were reported during the entire study.

[0294] Basic information of participants

[0295] Table 10

[0296] Basic information of participants used in the study

[0297]

[0298] Gastrointestinal health status at the screening visit:

[0299] Table 11

[0300] Gastrointestinal health at baseline (n=98). (How often did you experience the following problems in the past month?)

[0301]

[0302]

[0303] N* = number of participants included in the ITT population, *p-value of the Kruskal-Wallis test

[0304] There were no significant differences between the groups for any individual readings.

[0305] Stool consistency and regularity

[0306] Mean bowel movement frequency (regularity) among study participants ranged from 0.33 to 2.16 bowel movements / day. Comparisons of the various time periods and intervention groups yielded mixed results. There were no significant differences in bowel movement frequency compared with the placebo-treated group or the washout period (Table 12).

[0307] Table 12

[0308] Treatment had no effect on stool regularity over the 45-day trial period compared with placebo

[0309]

[0310] *p-value of ANOVA test

[0311] Stool consistency was reported as the proportion of participants with loose stools and the proportion of participants with hard stools during the total treatment period. The baseline questionnaire showed no difference in the incidence of loose or hard stools / constipation in the study group compared with the control group (Table 2). Participants were asked to report how often they had loose or hard stools / constipation in the past month. The scale was as follows: 0 = never, 1 = monthly, 2 = weekly, and 3 = daily.

[0312] Fig.26 It was shown that the probiotic mixture significantly reduced the incidence of loose stools during the study compared to the placebo control.

[0313] During the first 6 weeks of the study, the probiotic mixture significantly reduced the incidence of loose stools compared with the control group ( Fig.26 ), which was determined by repeated measures one-way ANOVA (treatment: F (2.615，13.08) =20.07; P<0.0001; time (F (5，15) =2.803, p=0.055). Among the study participants, 16 of 25 in the probiotic group reported no loose stools throughout the study, compared with only 8 in the placebo group, 10 in the Bacillus clausii group, and 10 in the Bacillus megaterium group.

[0314] Fig. 27 showed no effect of any treatment on the percentage of hard stools compared with placebo control.

[0315] There was no significant effect of any treatment group on the percentage of hard stools throughout the study ( Fig. 27 )(F (1.829,9.146) =2.831, P = 0.113; time (F (5，15) =1.121, p=0.391).

[0316] Incidence and duration of gastrointestinal symptoms

[0317] Table 13

[0318] The number of days during the study on which participants reported gastrointestinal symptoms in their diaries

[0319]

[0320]

[0321] N* = number of participants included in the ITT population, NC = not calculable, p* = p-value of the Kruskal-Wallis test, P = p-value of the Mann-Whitney U test.

[0322] The Kruskal-Wallis test showed no significant differences in the number of days with gastrointestinal symptoms between treatment groups. The study product containing probiotics did not show a statistically significant difference in the number of days with gastrointestinal symptoms compared with placebo.

[0323] Incidence and duration of urinary tract symptoms

[0324] Table 14

[0325] Number of days with urinary tract discomfort symptoms reported in the participants' diaries

[0326]

[0327] N* = number of participants included in the ITT population, NC = not calculable, p* = p-value of the Kruskal-Wallis test, P = p-value of the Mann-Whitney U test.

[0328] The Kruskal-Wallis test did not show any significant differences in the number of days with UTI symptoms between treatment groups. None of the study products, including the probiotic, showed a statistically significant difference in the number of days with UTI symptoms compared with placebo.

[0329] Incidence and duration of upper respiratory tract infections

[0330] Table 15

[0331] Number of days with respiratory symptoms reported in participants' diaries

[0332]

[0333]

[0334] N* = number of participants included in the ITT population, NC = not calculable, p* = p-value of Kruskal-Wallis test, p = p-value of Mann-Whitney U test.

[0335] The Kruskal-Wallis test did not reveal any significant differences in the number of days with respiratory tract infection symptoms between the treatment groups. The study product containing probiotics did not show a statistically significant difference in the number of days with symptoms compared with placebo.

[0336] Daily questionnaire analysis

[0337] Table 16 summarizes the responses to the mood questionnaire at baseline and at the end of the study for the three treatment groups and the placebo group. Changes in the mean with 95% confidence intervals are shown. Results of the ANOVA omnibus test (p*-value) and the one-sample T-test (p-value) are also provided. The normality test for the changes in the gut-brain axis scores showed that the data did not follow a normal distribution, which could affect the results with borderline significance (p-values ​​between 0.05 and 0.10). This affected two items: fatigue (loss of energy) and changes in appetite. A non-parametric Kruskal-Wallis test was performed on these items; the observed p-values ​​were 0.111 (fatigue) and 0.123 (changes in appetite). Overall, at the end of the treatment period, including the placebo group, the mean intensity of the scores decreased (participants were less bothered by these symptoms). Therefore, the one-sample T-test results show that statistically significant changes in the gut-brain axis questionnaire scores were observed in one-third of the tests (70 times in total). However, this was observed in all treatment groups, including the placebo group. Thus, the results of the ANOVA test showed that no significant differences in the changes in the gut-brain axis score were detected between the treatment groups, but borderline significant differences in the change in appetite and appetite items were observed. Participants in the Bacillus megaterium group experienced the greatest changes in both items. However, no statistically significant differences were observed for the pairwise comparisons of the probiotic groups with the placebo (Table 16 (below)).

