Bacillus megaterium strains, compositions and methods of use thereof

Abstract

The present invention provides a Bacillus megaterium strain 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 megaterium strain has at least 97% identity to SEQ ID NO: 3. The strains may be used in compositions and methods.

Description

Technical Field

[0001] The present invention relates to a novel Bacillus megaterium strain, which can be used as a probiotic or with prebiotics and synbiotics alone or in combination with other Bacillus strains. The present invention also relates to a composition, such as a pharmaceutical composition, a dairy product, a functional food, a nutraceutical and a product for personal care, comprising a Bacillus megaterium strain alone or in combination, and the use 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 of thirteen selected strains in humans. The strains adhering to Caco-2 cells were tested for resistance to pH 2.5 and 0.3% ox bile, as well as antimicrobial activity against enteric pathogens (Khochamit et al., 2015). In addition to having good adhesion and biotherapeutic properties, Bacillus also showed the first condition of having GIT stress tolerance (Thakur et al., 2016).

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

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

[0012] Although long considered soil microorganisms, species of the genus Bacillus have been used for more than 50 years in the form of fermentation products or spore-based supplements (Cutting et al., 2011). Bacillus are ubiquitous in nature and are continuously introduced into the gastrointestinal tract and respiratory tract of healthy humans through food, water, and air (Benno & Mitsuoka, 1986). They are isolated from the intestine and can reach up to 10 7CFU / g, and is therefore considered to be one of the main components of the normal intestinal microflora (microbiota) (Lakshmi et al., 2017).

[0013] Bacillus megaterium has been found in a variety of habitats, from soil to seawater, sediments, rice fields, honey, fish, milk, and dried foods (Alfoldi, 1957; Alippi & Reynaldi, 2006; Padgham & Sikora, 2007; Pelletier & Sygusch, 1990; Vary et al., 2007; Von Tersch & Carlton, 1983; Scholle et al., 2003; Kotb, 2014). Further qualitative analysis of microorganisms isolated from honey showed that one of the most common Bacillus species was Bacillus megaterium (Alippi, 1995; Alippi et al., 2004; Snowdon & Cliver, 1996; Tysset, Durand & Taliergio, 1970). Additional studies have isolated Bacillus megaterium in fish (Sumathi et al., 2017). Afrilasari et al., 2015 also successfully isolated B. megaterium from the digestive tract of catfish and identified the strain as PTB 1.4. The harmless nature of B. megaterium has led to the inclusion of the bacterium in the Qualified Presumption of Safety (QPS) list (EFSA, 2017). It has been confirmed through genomic analysis that B. megaterium strain ATCC 14581 is almost identical (>99%) to the currently requested B. megaterium MIT411. Health Canada has stated that the organism is not harmful to human health or the environment; exposure to the environment and Canadians is moderate. Therefore, it can be concluded that B. megaterium strain ATCC 14581 is not harmful to human health or the environment (Health Canada, 2018).

[0014] 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

[0015] The object of the present invention is to provide strains and compositions as described in the present application, such as pharmaceutical preparations or absorption products of suitable probiotic bacillus strains having the desired properties as described above. In an embodiment, the present invention relates to Bacillus megaterium MIT411 alone or with other strains such as Bacillus strains such as 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) and Bacillus clausii strain CSI08 (disclosed in corresponding PCT application PCT / US2022 / xxxxx, and claiming priority to Irish Patent Application No. 2021 / 0209, the contents of which are incorporated herein in their entirety). In embodiments, these strains have similar or substantially identical advantageous properties, such as the ability to colonize by adhering to mucous membranes / 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 megaterium strain MIT411 and compositions for fecal transplantation.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] Another object of the present invention is to provide compositions, such as dairy products, nutritional products and functional foods, comprising Bacillus megaterium MIT411 strain alone or in combination with other Bacillus strains such as Bacillus coagulans strain and / or Bacillus clausii strain, which have substantially the same properties, 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 composition 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.

[0022] Attached photos

[0023] In the attached picture:

[0024] Figure 1 Shown is the genome analysis of Bacillus megaterium MIT411.

[0025] Figure 2 Phylogenetic tree showing Bacillus species arranged by clade (16S).

[0026] Figure 3 Phylogenetic tree showing Bacillus species arranged by clade (gyrB).

[0027] Figure 4The stability of Bacillus megaterium in phosphate buffer during pasteurization is shown; the results show the mean concentration ± standard deviation.

[0028] Figure 5 The antimicrobial activity of Renuspore against intestinal, skin and urinary tract opportunistic pathogens in solid culture medium (TSA) is shown.

[0029] Figure 6 Shown is the antimicrobial activity of B. megaterium MIT411 in liquid TSB medium against opportunistic pathogens of the intestinal tract, skin, and urinary tract: Escherichia coli (*p<0.05), Salmonella enteritidis (****p<0.0001), Pseudomonas aeruginosa (****p<0.0001), and Staphylococcus aureus (S. aureus).

[0030] Figure 7 The total antioxidant capacity of PBS and Bacillus megaterium is shown.

[0031] Fig. 8A Heavy metal bioaccumulation of Renuspore in TSB medium supplemented with 1 ppm lead is shown.

[0032] Figure 8B Heavy metal bioaccumulation of Renuspore in TSB medium supplemented with 1 ppm mercury is shown.

[0033] Fig. 9 The iron concentration in the extracellular part of Renuspore is shown.

[0034] Fig.10 The calcium concentration in the extracellular part of Renuspore is shown.

[0035] Fig.11 The magnesium concentration in the extracellular part of Renuspore is shown.

[0036] Fig.12 It was shown that B. megaterium did not affect the concentration of bisphenol A in TST or MM medium.

[0037] Fig.13 Shown is the nitrite concentration in the Renuspore extracellular matrix.

[0038] Fig.14A Shown is the degradation of ammonia by Renuspore.

[0039] Fig. 14BShown is the remaining ammonia concentration in TSB + 1 mM ammonia after incubation of Renuspore and control at 37°C for 24 hours.

[0040] Fig.15 Shown is the adhesion of Bacillus megaterium MIT411 spores and vegetative cells to HT-29 and HT-29MTX cells at 37°C.

[0041] Fig.16 The casein decomposition activities of Bacillus megaterium MIT411 (positive) and Bacillus coagulans (negative) were shown, and were detected at 24 hours using a skim milk agar medium by a conventional method.

[0042] Fig.17 Shown are protease activities shown using the quantitative extracellular protease assay, Renuspore, using the EnzCheck kit.

