Compositions and Methods for Germinating Wild-Type or Super-Dormant Bacillus Strains
A germinant composition for activating one or more wild-type and/or super-dormant spores, particularly Bacillus spores, comprises L-isoleucine or L-valine or both. A germinant composition may also comprise a buffer, a source of potassium, an industrial preservative, and one or more other L-amino acids. Wild-type and/or super-dormant spores may be incubated with a germinant composition by heating to an incubation temperature of 26 to 60° C. for an incubation period of 30 minutes to 36 hours. Incubation may be a single step process without pre-activation or heat shock or a two-step process. A method of pre-activation comprises combining spores with a germinant composition and either bioprocessing or heat treating and then drying. An incubated bacteria solution preferably comprises at least 50% activated spores after an incubation period of 24 hours and more preferably after 1 hour.
1 . A germinant composition for activating one or more bacteria spores, the germinant composition comprising: L-isoleucine or L-valine or both; and
wherein the one or more bacteria spores are wild-type spores or super-dormant spores or both.
2 . The germinant composition of claim 1 further comprising: (1) a buffer and (2) a source of potassium.
3 . The germinant composition of claim 2 wherein the buffer comprises monosodium phosphate or disodium phosphate.
4 . The germinant composition of claim 2 wherein the buffer comprises monosodium phosphate and disodium phosphate.
5 . The germinant composition of claim 4 wherein the source of potassium comprises potassium chloride.
6 . The germinant composition of claim 5 further comprising the one or more bacteria spores.
7 . The germinant composition of claim 6 further comprising an industrial preservative and wherein the germinant composition is in a liquid form.
8 . The germinant composition of claim 7 further comprising a germination inhibitor.
9 . The germinant composition of claim 6 wherein the one or more bacteria spores are pre-activated.
10 . The germinant composition of claim 6 wherein the one or more bacteria spores are not pre-activated.
11 . The germinant composition of claim 2 wherein the germinant composition does not include any other L-amino acids.
12 . The germinant composition of claim 11 wherein the germinant composition does not include any aspartic acid or inosine.
13 . The germinant composition of claim 2 further comprising an industrial preservative, and wherein the germinant composition is in a liquid form.
14 . The germinant composition of claim 13 wherein the industrial preservative comprises one or more of methyl chloro isothiazolinone, methyl isothiazolinone, propylparaben, or methylparaben.
15 . The germinant composition of claim 14 wherein the germinant composition comprises 23 to 45 g/L of the source of potassium, 4 to 20 g/L of the buffer, 0.5-3.3 g/L of the industrial preservative, and water.
16 . The germinant composition of claim 15 wherein the germinant composition comprises 9.5 to 20 g/L of the buffer and the buffer comprises 7.5 to 15 g/L of disodium phosphate and 2.5 to 5 g/L of monosodium phosphate.
17 . The germinant composition of claim 1 further comprising one or more of L-arginine, L-tryptophan, L-tyrosine, L-phenylalanine, L-cysteine, L-cystine, L-glutamine, L-proline, or L-lysine.
18 . The germinant composition of claim 17 further comprising aspartic acid or inosine or both.
19 . The germinant composition of claim 1 further comprising (1) 62 to 159 g/L total of one or more of L-arginine, L-tryptophan, L-tyrosine, L-phenylalanine, L-cysteine, L-cystine, L-glutamine, L-proline, and L-lysine, (2) aspartic acid, (3) inosine, or (4) a combination thereof.
20 . The germinant composition of claim 2 wherein the germinant composition comprises around 55 to 95% of a nutrient-germinant composition and around 5 to 13% of a spore composition;
wherein the nutrient-germinant composition comprises the buffer, the source of potassium, and the L-isoleucine or L-valine or both; and
wherein the spore composition comprises the one or more bacteria spores.
21 . The germinant composition of claim 20 wherein the one or more bacteria spores comprises spores of Bacillus.
22 . The germinant composition of claim 21 wherein the germinant composition is in a dry form.
23 . The germinant composition of claim 1 wherein the one or more bacteria spores comprises spores of Bacillus.
24 . A method of pre-activating one or more bacteria spores, the method comprising the following steps:
forming a mixed liquid solution comprising a nutrient-germinant composition and the one or more bacteria spores;
treating the mixed liquid solution by (1) bioprocessing the mixed liquid solution for a bioprocessing period of time or (2) heat treating the mixed liquid solution for a heat treatment period of time; and
drying the mixed liquid solution after the treating step to form a dry nutrient-spore composition with the one or more bacteria spores in a pre-activated state;
wherein the nutrient-germinant composition comprises L-isoleucine or L-valine or both; and
wherein the one or more bacteria spores are wild-type spores or super-dormant spores or both.
25 . The method of claim 24 wherein the drying step comprises spray drying at a first temperature in a first range of around 117 to 130° C.
26 . The method of claim 25 wherein the treating step comprises the bioprocessing step and the bioprocessing period of time is around 10 minutes to 30 minutes.
