CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of and priority to U.S. Provisional Patent Application No. 62/946801, filed on December 11, 2019, the entire disclosure of which is hereby incorporated by reference herein.

TECHNICAL FIELD

[0002] The present technology relates generally to edible product compositions comprising bacterial strains and their use as dietary supplements. In particular, the present technology relates to fermented food products comprising strains of Bacillus amyloliquefaciens bacteria identified as ART24, ART4, and ART12 and their use as dietary supplements.

BACKGROUND

[0003] The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the compositions and methods disclosed herein.

[0004] Clostridium difficile is a spore-forming, Gram-positive, anaerobic bacillus that can colonize the large bowel. Upon particular clinical conditions, e.g. when the natural microbiota has been disrupted or depleted, C. difficile can overgrow and produce toxins that cause disease pathologies. C. difficile infection (CDI) can cause symptoms ranging from mild diarrhea to life-threatening pseudomembranous colitis and toxic megacolon. The prolonged use of antibiotics, especially broad-spectrum agents, can induce a change in the composition and function of the intestinal microbiota resulting in antibiotic associated diarrhea (AAD). CDI or Clostridium difficile -a.$$oc\ ated disease (CD AD) is used to describe a constellation of illnesses caused by toxins A and B produced by the C. difficile bacillus.

[0005] C. difficile is the most common cause of nosocomial infectious diarrhea and is becoming an increasing burden to the health care system, resulting in approximately 29,000 deaths and totaling more than $1 billion per year in the United States. The emergence of highly virulent strains of C. difficile has been linked to increased disease prevalence and severity as well as higher rates of treatment failure with metronidazole.

[0006] Current therapeutic approaches involve terminating treatment with the offending antibiotic(s) and initiating treatment with vancomycin or metronidazole or fidaxomicin. Treatment of patients with C. difficile infection with Standard of Care (SOC) antibiotics reduces morbidity and mortality. However, the number of patients who do not respond to metronidazole is increasing. Antibiotic treatments will disrupt and can inhibit reestablishment of the beneficial endogenous gastrointestinal tract (GIT) microbiota, which renders the patients effectively cured to become at risk for recurrence of the disease. Despite advances in the treatment of infections, CDI recurrence is seen in about 10-35% of patients after their initial case of CDI, and in 35-65% of patients after a primary recurrence. Accordingly, there is a need for more effective therapies for CDI, and, in particular, therapies that preserve the endogenous gastrointestinal microbiota.

[0007] Digestive enzymes are normally produced and secreted by the gastrointestinal system to degrade fats, proteins, and carbohydrates, to accomplish digestion and the absorption of nutrients. Their supplementation may provide a reliable therapy for disorders, such as exocrine pancreatic insufficiency (EPI) and celiac disease, which may be characterized by an impairment of digestive functions. Accordingly, there is a need to develop effective therapies for enzymatic supplementation in gastrointestinal diseases.

SUMMARY

[0008] In one aspect, the present disclosure provides an edible product comprising a bacterial strain selected from the group consisting of ART24 (NCIMB Accession No. 43088), ART4 (NCIMB Accession No. 43086), and ART 12 (NCIMB Accession No. 43087). In some embodiments, the edible product is a dietary supplement. In some embodiments, the bacterial strain is ART24 (NCIMB Accession No. 43088). In some embodiments, the bacterial strain is ART4 (NCIMB Accession No. 43086). In some embodiments, the bacterial strain is ART12 (NCIMB Accession No. 43087). In some embodiments, the bacterial strain is lyophilized. In some embodiments, the bacterial strain is in the form of a spore. In some embodiments, the bacterial strain is in vegetative form. In some embodiments, the edible product further comprises a carrier, vehicle, or excipient. In some embodiments, the edible product further comprises a fermented food product, soybean, mushroom, mung bean, locus bean, rice, or extracts thereof. In some embodiments, the soybean is fermented soybean or fermented soybean paste. In some embodiments, the fermented soybean or fermented soybean paste is Cheonggukjang, Douchi, Hawaijar, Bekang, Peruyaan, Tungrymbai, Eoyukjang, Kinema, Aakhone, Miso, Natto, or Thua-nao.

[0009] In some embodiments, the product exhibits anti-C. difficile activity, lipase activity, amylase activity, protease activity, and/or gliadinase activity.

[0010] In one aspect, the present disclosure provides a method for treating or preventing a Clostridium difficile infection (CDI) or a Clostridium £¾/zcz7e-associated disease (CD AD) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the edible product comprising a bacterial strain selected from the group consisting of ART24 (NCIMB Accession No. 43088), ART4 (NCIMB Accession No. 43086), and ART 12 (NCIMB Accession No. 43087). In some embodiments, the edible product is a dietary supplement. In some embodiments, the bacterial strain is ART4 (NCIMB Accession No. 43086). In some embodiments, the bacterial strain is ART24 (NCIMB Accession No. 43088). In some embodiments, the bacterial strain is ART12 (NCIMB Accession No. 43087). In some embodiments, the bacterial strain is lyophilized. In some embodiments, the bacterial strain is in the form of a spore. In some embodiments, the bacterial strain is in vegetative form. In some embodiments, the edible product further comprises a carrier, vehicle, or excipient. In some embodiments, the edible product further comprises a fermented food product, soybean, mushroom, mung bean, locus bean, rice, or extracts thereof. In some embodiments, the soybean is fermented soybean or fermented soybean paste. In some embodiments, the fermented soybean or fermented soybean paste is Cheonggukjang, Douchi, Hawaijar, Bekang, Peruyaan, Tungrymbai, Eoyukjang, Kinema, Aakhone, Miso, Natto, or Thua-nao. In some embodiments, the product exhibits anti-C. difficile activity.

[0011] In another aspect, the present disclosure provides a method for treating or preventing celiac disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the edible product comprising a bacterial strain selected from the group consisting of ART24 (NCIMB Accession No. 43088), ART4 (NCIMB Accession No. 43086), and ART12 (NCIMB Accession No. 43087). In some embodiments, the edible product is a dietary supplement. In some embodiments, the bacterial strain is ART4 (NCIMB Accession No. 43086). In some embodiments, the bacterial strain is ART24 (NCIMB Accession No. 43088). In some embodiments, the bacterial strain is ART12 (NCIMB Accession No. 43087). In some embodiments, the bacterial strain is lyophilized. In some embodiments, the bacterial strain is in the form of a spore. In some embodiments, the bacterial strain is in vegetative form. In some embodiments, the edible product further comprises a carrier, vehicle, or excipient. In some embodiments, the edible product further comprises a fermented food product, soybean, mushroom, mung bean, locus bean, rice, or extracts thereof. In some embodiments, the soybean is fermented soybean or fermented soybean paste. In some embodiments, the fermented soybean or fermented soybean paste is Cheonggukjang, Douchi, Hawaijar, Bekang, Peruyaan, Tungrymbai, Eoyukjang, Kinema, Aakhone, Miso, Natto, or Thua-nao. In some embodiments, the product exhibits gliadinase activity.

[0012] In another aspect, the present disclosure provides a method for treating or preventing non-celiac gluten intolerance in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the edible product comprising a bacterial strain selected from the group consisting of ART24 (NCIMB Accession No. 43088), ART4 (NCIMB Accession No. 43086), and ART12 (NCIMB Accession No. 43087). In some embodiments, the edible product is a dietary supplement. In some embodiments, the bacterial strain is ART4 (NCIMB Accession No. 43086). In some embodiments, the bacterial strain is ART24 (NCIMB Accession No. 43088). In some embodiments, the bacterial strain is ART12 (NCIMB Accession No. 43087). In some embodiments, the bacterial strain is lyophilized. In some embodiments, the bacterial strain is in the form of a spore. In some embodiments, the bacterial strain is in vegetative form. In some embodiments, the edible product further comprises a carrier, vehicle, or excipient. In some embodiments, the edible product further comprises a fermented food product, soybean, mushroom, mung bean, locus bean, rice, or extracts thereof. In some embodiments, the soybean is fermented soybean or fermented soybean paste. In some embodiments, the fermented soybean or fermented soybean paste is Cheonggukjang, Douchi, Hawaijar, Bekang, Peruyaan, Tungrymbai, Eoyukjang, Kinema, Aakhone, Miso, Natto, or Thua-nao. In some embodiments, the product exhibits gliadinase activity.