[0043] Fig.18 Shown is the increased FAA in the Renuspore UHT fermented milk samples.

[0044] Fig.19 Shown is the increased FAA in the Renuspore UHT fermented milk samples.

[0045] Fig. 20 Shown is the increased FAA in the Renuspore UHT fermented milk samples.

[0046] Fig.21 Shown is the increased FAA in the Renuspore UHT fermented milk samples.

[0047] Fig. 22 Shown are the increased SCFAs in Renuspore UHT fermented milk samples.

[0048] Fig.23 Compared with the control, after 24 hours of culture in the basic medium, There was no significant increase in the concentration of Renuspore (CFU / mL).

[0049] Fig.24 and 25 Renuspore was shown to increase the expression of cytokines in a human macrophage culture model.

[0050] Fig.26 Renuspore was shown to not increase the survival of C. elegans after exposure to H2O2.

[0051] Fig. 27The adhesion ability of Bacillus megaterium MIT411 propagated somatic cells and spores to the intestinal epithelial cell lines HT-29 and HT-29-MTX at 37°C is shown.

[0052] Fig.28 Graphical flow chart showing the study design.

[0053] Fig.29 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.

[0054] Fig.30 It was shown that any of the treatments given during the study had no effect on the percentage of hard stools compared to the placebo control.

[0055] Fig.31 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.

[0056] Fig.32 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.

[0057] Fig.33 Shows PCoA clustering performed on the Bray-Curtis dissimilarity matrix. DETAILED DESCRIPTION

[0058] Genotype identification

[0059] The applicant collaborated with Cornell University (Ithaca NY, USA) to conduct genome sequencing and characterization.

[0060] WGSDNA composition

[0061] Whole genome sequencing (WGS) was performed by Cornell University, including assembly and annotation. Bioinformatics analysis was performed at Cornell University and Deerland Probiotics and Enzymes (Kennesaw GA, USA). The applicant performed the identification of gyrB gene polymorphism.

[0062] The gyrB gene encodes DNA gyrase subunit B. DNA gyrase negatively supercoils closed circular double-stranded DNA in an ATP-dependent manner to maintain the underwound state of chromosomes. Gene sequencing analysis was performed using gyrB gene polymorphism, which is a well-established method for prokaryotic species identification (Bavykin et al., 2014; Wang et al., 2007). Representative genomes were reviewed and curated by NCBI and coordinated with the UniProtein Consortium (NCBI, 2016; UniProt, 2016). SequinR, an R software package, combined with UniProt Consortium analysis, was used to compare the whole genome sequence (WGS) and GyrB sequence of the currently claimed Bacillus coagulans CGI314 with other reference strains (Tables A, B, and C below).

[0063] Identification of genotype, gyrB and 16S rRNA of Bacillus megaterium MIT411

[0064] MIT411 was isolated and the genome was considered successful.

[0065] The genome size (5.4 MBP) and GC content (37.8%) of this isolate were comparable to those of Bacillus megaterium strains.

[0066] Table A

[0067] MIT411 whole genome sequencing indicators.

[0068]

[0069] Table B

[0070] Distance matrix of the gyrB gene.

[0071] Bacillus megaterium subsp. ATCC 14581 MIT411 99.9

[0072] Table C

[0073] Whole genome sequence comparison

[0074] Bacillus megaterium subsp. Accession Number %GC Sequence length MIT411 JABBNK000000000.1 37.81 5,416,213 ATCC 14581 GCA_000764085.1 37.73 5,727,066

[0075] 16S rRNA

[0076] Whole genome sequencing (WGS) and 16S rRNA analysis of MIT-411 compared to a reference strain showed an average nucleotide identity (ANI) score of >99% for 16S rRNA compared to B. megaterium strain ATCC-14581. The genome size (5.4 Mbp) and GC content (37.8%) of B. megaterium MIT-411 were comparable to those of the reference strain.

[0077] Further deposits and accession numbers

[0078] 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) .

[0079] The genome sequence data of Bacillus clausii strain CSI08 (Munispore) was deposited into the NCBI GenBank database and the genome sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The genome is publicly available, the strain's GenBank accession number is JABBNL000000000.1, and can be obtained through the following link: Alkalihalobacillus clausii strain CSI08, whole genome shotgun sequence–Nucleotide–NCBI (nih.gov) .

[0080] 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 .

[0081] Phylogenetic positioning was performed by DiLan Probiotics and Enzymes Ltd.

[0082] 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 analysis. GGDC confirmed that Bacillus megaterium MIT411 and ATCC14581 are closely related groups.

[0083] Although the conserved 16S rRNA sequence is a well-established method for comparing and studying bacterial phylogeny, the high ratio of sequence similarity between closely related species limits its usefulness (Wang et al., 2007). The high ratio of 16S rRNA sequence similarity in closely related bacterial species is due to the slower rate of molecular evolution. Past studies (Bavtlin et al., 2004; Wang et al., 2007) support the validity of using gyrB sequences as taxonomic biomarkers because of their base substitution rates and their significant and reliable correlation with DNA-DNA hybridization analysis (Dauga et al., 2002; Kasai et al., 1998; Wang et al., 2007). gyrB encodes DNA gyrase B and a type II topoisomerase that plays an important role in DNA replication. The gyrase B subunit is encoded by the gyrB gene.

[0084] Phylogenetic analysis using the neighbor-joining method (NJ) (Saitou & Nei, 1987) placed Bacillus megaterium MIT411 and Bacillus megaterium ATCC 15481 in the same branch ( Figure 2 ). This confirms all previous genome identity determinations. B. megaterium MIT411 has been assigned to the Bacillus megaterium group.

[0085] definition

[0086] "Excipient" refers to any inactive ingredient added to form part of the final formulation.

[0087] "Probiotics" refers to live microbial supplements that have a beneficial effect on a subject through action in the intestinal tract, urinary tract, vagina, skin, and / or other areas of the subject's body. The term may refer to live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Foods and food additives containing probiotics can help restore a healthy balance of the intestinal microflora. In addition, probiotics that supplement the intestinal flora can promote a healthy intestinal balance.

[0088] "Prebiotics" are used herein as substrates which have a beneficial effect on probiotics and thus on the individual subject taking (eg administering) the probiotics. Suitable prebiotics may be selected from inulin, oligosaccharides and / or vitamins.

[0089] 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 megaterium strains (e.g., MIT411) of the present invention. Optionally, the subject is a human, a patient, and / or a mammal.

[0090] A “synbiotic product” is a combination of probiotics and prebiotics that work synergistically to have a beneficial effect on patients.