27 . The method of claim 25 wherein the treating step comprises the heat treating step, and wherein the heat treating step comprises heating the mixed liquid solution to a second temperature in a second temperature range of around 35 to 45° C. and the heat treatment period of time is around 180 seconds to 10 minutes.
28 . The method of claim 25 wherein the heat treating step further comprises:
mixing the one or more bacteria spores with a buffer composition in a container;
adding a surface area enhancement media to the container;
coating the surface area enhancement media by shaking the container or stirring;
adding a surfactant to the container;
cooling the mixed liquid solution in an ice bath after the heat treatment period of time; and
wherein the forming step comprises adding the nutrient-germinant composition to the container.
29 . The method of claim 25 wherein the one or more bacteria spores comprises spores of Bacillus.
30 . A method of activating one or more bacteria spores, the method comprising the following steps:
heating a nutrient-spore solution to a temperature in a temperature range of around 26 to 60° C. for an incubation period of around 30 minutes to 36 hours to form an incubated bacteria solution;
wherein the nutrient-spore solution comprises a nutrient-germinant composition and a spore composition;
wherein the nutrient-germinant composition comprises L-isoleucine or L-valine or both;
wherein the spore composition comprises the one or more bacteria spores; and
wherein the one or more bacteria spores are wild-type spores or super-dormant spores or both.
31 . The method of claim 30 wherein the nutrient-germinant composition further comprises (1) a buffer and (2) a source of potassium.
32 . The method of claim 31 wherein the buffer comprises monosodium phosphate or disodium phosphate or both; and
wherein the source of potassium comprises potassium chloride.
33 . The method of claim 32 wherein the nutrient-germinant composition further comprises an industrial preservative and wherein the nutrient-germinant composition is in a liquid form.
34 . The method of claim 33 wherein the industrial preservative comprises one or more of methyl chloro isothiazolinone, methyl isothiazolinone, propylparaben, or methylparaben.
35 . The method of claim 33 wherein the nutrient-germinant composition further comprises a germination inhibitor.
36 . The method of claim 33 wherein the one or more bacteria spores are pre-activated.
37 . The method of claim 33 wherein the one or more bacteria spores are not pre-activated.
38 . The method of claim 32 wherein the nutrient-germinant composition does not include any other L-amino acids.
39 . The method of claim 32 wherein the nutrient-germinant composition does not include any aspartic acid or inosine.
40 . The method of claim 33 wherein the nutrient-germinant composition further comprises 23 to 45 g/L of the potassium chloride, 4 to 20 g/L total of the buffer, 0.5-3.3 g/L of the industrial preservative, and water.
41 . The method of claim 30 wherein the nutrient-germinant composition further comprises one or more of L-arginine, L-tryptophan, L-tyrosine, L-phenylalanine, L-cysteine, L-cystine, L-glutamine, L-proline, or L-lysine.
42 . The method of claim 41 wherein the nutrient-germinant composition further comprises aspartic acid or inosine or both.
43 . The method of claim 30 wherein the nutrient-germinant composition further comprises (1) 62 to 159 g/L total of one or more of L-arginine, L-tryptophan, L-tyrosine, L-phenylalanine, L-cysteine, L-cystine, L-glutamine, L-proline, and L-lysine, (2) aspartic acid, (3) inosine, or (4) a combination thereof.
44 . The method of claim 30 wherein the nutrient-spore solution comprises around 55 to 95% of the nutrient-germinant composition and around 5 to 13% of the spore composition.
45 . The method of claim 30 wherein the method is carried out at or near a point-of-use of the incubated bacteria solution.
46 . The method of claim 45 further comprising:
forming the nutrient-spore solution by mixing the nutrient-germinant composition and the spore composition.
47 . The method of claim 45 wherein the nutrient-germinant composition is in a dry or powdered form and the forming step further comprises adding and mixing water with the nutrient-germinant composition and the spore composition.
48 . The method of claim 45 wherein the nutrient-germinant composition is in a concentrated liquid form and the forming step further comprises adding and mixing water with the nutrient-germinant composition and the spore composition.
49 . The method of claim 45 wherein the nutrient-germinant composition further comprises an industrial preservative.
50 . The method of claim 30 wherein the nutrient-spore solution is a liquid nutrient-spore solution and the nutrient-spore solution further comprises an industrial preservative.
51 . The method of claim 50 wherein the nutrient-spore solution is in a concentrated liquid form and the method further comprises adding and mixing water with the nutrient-spore solution prior to or during the heating step.
52 . The method of claim 30 wherein the incubated bacteria solution comprises at least 50% activated bacteria when the incubation period is at least 24 hours.
53 . The method of claim 30 wherein the incubated bacteria solution comprises at least 70% activated bacteria when the incubation period is at least 24 hours.
54 . The method of claim 30 wherein the incubated bacteria solution comprises at least 90% activated bacteria when the incubation period is at least 24 hours.
55 . The method of claim 30 wherein the incubated bacteria solution comprises at least 50% activated bacteria when the incubation period is at least 1 hour and the temperature range is 40 to 60° C.