Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K2035/11—Medicinal preparations comprising living procariotic cells
  • A61K2035/115—Probiotics
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/07—Bacillus

Definitions

• This invention relates to a new Bacillus clausii strain, which alone or in combination with other Bacilli strains can be used as probiotics or together with a prebiotic and a symbiotic.
• the invention also relates to a composition such as a pharmaceutical composition, dairy product, functional food, nutraceutical, dietary supplement, and product for personal care comprising the new Bacillus clausii strain alone or in combination with other strain(s), as well as use of the strain for prevention or treatment of gastrointestinal, urinary tract, vaginal, and other infections and diseases, and other uses.
• Probiotics are live microorganisms or microbial mixtures administered to improve the patient’s microbial balance, particularly the environment of the respiratory and gastrointestinal tract.
• Bacillus strains have been employed for the treatment of respiratory infections, prevention of diarrhoea, as well as, for the treatment of immuno-related diseases (Elshaghabee et al., 2017).
• Dysbiosis is a condition that is characterized by a decrease of the certain bacterial species and an increased growth of pathogenic bacteria. Dysbiosis has been associated with the development of periodontal disease, inflammatory bowel disease, and chronic fatigue syndrome. Some studies have suggested patients with dysbiosis may have an increased risk of developing metabolic and cardiac disorders (Chan et al., 2013).
• Dysbiosis is a common gastrointestinal problem.
• Dysbiosis caused by Escherichia coli is also a common problem (Chan et al., 2013).
• Bacilli is important for the maintenance of the intestinal microbial ecosystem. Bacilli have been shown to possess inhibitory activity toward the growth of pathogenic bacteria such as Listeria monocytogenes, Escherichia coli, Salmonella spp. and others (Yilmaz et al., 2005). This inhibition could be due to the production of inhibitory compounds such as organic acids, hydrogen peroxide, bacteriocins or reuterin or to competitive adhesion to the epithelium (Abriouel et al., 2010).
• pathogenic bacteria such as Listeria monocytogenes, Escherichia coli, Salmonella spp. and others (Yilmaz et al., 2005). This inhibition could be due to the production of inhibitory compounds such as organic acids, hydrogen peroxide, bacteriocins or reuterin or to competitive adhesion to the epithelium (Abriouel et al., 2010).
• Bacilli have also been examined as a treatment of respiratory tract infections (Marseglia et al., 2007). For example, the installation of Bacilli, and stimulation of indigenous organisms has been employed to prevent recurrence of urinary tract infections (Marseglia et al., 2007). The role of Bacilli in preventing intestinal infections has also been investigated.
• Hyronimus et al., 2000 discloses the screening of probiotic activities of a number of Bacilli strains by in vitro techniques and evaluation of the colonization ability of thirteen selected strains in humans. The strains were examined for resistance to pH 2.5 and 0.3% Oxgall adhesion to Caco- 2 cells and antimicrobial activities against enteric pathogenic bacteria (KACHamit et al., 2015). Bacilli have been shown to possess the primary requirement of GIT stress tolerance, besides having good adhesion and bio-therapeutic properties (Thakur et al., 2016).
• compositions of Bacilli known in the art are not sufficiently efficient in recolonizing in vivo i.e., mammalian microbial ecosystems, and there is, therefore, a need to find Bacilli with an inherent ability to recolonize upon administering the Bacilli in the form of a pharmaceutical composition, a nutraceutical, a dairy product, a functional food or absorbent product.
• Bacilli isolated from soil may have the ability to recolonize in vivo upon administration because of their inherent ability to survive in the human microbial ecosystem. It is often a cumbersome process to identify Bacilli strains with enhanced abilities to colonize upon administration and it is therefore important to select useful test systems to predict their in vivo ability to colonize.
• Bacilli being ubiquitous in nature, consistently enter the gastrointestinal and respiratory tracts of healthy people through food, water, and air (Benno & Mitsuoka, 1986). They have been isolated from the gut and can reach up to 10 7 CFU/g and hence are considered to be one of the dominant components of the normal gut microbiota (Lakshmi et al., 2017). More recently, strains of Bacillus clausii have been isolated in order to provide more specific functions and its safety has been evaluated.
• Bacillus clausii has been previously used in diarrhoeal patients (Sudha et al., 2013, Horosheva et al., 2014) and children with recurrent respiratory infections (Marseglia et al., 2007) with no adverse events reported. Though the countries and strains are not specified, Bacillus clausii has been commercialized in 55 countries around the world (Nista et al. 2004; Gabrielli et al. 2009). The literature review for Bacillus clausii showed no adverse events related to the probiotic and the worldwide presence of bacteria in different countries supplements the narrative of its safety for human consumption.
• Bacilli strains with probiotic capabilities should be able to adhere to other suitable cells, such as the cell line Caco-2 cells. Moreover, it is also desirable that the Bacilli strains with probiotic capabilities show in vitro inhibitory activity against other bacterial species, produce acid after growth in liquid culture and/or produce hydrogen peroxide.
• the present invention concerns the Bacillus clausii strain CSI08 alone or in combination with other Bacilli strains such as Bacillus megaterium strain MIT411 (disclosed and claimed in corresponding PCT Application PCT/US2022/xxxxx claiming priority from Irish Patent Application No. 2021/0211, whose contents are incorporated herein in their entirety) and Bacillus coagulans strain CGI314 (disclosed and claimed in corresponding PCT Application PCT/US2022/xxxxx claiming priority from Irish Patent Application No.
• these strains have similar or essentially the same advantageous properties e.g. the ability to colonize by adherence to mucosal membranes/surfaces and which are therefore suited for the treatment or prevention of infections or diseases of the vaginal, urinary- tract, gastrointestinal, naso-sinal, pharyngeal, esophageal, oral, and/or other areas of the body with mucosal membranes, as well as, the treatment or prevention of infections or diseases of the skin and/or other areas of the body having epithelium; immune health, protection against oxidative stress, cleansing and detoxification, metabolic health and cardiovascular health amongst others such as providing antimicrobial activity, anti-inflammatory activity, suppression of pro- inflammatory response, activating and/or provoking immune response eg.
• the present invention allows the use of Bacillus clausii strain CSI08 and compositions for use in fecal transplants.
