Nutrient rich germinant composition and spore incubation method

Patent metadata

Field	Value
Publication number	US-10610552-B2
Application number	US-201715479773-A
Country	US
Kind code	B2
Filing date	Apr 5, 2017
Priority date	Apr 5, 2016
Publication date	Apr 7, 2020
Grant date	Apr 7, 2020

Abstract

A nutrient-germinant composition to aid in spore germination and a method for increased spore germination efficiency. The composition comprises L-amino acids, D-glucose and/or D-fructose, a phosphate buffer, an industrial preservative, and may include bacteria spores or they may be separately combined for germination. The method comprises providing a nutrient-germinant composition and bacteria spores, preferably of one or more Bacillus species, and heating to a preferred elevated temperature range of 41° C. to 44° C. for an incubation period of around 2 to 60 minutes. The nutrient-germinant composition is preferably in a concentrated liquid form that is diluted just prior to initiating the germination/incubation method at the point of use. The method may also include dispensing a germinated spore solution to a point-of-use/consumption, such as animal feed, water, or bedding, or a wastewater system or drain.

First claim

We claim:

1. A method of germinating bacterial spores at a point of use comprising the following steps:
providing a nutrient-germinant composition and spores of a bacteria species, if not included in the nutrient-germinant composition;
heating the nutrient-germinant composition and spores to a temperature in a range of around 35° C. to 60° C. at or near a point-of-use;
maintaining the temperature for an incubation period of around 2 to 60 minutes to form a germinated spore solution;
dispensing the germinated spore solution to the point-of-use and wherein the point of use comprises animal feed, animal water, animal bedding, a plant, a pond, a wastewater system, or a drain and
wherein the nutrient-germinant composition comprises: (1) one or more L-amino acids; (2) one or more buffers comprising a phosphate buffer, HEPES, Tris base, or a combination thereof; (3) an industrial preservative; and (4) optionally a source of potassium ions.

2. The method of claim 1 wherein the nutrient-germinant composition is in a concentrated liquid form and further comprising:
adding a diluent to the nutrient-germinant composition;
mixing the diluted nutrient-germinant composition and bacterial spores during the incubation period; and
wherein the concentration of the diluted nutrient-germinant composition is around 0.1% to 10%.

3. The method of claim 1 wherein the temperature range is around 41° C. to 44° C.

4. The method of claim 1 wherein the L-amino acid is L-alanine, L-asparagine, L-valine, L-cysteine, a hydrolysate of soy protein, or a combination thereof.

5. The method of claim 1 wherein the nutrient-germinant composition is in a concentrated liquid form comprising around 17.8 g/L to 89 g/L total of one or more L-amino acids, the method further comprising:
adding a diluent to the nutrient-germinant composition;
mixing the diluted nutrient-germinant composition and bacterial spores during the incubation period; and
wherein the concentration of the diluted nutrient-germinant composition is 0.01% to 10% of the concentrated nutrient-germinant composition.

6. The method of claim 5 wherein the nutrient-germinant composition further comprises spores of a Bacillus species and a germination inhibitor.

7. The method of claim 6 wherein the germination inhibitor or preservative comprises sodium chloride, D-alanine, or a combination thereof.

8. The method of claim 7 wherein the concentrated nutrient-germinant composition comprises around 29 g/L to 117 g/L sodium chloride.

9. The method of claim 7 wherein the concentrated nutrient-germinant composition comprises around 8 g/L to 116 g/L D-alanine.

10. The method of claim 6 wherein the Bacillus species is Bacillus licheniformis, Bacillus subtilis, Bacillus amyloliquiefaciens, Bacillus polymyxa, Bacillus thuringiensis, Bacillus megaterium, Bacillus coagulans, Bacillus lentus, Bacillus clausii, Bacillus circulans, Bacillus firmus, Bacillus lactis, Bacillus laterosporus, Bacillus laevolacticus, Bacillus pumilus, Bacillus simplex , and Bacillus sphaericus , or a combination thereof.

11. The method of claim 6 wherein the point of use comprises animal feed or animal water or both and wherein the Bacillus species are capable of producing enzymes that aid in the breakdown of organic matter in the digestive tract of a consuming animal.

