# US 10,308,909 B2 · Dried spore germinative compound mixtures > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,308,909 B2 - Document ID: US10308909B2 - Status: Granted - Kind: B2 - Application number: 15/248,328 - Filed: 2016-08-26 - Published: 2016-12-15 - Granted: 2019-06-04 - Earliest claimed: 2013-02-06 - Assignees: Envera LIC, LLC - Applicants: Envera, LLC - Inventors: Tommie Eugene Hashman; Michael Matheny - Web page: https://patentgrep.com/patent/US10308909B2 ## CPC - C12N 1/20: Bacteria; Culture media therefor - A61K 35/742: Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract In one aspect, the present invention is directed to a dried intimate mixture comprising a bacteria spore and a germinative compound, and methods for preparing the intimate mixture. In another aspect, this invention is directed to a composition comprising such an intimate mixture. The invention also relates to methods for increasing the germination, growth, metabolism, and/or enzyme activity of a bacteria spore comprising preparing an intimate mixture of a bacteria spore and a germinative compound. ## Claims ### Claim 1 A dried mixture comprising a bacterial spore and an L-amino acid, wherein the dried mixture is prepared by drying a solution comprising the L-amino acid and the bacterial spore, wherein the L-amino acid is adsorbed to or absorbed by the bacterial spore and binds to said bacterial spore's germination initiator sites when the mixture reaches an appropriate environment for germination, and wherein the bacterial spore in the dried mixture germinates more rapidly than a bacterial spore that is not dried with the L-amino acid. ### Claim 2 (depends on 1) The mixture of claim 1 wherein the spore is selected from the group consisting of B. alcalophilus, B. alvei, B. amyloliquefaciens, B. aneurinolyticus, B. anthracis, B. aquaemaris, B. atrophaeus, B. boronophilus, B. brevis, B. caldolyyicus, B. centrosporus, B. cereus, B. circulans, B. clausii, B. coagulans, B. firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, B. larvae, B. laterosporus, B. lentus, B. lentimorbus, B. licheniformis, B. megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B. pantothenicus, B. popilliae, B. polymyxa, B. pseudoanthracis, B. pumilus, B. schlegelii, B. simplex, B. sphaericus, B. sporothermodurans, B. stearothermophilus, B. subtilis, B. thermoglucosidasius, B. thuringiensis, B. vulgatis and B. weihenstephanensis. ### Claim 3 (depends on 1) The mixture of claim 1 wherein the L-amino acid is selected from the group consisting of L-alanine, L-valine, L-proline, L-leucine, L-cysteine, L-threonine, L-glutamine, L-asparagine, L-phenylalanine and analogues thereof. ### Claim 4 (depends on 1) The mixture of claim 1 wherein said composition is a mixture selected from the group consisting of L-alanine+Bacillus subtilis, L-alanine+Bacillus licheniformis, L-alanine+Bacillus pumilus, L-alanine+Bacillus amyloliquefaciens, L-alanine+Bacillus coagulans, L-alanine+Bacillus cereus, L-alanine+Bacillus clausii, L-valine+Bacillus licheniformis, L-valine+Bacillus pumilus, L-valine+Bacillus amyloliquefaciens, L-valine+Bacillus coagulans, L-valine+Bacillus cereus, L-valine+Bacillus clausii, L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus subtilis, L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus licheniformis, L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus pumilus, L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus amyloliquefaciens, L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus coagulans, L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus cereus, L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus clausii, L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus subtilis, L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus licheniformis, L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus pumilus, L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus amyloliquefaciens, L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus coagulans, L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus cereus, L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus clausii, L-alanine+inosine+Bacillus subtilis, L-alanine+inosine+Bacillus licheniformis, L-alanine+inosine+Bacillus pumilus, L-alanine+inosine+Bacillus amyloliquefaciens, L-alanine+inosine+Bacillus coagulans, L-alanine+inosine+Bacillus cereus, L-alanine+inosine+Bacillus clausii, L-proline+glucose+Bacillus megaterium, and L-proline+Bacillus megaterium. ### Claim 5 (depends on 1) The mixture of claim 1 wherein the mixture is produced by spray-drying or freeze-drying. ### Claim 6 (depends on 1) The mixture of claim 1 wherein the spore is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium, and B. pumilus; and the L-amino acid is selected from the group consisting of L-alanine, L-valine, and L-asparagine. ### Claim 7 (depends on 1) A composition comprising the dried mixture of claim 1. ### Claim 8 (depends on 7) The composition of claim 7 wherein the spore is selected from the group consisting of B. alcalophilus, B. alvei, B. amyloliquefaciens, B. aneurinolyticus, B. anthracis, B. aquaemaris, B. atrophaeus, B. boronophilus, B. brevis, B. caldolyyicus, B. centrosporus, B. cereus, B. circulans, B. clausii, B. coagulans, B. firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, B. larvae, B. laterosporus, B. lentus, B. lentimorbus, B. licheniformis, B. megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B. pantothenicus, B. popilliae, B. polymyxa, B. pseudoanthracis, B. pumilus, B. schlegelii, B. simplex, B. sphaericus, B. sporothermodurans, B. stearothermophilus, B. subtilis, B. thermoglucosidasius, B. thuringiensis, B. vulgatis