• Gastrointestinal diseases include, but are not limited to, treating gastrointestinal irregularity in an individual, wherein the individual has at least one 24-hour episode per month of bowel movements measuring 1 or 2 on the Bristol Stool Scale (i.e. treating constipation; or wherein the individual has at least one 24-hour episode per month of bowel movements measuring 6 to 7 on the Bristol Stool Scale (tending towards diarrhea), wherein the frequency of the individual's 24- hour episodes per month of bowel movements measuring 1 or 2 (or 6 to 7) on the Bristol Stool Scale decreases.
• the invention further includes maintaining healthy gut microflora, with Bacillus- containing composition(s).
• Bacillus- containing composition(s) can be used as probiotic supplementation of the gastrointestinal microflora, and may compete with or otherwise discourage pathogenic bacteria in the gut such as Escherichia coH, Listeria monocytogenes, Salmonella spp.
• Another object of the present invention is to provide pharmaceutical formulations with an increased ability to colonize by adherence to the mucosal membrane by employing mucous adhesive excipients.
• compositions such as dairy products, nutraceutical products and functional foods comprising Bacillus clausii strain CSI08 alone or in combination with other Bacilli strains such as Bacillus megaterium strain MIT411 and/or Bacillus coagulans strain CGI314, in an embodiment, having essentially the same properties, in an embodiment having the ability to colonize mucosal membranes and therefore adapt to treatment or prevention of vaginal infections, urinary -tract infections and gastrointestinal diseases.
• Bacillus clausii strain CSI08 alone or in combination with other Bacilli strains
• Bacillus megaterium strain MIT411 and/or Bacillus coagulans strain CGI31411 in an embodiment, having essentially the same properties, in an embodiment having the ability to colonize mucosal membranes and therefore adapt to treatment or prevention of vaginal infections, urinary -tract infections and gastrointestinal diseases.
• compositions of the present invention may 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 more, including any timeframe identified and/or falling within these ranges.
• Figure 1 illustrates the genome analysis of Bacillus clausii CSI08.
• Figure 2 illustrates the phylogenetic tree (16S) of Bacillus spp, arranged in clades.
• Figure 3 illustrates the phylogenetic tree (gyrB) of Bacillus spp., arranged in clades.
• Figure 4 shows the stability of Muni Spore in PBS during a pasteurization process.
• Figure 5 shows pH Survivability of B. clausii CSI08 spores at 0-, 3-, and 24-hour timepoints.
• Figure 6 shows bile salt survivability of B. clausii CSI08 spores at 0-, 3-, and 24-hour timepoints.
• Figure 7 shows survival of Bacillus clausii CSI08 spores and L. rhamnosus GG under gastric and small intestinal digestion conditions simulated in vitro.
• Figures 8 and 9 show antimicrobial activity of Munispore (B. clausii CSI08) in liquid culture.
• Figure 10 shows total antioxidant capacity of B. clausii CSI08 and L. rhamnosus GG.
• Figure 11 shows the cytoprotective effect of the vegetative form# clausii CSI08 on H2O2- exposed epithelium.
• Figure 12 shows the survival rate of C. fed with 10 8 and 10 9 CFU/ml B. clausii CSI08, followed by oxidative stress caused by H2O2. NGM - control-fed nematodes; Vit C - positive control.
• Figure 13 shows the effect of vegetative cells B. clausii CSI08 on HT-29 cell viability. B. clausii CSI08 compared with untreated cells (medium).
• Figure 14 shows adhesion of vegetative cells and spores B. clausii CSI08 to the HT-29- MTX. B. clausii CSI08 compared with L. fermentum.
• Figures 15A and 15B show the modulation of LPS-induced pro-inflammatory response by B. clausii CSI08 in HT-29 cell line, and the pattern of gene expression after co-incubation of HT- 29 cells with B. clausii CSI08 and its CFS in unstimulated conditions (LPS - ).
• Figure 16 shows modulation of LPS-induced IL8 gene expression by viable and heat-killed B. clausii CSI08.
• Figure 17 shows NF-KB levels in the nuclear fractions of control HT-29s, cells exposed to LPS, and cells treated with B. clausii CSI08 prior to adding LPS.
• Figures 18A and 18B show modulation of Polyl C-triggered pro-inflammatory response by B. clausii CSI08 in HT-29 cell line. qPCR analysis of IL-8, TNF-a, IL-17C, and CXCL10 gene expression 4 hours after exposure to PolyLC in HT-29 cells preincubated with B. clausii CSI08 or its cell free supernatants (CFS).
• CFS cell free supernatants
• Figure 19 shows immunostimulatory effect of CSI08. Cytokine levels in cell culture supernatants of U937-derived macrophages challenged by vegetative cells B. clausii CSI08 or LPS for 5 hours.
• Figure 20 shows the lifespan of C. elegans wild type N2 strain in control conditions (NGM) and lifespan of C. elegans wild type N2 fed with B. clausii CSI08.
• Figure 21 shows the lifespan of C. elegans daf-16 mutant worms in control conditions (NGM) and lifespan of C. elegans daf-16 mutants fed with B. clausii CSI08.
• Figure 22 shows B. clausii CSI08 is slightly caseolytic using streak method, as well as B. megaterium and B. clausii CSI08 caseolytic activity.
• Figure 23 shows proteolytic activity determined by EnzCheck® kit assay.
• Figure 24 shows concentrations of essential amino acids and vitamins in overnight cultures of B. clausii CSI08 determined by RP-HPLC-FLD and RP-HPLC-MS.
• Figure 25 shows a graphical flow chart of the study design.
• Figure 26 shows the probiotic cocktail administered during the study significantly decreased the incidence of loose stool over the course of the study as compared to placebo control.
• Figure 27 shows no effect of any treatments administered during the study on percentage of hard stools as compared to placebo control.
• Figure 28 is a boxplot showing the Chaol values distribution in each experimental group for Day 1 and Day 45 of the study. Dotted lines connect the paired samples. A paired Wilcoxon test was used to compare the distribution of the groups.
• Figure 29 is a boxplot showing the Chaol values distribution in each experimental group for Day 1 and Day 45 of the study. A Wilcoxon test was used to compare the distribution of each experimental group against the Placebo.
• Figure 30 illustrates PCoA clustering performed on the Bray-Curtis dissimilarity matrix.