[0013] In another aspect, the present disclosure provides a method for treating or preventing exocrine pancreatic insufficiency in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the edible product comprising a bacterial strain selected from the group consisting of ART24 (NCIMB Accession No. 43088), ART4 (NCIMB Accession No. 43086), and ART12 (NCIMB Accession No. 43087). In some embodiments, the edible product is a dietary supplement. In some embodiments, the bacterial strain is ART4 (NCIMB Accession No. 43086). In some embodiments, the bacterial strain is ART24 (NCIMB Accession No. 43088). In some embodiments, the bacterial strain is ART12 (NCIMB Accession No. 43087). In some embodiments, the bacterial strain is lyophilized. In some embodiments, the bacterial strain is in the form of a spore. In some embodiments, the bacterial strain is in vegetative form. In some embodiments, the edible product further comprises a carrier, vehicle, or excipient. In some embodiments, the edible product further comprises a fermented food product, soybean, mushroom, mung bean, locus bean, rice, or extracts thereof. In some embodiments, the soybean is fermented soybean or fermented soybean paste. In some embodiments, the fermented soybean or fermented soybean paste is Cheonggukjang, Douchi, Hawaijar,

Bekang, Peruyaan, Tungrymbai, Eoyukjang, Kinema, Aakhone, Miso, Natto, or Thua-nao. In some embodiments, the product exhibits amylase, protease, and/or lipase activity.

[0014] In one aspect, the edible product is for use as a pancreatic enzyme replacement therapy (PERT).

[0015] In one aspect, the present disclosure provides a method of producing a fermented edible product comprising a bacterial strain selected from the group consisting of ART24 (NCIMB Accession No. 43088), ART4 (NCIMB Accession No. 43086), and ART 12 (NCIMB Accession No. 43087), the method comprising inoculating a food with the bacterial strain and incubating the inoculated food for a period of time sufficient to produce a fermented food product. In some embodiments, the fermented edible product comprises fermented soybean and/or fermented soybean paste. [0016] In one aspect, the present disclosure provides a method for treating or preventing celiac disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising one or more bacterial strains selected from the group consisting of ART4 (NCIMB Accession No. 43086), ART24 (NCIMB Accession No. 43088), and ART12 (NCIMB Accession No. 43087). In some embodiments, the bacterial strain is ART4 (NCIMB Accession No. 43086). In some embodiments, the bacterial strain is ART24 (NCIMB Accession No. 43088). In some embodiments, the bacterial strain is ART12 (NCIMB Accession No. 43087). In some embodiments, the bacterial strain is lyophilized. In some embodiments, the bacterial strain is in the form of a spore. In some embodiments, the bacterial strain is in vegetative form. In some embodiments, the pharmaceutical composition is administered enterically. In some embodiments, the pharmaceutical composition is formulated for use as a dietary supplement.

[0017] In another aspect, the present disclosure provides a method for treating or preventing non-celiac gluten intolerance in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising one or more bacterial strains selected from the group consisting of ART4 (NCIMB Accession No. 43086), ART24 (NCIMB Accession No. 43088), and ART12 (NCIMB Accession No. 43087). In some embodiments, the bacterial strain is ART4

(NCIMB Accession No. 43086). In some embodiments, the bacterial strain is ART24

(NCIMB Accession No. 43088). In some embodiments, the bacterial strain is ART12

(NCIMB Accession No. 43087). In some embodiments, the bacterial strain is lyophilized. In some embodiments, the bacterial strain is in the form of a spore. In some embodiments, the bacterial strain is in vegetative form. In some embodiments, the pharmaceutical composition is administered enterically. In some embodiments, the pharmaceutical composition is formulated for use as a dietary supplement.

[0018] In another aspect, the present disclosure provides a method for treating or preventing exocrine pancreatic insufficiency in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising one or more bacterial strains selected from the group consisting of ART4 (NCIMB Accession No. 43086), ART24 (NCIMB Accession No. 43088), and ART12 (NCIMB Accession No. 43087). In some embodiments, the bacterial strain is ART4 (NCIMB Accession No. 43086). In some embodiments, the bacterial strain is ART24 (NCIMB Accession No. 43088). In some embodiments, the bacterial strain is ART12 (NCIMB Accession No. 43087). In some embodiments, the bacterial strain is lyophilized. In some embodiments, the bacterial strain is in the form of a spore. In some embodiments, the bacterial strain is in vegetative form. In some embodiments, the pharmaceutical composition is administered enterically. In some embodiments, the pharmaceutical composition is formulated for use as a dietary supplement.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGS. 1A-1H are photographs showing the growth progression of the un-inoculated and inoculated soybeans from Artujang 1.0. FIG. 1A shows the un-inoculated beans after 24h incubation. FIG. IB shows the un-inoculated beans after 48h incubation. FIG. 1C shows beans inoculated with 6xl0 ⁸ CFU/ml ART24 inoculum after 24h. FIG. ID shows beans inoculated with 6xl0 ⁸ CFU/ml ART24 inoculum after 48h. FIG. IE shows beans inoculated with 6xl0 ⁹ CFU/ml ART24 inoculum after 24h. FIG. IF shows beans inoculated with 6xl0 ⁹ CFU/ml ART24 inoculum after 48h FIG. 1G shows the bacterial colony morphology identified from beans inoculated with ART24 after 48h. FIG. 1H shows the bacterial colony morphology identified from un-inoculated beans.

[0020] FIG. 2 shows the vegetative colony forming units per mL (CFU/g) of Bacillus colonies and the spore count identified from the soybeans over 96 hours.

[0021] FIGS. 3A-3B show the effect of Artujang 1.0 on the growth of C. difficile using well diffusion assay (WDA). FIG. 3A shows the effect of IP A extracts from the bean surface after 24 hours incubation on C. difficile. FIG. 3B shows the effect of IP A extracts from the homogenised bean pastes after 24 hours incubation on C. difficile.

[0022] FIGS. 4A-4I show the MALDI-TOF mass spectrometry chromatographs identified from the cell surface extracts from each treatment across the different time points. FIG. 4A shows the trace file corresponding to the uninoculated beans at T24. FIG. 4B. shows the trace file corresponding to the beans inoculated with 6xl0 ⁸ CFU/ml ART24 at T24. FIG. 4C shows the trace file corresponding to the beans inoculated with 6xl0 ⁹ CFU/ml ART24 at T24. FIG. 4D shows the trace file corresponding to the uninoculated beans at T48. FIG. 4E shows the trace file corresponding to the beans inoculated with 6xl0 ⁸ CFU/ml ART24 at T48. FIG. 4F shows the trace file corresponding to the beans inoculated with 6xl0 ⁹ CFU/ml at T48. FIG. 4G shows the trace file corresponding to the uninoculated homogenised bean paste at T96. FIG. 4H shows the trace file corresponding to the paste from the beans inoculated with 6xl0 ⁸ CFU/ml ART24 at T96. FIG. 41 shows the trace file corresponding to the paste from the beans inoculated with 6xl0 ⁹ CFU/ml ART24 at T96.

[0023] FIGS. 5A-5G are photographs showing the growth progression of the un-inoculated and inoculated soybeans from Artujang 2.0. FIG. 5A shows beans that have not been autoclaved or inoculated. FIG. 5B shows beans and paste that have been autoclaved but not inoculated with ART24. FIG. 5C shows beans and paste that were incubated overnight at 37°C and was then inoculated with 10 ⁸ CFU/ml of ART24. FIG. 5D shows beans and paste that were inoculated 10 ⁹ CFU/ml of ART24. FIG. 5E shows beans and paste that were inoculated with 10 ⁸ CFU/ml of ART24. FIG. 5F shows beans and paste that were inoculated with 10 ⁶ CFU/ml of ART24. FIG. 5G shows beans and paste that were inoculated with 10 ⁴ CFU/ml of ART24.

[0024] FIG. 6 shows the vegetative colony forming units per ml (CFU/g) of Bacillus colonies identified from the soybeans over 48-72 hours.