[0338] Table 16

[0339] Gut-brain axis questionnaire responses at baseline (N=98) and at the end of the study (Post). (In the past 30 days (including today), how much did the following emotions or feelings bother you?)

[0340]

[0341]

[0342] N* = number of participants included in the ITT population; score: 0 - not bothered, 1 - slightly bothered, 2 - somewhat bothered, 3 - very bothered. * p-value of ANOVA (omnibus test).

[0343] Cholesterol and triglyceride levels

[0344] Blood samples were collected at the beginning of the study, before any treatment, and again at the end of the 45-day treatment period. There were no significant effects of treatment within the groups, nor were there any significant effects of treatment compared to baseline for high-density lipoprotein, low-density lipoprotein, total cholesterol, and triglyceride concentrations (Table 17).

[0345] Table 17

[0346] Cholesterol and triglyceride levels at baseline and end of study (n=98)

[0347]

[0348] N* = number of participants included in the ITT population, HDL = high-density lipoprotein, LDL = low-density lipoprotein, TC = total cholesterol, TG = triglycerides. *p-value of ANOVA test)

[0349] Blood cytokine levels

[0350] Table 18

[0351] IL-8 and TNFα levels at baseline and end of study (n=98)

[0352]

[0353] N* = number of participants included in the ITT population, *p-value for ANOVA (omnibus test)

[0354] Blood samples were collected at the beginning of the study, before any treatment, and again at the end of the 45-day treatment period. There were no significant effects of treatment within group, nor were there any significant effects of treatment compared to baseline for either IL-8 or TNFα (Table 18).

[0355] Blood antioxidant levels

[0356] Table 19

[0357] Antioxidant levels at baseline and end of study (n=98)

[0358]

[0359] N* = number of participants included in the ITT population, *p-value for ANOVA (omnibus test)

[0360] Blood samples were collected at the beginning of the study, before any treatment, and again at the end of the 45-day treatment period. There were no significant effects of treatment within the groups, nor were there any significant effects of treatment compared to baseline antioxidant levels (Table 10).

[0361] Metabolite levels

[0362] Table 20

[0363] Amino acid levels at baseline and end of study (n=98)

[0364]

[0365]

[0366] N* = number of participants included in the ITT population, *p-value for ANOVA (omnibus test)

[0367] Blood samples were collected at the beginning of the study, before any treatment, and again at the end of the 45-day treatment period. There were no significant effects of treatment within the groups, nor were there any significant effects of treatment compared to the baseline for the amino acids tested (Table 20).

[0368] Table 21

[0369] Mineral levels at baseline and end of study (n=98)

[0370]

[0371]

[0372] N* = number of participants included in the ITT population, *p-value for ANOVA (omnibus test)

[0373] Blood samples were collected at the beginning of the study, before any treatment, and again at the end of the 45-day treatment period. There were no significant effects of treatment within the groups, nor were there any significant effects of treatment on any mineral levels compared to baseline (Table 21).

[0374] Changes in microbial flora

[0375] Subject samples collected before and after treatment were selected for comprehensive microbiome analysis. After removing 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 ESPRIT-tree, 1,077 and 1,618 OTUs were retained at 95% and 98% similarity levels after removing OTUs containing less than 10 sequences.

[0376] Fig.28 is a box plot showing the distribution of Chao1 values ​​for each experimental group on day 1 and day 45. Dashed lines connect paired samples. Paired Wilcoxon test was used to compare the distribution of each group. p-value less than 0.05 should be considered statistically significant.

[0377] Fig.29 is a box plot showing the distribution of Chao1 values ​​for each experimental group on day 1 and day 45. The Wilcoxon test was used to compare the distribution of each experimental group with placebo. A p-value less than 0.05 should be considered statistically significant.

[0378] Fig.30 Shows PCoA clustering performed on a Bray-Curtis dissimilarity matrix. Each treatment is separated in a different label, while the color and shape are associated with the time point. For all treatments, samples from both time points tend to cluster together, and at the baseline reading on day 1, the data are not significantly different from each other. There are no significant differences between samples due to treatment effects within or between groups.