12. The method of claim 11 wherein the wherein the enzymes comprise amylase, protease, lipase, esterase, urease, cellulase, xylanase, or a combination thereof.

13. The method of claim 1 wherein the nutrient-germinant composition is a concentrated liquid comprising:
around 8.9-133.5 g/L of one or more L-amino acids;
around 0.8-3.3 g/L total of the one or more industrial preservatives;
around 10-36 g/L monosodium phosphate, or around 30-90 g/L disodium phosphate, or around 15-61 g/L Tris base, or around 32.5-97.5 g/L HEPES, or a combination thereof; and
optionally around 18-54 g/L of D-glucose, D-fructose, or a combination thereof.

14. The method of claim 13 further comprising:
adding a diluent to the nutrient-germinant composition; and
mixing the diluted nutrient-germinant composition and bacteria spores during the incubation period.

15. The method of claim 14 wherein the concentration of the diluted nutrient-germinant composition is around 4% to 10%.

16. The method of claim 1 wherein the nutrient-germinant composition is a concentrated liquid comprising:
around 13.2-111.25 g/L of L-alanine;
around 0.8-3.3 g/L total of propylparaben or methylparaben or both;
one or more buffers comprising a phosphate buffer, HEPES, Tris base, or a combination thereof;
a source of potassium ions;
optionally spores of one or more Bacillus species; and
wherein if spores are included, the concentrated nutrient-germinant composition further comprises around 29 g/L to 117 g/L sodium chloride or around 8 g/L to 116 g/L D-alanine or both.

17. The method of claim 16 further comprising:
adding a diluent to the nutrient-germinant composition prior to or during the heating step; and
wherein the concentration of the diluted nutrient-germinant composition is around 4% of the concentrated liquid.

18. The method of claim 1 wherein the nutrient-germinant composition does not include fructose or glucose.

19. The method of claim 13 wherein the nutrient-germinant composition does not include fructose or glucose.

20. The method of claim 16 wherein the nutrient-germinant composition does not include fructose or glucose.

21. The method of claim 1 wherein the incubation period is 2 to 5 minutes.

22. The method of claim 13 wherein the incubation period is 2 to 5 minutes.

23. The method of claim 16 wherein the incubation period is 2 to 5 minutes.

24. The method of claim 1 wherein the industrial preservative comprises propylparaben, methylparaben, methyl chloro isothiazolinone, methyl isothiazolinone or a combination thereof and wherein the nutrient-germinant composition does not include fructose or glucose.

25. The method of claim 1 wherein the spores are not heat activated prior to heating with the nutrient-germinant composition.

26. The method of 18 wherein the spores are not heat activated prior to heating with the nutrient-germinant composition.

27. The method of claim 1 wherein the spores are Bacillus subtilis , or Bacillus licheniformis , or both.

28. A method of germinating bacterial spores at a point of use comprising the following steps:
providing a nutrient-germinant composition and spores of a bacteria species, if not included in the nutrient-germinant composition;
heating the nutrient-germinant composition and spores to a temperature in a range of 38° C. to 60° C.;
maintaining the temperature in the range for an incubation period of around 2 to 60 minutes to form a germinated spore solution; and
dispensing the germinated spore solution to the point-of-use and wherein the point of use comprises animal feed, animal water, animal bedding, a plant, a pond, a wastewater system, or a drain; and
wherein the spores are not heat activated prior to heating with the nutrient-germinant composition.

29. The method of claim 27 wherein the nutrient-germinant composition wherein the nutrient-germinant composition comprises: (1) one or more L-amino acids; (2) one or more buffers comprising a phosphate buffer, HEPES, Tris base, or a combination thereof; (3) an i

Assignees

• Nch Corp

Inventors

• Everett Gabriel F K
• Greenwald Charles
• Pruitt Judy
• Rosmarin Amanda
• Church Jordan
• Aberle Daniel
• Aboagye George

Classifications

• A61K35/742 Primary

  Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes · CPC title

• C12N1/20 Primary

  Bacteria; Culture media therefor · CPC title

• A23L29/065

  Microorganisms (addition of bacteria for nutritional purposes A23L33/135) · CPC title

• C02F3/348

  characterised by the way or the form in which the microorganisms are added or dosed · CPC title

• C12N3/00

  Spore forming or isolating processes · CPC title