[0025] FIGS. 7A-7N shows the MALDI-TOF mass spectrometry chromatographs identified from the cell surface extracts from each treatment across the different time points. FIG. 7A shows the trace file for the beans inoculated with 10 ⁹ CFU/ml ART24 at T24. FIG. 7B shows the trace file for the paste inoculated with 10 ⁹ CFU/ml ART24 at T24. FIG. 7C shows the trace file for the beans inoculated with 10 ⁸ CFU/ml ART24 at T24. FIG. 7D shows the trace file for the paste inoculated with 10 ⁸ CFU/ ART24 at T24. FIG. 7E shows the trace file for the beans inoculated with 10 ⁶ CFU/ml ART24 at T24. FIG. 7F shows the trace file for the paste inoculated with 10 ⁶ CFU/ml ART24 at T24. FIG. 7G shows the trace file for the beans inoculated with 10 ⁴ CFU/ml ART24 at T24. FIG. 7H shows the trace file for the paste inoculated with 10 ⁴ CFU/ml ART24 at T24. FIG. 71 shows the trace file for the uninoculated beans at T24. FIG. 7J shows the trace file for the uninoculated pastes at T24. FIG. 7K shows the trace file for the beans that were not autoclaved or inoculated at T24. FIG. 7L shows the trace file for the paste that was not autoclaved or inoculated at T24. FIG. 7M shows the trace file for the beans inoculated with 10 ⁸ CFU/ml ART24 after 24h at T24. FIG. 7N shows the trace file for the paste inoculated with 10 ⁸ CFU/ml ART24 after 24h at T24.

[0026] FIGS. 8A-8N shows the MALDI-TOF mass spectrometry chromatographs identified from the cell surface extracts from each treatment across the different time points. FIG. 8A shows the trace file for the beans inoculated with 10 ⁹ CFU/ml at T48. FIG. 8B shows the trace file for the paste inoculated with 10 ⁹ CFU/ml at T48. FIG. 8C shows the trace file for the beans inoculated with 10 ⁸ CFU/ml at T48. FIG. 8D shows the trace file for the paste inoculated with 10 ⁸ CFU/ml at T48. FIG. 8E shows the trace file for the beans inoculated with 10 ⁶ CFU/ml at T48. FIG. 8F shows the trace file for the paste inoculated with 10 ⁶ CFU/ml at T48. FIG. 8G shows the trace file for the beans inoculated with 10 ⁴ CFU/ml at T48. FIG. 8H shows the trace file for the paste inoculated with 10 ⁴ CFU/ml at T48. FIG. 81 shows the trace file for the un-inoculated beans at T48. FIG. 8J shows the trace file for the un-inoculated pastes at T48. FIG. 8K shows the trace file for the beans that were not autoclaved or inoculated at T48. FIG. 8L shows the trace file for the paste that was not autoclaved or inoculated at T48. FIG. 8M shows the trace file for the beans inoculated with 10 ⁸ CFU/ml ART24 after 24h at T48. FIG. 8N shows the trace file for the paste inoculated with 10 ⁸ CFU/ml ART24 after 24h at T48.

[0027] FIG. 9 shows the zones of inhibition against C. difficile from the T24 IPA extracts from beans and pastes from Artujang 2.0. A = lxlO ⁹ Beans; B= lxlO ⁹ Paste; C = lxlO ⁸ Beans; D = lxlO ⁸ Paste; E = lxlO ⁶ Beans; F = lxlO ⁶ Paste; G = lxlO ⁴ Beans; H = lxlO ⁴ Paste; I = No Inoculum Beans; J = No Inoculum Paste* (*Cross contaminated with ART24); K = No autoclaving Beans; L = No autoclaving Paste; M = lxlO ⁸ 24h Beans; N = lxlO ⁸ 24h Paste.

[0028] FIG 10 shows the zones of inhibition against C. difficile from the T48 IPA extracts from beans and pastes from Artujang 2.0. A = lxlO ⁹ Beans; B= lxlO ⁹ Paste; C = lxlO ⁸ Beans; D = lxlO ⁸ Paste; E = lxlO ⁶ Beans; F = lxlO ⁶ Paste; G = lxlO ⁴ Beans; H = lxlO ⁴ Paste; I = No Inoculum Beans; J = No Inoculum Paste* (*Cross contaminated with ART24); K = No autoclaving Beans; L = No autoclaving Paste; M = lxlO ⁸ 48h Beans; N = lxlO ⁸ 48h Paste. [0029] FIGS. 11A-11E are photographs showing the growth progression of the inoculated soybeans from Artujang 3.0. FIG. 11A shows the beans and paste at TO. FIG. 11B shows the beans and paste at T24. FIG. 11C shows the beans and paste at T24 post mixing. FIG. 11D shows the beans and paste at T48. FIG. HE shows the beans and paste at T48 post mixing.

[0030] FIG. 12 shows the vegetative colony forming units per ml (CFU/g) of Bacillus colonies identified from the soybeans over 48 hours from Artujang 3.0.

[0031] FIG. 13 shows the zones of inhibition against C. difficile from the T24 IPA extracts from beans and pastes from Artujang 3.0.

[0032] FIG. 14 shows the zones of inhibition against C. difficile from the T48 IPA extracts from beans and pastes from Artujang 4.0.

[0033] FIG. 15 show photographs of the Artujang 4.0 flasks at TO.

[0034] FIG. 16 show photographs of the Artujang 4.0 flasks at T24.

[0035] FIG. 17 show photographs comparing the beans static and shaking flasks after 24h.

[0036] FIG. 18 is an image of the zones of degradation of extracts from Artujang 4.0 treatments on starch agar plates. A = Beans shaking; B = Beans static; C = No soak milled beans shaking; D = No soak milled beans static; E = Milled beans shaking; F = Milled beans static.

[0037] FIG. 19 is an image of the zones of degradation of extracts from Artujang 4.0 treatments on gliadin agar plates. A = Beans shaking; B = Beans static; C = No soak milled beans shaking; D = No soak milled beans static; E = Milled beans shaking; F = Milled beans static.

[0038] FIG. 20 is an image of the zones of degradation of tributyrin from Artujang 4.0 extracts. A = Beans shaking; B = Beans static; C = No soak milled beans shaking; D = No soak milled beans static; E = Milled beans shaking; F = Milled beans static.

[0039] FIGS. 21A-21B are GIP ELISA charts showing the gliadinase activity of ART4 supernatant against 33mer gliadin peptide (FIG. 21A) and full gliadin (FIG. 21B). [0040] FIGS. 22A-22B are lipase assay charts showing the lipase activity of ART4 supernatant (FIG. 22 A) and that of CREON ® (pancrelipase) (FIG. 22B). For FIG. 22 A: Slope = 0.65 mM/min; Specific activity = 108-117 pmol/hour/mL; Final = 117 U/mL. For FIG. 22B: Slope = 0.475 mM/min; Specific activity = 86-135 pmol/hour/mL; Final = 19922- 31455 U/capsule.

DETAILED DESCRIPTION

[0041] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present technology are described below in various levels of detail in order to provide a substantial understanding of the present technology. The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this present technology belongs.

I. Definitions

[0042] The following terms are used herein, the definitions of which are provided for guidance.

[0043] As used herein, the singular forms “a,” “an,” and “the” designate both the singular and the plural, unless expressly stated to designate the singular only.

[0044] The term “about” and the use of ranges in general, whether or not qualified by the term about, means that the number comprehended is not limited to the exact number set forth herein, and is intended to refer to ranges substantially within the quoted range while not departing from the scope of the invention. As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term.

[0045] As used herein, “administration” of an edible product or composition of the present technology to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. In particular embodiments, the compositions of the present technology are formulated for enteric administration. In some embodiments, the compositions are formulated for oral or sublingual delivery. In some embodiments, the compositions are formulated for use as a probiotic. In some embodiments, the compositions are formulated for use as a live biotherapeutic. As used herein, administration includes self- administration and administration by another.

[0046] As used herein, “ART24” refers to a bacterial strain, or spore thereof, having been deposited under NCIMB Accession No. 43088, or compositions comprising the strain.

[0047] As used herein, “ART4” refers to a bacterial strain, or spore thereof, having been deposited under NCIMB Accession No. 43086, or compositions comprising the strain.

[0048] As used herein, “ART 12” refers to a bacterial strain, or spore thereof, having been deposited under NCIMB Accession No. 43087, or compositions comprising the strain.

[0049] As used herein, “Artujang” is a collective term for whole soybeans and/or soybean pastes inoculated with ART24. In some embodiments, “Artujang,” may also refer to whole soybeans and/or soybean pastes inoculated with any one or more of ART24, ART4, or ART12.

[0050] As used herein, “Cheonggukjang” refers to a fermented soybean paste used in Korean cuisine. Cheonggukjang is produced during the short-term fermentation (2-3 days) of soybeans using the natural microbiota of the air or from rice straw such as Bacillus subtilis. Following fermentation, the final Cheonggukjang product contains enzymes and bioactive compounds that are absent from unfermented soybeans.