[0091] "Hardy growth" means that the bacteria show very good growth.

[0092] The abbreviation "CFU" means colony forming units.

[0093] 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.

[0094] According to a first aspect, the present invention includes Bacillus megaterium strain MIT411 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 clausii strains and Bacillus coagulans strains. Such other bacillus strains also include Bacillus clausii strains and Bacillus coagulans strains, each of which is filed today under its own title, the contents of which are incorporated herein in their entirety.

[0095] As described in the appended claims, SEQ ID NO: 1 includes gyrB of Bacillus megaterium MIT411.

[0096] As described in the appended claims, SEQ ID NO: 2 includes the 16S rRNA of Bacillus megaterium MIT411.

[0097] As described in the appended claims, SEQ ID NO: 3 comprises the assembled complete genome sequence of Bacillus megaterium MIT411.

[0098] The Bacillus strain claimed herein has at least 97% identity to SEQ ID NO: 1 and / or 2; or at least 97% identity to SEQ ID NO: 3, having the following properties:

[0099] Bacillus megaterium MIT411:

[0100] This strain showed bile stability.

[0101] This strain showed acid stability.

[0102] This strain showed heat tolerance.

[0103] This strain produces natural antimicrobial substances in the form of bacteriocins.

[0104] 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.

[0105] The strain was shown to have little or no antibiotic resistance and presented no safety concerns.

[0106] The strain was found to exhibit stability to acid and bile.

[0107] According to a second aspect, the Bacillus strains of the invention are suitable for the prevention or treatment of vaginal infections, urinary tract infections and gastrointestinal diseases (including gastrointestinal infections), as well as medical uses for improving immune health, protection from oxidative stress, cleansing and detoxification, metabolic health and cardiovascular health.

[0108] In another preferred embodiment, a composition, such as a pharmaceutical composition, is provided, which includes Bacillus megaterium MIT411 alone or in combination with other probiotic bacillus 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 clausii strains and Bacillus coagulans 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.

[0109] 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.

[0110] 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.

[0111] Bacillus megaterium has protective effects 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.

[0112] and chelate heavy metals, all of which were performed in vitro (Ferreira et al., 2019).

[0113] Preferably, the probiotic bacteria used in the present invention are present in an amount of 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 megaterium MIT411, basically composed of about 10 6 - Approx. 10 13 CFU, such as about 10 9Bacillus megaterium MIT411, composed of about 10 6 - Approx. 10 13 CFU, such as about 10 9 Bacillus megaterium MIT411 is composed of 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 megaterium MIT411 (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 megaterium MIT411 is in spore form, or is not in spore form.

[0114] In certain embodiments, the composition comprising Bacillus megaterium MIT411 may include one or more dry carriers selected from the group consisting of trehalose, maltodextrin, rice flour, microcrystalline cellulose, magnesium stearate, inositol, oligofructose, oligogalactose, dextrose, dry dairy products, etc. In certain embodiments, the dry carrier may be added to the composition comprising Bacillus megaterium MIT411 at a weight percentage of about 1% to about 95% by weight of the composition.

[0115] In certain embodiments, the composition comprising Bacillus megaterium MIT411 may include one or more liquid or gel-based carriers selected from the group consisting of water and physiological salt solutions, urea, 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 megaterium MIT411 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 megaterium MIT411 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 megaterium MIT411 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 megaterium MIT411 at a weight / volume percentage of about 2% weight / volume of the composition. In certain embodiments, one or more spore germination inhibitors may be added to a composition including Bacillus megaterium MIT411 at a weight / volume percentage of about 1% weight / volume of the composition.

[0116] Delivery System

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

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

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

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

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

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

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

[0124] Liquid preparations include but are not limited to solutions, suspensions and emulsions, such as water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in polyethylene glycol aqueous solutions. In certain embodiments, the compound used according to the method of the present disclosure can be formulated for parenteral administration (for example, by injection, for example, bolus injection or continuous infusion), and can be present in unit doses, for use in ampoule, prefilled syringe, small volume infusion or 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.

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

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

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

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

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

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

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

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

[0133] Route of administration

[0134] Compounds can be administered by any route, including but not limited to oral, sublingual, oral, ocular, pulmonary, rectal and parenteral administration, or as oral or nasal sprays (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 into a local location.

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

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

[0137] Example

[0138] Example 1

[0139] Further characterization of Bacillus megaterium MIT411 (hereinafter also referred to as Renuspore)

[0140] Temperature stability

[0141] Bacillus megaterium MIT411 in PBS - pH 7.51 - Stable in PBS from 30 seconds to 3 minutes at 45°C, 75°C and 90°C ( Figure 4 ).

[0142] Renuspore is stable during pasteurization and other production methods for food, beverages and other applications.

[0143] Figure 4 The stability of Bacillus megaterium in phosphate buffer during pasteurization is shown; the results show the mean concentration ± standard deviation.

[0144] Antimicrobial activity against enteric and skin pathogens in solid environments:

[0145] Bacillus megaterium MIT411 (Renuspore) has weak antimicrobial activity, and a vague inhibition zone was observed against Escherichia coli, Salmonella enteritidis, and Staphylococcus aureus on TSA overlaid with 0.4% TSA agar ( Figure 5 and Table 1). No antimicrobial activity was observed against Pseudomonas aeruginosa (P. aeruginosa) in solid medium.

[0146] Table 1

[0147]

[0148] Table 1. Antimicrobial activity of Renuspore against opportunistic pathogens of the intestinal tract, skin and urinary tract in solid medium (TSA). Antimicrobial activity is expressed as inhibition zone (mm) ± standard deviation.

[0149] Figure 5 The antimicrobial activity of Renuspore against intestinal, skin and urinary tract opportunistic pathogens in solid medium (TSA) is shown. A fuzzy inhibition zone was observed around the growth of Bacillus megaterium MIT411. The antimicrobial activity is expressed as the standard deviation of the inhibition zone (mm). A-Escherichia coli (E.coli) and B-Salmonella enteritidis (S.enteritidis) and C-Staphylococcus aureus (S.aureus).

[0150] In solid media, Renuspore exhibited broad antimicrobial properties, with activity against the enteric pathogen Salmonella enterica and the enteric and urinary tract opportunistic pathogen Escherichia coli. Renuspore was also active against the skin opportunistic pathogen Staphylococcus aureus (S. aureus).

[0151] Renuspore therefore has the potential to crowd out bacterial pathogens and maintain a healthy intestinal and skin microflora.