• Identifying gyrB gene polymorphism was carried out by the Applicant.
• the gyrB gene encodes DNA gyrase subunit B.
• DNA gyrase negatively supercoils closed circular doublestranded DNA in an ATP-dependent manner to maintain chromosomes in an underwound state.
• Genome sequence data of Bacillus clausii strain CSI08 was deposited into NCBI GenBank database, and the genome sequence was annotated with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).
• the genome is publicly available, with GenBank Accession Number JABBNL000000000.1 for the strain, and available for instance at the link: Alkalihalobacillus clausii strain CSI08, whole genome shotgun sequenci - Nucleotide - NCBI nih. ov).
• Genome sequence data of Bacillus megaterium strain MIT411 was deposited into NCBI GenBank database, and the genome sequence was annotated with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).
• the genome is publicly available, with GenBank Accession Number JABBNK000000000.1 for the strain, and available for instance at the link: Priestia megaterium strain MIT41 L whole genome shotgun sequencing pro - Nucleotide - NCBI nih. ov).
• Genome sequence data of Bacillus coagulans strain CGI314 was deposited into NCBI GenBank database, and the genome sequence was annotated with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).
• the genome is publicly available, with GenBank Accession Number JABBFU000000000.1 for the strain, and available for instance at the link: https://www.ncbi.nlm.nih.gov/nuccore/JABBFU00000000Q.1.
• Genome-to-genome distance calculation (GGDC), a digital gold standard, is as reliable as DNA-DNA hybridization (DDH) (Auch et al., 2010). GGDC holds more discriminatory power for subspecies delineation and subsequently, was used as a confirmation of multiple alignment and phylogenetic analyses. GGDC yielded three calculation-based models that further verified Bacillus clausii CSI08 is a close relative to Bacillus clausii Bl 06 (Table 3).
• excipient is meant any non-active ingredient that is added to form part of the final formulation.
• probiotic is meant a viable microbial supplement, which has a beneficial influence on a subject through its effects in the intestinal tract, urinary tract, vaginal tract, and/or other areas of a subject’s body.
• a “prebiotic” is used herein as a substrate, which has a beneficial effect on a probiotic and thus on a subject taking (e.g. administered) the probiotic. Suitable prebiotics may be selected from an inulin, an oligosaccharide, and/or a vitamin.
• a “subject” as used herein includes a person suffering from any clinical condition related to a microbial imbalance as well as a person using bacterial preparations prophylactically, for wellness, or any other purpose including for instance benefitting from the administration of Bacillus clausii strain of this invention (e.g. CSI08).
• the subject is a human, a patient, and/or a mammal.
• a “symbiotic product” is meant a combination of probiotic and prebiotic, which is synergy, have a beneficial influence on the patient.
• CFU colony forming units
• the present invention comprises Bacillus clausii strain CSI08 alone or in combination with other probiotic Bacilli strains with essentially the same properties.
• Such other probiotic Bacilli strains include, but are not limited to a Bacillus coagulans strain and a Bacillus megaterium strain.
• Such other Bacilli strains further include a Bacillus coagulans strain and a Bacillus megaterium strain each filed today under these respective titles - their contents are incorporated herein in their entirety.
• SEQ ID NO: 1 as recited in the claims attached hereto, comprises gyrB of Bacillus clausii CSI08.
• SEQ ID NO: 2 comprises 16S rRNA of Bacillus clausii CSI08.
• SEQ ID NO: 3 as recited in the claims attached hereto, comprises the assembled whole genome sequence of Bacillus clausii CSI08.
• the Bacillus clausii CSI08 strain claimed herein, with reference to at least 97% identity to SEQ ID NO: 1 and / or 2; or to at least 97% identity to SEQ ID NO: 3, has the following properties: [0083] In order to determine the genus and species of the strains disclosed herein, the whole genome was sequenced. The amount and composition of the strains were identified and determined. [0084] The strain was shown to possess little to no antibiotic resistance and no safety concerns.
• the strain was found to show stability toward acid and bile and showed heat tolerance.
• the strain produced a natural antibiotic substance in the form of bacteriocins.
• the Bacilli strain of the present invention is suitable for medical use in preventing or treating vaginal infections, urinary tract infections and gastrointestinal diseases, as well as, improving immune health, protection against oxidative stress, cleansing and detoxification, metabolic health and cardiovascular health.
• a composition such as a pharmaceutical composition
• a composition comprising Bacillus clausii CSI08 alone or in combination with other probiotic Bacilli strains with similar and/or essentially the same properties, together with a pharmaceutically acceptable carrier and/or diluent.
• probiotic Bacilli strains include, but are not limited to a Bacillus coagulans strain and a Bacillus megaterium strain.
• the bacterial strains are formulated into compositions such as pharmaceutical formulations in order to allow the easy administration of the probiotic strains and by means known to the man skilled in the art.
• Bacillus coagulans has been proven able to alleviate symptoms of irritable bowel syndrome (Sudha et al., 2018), improve muscle integrity and cytokine response (Gepner et al., 2017; Jager et al., 2018), modulate the gut microbiome and the immune response (Kimmel et al., 2010), reduce function intestinal gas symptoms (Kalman et al., 2009), reduce the instance and duration of diarrhea (Dolin et al., 2009), improve the 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 severity of bacterial vaginosis (Sudha et al., 2012), and reduce cholesterol (Sudha et al., 2012) all in vivo.
• Bacillus coagulans has also shown to induce immune response and anti-inflammatory action (Jensen et al., 2017), improve plant protein digestion (Keller et al., 2017), adhere to Caco-2 cells (Sharma & Kanwar, 2017), improve colonic microenvironment in patients with ulcerative colitis (Sasaki et al., 2020), reduce the adhesion, cytotoxicity and induction of apoptosis caused by S. typhimurium in HT-29 cells (Kawarizadeh et al., 2019), hydrolyze lactose from whey protein (Liu et al., 2019), and enhancing t-cell response (Baron, 2009) all in vitro.