and B. weihenstephanensis. ### Claim 9 (depends on 7) The composition of claim 7 wherein the L-amino acid is selected from the group consisting of L-alanine, L-valine, L-proline, L-leucine, L-cysteine, L-threonine, L-glutamine, L-asparagine, L-phenylalanine and analogues thereof. ### Claim 10 (depends on 7) The composition of claim 7 wherein said composition is a mixture selected from the group consisting of L-alanine+Bacillus subtilis, L-alanine+Bacillus licheniformis, L-alanine+Bacillus pumilus, L-alanine+Bacillus amyloliquefaciens, L-alanine+Bacillus coagulans, L-alanine+Bacillus cereus, L-alanine+Bacillus clausii, L-valine+Bacillus subtilis, L-valine+Bacillus licheniformis, L-valine+Bacillus pumilus, L-valine+Bacillus amyloliquefaciens, L-valine+Bacillus coagulans, L-valine+Bacillus cereus, L-valine+Bacillus clausii, L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus subtilis, L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus licheniformis, L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus pumilus, L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus amyloliquefaciens, L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus coagulans, L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus cereus, L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus clausii, L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus subtilis, L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus licheniformis, L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus pumilus, L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus amyloliquefaciens, L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus coagulans, L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus cereus, L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus clausii, L-alanine+inosine+Bacillus subtilis, L-alanine+inosine+Bacillus licheniformis, L-alanine+inosine+Bacillus pumilus, L-alanine+inosine+Bacillus amyloliquefaciens, L-alanine+inosine+Bacillus coagulans, L-alanine+inosine+Bacillus cereus, L-alanine+inosine+Bacillus clausii, L-proline+glucose+Bacillus megaterium, and L-proline+Bacillus megaterium. ### Claim 11 (depends on 7) The composition of claim 7 wherein the mixture is produced by spray-drying or freeze-drying. ### Claim 12 (depends on 7) The composition of claim 7 wherein the composition further comprises an enteric coating. ### Claim 13 (depends on 7) The composition of claim 7 wherein the spore is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium, and B. pumilus; and the L-amino acid is selected from the group consisting of L-alanine, L-valine, and L-asparagine. ### Claim 14 (depends on 1) The mixture of claim 1, wherein the L-amino acid is formulated, prior to drying, at a concentration of 0.0003 mg/mL to 30 mg/mL. ### Claim 15 A method for preparing a dried mixture comprising a bacterial spore and an L-amino acid, the method comprising: a) preparing a solution comprising a bacterial spore and an L-amino acid; and b) drying the solution to obtain a dried mixture comprising the bacterial spore and the L-amino acid, wherein the L-amino acid is adsorbed to or absorbed by the bacterial spore and binds to said bacterial spore's germination initiator sites when the mixture reaches an appropriate environment for germination, and wherein the bacterial spore in the dried mixture germinates more rapidly than a bacterial spore that is not dried with the L-amino acid. ### Claim 16 (depends on 15) The method of claim 15, wherein the drying is spray-drying or freeze-drying. ### Claim 17 (depends on 15) The method of claim 15, wherein the spore is selected from the group consisting of B. alcalophilus, B. alvei, B. amyloliquefaciens, B. aneurinolyticus, B. anthracis, B. aquaemaris, B. atrophaeus, B. boronophilus, B. brevis, B. caldolyyicus, B. centrosporus, B. cereus, B. circulans, B. clausii, B. coagulans, B. firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, B. larvae, B. laterosporus, B. lentus, B. lentimorbus, B. licheniformis, B. megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B. pantothenicus, B. popilliae, B. polymyxa, B. pseudoanthracis, B. pumilus, B. schlegelii, B. simplex, B. sphaericus, B. sporothermodurans, B. stearothermophilus, B. subtilis, B. thermoglucosidasius, B. thuringiensis, B. vulgatis and B. weihenstephanensis. ### Claim 18 (depends on 15) The method of claim 15, wherein the spore is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium, and B. pumilus; and the L-amino acid is selected from the group consisting of L-alanine, L-valine, and L-asparagine. ### Claim 19 (depends on 15) A dried mixture produced by the method of claim 15. ### Claim 20 A method for increasing the germination of bacterial spores, comprising: a) preparing a solution comprising a bacterial spore and an L-amino acid; and b) drying the solution to obtain a dried mixture comprising the bacterial spore and the L-amino acid, wherein the L-amino acid is adsorbed to or absorbed by the bacterial spore and binds to said bacterial spore's germination initiator sites when the mixture reaches an appropriate environment for germination; and c) exposing the mixture to an environment conducive to germination of the bacterial spores, wherein the germination of the bacterial spores in the dried mixture is increased relative to a corresponding bacterial spore formulation that lacks the L-amino acid.