[0051] As used herein, the term “dietary supplement” refers to a product intended to supplement the diet. Typically, a dietary supplement is a product that is labeled as a dietary supplement and is not represented for use as a conventional food or as a sole item of a meal or the diet. A dietary supplement can be consumed by a subject independent of any food, unlike a food additive, which is incorporated into a food or food composition during the processing, manufacture, preparation, or delivery of the food or food composition, or just prior to its consumption. [0052] As used herein, the term “edible product” refers to any substance, whether processed, semi-processed, or raw, which is intended for consumption by animals including humans. In some embodiments, the term “edible product” refers to a food that comprises one or more of the bacterial strains of the present technology. Any food to which the bacterial strains of the present technology is added is an edible product of the present technology. Any food in which a bacterial strain of the present technology is made to be present at a greater level is also an edible product of the present technology. Edible products comprising one or more bacterial strains of the present technology include dietary supplements, nutraceutical compositions, food additives, food compositions, food compositions in bulk, food additives in bulk, medical foods, and foods for special dietary use. Edible products of the present technology include, but are not limited to, fermented food products, soybean, soybean-based food products, fermented soybean, fermented soybean paste, mushroom, mung bean, locus bean, rice, or extracts thereof. In some embodiments, edible products of the present technology refer to a food that is formulated to be consumed or administered enterally to a subject.

[0053] As used herein, the terms “effective amount,” or “therapeutically effective amount,” and “pharmaceutically effective amount” refer to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g ., an amount which results in the prevention of a disease, condition, and/or symptom(s) thereof. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the subject, such as general health, age, sex, body weight, and tolerance to the composition drugs. It will also depend on the degree, severity, and type of disease or condition. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. In some embodiments, multiple doses are administered. Additionally or alternatively, in some embodiments, multiple therapeutic compositions or compounds (e.g, pharmaceutical compositions comprising multiple bacterial strains alone or in combination with additional active agents, such as antibiotics) are administered. In some embodiments, in the methods described herein, compositions comprising the bacterial strains of the present technology, or spores thereof, may be administered to a subject having one or more signs, symptoms, or risk factors of C. difficile infection (CDI) or Clostridium difficile -associated disease (CD AD), including, but not limited to diarrhea, weight loss, appetite loss, bloating, flu-like symptoms, fever, abdominal pain, nausea, dehydration, colitis, and pseudomembraneous colitis. In some embodiments, in the methods described herein, compositions comprising the bacterial strains of the present technology, or spores thereof, may be administered to a subject having one or more signs, symptoms, or risk factors of celiac disease or non-celiac gluten intolerance, including, but not limited to abdominal pain, bloating, gas, diarrhea, foggy mind, lethargy and fatigue. In some embodiments, in the methods described herein, compositions comprising the bacterial strains of the present technology, or spores thereof, may be administered to a subject having one or more signs, symptoms, or risk factors of exocrine pancreatic insufficiency (EPI), including, but not limited to malabsorption, steatorrhea, weight loss, and malnutrition. For example, a “therapeutically effective amount” of the compositions of the present technology, includes levels at which the presence, frequency, or severity of one or more signs, symptoms, or risk factors of CDI, CD AD, celiac disease, non-celiac gluten intolerance, and EPI are, at a minimum, ameliorated. In some embodiments, a therapeutically effective amount reduces or ameliorates the physiological effects of CDI, CD AD, celiac disease, non-celiac gluten intolerance, and EPI and/or the risk factors of CDI, CD AD, celiac disease, non-celiac gluten intolerance, and EPI, and/or the likelihood of developing CDI, CD AD, celiac disease, non-celiac gluten intolerance, and EPI. In some embodiments, a therapeutically effective amount is achieved by multiple administrations. In some embodiments, a therapeutically effective amount is achieved with a single administration.

[0054] As used herein, the term “food additive” refers to any substance not normally consumed as a food by itself and not normally used as a typical ingredient of the food, whether or not it has nutritive value, but which is intentionally added to food.

[0055] As used herein, the term “food for special dietary use” refers to a food that purports or is represented to be used for at least one of the following: supplying a special dietary need that exists by reason of physical, physiological, pathological of other condition, including CDI, CD AD, celiac disease, non-celiac gluten intolerance, or EPI. [0056] As used herein, the terms “freeze-dried” or “freeze-drying” and “lyophilized” or “lyophilization” are used interchangeably and refer to a process that removes water from a product after it is frozen and placed under a vacuum and the products produced therefrom.

[0057] As used herein, the term “medical food” refers to a food that is formulated to be consumed or administered enterally under the supervision of a physician and which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements are established.

[0058] As used herein, “pharmaceutically acceptable carrier and/or diluent” or “pharmaceutically acceptable excipient” includes but is not limited to solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In some embodiments, the pharmaceutically acceptable carrier comprises a polysaccharide, locust bean gum, an anionic polysaccharide, a starch, a protein, sodium ascorbate, glutathione, trehalose, sucrose, or pectin. In some embodiments, the polysaccharide comprises a plant, animal, algal, or microbial polysaccharide. In some embodiments, the polysaccharide comprises guar gum, inulin, amylose, chitosan, chondroitin sulphate, an alginate, or dextran. In some embodiments, the starch comprises rice starch.

The use of such media and agents for biologically active substances is well known in the art. Further details of excipients are provided below. Supplementary active ingredients, such as antimicrobials, for example antifungal agents, can also be incorporated into the compositions.

[0059] As used herein, “pharmaceutically acceptable excipient” refers to substances and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or a human. As used herein, the term includes all inert, non-toxic, liquid or solid fillers, or diluents that do not react with the therapeutic substance of the invention in an inappropriate negative manner, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, preservatives and the like, for example liquid pharmaceutical carriers e.g., sterile water, saline, sugar solutions, Tris buffer, ethanol and/or certain oils.

[0060] As used herein, “probiotic” refers to bacteria comprising a component of the transient or endogenous flora of a subject administered to confer a beneficial prophylactic and/or therapeutic effect on the subject. Probiotics are generally known to be safe by those skilled in the art. Although not wishing to be bound by any particular mechanism, it is possible that the prophylactic and/or therapeutic effect of the edible products comprising the B. amyloliquefaciens strains of the present technology results from inhibition of pathogenic bacterial growth due to the production of extracellular products having antimicrobial activity. In other embodiments, although not wishing to be bound by any particular mechanism, it is possible that the prophylactic and/or therapeutic effect of the edible products comprising the B. amyloliquefaciens strains of the present technology results from production of enzymatic activity due to the production of extracellular products having enzymatic activity ( e.g ., gliadinase, amylase, protease, and/or lipase activity). In some embodiments, “probiotics” include “live biotherapeutic products.”

[0061] As used herein, “prevention,” “prevent,” or “preventing” of a disorder or condition refers to, in a statistical sample, reduction in the occurrence or recurrence of the disorder or condition in treated subjects/samples relative to an untreated controls, or refers delays the onset of one or more symptoms of the disorder or condition relative to the untreated controls.

[0062] As used herein “subject” and “patient” are used interchangeably. In some embodiments, the subject is an animal subject. In some embodiments, the animal subject is a mammal. In some embodiments, the mammalian subject is a human.

[0063] As used herein, the term “simultaneous” administration refers to the administration of at least two agents by the same route and at the same time or at substantially the same time.

[0064] As used herein, the term “separate” administration refers to an administration of at least two agents at the same time or at substantially the same time by different routes.

[0065] As used herein, the term “sequential” administration refers to administration of at least two agents at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one agent before administration of the other agent(s) commences. It is thus possible to administer one of the agents over several minutes, hours, or days before administering another. [0066] A “synergistic therapeutic effect” refers to a greater-than-additive therapeutic effect which is produced by a combination of at least two therapeutic agents, and which exceeds that which would otherwise result from the individual administration of the agents. For example, use of bacterial strains of the present technology in conjunction with other agents for the treatment of CDI, CD AD, celiac disease, non-celiac gluten intolerance, or exocrine pancreatic insufficiency may result in a greater than additive therapeutic effect. In some embodiments, the synergistic effect may permit the use of lower doses of bacterial strains of the present technology and/or other agents than would be required if each were used alone.