• Bacillus clausii has been proven efficacious in preventing recurrent respiratory infections (Marseglia et al., 2007), reducing duration and severity of diarrhoea (Sudha et al., 2019) in vivo. Bacillus clausii has also been proven capable to produce protein hydrolysates with antimicrobial and antioxidant capacity (Rochin-Medina et al., 2017), protect against acetaminophen induced acute liver injury (Neag et al., 2020), inhibit cytotoxic effects induced by Clostridium difficile and Bacillus cereus toxins (Ripert et al., 2016) in vitro.
• Bacillus megaterium has been shown to exert protective effects against oxidative stress both in vitro and in vivo (Mazzoli et al., 2019). Bacillus megaterium has also been shown capable of adapting and surviving in acid stress conditions and chelating heavy metals in vitro (Ferreira et al., 2019).
• the probiotic bacteria employed in this invention are used in bacterial concentration of 10 6 - 10 13 CFU (colony forming units), for instance as a daily dose, including any amount or range that is included in said range.
• the bacteria are employed in an amount of 10 7 - 10 12 CFU, or 10 8 - 10 11 CFU, or 10 9 - 10 10 CFU, or for instance in an amount of 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.
• a composition of this invention comprises, consists essentially of, consists of, and/or is characterized by about 10 6 - about 10 13 CFU such as about 10 9 Bacillus clausii CSI08.
• a composition of this invention comprises Bacillus clausii CSI08 (for instance about 10 9 CFU) in combination with Bacillus megaterium MIT411 and/or Bacillus coagulans CGI314.
• a composition of this invention is orally administered in capsule form.
• Bacillus clausii CSI08 is in spore form, or is not in spore form.
• compositions comprising Bacillus clausii CSI08 can include one or more dry carriers selected from the group consisting of trehalose, maltodextrin, rice flour, microcrystalline cellulose, magnesium stearate, inositol, fructooligosaccharide, galactooligosaccharide, dextrose, dried dairy products, and the like.
• the dry carrier can be added to the compositions comprising Bacillus clausii CSI08 in a weight percentage of from about 1% to about 95% by weight of the composition.
• the compositions comprising Bacillus clausii CSI08 can include one or more liquid or gel-based carriers, selected from the group consisting of water and physiological salt solutions, urea, alcohols and derivatives thereof (e.g., methanol, ethanol, propanol, butanol), glycols (e.g., ethylene glycol, propylene glycol), and the like; natural or synthetic flavorings and food-quality coloring agents, all compatible with the organism; thickening agents selected from the group consisting of com starch, guar gum, xanthan gum, and the like; one or more spore germination inhibitors selected from the group consisting of hyper-saline carriers, methylparaben, guargum, polysorbate, preservatives, and the like.
• liquid or gel-based carriers selected from the group consisting of water and physiological salt solutions, urea, alcohols and derivatives thereof (e.g., methanol, ethanol, propanol, butanol), glycols
• the one or more liquid or gel-based carrier(s) can be added to the compositions comprising Bacillus clausii CSI08 in a weight/volume percentage of from about 0.6% to about 95% weight/volume of the composition.
• the natural or synthetic flavoring(s) can be added to the compositions comprising Bacillus clausii CSI08 in a weight/volume percentage of from about 3.0% to about 10.0% weight/volume of the composition.
• the coloring agent(s) can be added to the compositions comprising Bacillus clausii CSI08 in a weight/volume percentage of from about 1.0% to about 10.0% weight/volume of the composition.
• the thickening agent(s) can be added to the compositions comprising Bacillus clausii CSI08 in a weight/volume percentage of about 2% weight/volume of the composition.
• the one or more spore germination inhibitors can be added to the compositions comprising Bacillus clausii CSI08 in a weight/volume percentage of about 1% weight/volume of the composition.
• Suitable dosage forms include tablets, capsules, solutions, suspensions, powders, gums, and confectionaries.
• Sublingual delivery systems include, but are not limited to, dissolvable tabs under and on the tongue, liquid drops, and beverages. Edible films, hydrophilic polymers, oral dissolvable films, or oral dissolvable strips can be used. Other useful delivery systems comprise oral or nasal sprays or inhalers, and the like.
• Suitable dosage forms include tablets, capsules, solutions, suspensions, powders, gums, and confectionaries.
• Sublingual delivery systems include, but are not limited to, dissolvable tabs under and on the tongue, liquid drops, and beverages. Edible films, hydrophilic polymers, oral dissolvable films, or oral dissolvable strips can be used. Other useful delivery systems comprise oral or nasal sprays or inhalers, and the like.
• probiotics may be further combined with one or more solid inactive ingredients for the preparation of tablets, capsules, pills, powders, granules, or other suitable dosage forms.
• the active agent may be combined with at least one excipient selected from the group consisting of fillers, binders, humectants, distintegrating agents, solution retarders, absorption accelerators, wetting agents, absorbents, and lubricating agents.
• excipients include, but are not limited to, magnesium stearate, calcium stearate, mannitol, xylitol, sweeteners, starch, carboxymethylcellulose, microcrystalline cellulose, silica, gelatin, silicon dioxide, and the like
• compositions administered according to the methods of the present disclosure may thus be placed into the form of pharmaceutical compositions and unit dosages thereof.
• forms include: solids, and in particular, tablets, filled capsules, powder and pellet forms; liquids, and in particular, aqueous or non-aqueous solutions, suspensions, emulsions, elixirs; and capsules filled with the same; all for oral use, suppositories for rectal administration, and sterile injectable solutions for parenteral use.
• Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.
• compositions administered according to the methods of the present disclosure can be administered in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, in certain embodiments, as the active component, either a chemical compound of the present disclosure or a pharmaceutically acceptable salt of a chemical compound of the present disclosure.
• pharmaceutically acceptable carriers can be either solid or liquid.
• Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
• a solid carrier can be one or more substances that may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials.
• the carrier is a finely divided solid, which is in a mixture with the finely divided active component.
• the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.
• powders and tablets administered according to methods of the present disclosure preferably may contain from five or ten to about seventy percent of the active compound.
• Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
• preparation is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without additional carriers, is surrounded by a carrier, which is thus in association with it.
• carrier providing a capsule in which the active component, with or without additional carriers, is surrounded by a carrier, which is thus in association with it.
• cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.
• Liquid preparations include, but are not limited to, solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions.
• parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.
• chemical compounds administered according to methods of the present disclosure may thus be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose for administration in ampoules, pre-filled syringes, small-volume infusion, or in multi-dose containers with an added preservative.
• compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilizing, and/or dispersing agents.
• the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
• Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents, as desired.
• Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well-known suspending agents.
• compositions suitable 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 glycerine or sucrose and acacia; and mouthwashes comprising the active ingredient in suitable liquid carrier.
• 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 glycerine or sucrose and acacia
• mouthwashes comprising the active ingredient in suitable liquid carrier.
• Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example, with a dropper, pipette, or spray.
• the compositions may be provided in single or multidose form.
• the compound In compositions intended for administration to the respiratory tract, including intranasal compositions, the compound will generally have a small particle size, for example, of the order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example, by micronization.
• the pharmaceutical preparations are preferably in unit dosage forms.
• the preparation is subdivided into unit doses containing appropriate quantities of the active component.
• the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packaged tablets, capsules, and powders in vials or ampoules.
• the unit dosage form can be a capsule, tablet, cachet, or lozenge itself; or it can be the appropriate number of any of these in packaged form.
• Tablets, capsules, and lozenges for oral administration and liquids for oral use are preferred compositions. Solutions or suspensions for application to the nasal cavity or to the respiratory tract are preferred compositions. Transdermal patches for topical administration to the epidermis are preferred.
• compositions of the present invention including compositions administered according to the methods of the present disclosure may also include one or more excipients, most preferably one or more nutraceutical or pharmaceutical excipients.
• compositions containing one or more excipients and incorporating one or more probiotics can be prepared by procedures known in the art.
• compositions can include one or more adjuvants, excipients, carriers, buffers, diluents, and/or other customary pharmaceutical auxiliaries.
• 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 onto patches for transdermal application with common and conventional barriers, binders, diluents, and excipients.
• nutraceutical compositions including nutraceutical compositions administered according to the methods of the present disclosure may include and may be administered in combination with a pharmaceutically acceptable carrier.
• the active ingredients in such formulations may comprise from about 1% by weight to about 99% by weight. In other embodiments, the active ingredients in such formulations may comprise from about 0.1% by weight to about 99.9% by weight.
• “Pharmaceutically acceptable carrier” means any carrier, diluent, or excipient that is compatible with the other ingredients of the formulation and not deleterious to the user.
• Useful excipients include, but are not limited to, microcrystalline cellulose, magnesium stearate, calcium stearate, any acceptable sugar (e.g., mannitol, xylitol), and the like, and for cosmetic use, a water or an oil base may be used, or mixture thereof including such as an emulsion.
• the strain Bacillus clausii CSI08 or a composition comprising a strain of the present invention may be administered by any route, including, but not limited to, oral, sublingual, buccal, ocular, pulmonary, rectal, vaginal, urethral, ureteral, and parenteral administration, or as an oral or nasal spray (e.g., inhalation of nebulized vapors, 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.
• a pharmaceutical composition in the body of the patient in a controlled formulation, with systemic or local release of the drug to occur at a later time.
• the drug may be localized in a depot for controlled release to the circulation, or for release to a local site.
• compositions of the invention may be those suitable for, and formulated for, any of the routes identified above, including for instance oral, rectal, bronchial, nasal, pulmonal, topical (including buccal and sub-lingual), transdermal, vaginal, urethral, ureteral, or parenteral (including cutaneous, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intracerebral, intraocular injection, or influsion) administration, or those in a form suitable for administration by inhalation or insufflation, including powders and liquid aerosol administration, or by sustained release systems.
• sustained release systems include semipermeable matrices of solid hydrophobic polymers containing the compound of the invention, which matrices may be in the form of shaped articles, e.g., films or microcapsules.
• Bacillus clausii CSI08 spores in PBS (pH 7.72) are stable at 45°C, and 75°C from 30 seconds to 3 minutes, with some reduction in spore counts after treatment at 90°C for 3 minutes.
• Figure 4 shows the stability of MuniSpore in PBS during a pasteurization process. Data represent the mean ⁇ SEM. Spore counts compared to 0 min. * P ⁇ 0.0001.
• Bacillus clausii CSI08 spores are stable in a pasteurization process and during other manufacturing methodologies used in food & beverage.
• the strain shows bile stability and the strain shows acid stability.
• the survivability of B. clausii CSI08 spores was determined in acidic and bile salt conditions in nutrient broth media for 24 hours. Total spore count for B. clausii CSI08 does not show a significant reduction in viability or concentration after contact with acidic conditions or bile salt concentrated nutrient broth for 3 hours. At the most extreme conditions, pH 1.2 and 0.45% bile salt concentration a significant decrease in counts was observed after 24 hours. Both pH and bile salts at these concentrations and exposures greatly exceed the conditions in the stomach and intestinal tract.
• Figure 5 shows pH Survivability of B. clausii CSI08 spores at 0-, 3-, and 24-hour timepoints.
• Figure 6 shows bile salt survivability of B. clausii CSI08 spores at 0-, 3-, and 24-hour timepoints.
• B. clausii CSI08 spores are not sensitive to acid conditions above 1.2 for 24h and are resistant to bile salts concentrations up to 0.30% for 24h. Therefore, B. clausii CSI08 spores can survive in low pH food and beverages (e.g., fruit juices) and in the stomach and intestine conditions. [0123] The strain shows heat tolerance. Bacillus clausii CSI08 spores in PBS (pH 7.72) are stable at 45°C, and 75°C from 30 seconds to 3 minutes, with some reduction in spore counts after treatment at 90°C for 3 min.
• Bacillus clausii CSI08 spores are stable in a pasteurization process and during other manufacturing methodologies in food & beverage.
• Results show the ability of Bacillus clausii CSI08 spores to efficiently survive the transit through the upper digestive tract.
• Figure 7 shows survival of Bacillus clausii CSI08 spores and L. rhamnosus GG under gastric and small intestinal digestion conditions simulated in vitro. Data represent the mean ⁇ SEM. Survival compared to 0 h * P ⁇ 0.0001.
• the strain produces a natural antibiotic substance in the form of bacteriocins.
• the vegetative form B. clausii CSI08 is active against the common intestinal pathogens E. coli and Salmonella having potential to protect and restore gut microbial communities.
• the vegetative form# clausii CSI08 has also proven to have antimicrobial activity against the well-known skin and urinary tract pathogen Pseudomonas aeruginosa in liquid media.