[0152] Antimicrobial Activity Against Enteric and Skin Pathogens in Liquid Environments:

[0153] Figure 6 Shown is the antimicrobial activity of Bacillus megaterium MIT411 against opportunistic pathogens of the intestinal tract, skin and urinary tract in liquid TSB medium: Escherichia coli, Salmonella enteritidis, Pseudomonas aeruginosa and Staphylococcus aureus. Controls represent the growth of pathogens respectively; treatments represent the growth of pathogens in the presence of Bacillus megaterium MIT411. *p<0.05 and ****p<0.0001.

[0154] In liquid culture, Renuspore inhibits the growth of the enteric pathogen Salmonella Enteritidis and the intestinal and urinary tract opportunistic pathogen Escherichia coli. In liquid culture conditions, Renuspore is active against the skin and urinary tract opportunistic pathogen Pseudomonas aeruginosa.

[0155] Renuspore has the potential to crowd out bacterial pathogens and maintain a healthy intestinal and skin microflora.

[0156] Antimicrobial activity against enteric and skin pathogens in liquid environments:

[0157] Bacillus megaterium MIT411 (Renuspore) has significant antimicrobial activity against Salmonella enteritidis and Pseudomonas aeruginosa in liquid TSB medium ( Figure 6 and Table 2). Under these conditions, weak antimicrobial activity was detected against Escherichia coli, and no antimicrobial activity was observed against Staphylococcus aureus.

[0158] Table 2

[0159]

[0160] Table 2 Summary of the antimicrobial activity of Bacillus megaterium MIT411 against opportunistic pathogens of the intestinal tract, skin and urinary tract. Antimicrobial activity was detected (+), and no antimicrobial activity was observed (-).

[0161] Renuspore therefore has the potential to crowd out bacterial pathogens and maintain a healthy intestinal and skin microflora.

[0162] · Antioxidant activity The antioxidant activities of Bacillus megaterium Renuspore and Lactobacillus rhamnosus were compared.

[0163] Figure 7 The total antioxidant capacity of PBS and Bacillus megaterium is shown. The results show the mean concentration of Trolox equivalents in nmole / g (n=3) ± standard error. Renuspore had higher antioxidant levels compared to the potential probiotic Lactobacillus rhamnosus (not shown).

[0164] According to Tukey's multiple comparison test, Renuspore showed significant antioxidant levels and was not significantly different from Fortispore (Bacillus coagulans CGI314).

[0165] Renuspore bioaccumulates lead and removes it from the environment:

[0166] Renuspore can eliminate 37.97% of bioavailable lead ( Fig. 8A ). Renuspore has also been shown to be effective in bioaccumulating heavy metals.

[0167] Fig. 8A Heavy metal bioaccumulation of Renuspore in TSB medium supplemented with 1 ppm lead is shown. Results show mean concentrations in ppm (n=5) ± standard error. Significant reduction was observed between Renuspore and control: ***p=0.001.

[0168] Renuspore is able to bioaccumulate the most common heavy metal in our environment - lead. These data show the potential of Renuspore to bioaccumulate environmental pollutants such as heavy metals present in the environment and prevent their harmful effects.

[0169] Renuspore can act as a potential probiotic for the biological removal of heavy metals, thereby alleviating the effects of heavy metals on the human body.

[0170] Heavy Metal Bioaccumulation - Mercury

[0171] Renuspore can effectively bioaccumulate mercury, reducing free bioavailable mercury by 85.80%. Figure 8B ).

[0172] Figure 8BShown is the heavy metal bioaccumulation of Renuspore in TSB medium supplemented with 1 ppm mercury. The results show the mean concentration in ppm (n=5) ± standard error. It is shown that a significant reduction was observed between Renuspore and the control: ****p<0.0001.

[0173] Renuspore is able to bioaccumulate two of the most common heavy metals in our environment - lead and mercury. In summary, these data show the potential of Renuspore to bioaccumulate environmental pollutants such as heavy metals present in the environment and prevent their harmful effects.

[0174] Renuspore can act as a potential probiotic for the biological removal of heavy metals, thereby alleviating the effects of heavy metals on the human body.

[0175] Iron bioaccumulation

[0176] Renuspores were cultured in TSB medium in the presence of iron and their supernatants were assayed. TSB medium + iron was used as a control. The results showed that Renuspores did not bioaccumulate iron in TSB medium, as the iron concentration in the extracellular part remained unchanged ( Fig. 9 ).

[0177] Fig. 9 The iron concentration in the extracellular part of Renuspore is shown. The results show the average concentration of total iron concentration in nanomoles / ml (n=3) ± standard error.

[0178] Renuspore can act as a potential probiotic for the bioremoval of toxic heavy metals without affecting the body's natural absorption of essential minerals such as iron.

[0179] Renuspore does not bioaccumulate calcium:

[0180] Renuspore was cultured in TSB medium in the presence of calcium and its supernatant was assayed. TSB medium + calcium was used as a control. Renuspore did not bioaccumulate calcium in TSB medium because the calcium concentration in the supernatant remained unchanged ( Fig.10 ).

[0181] Fig.10 The calcium concentration in the Renuspore extracellular part is shown. The results show the mean concentration of calcium concentration in μM (n=3) ± standard error using Dunnett's t test.

[0182] Renuspore can act as a potential probiotic for the bioremoval of toxic heavy metals without affecting the body's natural absorption of essential minerals such as calcium.

[0183] Renuspore does not bioaccumulate magnesium:

[0184] Renuspore was cultured in TSB medium in the presence of magnesium and its supernatant was assayed. TSB medium + magnesium was used as a control. Using Dunnett's t-test, the results showed no significant difference between Renuspore and the control (TSB medium). This suggests that Renuspore does not bioaccumulate magnesium from the environment ( Fig.11 ).

[0185] Fig.11 The magnesium concentration in the extracellular part of Renuspore is shown. The results show the mean concentration of magnesium concentration in mmol / L (n=3) ± standard error, using Dunnett's t-test.

[0186] This study demonstrates that Renuspore does not bioaccumulate magnesium or compete with the intestine for the absorption of this essential mineral.

[0187] Renuspore can act as a potential probiotic for the bioremoval of toxic heavy metals without affecting the body's natural absorption of essential minerals such as magnesium, iron and calcium.