[0379] Table 22

[0380] Proportion of participants reporting respiratory infection symptoms in Diary 1 (n=123).

[0381]

[0382] N* = number of participants included in the ITT population

[0383] The only significant difference between the treatment groups was in the number of days with runny nose (p*=0.018), which may be due to the fact that only three participants in the probiotic mixture group reported this symptom, while no participants in the other four treatment groups reported this symptom. However, further analysis (Mann-Whitney U test with Holm correction) comparing the number of days with runny nose between the probiotic mixture group and the placebo group did not show a significant difference, which may be due to the small sample size.

[0384] Table 23

[0385] Proportion of clinically relevant infections reported in participant diary 1 (n=123).

[0386]

[0387] N* = number of participants included in the ITT population

[0388] The Kruskal-Wallis test did not reveal any significant differences in the number of days with clinically relevant infections between the treatment groups. However, a borderline statistically significant result was observed for clinically relevant gastrointestinal infections. This may have occurred by chance because no participant in any of the four probiotic treatment groups developed a clinically relevant gastrointestinal infection, whereas a total of 2 days of such infections were observed in the probiotic treatment groups.

[0389] Despite this, none of the study products that included probiotics showed a statistically significant difference compared with placebo.

[0390] Table 24

[0391] Ratio of loose stools to hard stools in all stools at the 6th and 7th weeks of treatment (n=121)

[0392]

[0393] *p-value of the Kruskal-Wallis test. If P < 0.05, individual comparisons to placebo were calculated (Mann-Whitney U test with Holm correction).

[0394] The proportion of loose stools differed significantly between groups throughout the 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, possibly due to the small sample size. Participants in the probiotic mixture group had the smallest proportion of all stools that were loose.

[0395] Table 25

[0396] Proportion of participants reporting gastrointestinal infection symptoms in Diary 2 (n=118)

[0397]

[0398] N* = number of participants included in the ITT population

[0399] A significant difference was detected only in the number of constipation days between the groups (p*=0.013), which may be due to the fact that only three participants in the placebo group reported this symptom, while no participants in the other four treatment groups reported this symptom in participant diary 2. However, further analysis (Mann-Whitney U test with Holm correction) comparing the number of constipation days in the individual probiotic groups with the placebo group did not show a significant difference, which may be due to the small sample size.

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

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

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

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

[0404] In conclusion, probiotic products are safe for use in adults and show some favorable data on the gut-brain axis and stool consistency.

[0405] discuss

[0406] In the past few years, the use of Bacillus probiotics in maintaining intestinal health has gained wide support and promoted its clinical application. Their beneficial effects are related to several properties, such as antimicrobial and immunomodulatory activities, regulation of cell growth and differentiation, cell-cell signaling, cell adhesion, signal transcription and transduction, vitamin production and intestinal protection against genotoxic agents.

[0407] The trial was designed to evaluate the effects of three probiotic treatments on general health and gastrointestinal symptoms in healthy adults. There were no safety or tolerability issues and no adverse events. In this small study conducted in healthy individuals without any gastrointestinal problems, there was no negative effect on stool regularity and consistency, nor on sadness, irritability, energy, appetite, nervousness, stress, sleep, cardiovascular events, pain, and dizziness. In fact, we report a decrease in the incidence of loose stools throughout the intervention period as a result of the administration of the probiotic mixture.

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[0461] The invention is not limited to the embodiments described herein and modifications or variations may be made without departing from the scope of the invention.

[0462] The terms "a", "an", "the" and similar references used in the context of describing the invention (especially in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Unless otherwise indicated herein, the recitation of numerical ranges herein is intended merely to serve as a shorthand method of individually referring to each individual value falling within the range, and each individual value is incorporated into the specification as if it were individually recited herein. The use of the term "about" is intended to describe values ​​within a range of about ±10% above or below the stated value; in other embodiments, these values ​​may be within a range of about ±5% above or below the stated value; in other embodiments, these values ​​may be within a range of about ±2% above or below the stated value; in other embodiments, these values ​​may be within a range of about ±1% above or below the stated value. The foregoing ranges are intended to be made clear by the context and are not meant to be further limited. All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Unless otherwise specified, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention rather than to limit the scope of the present invention. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the present invention.

[0463] Although in the foregoing description, the present invention has been described in conjunction with certain embodiments of the present invention and many details have been presented for illustrative purposes, it is obvious to those skilled in the art that the present invention may have additional embodiments and that certain details described herein may be considerably changed without departing from the basic principles of the present invention.

[0464] 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 attributes thereof, and therefore, reference should be made to the appended claims rather than to the foregoing specification to indicate the scope of the invention.

Claims

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

2.

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

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

2.

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

1.

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

1.

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

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

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

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

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

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

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

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

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

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

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

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

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

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