[0067] “Treating,” “treat,” “treated,” or “treatment” of a disease or disorder includes: (i) inhibiting the disease or disorder, arresting its development; (ii) relieving the disease or disorder, i.e., causing its regression; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder.

[0068] It is to be appreciated that the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean “substantial,” which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.

II. Clostridium difficile Infection (CDI)

[0069] Clostridium difficile infection (CDI) or Clostridium i¾/zcz7e-associated disease (CD AD) is a symptomatic infection caused by the anaerobic, spore forming, Gram-positive bacterium, Clostridium difficile. Symptoms of CDI include watery diarrhea, fever, nausea, and abdominal pain. CDI underlies about 20% of cases of antibiotic-associated diarrhea (AAD). Complications of CDI may include pseudomembranous colitis, toxic megacolon, perforation of the colon, and sepsis and death.

[0070] CDI is transmitted by bacterial spores. The spores are resistant to many types of disinfectants, and may persist on contaminated surfaces, including food surfaces, for a period of months. Risk factors for CDI include antibiotic or proton pump inhibitor use, hospitalization, and advanced age. In healthy individuals, the growth of C. difficile is kept in check by normal microbiota present in the gastrointestinal tract. The use of broad-spectrum antibiotics and medications that decrease stomach acidity, such as proton pump inhibitors, can allow C. difficile bacteria to proliferate and promote infection. In the colon, C. difficile produces an enterotoxin (toxin A) and a cytotoxin (toxin B). The toxins stimulate the production of inflammatory mediators, which increase intestinal wall permeability leading to diarrhea, and cause degradation of colon epithelial cells. Diagnosis of CDI is by stool culture or testing for the presence of bacterial DNA or toxins.

[0071] In some embodiments, the present technology provides methods and compositions for the treating or preventing C. difficile infection, including reducing the severity of one or more risk factors, signs, or symptoms associated with C. difficile infections. In some embodiments the compositions comprise the novel Bacillus amyloliquefaciens strains ART24, ART4, and ART12, or spores thereof.

III. Celiac Disease and Non-Celiac Gluten Intolerance

[0072] Celiac disease is a multifactorial disease characterized by an inflammatory response to ingested gluten in the small intestine. Gluten peptides rich in proline and glutamine, which are found primarily in wheat, barley, and rye, elicit an immune reaction in genetically predisposed subjects who carry the HLA-DQ2 or DQ8 genes. A gluten-free diet is the only known treatment for celiac disease. Some patients have symptoms of celiac disease but their blood tests and endoscopies are negative or normal. This condition has been called non-celiac gluten intolerance. The symptoms may be similar to celiac disease including abdominal pain, bloating, gas, diarrhea, foggy mind, lethargy or fatigue. Symptoms improve when gluten is eliminated from the diet and return when gluten containing foods are reintroduced. Signs and symptoms of celiac disease and non-celiac gluten intolerance include abdominal pain, bloating, gas, diarrhea, foggy mind, lethargy and fatigue. Left untreated, celiac disease may lead to non-intestinal symptoms including anemia, chronic fatigue, osteoporosis, impaired spleen, infertility, neurologic disorders, skin rashes and cancer.

[0073] In some embodiments, the present technology provides methods and compositions for the treating or preventing celiac disease or non-celiac gluten intolerance, including reducing the severity of one or more risk factors, signs, or symptoms associated with celiac disease or non-celiac gluten intolerance. In some embodiments the compositions comprise edible products comprising Bacillus amyloliquefaciens strains ART24, ART4, and ART12, or spores thereof. In some embodiments the edible products comprising one or more of the B. amyloliquefaciens strains of the present technology, are characterized by gliadinase activity, and may be useful in methods of treating or preventing celiac disease or non-celiac gluten intolerance.

IV. Exocrine Pancreatic Insufficiency

[0074] Pancreatic enzymes ( e.g ., lipase, protease, amylase) help break down fats, proteins and carbohydrates. Pancreatic insufficiency is the inability of the pancreas to secrete the enzymes needed for digestion. In exocrine pancreatic insufficiency (EPI), the pancreas is unable to secrete sufficient quantities of digestive enzymes needed for digestion. After pancreatic surgery, up to 80% of patients will develop EPI. EPI is a life-threatening condition associated with several pancreatic and extra-pancreatic diseases, such as chronic pancreatitis, acute pancreatitis, pancreatic cancer, cystic fibrosis, and as a consequence of gastrointestinal and pancreatic surgery. Signs and symptoms of EPI include feelings of indigestion, cramping after meals, large amounts of gas, foul smelling gas or stools, floating or fatty stools, light-colored, yellow or orange stools, frequent stools, loose stools, and weight loss.

[0075] In some embodiments, the present technology provides methods and compositions for the treating or preventing EPI, including reducing the severity of one or more risk factors, signs, or symptoms associated with EPI. In some embodiments the compositions comprise edible products comprising Bacillus amyloliquefaciens strains ART24, ART4, and ART12, or spores thereof. In some embodiments the edible products comprising one or more of the B. amyloliquefaciens strains of the present technology, which are characterized by one or more of amylase, lipase, or protease activity, may be used as a pancreatic enzyme replacement therapy (PERT).

V. Bacillus amyloliquefaciens Strains of the Present Technology

[0076] The technology of the present disclosure relates to the use of several strains of Bacillus amyloliquefaciens bacteria, or spores thereof, in the production of edible products, such as a dietary supplement. The bacterial strains were isolated from human stool samples and analyzed for anti-C. difficile activity. A primary screen was done for proteolytic activity, with a secondary screen for anti-C. difficile activity. Colonies showing antimicrobial activity against C. difficile strain were purified and designated as “ART24,” “ART4,” and “ART12.” ART24 refers to a bacterial strain, or spore thereof, characterized by NCIMB Accession No. 43088, or compositions comprising the strain. ART4 refers to a bacterial strain, or spore thereof, characterized by NCIMB Accession No. 43086, or compositions comprising the strain. ART12 refers to a bacterial strain, or spore thereof, characterized by NCIMB Accession No. 43087, or compositions comprising the strain.

[0077] In some embodiments, bacterial strains of the present technology, or spores thereof, are used in methods and edible product compositions for treating or preventing C. difficile infections in a subject in need thereof. In some embodiments, the bacterial strains comprise a dietary supplement for preventing or controlling C. difficile infections in a subject in need thereof.

[0078] In some embodiments, bacterial strains of the present technology, or spores thereof, are used in methods and edible product compositions for treating or preventing celiac disease in a subject in need thereof. In some embodiments, the bacterial strains comprise a dietary supplement for treating or preventing celiac disease in a subject in need thereof.

[0079] In some embodiments, bacterial strains of the present technology, or spores thereof, are used in methods and edible product compositions for treating or preventing non-celiac gluten intolerance in a subject in need thereof. In some embodiments, the bacterial strains comprise a dietary supplement for treating or preventing non-celiac gluten intolerance in a subject in need thereof.

[0080] In some embodiments, bacterial strains of the present technology, or spores thereof, are used in methods and edible product compositions for treating or preventing exocrine pancreatic insufficiency in a subject in need thereof. In some embodiments, the bacterial strains comprise a dietary supplement for treating or preventing exocrine pancreatic insufficiency in a subject in need thereof. In some embodiments, the bacterial strains comprise an edible food product or dietary supplement for use as a pancreatic enzyme replacement therapy (PERT) in a subject in need thereof. [0081] In some embodiments, compositions of the present technology comprise vegetative bacterial cells. In some embodiments, compositions of the present technology comprise bacterial spores. In some embodiments, compositions of the present technology comprise a combination of vegetative bacterial cells and bacterial spores.

VII. Therapeutic and Prophylactic Methods

[0082] The following discussion is presented by way of example only, and is not intended to be limiting.

[0083] One aspect of the present technology includes methods of treating or preventing CDI, CD AD, celiac disease, non-celiac gluten intolerance, or EPI in a subject diagnosed as having, suspected as having, or at risk of having CDI, CD AD, celiac disease, non-celiac gluten intolerance, or EPI. In therapeutic applications, edible products or dietary supplements comprising a bacterial strain, or spore thereof, selected from the group consisting of ART24, ART4, ART12, and combinations thereof, are administered to a subject suspected of, or already suffering from such a disease, in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease, including its complications and intermediate pathological phenotypes in development of the disease.

[0084] Subjects suffering from CDI, CD AD, celiac disease, non-celiac gluten intolerance, or EPI can be identified by any or a combination of diagnostic or prognostic assays known in the art.