[0188] Renuspore does not utilize or degrade bisphenol A (BPA):

[0189] Bacillus megaterium MIT411 (also known as Renuspore) was unable to utilize BPA as a sole carbon source, as no growth was observed at all BPA concentrations analyzed (5 mg / L to 100 mg / L) in minimal medium (MM) agar and broth. Cell growth of B. megaterium decreased with increasing BPA concentrations (Table 3). In both MM and TSB broth, B. megaterium did not decrease the concentration of 5 mg / L BPA over time ( Fig.12 ).

[0190] Fig.12 It is shown that B. megaterium does not affect the concentration of bisphenol A in TST or MM medium (leftmost vertical bar: control; T=24, 48, 72, 96, 120 hours, vertical bars from left to right).

[0191] Table 3

[0192]

[0193] Table 3 Bacillus megaterium was grown in TSB broth and the absorbance was measured at 600 nm.

[0194] Renuspore does not have the ability to degrade BPA present in the environment: Renuspore does not utilize or degrade DEET (N,N-diethyl-m-toluamide):

[0195] No genes for DEET hydrolase were detected in the Renuspore genome. Renuspore cannot utilize DEET as an energy source using minimal medium. In addition, increasing the concentration of this synthetic chemical in nutrient-rich medium had a toxic effect on the growth of Renuspore (Tables 4, 5, and 6). Therefore, Renuspore cannot use DEET as a food source or break it down into less toxic products.

[0196]

[0197] Table 4 CFU / ml of Renuspore in TSB with added DEET at T24.

[0198]

[0199] Table 5 Renuspore (OD600) cell growth in mineral salt medium (MM) supplemented with DEET at T24

[0200]

[0201] Table 6 Renuspore cell growth (OD600) in TSB supplemented with DEET at T24

[0202] Renuspore does not have the ability to degrade DEET present in the environment.

[0203] Renuspore can detoxify nitrite in the environment:

[0204] Renuspores were cultured in TSB medium in the presence of nitrite and their supernatants were assayed. TSB medium + nitrite was used as a control. Renuspores completely removed nitrite from the environment and began to convert it to nitrate or nitric oxide ( Fig.13 ). Renuspore can reduce nitrite to nitric oxide using nitrite reductase or oxidize nitrite to nitrate using oxidoreductase - both enzymes are found in its genome.

[0205] Fig.13The nitrite concentration in the Renuspore extracellular matrix is ​​shown. The results show the mean concentration of nitrite in nanomoles / ml (n=3) ± standard error. It is shown that a significant reduction was observed between the control and Renuspore: ****p<0.0001.

[0206] Renuspore can remove nitrite from the environment and play an important role in reducing the toxic level of nitrite in the human body.

[0207] In conclusion, Renuspore can act as a potential probiotic for the bioremoval of toxic nitrite, oxidizing it to less harmful products such as nitrate.

[0208] Renuspore cannot biodegrade ammonia:

[0209] Minimal salts medium containing ammonium chloride as the sole nitrogen source was used to evaluate whether Renuspore could use ammonia as a nitrogen source. Renuspore was able to grow in minimal medium in the presence of glucose, magnesium sulfate, and calcium chloride, thereby using 30% of the ammonia from the medium ( Fig.14A In TSB medium, Renuspore was able to synthesize ammonia, probably from the peptide source present in TSB medium, as a 47.9% increase in ammonia concentration was observed compared to the control ( Fig. 14B ).

[0210] Fig.14A The degradation of ammonia by Renuspore is shown. The results show the average concentration of ammonia in μmol / L (n=3) ± standard error, using Tukey's test. Note: The symbol ** indicates the significance between Renuspore and the control (P<0.05).

[0211] Fig. 14B The remaining ammonia concentration in TSB + 1 mM ammonia after Renuspore and control were incubated at 37°C for 24 hours is shown. Note: The symbol ** indicates the significance between Renuspore and control (P<0.05)

[0212] Renuspore can utilize ammonia as a nutrient source.

[0213] Renuspore adheres to intestinal epithelial cells:

[0214] Fig.15 Shown is the adhesion of Bacillus megaterium MIT411 spores and vegetative cells to HT-29 and HT-29MTX cells at 37°C.

[0215] Bacillus megaterium MIT411 vegetative cells do not adhere to HT-29 and HT-29-MTX enterocyte cell lines. Bacillus megaterium MIT411 spores adhere to HT-29 and the mucus-producing HT-29-MTX cell lines; therefore, it can attach to enterocytes and germinate into vegetative cells.

[0216] The reproductive somatic cells can bioaccumulate toxic environmental pollutants and remove them from the body without attaching to the intestine.

[0217] Renuspore shows high protease activity:

[0218] Renuspore showed caseinolytic activity on skim milk agar plates (see Fig.16 ). Quantitative analysis of Renuspore caseinolytic activity was assessed by using a commercial kit employing fluorescently labeled casein derivatives. Renuspore exhibits extracellular protease activity. Genomic analysis of Renuspore revealed the presence of multiple genes encoding the caseinolytic protease CEP (prtP), which explains this high protease activity of Renuspore.

[0219] Fig.16 Shown is the caseinolytic activity of Bacillus megaterium MIT411 (positive) and Bacillus coagulans (negative), detected by conventional methods using skim milk agar medium at 24 hours. The clearing zone indicates the extent of casein degradation. The plate on the left shows the streaked plate, and the plate on the right shows an inoculation of the MIT411 strain in TSB overnight.

[0220] Fig.17 Shown are protease activities shown using the quantitative extracellular protease assay, Renuspore, using the EnzCheck kit.

[0221] Both in silico and in vitro analyses have shown that Renuspore is able to hydrolyze milk proteins, particularly casein.

[0222] Renuspore has a diverse carbohydrate profile: Renuspore metabolizes a range of monosaccharides, sugar alcohols, amine sugars, and glycosides:

[0223] Using the commercial API 50CH test, Renuspore was positive for 11 of the 49 carbohydrates. Most of these carbohydrates are monosaccharides, such as D-ribose, L-arabinose, D-xylose, D-glucose, D-fructose, and D-sucrose. The genome analysis of Renuspore revealed the presence of transporters and enzymes involved in the metabolism of most of these sugars. In addition, genes involved in polysaccharide metabolism, amylase A involved in starch metabolism, were also identified in the genome of Renuspore.

[0224]

[0225] Table 7 List of carbohydrates efficiently fermented by Bacillus megaterium MIT411 using API 50Ch strips.

[0226] Both computer and in vitro analyses showed that Renuspores vary in their ability to ferment a range of carbohydrates.