• B. clausii CSI08 revealed antimicrobial activity against E. coli, Salmonella, S. aureus and Pseudomonas aeruginosa in liquid medium.
• Figures 8 and 9 show antimicrobial activity of Munispore (B. clausii CSI08) in liquid culture. Values represent average concentration lofio CFU/ml ⁇ standard deviation. * p ⁇ 0.05, **** p ⁇ 0.0001.
• the vegetative form B. clausii CSI08 has the potential to crowd out bacterial pathogens and maintain a healthy gut and skin microbiome.
• Antioxidant activity The total antioxidant activity of the vegetative form B. clausii CSI08 was compared with L. rhamnosus GG (DI AS 030).
• Munispore has higher amount of antioxidant activity than /.. rhamnosus GG.
• Bacillus clausii CSI08 spores are a potential probiotic with strong antioxidant properties
• the vegetative form B. clausii CSI08 attenuates hydrogen peroxide-induced reduction in cell viability of epithelial cells (HT-29 cell line).
• the vegetative form B. clausii CSI08 attenuates hydrogen peroxide-induced decrease in survival rate of C. elegans N2 after oxidative stress.
• Figure 11 shows the cytoprotective effect of thevegetative form B. clausii CSI08 on H2O2- exposed epithelium. Values are the means ⁇ SEM. * - P ⁇ 0.0001 vs H2O2
• Figure 12 shows the survival rate of C. elegans W2 fed with 10 8 and 10 9 CFU/ml B. clausii CSI08, followed by oxidative stress caused by H2O2.
• NGM - control-fed nematodes; Vit C - positive control. Values are the average of two independent experiments (n 100/condition ). * p ⁇ 0.05 vs NGM (+H 2 O 2 ).
• Bacillus clausii CSI08 spores demonstrate a reversal of oxidative stress in mammalian cell lines and a host organism.
• Adhesion ability and cytotoxic effect the vegetative form B. clausii CSI08 doesn’t influence the viability of the intestinal epithelial cells (HT-29 model).
• Adhesion of the vegetative form and spores of B. clausii CSI08 to the non-mucous producing intestinal epithelial cell line (HT-29) is negligible, graph not shown.
• Figure 13 shows the effect of vegetative cells B. clausii CSI08 on HT-29 cell viability.
• Figure 14 shows adhesion of vegetative cells and spores B. clausii CSI08 to the HT-29- MTX.
• B. clausii CSI08 compared with L. fermentum. Values are means ⁇ SEM
• Bacillus clausii CSI08 does not negatively impact mammalian cell viability and can adhere to the gut lining.
• Anti-inflammatory activity the vegetative form of B. clausii CSI08 and its cell-free supernatants ability to attenuate LPS-triggered pro-inflammatory response was investigated in an in vitro model of intestinal epithelium (HT-29 cell line).
• Figures 15A and 15B show the modulation of LPS-induced pro-inflammatory response by B. clausii CSI08 in HT-29 cell line. Also shown the pattern of gene expression after co-incubation of HT-29 cells with B. clausii CSI08 and its CFS in unstimulated conditions (LPS - ). $ p ⁇ 0.05 vs LPS, # p ⁇ 0.001 vs LPS, * p ⁇ 0.0001 vs. LPS.
• B. clausii CSI08 partially retains the ability to supress pro-inflammatory response triggered by LPS in HT-29 cells after heat-inactivation.
• Figure 16 shows modulation of LPS-induced IL8 gene expression by viable and heat-killed B. clausii CSI08. * p ⁇ 0.0001.
• B. clausii CSI08 can down regulate LPS-induced NF-KB activation in HT-29 cell line exposed to LPS.
• Figure 17 shows NF-KB levels in the nuclear fractions of control HT-29s, cells exposed to LPS, and cells treated with B. clausii CSI08 prior to adding LPS. LPS versus LPS + B. clausii * p ⁇ 0.001 according to t-test.
• B. clausii CSI08’s immunomodulatory efficacy is mediated in part via Nf-KB pathway.
• Figures 18A and 18B show modulation of Polyl C-triggered pro-inflammatory response by B. clausii CSI08 in HT-29 cell line.
• the vegetative form of B. clausii CSI08, and its cell free supernatants, can reduce pro- inflammatory response in HT-29 cells triggered by Polyl-C, a viral mimetic.
• B. clausii CSI08 stimulated a robust immune response, resulted in secretion of high levels of pro-inflammatory (TNF-a, IL-ip, IL-18), regulatory (G-CSF, GM- CSF, IL-6) and anti-inflammatory (IL-10, IL-IRA, EGF) cytokines by macrophages.
• Figure 19 shows immunostimulatory effect of CSI08.
• B. clausii CS108 prolongs the lifespan of C. elegans N2 worms compared with control condition - NGM medium. This effect is DAF-16-dependent.
• the data indicates the role of B. clausii CSI08 as a potential anti-inflammatory effector or immune activator.
• B. clausii CSI08 demonstrates strong immunoprotective effects in a host organism. [0175] B. clausii CSI08 exhibits moderate caseolytic/protease activity.
• B. clausii CSI08 was weakly positive for casein degradation / Caseolytic activity on skim milk agar plates.
• B. clausii CSI08 displays moderate protease activity (Lcrhamnosus as a negative control).
• Figure 22 shows B. clausii CSI08 is slightly caseolytic using streak method.
• the figure shows B. megaterium and B. clausii CSI08 caseolytic activity.
• Figure 23 shows proteolytic activity determined by EnzCheck® kit assay. Data represent the mean ⁇ SEM.
• B. clausii CSI08 s diverse carbohydrate assimilation profile: B. clausii CSI08 was positive for 20 carbohydrates out of 49 tested, using commercial API 50 CH strip.
• Table 5 List of Carbohydrates that are effectively fermented using API 50 Ch strips.
• B. clausii CSI08 is Esteroloytic and has Phosphatase and P-Galactosidase Enzymes: B. clausii CSI08 was positive for esterase, phosphohydrolase, and p-galactosidase activity using API ZYM kit which implies that
• B. clausii CSI08 may generate free fatty acids from the action of esterase in the presence of an appropriate lipid source.