[0085] In one aspect, the present technology provides a method for preventing or delaying the onset of CDI, CD AD, celiac disease, non-celiac gluten intolerance, or EPI, or symptoms of CDI, CD AD, celiac disease, non-celiac gluten intolerance, or EPI, in a subject at risk of having CDI, CD AD, celiac disease, non-celiac gluten intolerance, or EPI. In some embodiments, the edible products of the present technology are formulated as a probiotic useful as a food or dietary supplement and for re-establishing beneficial bacteria in the intestinal tract. In some embodiments, the bacterial strains of the present technology are formulated as a live biotherapeutic edible product useful in pharmaceutical applications. VIII. Modes of Administration and Effective Dosages

[0086] Compositions of the present technology for use in preventing, ameliorating, or treating CDI, CD AD, celiac disease, non-celiac gluten intolerance, or EPI and/or reducing the severity of one or more risk factors, signs, or symptoms associated with CDI, CD AD, celiac disease, non-celiac gluten intolerance, or EPI include edible products comprising B. amyloliquefaciens bacteria according to the present technology. The edible products of the present technology are administered to the subject in effective amounts (i.e., amounts that have desired therapeutic effect). The dose and dosage regimen will depend upon the degree of the infection in the subject, the characteristics of the particular B. amyloliquefaciens strain used, e.g ., its therapeutic index, the subject, and the subject’s history. The effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians.

[0087] Edible products of the present technology may be formulated for adding to food, or used directly as a dietary supplement. In some embodiments, the edible product comprises a fermented food product, soybean, mushroom, mung bean, locus bean, or rice. In some embodiments, the soybean is fermented soybean or fermented soybean paste. The formulation may further include other probiotic agents or nutrients for promoting spore germination and/or bacterial growth.

[0088] Additional components of the compositions of the present technology may include a preservative selected from the group consisting of sucrose, sodium ascorbate, and glutathione. In some embodiments the preservative is a cryoprotectant selected from the group consisting of a nucleotide, a disaccharide, a polyol, and a polysaccharide. In some embodiments, the cryoprotectant is selected from the group consisting of inosine-5’- monophosphate (IMP), guanosine-5’ -monophosphate (GMP), adenosine-5 ’-monophosphate (AMP), uranosine-5’ -monophosphate (UMP), cytidine-5’ -monophosphate (CMP), adenine, guanine, uracil, cytosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, orotidine, thymidine, inosine, trehalose, maltose, lactose, sucrose, sorbitol, mannitol, dextrin, inulin, sodium ascorbate, glutathione, skim milk, and cryoprotectant 18. [0089] The edible products comprising B. amyloliquefaciens bacterial strains described herein (i.e., ART24, ART4, and ART 12) can be incorporated into food compositions or dietary supplements for administration, singly or in combination, and given to a subject for the treatment or prevention of a disorder described herein. Such compositions may include the active agent and a carrier, such as saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. Carriers can be solid-based dry materials for formulations in powdered form, and can be liquid or gel-based materials for formulations in liquid or gel forms. In some embodiments, the pharmaceutically acceptable carrier comprises a polysaccharide, locust bean gum, an anionic polysaccharide, a starch, a protein, sodium ascorbate, glutathione, trehalose, sucrose, or pectin. In some embodiments, the polysaccharide comprises a plant, animal, algal, or microbial polysaccharide. In some embodiments, the polysaccharide comprises guar gum, inulin, amylose, chitosan, chondroitin sulphate, an alginate, or dextran. In some embodiments, the starch comprises rice starch.

[0090] For the edible products described herein, pharmaceutical compositions of the present technology may be formulated as dietary supplements, nutraceutical compositions, food additives, food compositions, food compositions in bulk, food additives in bulk, medical foods, or foods for special dietary use, for enteric ( e.g ., oral, sublingual) administration.

[0091] Additionally or alternatively, the B. amyloliquefaciens strains described herein (i.e., ART24, ART4, and ART12) can be incorporated into pharmaceutical compositions for administration, singly or in combination, and given to a subject for the treatment or prevention of a disorder described herein. Such compositions typically include the active agent and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. Carriers can be solid-based dry materials for formulations in powdered form, and can be liquid or gel-based materials for formulations in liquid or gel forms, which forms depend, in part, upon the routes or modes of administration. In some embodiments, the pharmaceutically acceptable carrier comprises a polysaccharide, locust bean gum, an anionic polysaccharide, a starch, a protein, sodium ascorbate, glutathione, trehalose, sucrose, or pectin. In some embodiments, the polysaccharide comprises a plant, animal, algal, or microbial polysaccharide. In some embodiments, the polysaccharide comprises guar gum, inulin, amylose, chitosan, chondroitin sulphate, an alginate, or dextran. In some embodiments, the starch comprises rice starch.

[0092] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include enteric ( e.g ., oral, sublingual, rectal) administration. A therapeutic composition can be formulated to be suitable for oral administration in a variety of ways, for example in a liquid, a powdered food supplement, a solid food, a packaged food, a wafer, tablets, troches, or capsules, e.g., gelatin capsules, and the like. In some embodiments, the therapeutic compositions of the present technology comprise lyophilized ART4, ART12, and/or ART24. In some embodiments, the lyophilized ART4, ART12, and/or ART24 is encapsulated. A therapeutic composition can be formulated to be suitable for rectal administration in a variety of ways, for example in a suppository, liquid enema, or foam. Other formulations will be readily apparent to one skilled in the art. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.

[0093] Dosage, toxicity and therapeutic efficacy of any therapeutic agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. For any compound used in the methods, the therapeutically effective dose can be estimated initially from cell culture assays and in animal models. Such information can be used to determine useful doses in humans accurately.

[0094] As used herein, “Artujang” is a collective term for whole soybeans and/or soybean pastes inoculated with ART24. In some embodiments, “Artujang,” may also refer to whole soybeans and/or soybean pastes inoculated with any one or more of ART24, ART4, or ART12. [0095] In some embodiments, the edible products of the present technology, such as “Artujang” whole soybeans and/or soybean pastes, are inoculated with a concentration of ART24, ART12, and/ART4 ranging from at least about 1 x 10 ² colony forming units (CFU)/mL to at least about 1 x 10 ¹⁰ CFU/mL, or any value in between. For example, in some embodiments, the edible products are inoculated with at least about 1 x 10 ³ CFU/mL to at least about 1 x 10 ¹⁰ CFU/mL ART24, ART12, and/or ART4, at least about 1 x 10 ⁴ CFU/mL to at least about 1 x 10 ¹⁰ CFU/mL ART24, ART12, and/or ART4, at least about 1 x 10 ⁵ CFU/mL to at least about 1 x 10 ¹⁰ CFU/mL ART24, ART12, and/or ART4, at least about 1 x 10 ⁶ CFU/mL to at least about 1 x 10 ¹⁰ CFU/mL ART24, ART12, and/or ART4, at least about 1 x 10 ⁷ CFU/mL to at least about 1 x 10 ¹⁰ CFU/mL ART24, ART12, and/or ART4, at least about 1 x 10 ⁸ CFU/mL to at least about 1 x 10 ¹⁰ CFU/mL ART24, ART12, and/or ART4, at least about 1 x 10 ⁹ CFU/mL to at least about 1 x 10 ¹⁰ CFU/mL ART24, ART12, and/or ART4. In some embodiments, the edible products are inoculated with at least about 6 x 10 ⁸ CFU/mL ART24, ART12, and/or ART4. In some embodiments, the edible products are inoculated with at least about 6 x 10 ⁹ CFU/mL ART24, ART12, and/or ART4. In some embodiments, the edible products are inoculated with at least about 1 x 10 ⁴ CFU/mL ART24, ART12, and/or ART4. In some embodiments, the edible products are inoculated with at least about 1 x 10 ⁴ CFU/mL ART24, ART12, and/or ART4. In some embodiments, the edible products are inoculated with at least about 1 x 10 ⁶ CFU/mL ART24, ART12, and/or ART4.

In some embodiments, the edible products are inoculated with at least about 1 x 10 ⁸ CFU/mL ART24, ART12, and/or ART4. In some embodiments, the edible products are inoculated with at least about 1 x 10 ⁹ CFU/mL ART24, ART12, and/or ART4.