[0227] Renuspore has enzymatic activity towards esters, proteins and carbohydrates:

[0228] Using the API ZYM kit, Renuspore tested positive for esterase, α-chymotrypsin, alkaline phosphatase (ALP), and galactosidase activities, indicating that:

[0229] In the presence of an appropriate lipid source, Renuspore has a high probability of producing free fatty acids through the action of esterases.

[0230] - The α-chymotrypsin activity of Renuspore is capable of hydrolyzing amide bonds where the amino acid N-terminal to the bond is tryptophan, tyrosine, phenylalanine or leucine, and this activity should increase the proteolytic capacity of Renuspore.

[0231] Galactosidase increases the carbohydrate catabolic potential of the Renuspore, as they are active against various oligosaccharides, lactosylceramide, lactose, and many glycoproteins.

[0232] Indeed, computer analysis has identified genes encoding esterase, ALP, protease and galactosidase.

[0233]

[0234] Table 8. Enzyme profile of Renuspore using API ZYM kit.

[0235] This study confirmed Renuspore's ability to hydrolyze proteins and oligosaccharides and showed its potential to decompose fat.

[0236] These data suggest that Renuspore may aid digestion of these molecules in the intestine.

[0237] Renuspore produces a variety of amino acids from milk protein hydrolysis:

[0238] The proteolytic capacity of Renuspore was analyzed using a UHT milk model. Computer analysis of Renuspore revealed the presence of a series of proteases, peptide transporters, and peptidases, indicating the presence of a powerful proteolytic system in Renuspore. In Renuspore, GC-MS analysis identified a total of 38 free amino acid (FAA) compounds, of which 28 were statistically significant. The results of this analysis confirmed the presence of a highly active proteolytic system in Renuspore that can completely degrade milk proteins to release FAA. In addition, the proteolytic system in Renuspore showed the potential to further catabolize these amino acids to produce aromatic carboxylic acids (4-methyl-2-oxopentanoic acid, benzoic acid, octavalic acid, and propionic acid).

[0239]

[0240] Table 9 Statistically significant compounds analyzed by GC-MS using the FAA method are listed together with potential precursors.

[0241] Both computer analysis and in vitro analysis showed that Renuspore has a strong and active proteolytic system, releasing a large amount of amino acids and obtaining their downstream products in Renuspore fermented UHT milk.

[0242] Renuspore produces various amino acids from milk protein hydrolysis: Renuspore FAA analysis is presented as a bar graph (mean + SEM):

[0243] Fig.18 Shown is the increased FAA in Renuspore UHT fermented milk samples. Statistical analysis was performed using multiple T-tests - using unpaired parameters, two-stage step-up method (Benjamini, Krieger and Yekutieli, and P-value ≤ 0.01 = *. White bars represent controls.

[0244] Fig.19 Shown is the increased FAA in Renuspore UHT fermented milk samples. Statistical analysis was performed using multiple T-tests - using unpaired parameters, two-stage step-up method (Benjamini, Krieger and Yekutieli, and P-value ≤ 0.01 = *. White bars represent controls.

[0245] Fig. 20Shown is the increased FAA in Renuspore UHT fermented milk samples. Statistical analysis was performed using multiple T-tests - using unpaired parameters, two-stage step-up method (Benjamini, Krieger and Yekutieli, and P-value ≤ 0.01 = *. White bars represent controls.

[0246] Fig.21 Shown is the increased FAA in Renuspore UHT fermented milk samples. Statistical analysis was performed using multiple T-tests - using unpaired parameters, two-stage step-up method (Benjamini, Krieger and Yekutieli, and P-value ≤ 0.01 = *. White bars represent controls.

[0247] Renuspore showed weak lipolytic activity: Renuspore fermentation of UHT milk produced limited short-chain fatty acids (SCFA):

[0248] Although Renuspore showed esterase decomposition activity and possessed genes encoding esterase A and lipase, only two SCFAs were significantly increased in the Renuspore-fermented UHT milk samples.

[0249] Fig. 22 Increased SCFAs in Renuspore UHT fermented milk samples are shown. Statistical analysis was performed using multiple T-tests - using unpaired parameters, two-stage step-up method (Benjamini, Krieger and Yekutieli, and P-value ≤ 0.01 = *. White bars represent controls.

[0250] The only two SCFAs associated with the Renuspore are propionate and 2-methylpropionate, and they are generally associated with amino acid metabolism, particularly alanine and valine, respectively. Taken together, these data suggest that the lipolytic activity of the Renuspore is low.

[0251] Renuspore proteomic analysis identifies proteins with potential probiotic benefits: Proteomic studies - Renuspore secretome:

[0252] The extracellular secretions of Renuspore grown in TSB broth for 24 h were sent to a mass spectrometer to identify proteins released by the probiotic strains. A total of 23 proteins were detected, of which 4 had potential probiotic benefits (Table 9A):

[0253] Table 9A

[0254] Target protein Potential role Arginase Participate in the urea cycle to increase ammonia detoxification Metal-dependent hydrolases Involved in the digestion of proteins and carbohydrates Thiol peroxidase Participates in the reduction of hydrogen peroxide-antioxidant LysM - protein including a peptidoglycan binding domain Potential antimicrobial properties

[0255] These data confirm previous in vitro findings showing that Renuspore can aid in the digestion of proteins and carbohydrates, can detoxify harmful compounds, and has antimicrobial properties against pathogens.

[0256] ·Present in basic culture medium (F) Case of Renuspore:

[0257] Fig.23 Compared with the control, after 24 hours of incubation in minimal medium, There was no significant increase in the concentration of Renuspore (CFU / mL).

[0258] Immunomodulatory capacity of Renuspore in an in vitro human macrophage model:

[0259] Fig.24 and 25 Renuspore was shown to increase the expression of cytokines in a human macrophage culture model. Unlike the LPS positive control, Renuspore increased the expression of all cytokines tested (TNF-α, IL-1β, IL-18, IL-6, GM-CSF, IL-10, IL-1RA, and EGF). Renuspore was more effective than LPS in inducing the expression of TNF-α, GM-CSF, and EGF. Therefore, Renuspore can be considered a strong stimulator of the innate immune system. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 were significantly higher than the negative control; + p<0.05, ++ p<0.01, ++++ p<0.0001, significantly higher than the positive control.

[0260] Antioxidant capacity of Renuspore in the H2O2 oxidative stress C.elegans model:

[0261] Fig.26 Renuspore was shown to not increase the survival of C. elegans after exposure to H2O2. Vitamin C was used as a positive control for the assay. *P<0.05 significantly higher than control.

[0262] Example 2

[0263] Assessing adhesion capacity on an in vitro model of intestinal epithelium

[0264] 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.