• Beta-galactosidase- enzyme that catalyzes lactose hydrolysis into glucose and galactose and, importantly, is responsible for formation of galacto-oligosaccharides (GOS) promoting the growth of Bifidobacterium and Lactobacillus species
• Phosphohydrolase catalyzes the hydrolysis of phosphates
• Table 6 Enzymatic profile of B. clausii CSI08 using API ZYM kit [0188] This indicates the potential ability of B. clausii CSI08 to break down lactose and fats.
• Extracellular secretions of B. clausii CSI08 grown in TSB broth for 24h were sent to mass spectrometry to identify proteins released by the probiotic strain. A total of 29 proteins were detected of which 5 had potential probiotic benefits.
• B. clausii CSI08 can produce essential amino acids and group B vitamins: The ability of B. clausii CSI08 to synthesize essential amino acids and vitamins during cultivation in liquid medium (TSB) was assessed. The results suggest the production of several amino acids (alanine, glutamine and glutamic acid, histidine, methionine, proline, tyrosine, and threonine) and two group B vitamins (Pantothenic acid/B5 and Cyanocobalamin/B12) by B. clausii CSI08.
• TTB liquid medium
• Figure 24 shows concentrations of essential amino acids and vitamins in overnight cultures of B. clausii CSI08 determined by RP-HPLC-FLD and RP-HPLC-MS correspondingly. Values are means ⁇ SEM. * p ⁇ 0.05, & p ⁇ 0.01, $ p ⁇ 0.0001.
• B. clausii CSI08 can produce essential amino acids and group B vitamins
• Cell lines Human Colorectal Adenocarcinoma Cell Line HT-29 and mucous-secreting cell line HT-29-MTX were propagated using low glucose DMEM medium supplemented with 10% Fetal Bovine Serum, 2 mM glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin, and 2 pg/ml amphotericin B in a 5% CO2 atmosphere at 37°C.
• Cells were seeded onto 24-well plates at a density 5xl0 5 cell/well and cultured for 21-28 days to complete maturation. Media was replaced every 2-3 days.
• Adhesion assay 500 pl of spores suspensions (1.3xl0 7 - 9.2xl0 7 CFU/ml) were added to HT-29 and HT-29-MTX cells, mixed by a gentle swirl, and incubated for 2.5 h at 37°C in the CO2 incubator. Control wells not containing mammalian cells were prepared and incubated in parallel in the same way (0.5 ml of spores’ suspensions).
• HT-29 and HT-29-MTX cells were washed 4 times with 0.5 ml PBS. After that 50 pl of Trypsin/EDTA solution and 50 pl of PBS were added to the wells and incubated for 10 min with gentle shaking (-100 rpm) at 37°C. Fifty microliters of Trypsin/EDTA solution were added to control wells.
• Results set out above demonstrate higher ability of spores to adhere to the mucous- secreting cell line HT-29-MTX compared to non-mucus secreting cells, possibly due to spores’ physical properties.
• B. megaterium MIT411 and B. coagulans CGI314 spores have higher (but overall low) ability to adhere to non-mucus producing cell line HT-29 compared to B. clausii CSI08 spores.
• the probiotic cocktail significantly decreased the incidence of loose stools throughout the entire study.
• Probiotics are live microorganisms residing in the human gut with low or no pathogenicity and exhibit beneficial effects for the host.
• Common products containing probiotic bacteria include dietary supplements and foodstuffs such as fermented dairy products, sauerkraut, and salami.
• Probiotic supplementation has shown positive results for relief of various ailments such as: antibiotic associated diarrhea, constipation, allergies, and diabetes.
• Probiotics have also exhibited protective properties.
• Probiotic supplements can contain one or more different bacterial strains that exert different effects on the human gut.
• Common probiotic strains are lactic acid producers such as Lactobacillus, Bifidobacterium, and Streptococcus due to their resistance to gastric acids, bile salts, and pancreatic enzymes.
• lactic acid bacteria are effective inhibitors of pathogenic, gram-negative, bacterial colonization (e.g. Salmonella typhimurium, Clostridium difficile, and Escherichia coli) in vitro.
• Not all probiotic supplements are lactic acid producers however. Bacillus subtilis spores have been used as probiotics, competitive exclusion agents, and prophylactics for human and animal consumption.
• Bacilli strains are gram-positive, spore forming, rod-shaped bacterium. Under nutrient limiting conditions, Bacillus sp. can form resistant dormant endospores to environmental stressors and nutrient deprivation, making these bacteria a viable option for a probiotic supplement.
• DEI 11, CSI08, CGI314, and MIT411 are unique strain of probiotics. Being Bacillus strains of probiotics, they are able to resist the harsh digestive environment and colonise the gut, thus supporting a healthy GI tract. To date, DEI 11 is sold in both the USA and Canada as a probiotic food ingredient and as a probiotic capsule for adults. The other three Bacillus probiotics CSI08, CGI314, and MIT411 used in this trial are not currently on the market and are claimed herein.
• Treatment groups were 1 x 10 9 CFU/dose of Bacillus clausii CSI08, 1 x 10 9 CFU/dose of Bacillus megaterium MIT411, and a probiotic cocktail containing Bacillus subtilis DEI 11®, Bacillus megaterium MIT411, Bacillus coagulans CGI314, and Bacillus clausii CSI08 with a total count of 2.0 x 10 9 CFU/dose administered daily.
• Placebo was rice maltodextrin.
• a randomisation scheme was performed by CRO Vizera d.o.o., Slovenia with the allocation sequence being concealed from study personnel and participants until randomisation day in sealed, opaque envelopes. After assessment of baseline characteristics (age, sex, height, weight by digital scale) and collection of an initial stool sample, an envelope was unsealed and participants were assigned to an intervention. Investigators received individually closed envelopes containing the link between the randomization number and the treatment group for a specific participant. The sealed envelopes could only be opened in case of emergency. The Sponsor was immediately notified if a participant’s treatment was unblinded during the course of the study. Information regarding the un-blinding had to be recorded in the data source document and in the Case Report Form (CRF) of the participant. Participants were then instructed to consume one capsule per day at the end of a meal.
• CRF Case Report Form
• Figure 25 shows a graphical flow chart of the study design.
• Deerland Probiotics and Enzymes provided investigational products as identical, oblong 300mg capsules and placebo was indistinguishable by appearance.