[0096] An exemplary treatment regimen entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the subject can be administered a prophylactic regime. In some embodiments, compositions of the present technology are administered to a subject once, twice, or three times per day for 10 to 14 days or until the subject is deemed cured of primary disease, not to be at risk for recurrence of primary disease, or not to be at risk for contracting the disease. In some embodiments, administration is paired with a shortened exposure to agents known in the art for the treatment or amelioration of indications described herein, followed by once, twice, or three times daily dosing for 10 to 14 days or until the patient is deemed cured of primary disease or not to be at risk for recurrence of the disease. In some embodiments, methods of prophylaxis comprise administration of compositions of the present technology once, twice, or three times daily for 10 to 14 days or until the patient is deemed not to be at risk of contracting the disease in the case of patients known to be at risk for CDI ( e.g . PPI use or immunosuppression). In some embodiments, the edible products of the present technology are administered to a subject as a part of the subject’s regular diet to maintain levels of digestive enzymes (e.g, gliadinase, amylase, protease, lipase).

[0097] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.

EXAMPLES

[0098] The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. For each of the examples below, any bacterial strain described herein could be used. By way of example, the bacterial strain used in the examples below could be ART24, ART4, ART 12, or combinations thereof.

Materials and Methods

[0099] Soya beans. Organic soya beans (5 kg) were purchased online from buywholefoodsonline.eu (product SKU192547).

[0100] Enumerating Bacillus-like bacteria from foods. Five grams of soybeans were weighed into a filtered stomacher bag and then 45mL of MRD was added to the bag. The stomacher bag was inserted into a stomacher and homogenised for 1 minute on a medium setting. The liquid was transferred from the bag into a test tube. Serial dilutions (1:10) were carried out on the samples foods diluted to 10 ⁸ . A 100 mΐ aliquot of each dilution was transferred to the corresponding labelled BHI 2.5% agar plate and the dilutions were spread plated using sterile spreaders/hockey sticks. In plating for spores, each dilution tube was heated to 80°C for 10 minutes and then spread plated. The plates were incubated in a 37°C incubator for 18-24h. The CFU count for both the total count and spore count plates was recorded. The following calculation was used to identify the CFU/mL or CFU/g of the starting material CFU/g = [CFU counted / (dilution factor*0.1)]. To report on the true vegetative count, the spore count (CFU/g) was subtracted numbers from the total count CFU/g numbers.

[0101] Extraction of cell surface metabolites using Isopropanol (IP A). A gram of fermented soybeans was re-suspended 5 mL 70% IPA. The beans were vortexed for 10 minutes. The IPA was centrifuged at 4,000 rpm for 20 minutes. The IPA extracts were filter sterilized using 0.22 pm syringe filters.

[0102] Identification of anti-Clostridium difficile growth conditions (Well Diffusion Assay (WDA)). IPA extracts isolated from the samples and were analyzed for anti -Clostridium difficile activity. C. difficile VPI 10463 was inoculated at 1% (v/v) into Reinforced Clostridium Media (RCM) (1.015410.0500 Merck) agar and the plates were allowed to harden. Wells were made in the hardened agar plates using sterile Pasteur pipettes. Fifty microliters of the IPA extracts were dispensed into the wells of the RCM plate anaerobically and the plate was allowed to incubate for 24 h. Anti-C. difficile activity was assessed by examining the presence or absence of zones of inhibition surrounding the wells.

[0103] Preparation of starch agar. Starch agar was prepared using beef extract 3 g/L, soluble starch 10 g/L, agar 12 g/L at pH 7.5.

[0104] Preparation of gliadin agar. A gram of gliadin was added to 22 mL of 60% ethanol and incubated overnight on a shaker at 200 rpm. The solution was then centrifuged at 300 rpm for 2min. 850uL of supernatant was added to 20 mL of LB agar and plates are poured. [0105] Preparation of tributyrin agar. 20g of Tributyrin agar (Sigma 91015-500G) was dissolved in 1 liter of distilled water. Post-autoclaving the media was cooled to 80°C and then 10 mL of tributyrin (Sigma 91010) was added to the bottles and mixed with an Ultra- Turrax before dispensing into plates and allowed to solidify.

[0106] Amylase activity assay. IPA extracts were isolated from the samples and analyzed for enzymatic activity. Starch agar plates were poured and allowed to harden. Wells were made in the hardened agar plates using sterile Pasteur pipettes. Fifty microliters of the IPA extracts were dispensed into the wells of the starch agar plate and the plate was allowed to incubate for 24 h. Amylase activity was assessed by examining the presence or absence of zones of clearing surrounding the wells following the addition of iodine to the surface of the agar.

[0107] Gliadinase activity assay. IPA extracts were isolated from the samples and analyzed for enzymatic activity. Gliadin agar plates were poured and allowed to harden. Wells were made in the hardened agar plates using sterile Pasteur pipettes. Fifty microliters of the IPA extracts were dispensed into the wells of the gliadin agar plate and the plate was allowed to incubate for 24 h. Protease activity was assessed by examining the presence or absence of zones of clearing surrounding the wells.

[0108] Lipase activity assay. IPA extracts were isolated from the samples and analyzed for enzymatic activity. Tributyrin agar plates were poured and allowed to harden. Wells were made in the hardened agar plates using sterile Pasteur pipettes. Fifty microliters of the IPA extracts were dispensed into the wells of the tributryin agar plate and the plate was allowed to incubate for 24 h. Lipase activity was assessed by examining the presence or absence of zones of clearing surrounding the wells.

[0109] Artujang version 1.0. Fifteen grams of dry beans were weighed into 3x500 mL beakers and filled with water. Beans were soaked overnight, and the water was replaced twice during the day. ART24 was grown overnight in a 100 mL shake flask. The rehydrated beans were autoclaved at 123°C for 30 minutes. The beans were evenly dispersed into Styrofoam boxes (Sarstedt - 95.064.249). One box was left un-inoculated, 1 box was inoculated with 1 mL of culture 6xl0 ⁸ CFU and the remaining box was inoculated with 10 mL of culture 6xl0 ⁹ CFU. Each box was covered with aluminum foil and incubated for 48 hours at 37°C. After 48h the beans were homogenized in a commercial blender and stored at 4°C and samples were taken 96h post inoculation.

[0110] Artujang version 2.0. Thirty grams of dry beans were weighed into fourteen 500 mL beakers and filled with water. Beans were soaked overnight and the water was replaced twice during the day. ART24 was grown overnight in a 100 mL shake flask. One set of beans and homogenized beans were not autoclaved or inoculated. The rehydrated beans were autoclaved at 123°C for 30 minutes. Half of the beans (6x30g) were homogenized post autoclaving. Beans were distributed into sterilized plastic containers. A container of both the beans and paste remained un-inoculated for the duration of the experiment. A container of beans and paste were inoculated with lxlO ⁴ CFU/mL, lxlO ⁶ CFU/mL, lxlO ⁸ CFU/mL, lxlO ⁹ CFU/mL of ART24, respectively. One container of beans and paste was autoclaved after 24h incubation with lxlO ⁸ CFU/mL ART24.

[0111] Artujang version 3.0. Thirty grams of dry beans were weighed into four 500 mL beakers and filled with water. The beans were soaked overnight, replacing the water twice during the day. ART24 was grown overnight in a 100 mL shake flask. Prior to autoclaving the two beakers of soybeans were homogenized using a commercial blender. The rehydrated beans were autoclaved at 123°C for 30 minutes. Post-autoclaving one lot of beans and paste were mixed with 15 mL of water. All of the samples were then inoculated lxlO ⁴ CFU/mL ART24 and incubated for two days.

[0112] Artujang version 4.0. Thirty grams of dry beans were weighed into twelve 300 mL flasks and filled with water. Prior to autoclaving some of the soybeans were homogenized using a commercial blender. The two flasks of beans and two homogenized beans were soaked overnight, replacing the water twice during the day. Two flasks of homogenized beans were not soaked overnight. ART24 was grown overnight in a 100 mL shake flask.

Two flasks of beans were re-suspended in 17.5 mL of water (25% v/w) and another two flasks were re-suspended in 35 mL of water (50% v/w). Two flasks of milled soaked beans were re-suspended in 17.5 mL of water (25% v/w) and two flasks were re-suspended in 35 mL of water (50% v/w). Two flasks of milled un-soaked beans were re-suspended in 17.5 mL of water (25% v/w) and two flasks were re-suspended in 35 mL of water (50% v/w). The flasks were autoclaved at 123°C for 30 minutes. Post-autoclaving beans that were milled but not soaked overnight had absorbed all of the water and were re-suspended in an additional volume of 35 mL (100% v/w) or 70 mL (200% v/w). Each flask was inoculated with 3 mL of an ART24 overnight culture (10 ⁸ CFU/mL). One of each of the flasks was incubated static at 37°C and the remaining flasks were incubated shaking at 37°C at 200rpm.