[0265] The cells were plated at 5x10 5 Cells were seeded into 24-well plates at a density of 10 cells / well and cultured for 21-28 days to complete maturation. The culture medium was changed every 2-3 days.

[0266] 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.

[0267] 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.

[0268] 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).

[0269] After incubation, HT-29 and HT-29-MTX cells were washed 4 times with 0.5 ml PBS. Afterwards, 50 μl of trypsin / EDTA solution and 50 μl of PBS were added to the wells and incubated for 10 min at 37° C. with gentle shaking (~100 rpm). Fifty microliters of trypsin / EDTA solution were added to the control wells.

[0270] 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.

[0271] 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). The plates were incubated at 37°C for 48 h and then counted, while the PetriFilm was incubated at 37°C for 24 h and then counted.

[0272] Experiments were performed twice or three times with three technical replicates per experiment. Results are expressed as mean ± SEM.

[0273] Fig. 27 The adhesion capacity of Bacillus megaterium MIT411 propagated cells and spores to the intestinal epithelial cell lines HT-29 and HT-29-MTX at 37°C is shown (DI_EK_03). Fig. 27 , showing (left panel) the percentage of bacteria adhered on HT-29 using Bacillus megaterium MIT411 spores (left column) and Bacillus megaterium MIT411 propagules (right column). See also Fig. 27 , showing (right panel) the percentage of bacteria adhered on HT-29-MTX using B. megaterium MIT411 spores (left column) and B. megaterium MIT411 propagules (right column).

[0274] In a comparative study, the adhesion of Bacillus clausii CSI08, Bacillus megaterium MIT411 and Bacillus coagulans CGI314 spores to the HT-29-MTX cell line was as follows (Table D):

[0275] Table D

[0276]

[0277] Adhesion of Bacillus clausii CSI08, Bacillus megaterium MIT411 and Bacillus coagulans CGI314 spores to HT-29 cell line (Table E):

[0278] Table E

[0279]

[0280] in conclusion

[0281] 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.

[0282] 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.

[0283] Example 3

[0284] 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.

[0285] 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 of Bacillus subitilis 0.5x10 9 CFU of Bacillus megaterium MIT411, 0.5 x 10 9 CFU of Bacillus coagulans CGI314, 0.5x 10 9 CFU of Bacillus clausii CSI08 (Bacillus subtilis The total counts of Bacillus megaterium MIT411, Bacillus coagulans CGI314, and Bacillus clausii CSI08 were 2.0 x 10 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.

[0286] 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.

[0287] Probiotic supplements can contain one or more different strains that have different effects on the human gut. Common probiotics are lactic acid producers such as Lactobacillus, Bifidobacterium, and Streptococcus because of their tolerance to gastric acid, bile salts, and pancreatic enzymes. Studies have shown that lactic acid bacteria are effective inhibitors of colonization of pathogenic Gram-negative bacteria (e.g., Salmonella typhimurium, Clostridium difficile, and Escherichia coli) in vitro.

[0288] 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.

[0289] 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.

[0290] 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.

[0291] Materials and methods

[0292] Subjects

[0293] Healthy adult volunteers aged 18-65 years were recruited from February to July 2021 through flyers, posters, and their 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 medications, 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).

[0294] 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.

[0295] Experimental design

[0296] This study was a double-blind, placebo-controlled, randomized, parallel trial. The study was conducted at 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 a placebo administered daily. 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.

[0297] 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.

[0298] 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.28 shown.

[0299] 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.

[0300] Fig.28 Graphical flow chart showing the study design.

[0301] Probiotic administration regimen

[0302] The study product was provided by DiLan Probiotics & 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.

[0303] Study plan

[0304] 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 tract infection symptoms, headache, ENT, behavior, 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 researchers.

[0305] Blood sample collection and preparation

[0306] For safety blood, 3-mL red-cap serum coagulant tubes (Greiner Bio-One, 454029) were used for blood collection. Biochemical blood samples were measured for high-density lipoprotein and low-density lipoprotein, total cholesterol and triglycerides 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 15min. The supernatant was aliquoted and stored at -80°C for subsequent analysis.

[0307] LDL, HDL, total cholesterol and triglyceride measurements

[0308] Hematological and biochemical evaluations were performed at the University Clinical Center in Maribor, Slovenia. Safety blood was run with a Sysmex EN-1000, while biochemical determinations of LDL, HDL, total cholesterol, and triglycerides were performed according to the manufacturer's instructions and analyzed with an Abbott Allinity C.

[0309] Cytokine quantification

[0310] 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.

[0311] Antioxidant activity assay

[0312] 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.

[0313] Feces collection

[0314] Using Zymokit DNA / RNA Shield TM Fecal collection tubes (Zymo Research, 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.

[0315] DNA extraction and 16S rRNA sequencing

[0316] Total fecal DNA was extracted from approximately 200 mg of sample using the Zymo BIOMICS DNA Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's instructions. Briefly, fecal samples were placed in a 4% paraformaldehyde (5% dHO) containing 750 μl of Zymo BIOMICS 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 was further purified by using a III-HRC filter. The DNA concentration was determined using the Qubit dsDNA BR assay kit (ThermoFisherScientific).

[0317] Data Generation

[0318] 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.

[0319] Statistical analysis

[0320] Twenty-five participants per arm were determined to be sufficient to assess the incidence and nature of possible adverse events, including the incidence 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 correction was used for pairwise comparisons of the three probiotic product groups with the placebo group.

[0321] 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.

[0322] 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.

[0323] 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.

[0324] 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.

[0325] result

[0326] Participants

[0327] Ninety-eight participants completed the 45-day intervention ( Fig.28 ). 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.

[0328] Causality assessment showed no relationship between the reported AEs and the study product.

[0329] No serious adverse events were reported during the entire study.

[0330] Basic information of participants

[0331] Table 10

[0332] Basic information of participants used in the study

[0333]

[0334]

[0335] Gastrointestinal health status at the screening visit:

[0336] Table 11 Gastrointestinal health at baseline (n=98) (How often did you encounter the following problems in the past month?)

[0337]

[0338]

[0339] N* = number of participants included in the ITT population, *p-value from Kruskal-Wallis test No significant differences were found between the two groups for any individual reading.

[0340] Stool consistency and regularity

[0341] 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).

[0342] Table 12

[0343] Treatment had no effect on stool regularity over the 45-day trial period compared with placebo

[0344]

[0345]

[0346] *p-value of ANOVA test

[0347] 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.