• the study capsules were provided in bottles labelled with a treatment code by a study collaborator who did not have contact with study personnel or participants.
• Participants completed the questionnaire daily to monitor time of defaecation and type of stool samples based on the Bristol stool chart index and if there were any symptoms including: gastrointestinal distress, respiratory distress, urinary tract symptoms, cephalic, ear-nose-throat, behavioural, emetic, loss of appetite, fever and epidermal. If any visits to their GP or any medication was prescribed during the trial this was also captured and reported.
• a mood questionnaire was administered to participants at baseline and at the end of the treatment period to assess their experience over the previous month. This questionnaire consisted of 14 captured symptoms including sadness, irritability, energy, appetite, tension, stress, sleep, cardiovascular events, aches and pains and dizziness on a scale of 1 (no noticeable symptoms) to 3 (severe). Any adverse events were reported to study staff.
• a 3-mL red cap serum clot activator tube was used (Greiner Bio-One, 454029) for blood collection.
• Reiner Bio-One, 454029 For biochemistry blood panel high- and low-density lipoproteins, total cholesterol and triglyceride determination, 3.5mL SST II Advanced/gel yellow cap vials (Greiner Bio-One, 454029) were used.
• SST II Advanced/gel yellow cap vials for antioxidants and cytokine determination, whole blood was collected into 4-mL lithium-heparin containing tubes (Greiner Bio-One, 454029). Plasma samples were prepared by centrifugation at 2000 G for 15 min. The supernatant was aliquoted and stored at - 80 °C for later analysis.
• LDL, HDL, Total Cholesterol and Triglyceride determination [0226] Hematology and Biochmistry assessment were run in University Clinical Centre Maribor, Slovenia. Safety bloods were run with a Sysmex EN-1000, while Biochemical assays for LDL, HDL, total cholesterol and triglyceride were assayed according to manufacturer’s instructions and analysed with an Abbott Allinity C.
• IL-8 and TNF-alpha in serum samples were determined by sandwich ELISAs: Human IL-8 (CXCL8) ELISA Kit (ELH-IL8-1, RayBiotech) and Human TNF alpha ELISA Kit (ELH-TNFa-1, RayBiotech) according to the manufacturer’s instruction. Prior to ELISAs serum samples were diluted 1 :2 using dilution buffers supplied with the kits.
• Total antioxidant activity was assessed using the total antioxidant capacity assay kit (Sigma, Ireland) according to manufacturer’s instructions and the absorbance was measured at 340nm.
• Total fecal DNA from approximately 200mg sample was extracted using ZymoBIOMICS DNA Miniprep Kit (Zymo Research, Irvine, CA, USA) in accordance with manufacturer’s instructions. Briefly, the stool samples were placed in the ZR BashingBeadTM Lysis tubes containing 750 pl ZymoBIOMICSTM Lysis Solution and processed in a BeadBugTM 6 homogenizer (Benchmark Scientific, China): 5x 1 min beating at 4350 rpm with 1 min intermittent step between beating cycles. After that, the lysis tubes were centrifuged at 10,000 g for 1 minute.
• alpha diversity indices were calculated including Observed, Chaol, ACE, Shannon and Simpson index. The alpha diversity was then compared among the experimental groups and against the Placebo in order to detect differences due to the treatments - or within treatments from baseline to the post-treatment timepoint.
• N* number of participants included in the ITT population, *p-value for Kruskal-Wallis test
• Stool consistency is reported as the proportion of participants with loose stool and the proportion of participants with hard stool in the total treatment period.
• Figure 26 shows the probiotic cocktail significantly decreased the incidence of loose stool over the course of the study as compared to placebo control.
• Figure 27 shows no effect of any treatments on percentage of hard stools as compared to placebo control.
• N* number of participants included in the ITT population
• NC not calculable
• /?* p-value for Kruskal-Wallis test
• p p- value for Mann-Whitney U test.
• Table 16 summarizes the answers to the Mood questionnaire at baseline and at the end of the study for the 3 treatment groups and the placebo. Mean changes with 95 % confidence interval are shown. Results of the ANOVA omnibus test (p*-value) and one-sample T test (p- value) are also presented. Test of normality for the change in scores of the Gut-brain axis show that the data do not follow normal distribution, which could affect the results with borderline significance (p-values between 0.05 and 0.10). This affects two items: Loss of energy and Changes in appetite. An alternative nonparametric Kruskal Wallis test was applied to these items; p-values of 0.111 (Loss of energy) and 0.123 (Changes in appetite) were observed.
• N* number of participants included in the ITT population
• HDL high-density lipoprotein
• LDL low-density lipoprotein
• TC TC
• N* number of participants included in the ITT population, *p-value for ANOVA (omnibus test)
• N* number of participants included in the ITT population, *p-value for ANOVA (omnibus test)
• N* number of participants included in the ITT population, *p-value for ANOVA (omnibus test)
• N* number of participants included in the ITT population, *p-value for ANOVA (omnibus test)
• Figure 28 is a boxplot showing the Chaol values distribution in each experimental group for Day 1 and Day 45. Dotted lines connect the paired samples. A paired Wilcoxon test was used to compare the distribution of the groups. A p-value less than 0.05 should be considered as statistically significant.
• Figure 29 is a boxplot showing the Chaol values distribution in each experimental group for Day 1 and Day 45. A Wilcoxon test was used to compare the distribution of each experimental group against the Placebo. A p-value less than 0.05 should be considered as statistically significant.
• Figure 30 illustrates PCoA clustering performed on the Bray-Curtis dissimilarity matrix. Each treatment is separated in a different tab while colours and shape are associated with the time points. Samples from the two time points tend to cluster together for all the treatments, and the data are not significantly different from each other at day 1 baseline readings. Samples were not significantly different from each other as a consequence of treatment within or between groups.
• N* number of participants included in the ITT population
• N* number of participants included in the ITT population
• N* number of participants included in the ITT population
• Bacillus probiotics in maintenance of gut health has been largely supported in the last years and has driven its clinical applications. Their favorable effects have been linked to several properties, such as antimicrobial and immunomodulatory activity, regulation of cell growth and differentiation, cell-cell signaling, cell adhesion, signal transcription and transduction, production of vitamins and gut protection from genotoxic agents.

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