Example 1 : Bacillus amylolicwefaciens ART24 ability to ferment soybeans (Artuiang version

10)

[0113] This example demonstrates that ART24 can be used as a starter culture to inoculate and ferment soybeans similar to Cheonggukjang (FIGS. 1A-1F).

[0114] Using colony morphology, the effect of the inoculated strain ART24 compared to a natural Bacillus isolate that contaminated the surface of un-inoculated control beans was identified (FIGS 1G and 1H).

[0115] Using the plating methodology described above, the level of Bacillus on the surface of the beans was enumerated. FIG. 2 shows the increase in both vegetative and spore cell counts over the incubation time.

[0116] Using the well diffusion assay (WDA) method described above, the prepared extracts of each soybean inoculum and time point were tested against C. difficile VPI 10463. FIG. 3A and 3B show the zones of activity achieved against this strain.

[0117] Each sample was compared using MALDI-TOF mass spectrometry. FIG 4F and 41 were identified as containing ART24-related antimicrobial lipopeptides which may play a role in the anti-C. difficile effects identified. Beans that were not inoculated did FIG. 4A and 4D did not show these masses. Masses of interest occur at m/z 1044-1096Da and 1463- 1505Da corresponding to surfactin and fengycin, respectively.

[0118] Accordingly, these results show that ART24 can grow on and ferment soybeans and is useful in methods of inhibiting C. difficile growth and in methods for treating or preventing C. difficile infections. Example 2: Anti-C. difficile activity of the B. amyloliguefaciens inoculated soybeans (Artuiang version 2.0)

[0119] This example demonstrates that ART24 can be used as a starter culture to inoculate and ferment soybeans similar to Cheonggukjang (FIGS. 5C-5G).

[0120] Using the plating methodology described above, the level of Bacillus on the surface of the beans was enumerated. FIG. 6 shows the increase in vegetative cell counts over the incubation time.

[0121] Each sample was compared using MALDI-TOF mass spectrometry. FIG 7A-7H and 7J were identified from samples at T24 as containing ART24-related antimicrobial lipopeptides which may play a role in the anti-C. difficile effects identified. Beans that were not inoculated or autoclaved do not contain masses of interest (FIG. 71, 7K and 7L). Masses of interest occur at m/z 1044-1096Da and 1463-1505Da corresponding to surfactin and fengycin, respectively.

[0122] Each sample was compared using MALDI-TOF mass spectrometry. FIG8A-8H and 8J were identified from samples at T48 as containing ART24-related antimicrobial lipopeptides which may play a role in the anti-C. difficile effects identified. Beans that were not inoculated or autoclaved do not contain masses of interest (FIG. 81, 8K and 8L). Masses of interest occur at m/z 1044-1096Da and 1463-1505Da corresponding to surfactin and fengycin, respectively.

[0123] Using the well diffusion assay (WDA) method described above, the prepared extracts of each soybean inoculum were tested against C. difficile VPI10463. FIG. 9 shows the zones of activity achieved against this strain for samples at T24. Samples that were not autoclaved or not inoculated did not have any activity against C. difficile.

[0124] Using the well diffusion assay (WDA) method described above, the prepared extracts of each soybean inoculum were tested against C. difficile VPI10463. FIG. 10 shows the zones of activity achieved against this strain for samples at T48. Samples that were not autoclaved or not inoculated did not have any activity against C. difficile. [0125] Accordingly, these results show that ART24 can grow on and ferment soybeans and may present another method of inhibiting C. difficile growth.

Example 3 : Anti-C. difficile activity of the B. amyloliquefaciens inoculated soybeans (Artuiang version 3 0)

[0126] This example demonstrates that ART24 can be used as a starter culture to inoculate and ferment soybeans similar to Cheonggukjang (FIGS. 11A-11E).

[0127] Using the plating methodology described above, the level of Bacillus on the surface of the beans was enumerated. FIG. 12 shows the increase in vegetative cell counts over the incubation time. High Bacillus counts were identified in all samples, directly from the beans or paste or from the water surrounding the beans and paste.

[0128] Using the well diffusion assay (WDA) method described above, the prepared extracts of each soybean inoculum were tested against C. difficile VPI10463. FIG. 13 shows the zones of activity achieved against this strain for samples at T24. No activity was identified from the paste at T24. Varying levels of activity was identified from the beans, beans/paste in water, cells extracted from the water surrounding the beans/paste and from the water directly (not IPA extracted).

[0129] Using the well diffusion assay (WDA) method described above, the prepared extracts of each soybean inoculum were tested against C. difficile VPI10463. FIG. 14 shows the zones of activity achieved against this strain for samples at T48. Varying levels of activity was identified from the beans/paste, beans/paste in water, cells extracted from the water surrounding the beans/paste and from the water directly (not IPA extracted).

[0130] Accordingly, these results show that ART24 can grow on and ferment soybeans and may present another method of inhibiting C. difficile growth.

Example 4: Enzymatic activity of the B. amyloliquefaciens inoculated soybeans (Artuiang version 4 0)

[0131] This example demonstrates that ART24 can be used as a starter culture to inoculate and ferment soybeans similar to Cheonggukjang (FIGS. 15-17). [0132] Using the amylase activity assay described above, filtered extracts of each soybean test was tested for the production of the amylase enzyme. Each filtered sample resulted in a zone of clearing around the well (FIG. 18). Creon was used as a commercial positive control/comparator for enzymatic activity. Amylase activity was also identified from extracts of LotlOA2 capsules used again as comparators.

[0133] Using the gliadinase activity assay described above, filtered extracts of each soybean test was tested for the production of the protease enzymes capable of degrading gliadin. Each filtered sample resulted in a zone of clearing around the well (FIG. 19). Creon was used as a commercial positive control/comparator for enzymatic activity. Protease/gliadinase activity was not identified from extracts of LotlOA2 capsules used again as comparators.

[0134] Using the lipase activity assay described above, filtered extracts of each soybean test was tested for the production of lipase. None of the filtered sample resulted in a zone of clearing around the well (FIG. 20). Creon was used as a commercial positive control/comparator for enzymatic activity. Lipase activity was identified from extracts of LotlOA2 capsules used again as comparators after 48h.

[0135] Accordingly, these results show that ART24 can grow on and ferment soybeans and may present another method of for producing amylase and protease, and are useful in methods of treating or preventing exocrine pancreatic insufficiency and/or as a form of pancreatic enzyme replacement therapy (PERT).

Example 5: B. amyloliquefaciens strain supernatant exhibits gliadinase and lipase activities.

[0136] This example demonstrates that B. amyloliquefaciens strains of the present technology exhibit gliadinase and lipase activity.

Gliadinase activity

[0137] ART4 prepared as a pH-neutralized supernatant fraction was examined for gliadinase activity. A 4: 1 mixture of ART4 supernatant to 33mer gliadin peptide (0.2 mg/mL) was prepared. As shown in FIGS. 21A and 21B, ART4 exhibits gliadinase activity as assessed by Gastric Inhibitory Polypeptide (GIP) ELISA. As shown in FIG. 21A, pH neutralized ART4 supernatant degrades 33mer gliadin peptide (the pro-inflammatory component of gluten) within 28 hours of incubation at pH7, room temperature. As shown in FIG. 21B, pH neutralized ART4 supernatant degrades full-length gliadin within 4 hours of incubation.

Lipase activity

[0138] ART4 prepared as a pH-neutralized supernatant fraction was examined for lipase activity. As shown in FIGS. 22A and 22B, 100 pL of ART4 supernatant exhibits lipase activity that is equivalent to 2 mg of CREON ® (pancrelipase) (24,000 USP; 46 mg pill).

[0139] Overall, these results demonstrate that the B. amyloliquefaciens strains of the present technology possess gliadinase and lipase activities and are useful in methods for treating or preventing celiac disease, non-celiac gluten intolerance, pancreatic exocrine insufficiency, and may be useful as a pancreatic enzyme replacement therapy (PERT).

EQUIVALENTS

[0140] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present technology is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. [0141] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0142] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,”

“at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0143] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0144] Other embodiments are set forth within the following claims.