[0348] Fig.29 It was shown that the probiotic mixture significantly reduced the incidence of loose stools during the study compared to the placebo control.

[0349] During the first 6 weeks of the study, the probiotic mixture significantly reduced the incidence of loose stools compared with the control group ( Fig.29 ), 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.

[0350] Fig.30 showed no effect of any treatment on the percentage of hard stools compared with placebo control.

[0351] There was no significant effect of any treatment group on the percentage of hard stools throughout the study ( Fig.30 )(F (1.829,9.146) =2.831,P=0.113; time (F (5,15) =1.121, p=0.391).

[0352] Incidence and duration of gastrointestinal symptoms

[0353] Table 13

[0354] The number of days during the study on which participants reported gastrointestinal symptoms in their diaries

[0355]

[0356]

[0357] 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.

[0358] The Kruskal-Wallis test showed no significant differences in the number of days with gastrointestinal symptoms between treatment groups. The study products including probiotics did not show a statistically significant difference in the number of days with gastrointestinal symptoms compared with placebo.

[0359] Incidence and duration of urinary tract symptoms

[0360] Table 14

[0361] Number of days with urinary tract discomfort symptoms reported in the participants' diaries

[0362]

[0363]

[0364] 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.

[0365] The Kruskal-Wallis test did not show any significant differences in the number of days with UTI symptoms between treatment groups. The study product including the probiotic did not show a statistically significant difference in the number of days with UTI symptoms compared to placebo.

[0366] Incidence and duration of upper respiratory tract infections

[0367] Table 15

[0368] Number of days with respiratory symptoms reported in participants' diaries

[0369]

[0370]

[0371] 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.

[0372] The Kruskal-Wallis test did not reveal any significant differences in the number of days with respiratory tract infection symptoms between treatment groups. The study product containing probiotics did not show a statistically significant difference in the number of days with symptoms compared with placebo.

[0373] Daily questionnaire analysis

[0374] 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 significance was observed for the fatigue and appetite change items. Participants in the Bacillus megaterium group experienced the greatest changes in these two items. However, no statistically significant differences were observed for the pairwise comparisons of the probiotic groups with the placebo (Table 16 (below)).

[0375] Table 16

[0376] 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?)

[0377]

[0378]

[0379] 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).

[0380] Cholesterol and triglyceride levels

[0381] 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).

[0382] Table 17

[0383] Cholesterol and triglyceride levels at baseline and end of study (n=98)

[0384]

[0385] N* = number of participants included in the ITT population, HDL = high-density lipoprotein, LDL = low-density lipoprotein, TC

[0386] = total cholesterol, TG = triglycerides. * p-value of ANOVA (comprehensive test)

[0387] Blood cytokine levels

[0388] Table 18

[0389] IL-8 and TNFα levels at baseline and end of study (n=98)

[0390]

[0391] N* = number of participants included in the ITT population, *p-value for ANOVA (omnibus test)

[0392] 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).

[0393] Blood antioxidant levels

[0394] Table 19

[0395] Antioxidant levels at baseline and end of study (n=98)

[0396]

[0397] N* = number of participants included in the ITT population, *p-value for ANOVA (omnibus test)

[0398] 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).

[0399] Metabolite levels

[0400] Table 20

[0401] Amino acid levels at baseline and end of study (n=98)

[0402]

[0403]

[0404] N* = number of participants included in the ITT population, *p-value for ANOVA (omnibus test)

[0405] 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).

[0406] Table 21

[0407] Mineral levels at baseline and end of study (n=98)

[0408]

[0409]

[0410] N* = number of participants included in the ITT population, *p-value for ANOVA (omnibus test)

[0411] 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).

[0412] Changes in microbial flora

[0413] 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.

[0414] Fig.31 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-values ​​less than 0.05 should be considered statistically significant.

[0415] Fig.32 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.

[0416] Fig.33Shows 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.

[0417] Table 22

[0418] Proportion of participants reporting respiratory infection symptoms in Diary 1 (n=123).

[0419]

[0420] N* = number of participants included in the ITT population

[0421] 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.

[0422] Table 23

[0423] Proportion of clinically relevant infections reported in participant diary 1 (n=123).

[0424]

[0425] N* = number of participants included in the ITT population

[0426] 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.

[0427] Despite this, none of the study products that included probiotics showed a statistically significant difference compared with placebo.

[0428] Table 24

[0429] Ratio of loose stools to hard stools in all stools at the 6th and 7th weeks of treatment (n=121)

[0430]

[0431] *p-value of the Kruskal-Wallis test. If p<0.05, individual comparisons to placebo were calculated (Mann-Whitney U test with Holm correction).

[0432] The proportion of loose stools differed significantly between groups throughout the treatment period and at 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.

[0433] Table 25

[0434] Proportion of participants reporting gastrointestinal infection symptoms in Diary 2 (n=118)

[0435]

[0436]

[0437] N* = number of participants included in the ITT population

[0438] 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.

[0439] 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.

[0440] There were no differences in gastrointestinal health conditions at baseline among participants in the treatment groups, as a result of randomization.

[0441] 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.

[0442] 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.

[0443] In conclusion, probiotic products are safe for use in adults and show some favorable data on the gut-brain axis and stool consistency.

[0444] discuss

[0445] 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.

[0446] 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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[0500] 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.

[0501] 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.

[0502] 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.

[0503] 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.

[0504] Bacillus megaterium strain MIT411 is deposited in the Agricultural Research Culture Collection (NRRL), 1815N: University Street, Peoria, Illinois 61604 U.S.A., with the deposit number NRRL B-68213 (deposit date: October 27, 2022). Depositor: Deerland Probiotics and Enzymes, 2995 Cobb International Boulevard, Kennesaw, GA 30152, United States.

Claims

1. A Bacillus megaterium 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 megaterium strain of claim 1, which has at least 97% identity to SEQ ID NO:

3.

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

2.

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

1.

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

3.

6. A microbial composition comprising the Bacillus megaterium 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 megaterium 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 megaterium strain according to any one of claims 1 to 5, for use in preventing or treating vaginal infections, urinary tract infections, gastrointestinal infections, gastrointestinal diseases, improving immune health, protecting against oxidative stress, cleansing and detoxification, metabolic health and cardiovascular health.

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

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

15. A method for preventing or treating vaginal infections, urinary tract infections, gastrointestinal infections, gastrointestinal diseases, improving immune health, protection from 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 megaterium strain according to any one of claims 1 to 5.

17. The Bacillus megaterium 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 in separate compositions comprising a mixture of different bacterial strains.

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

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