METHOD FOR FACILITATING MATURATION OF THE MAMMALIAN IMMUNE SYSTEM

• US 20190224254A1
• Filed: 06/30/2017
• Published: 07/25/2019
• Est. Priority Date: 07/01/2016
• Status: Active Grant

• Current Assignee: Evolve Biosystems Inc.
• Original Assignee: Evolve Biosystems Inc.
• Inventors: KYLE, David, FRESE, Steven, FREEMAN-SHARKEY, Samara, HENRICK, Bethany
• Granted Patent: US 11,318,175 B2
• Time in Patent Office: Days
• CPC Class Codes: A23K 10/18 of live microorganisms
• CPC Class Codes: A23L 33/125 containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates indigestible substances A23L33/21
• CPC Class Codes: A23L 33/135 Bacteria or derivatives thereof, e.g. probiotics
• CPC Class Codes: A23L 33/21 Addition of substantially indigestible substances, e.g. dietary fibres
• CPC Class Codes: A23L 33/40 Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
• CPC Class Codes: A23V 2002/00 Food compositions, function of food ingredients or processes for food or foodstuffs
• CPC Class Codes: A23V 2200/3202 Prebiotics, ingredients fermented in the gastrointestinal tract by beneficial microflora
• CPC Class Codes: A23V 2200/3204 Probiotics, living bacteria to be ingested for action in the digestive tract
• CPC Class Codes: A23V 2250/284 Oligosaccharides, non digestible
• CPC Class Codes: A23V 2400/529 Infantis
• CPC Class Codes: A23V 2400/533 Longum
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 31/702 Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
• CPC Class Codes: A61K 35/741 Probiotics probiotic yeast, e.g. saccharomyces A61K36/06
• CPC Class Codes: A61K 35/744 Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
• CPC Class Codes: A61K 35/745 Bifidobacteria
• CPC Class Codes: A61K 35/747 Lactobacilli, e.g. L. acidophilus or L. brevis
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The inventions described herein relate generally to the use of compositions to increase output of acetate and lactate while reducing pH and the levels of pathogenic bacteria and inflammation in the gut of a nursing infant mammal including humans. These compositions generally comprise one or more bacterial strains selected for their growth on mammalian milk oligosaccharides, a source of mammalian milk oligosaccharides, and, optionally, nutritive components required for the growth of that infant mammal.

Claims

1. A method of monitoring the health of a mammal, comprising:

a) obtaining a fecal sample from the mammal;

b) determining the level of dysbiotic indicators in the sample;

c) identifying a dysbiotic state in the mammal based on the level and/or content of the dysbiotic indicator in the sample.

2. The method of claim 1, further comprising treating the dysbiotic mammal by:

i) administering a bacterial composition comprising bacteria capable of colonization of the colon;

ii) administering MMO;

or

iii) both (i) and (ii);

wherein the bacteria and/or the MMO are administered in respective amounts sufficient to change the level and/or content of the dysbiotic indicator in the feces of said mammal to that of a non-dysbiotic level and/or content.

4. The method of any one of claims 1-3, wherein the dysbiotic indicator is stool frequency, stool consistency, amount of short-chain fatty acid (SCFA), SCFA content, pH, amount of bifidobacteria, amount of B. infantis, amount of pathogenic bacteria, amount of lipopolysaccharide (LPS), amount of antibiotic resistance genes, amount of human milk oligosaccharides (HMO), and/or amount of proinflammatory cytokines.

5. The method of claim 4, wherein the dysbiotic indicator is (a) an increase in the lactate:

acetate ratio to around 2;

3 in the feces;

(b) decreased frequency of bowel movements as compared to a dysbiotic infant;

(c) more mature stool consistency (i.e., more firm and/or less watery);

(d) decreased pro-inflammatory cytokines (e.g., IL-8 and IL-10) by around 10×

in the feces;

(e) decreased inflammatory LPS by around 4×

in the feces;

(f) decreased pathogenic microbe levels in the feces;

(g) decreased antibiotic resistance gene load by around 3×

in the feces;

(g) titratable acidity above 2 mmol/g feces, preferably above 5 mmol/g feces;

(h) bifidobacteria levels of greater than 10⁶, preferably greater than 10⁸, more preferably greater than 10⁹ or 10¹⁰ in the feces;

(i) B. infantis levels of greater than 10⁶, preferably greater than 10⁸, more preferably greater than 10⁹ or 10¹⁰ in the feces; and

/or (j) decreased HMO levels present in the feces of at least an order of magnitude, compared to dysbiotic infants.

6. The method of any one of claims 2-5, wherein the step of treating the mammal and/or the step of administering the bacterial composition and/or MMO results in the proportion of one or more pathogenic bacteria in the microbiome of the mammal to be reduced.

7. The method of any one of claims 4-6, wherein the pathogenic bacteria is Salmonella, E. coli, Enterobacteria, Clostridium, Klebsiella, or combinations thereof.

8. The method of claim 6 or 7, wherein the pathogenic bacteria is reduced by 20% by the treatment.

9. The method of any one of claims 2-8, wherein the mammal'"'"'s colon has antibiotic resistance genes, and wherein the step of treating the mammal and/or the step of administering the bacterial composition and/or MMO results in the antibiotic resistance gene load of at least one gene being reduced by greater than 10%, 15%, 20%, 25%, 50%, 75% 80%, or 85%.

10. The method of any one of claims 4-9, wherein the SCFA comprises one or more of acetic, propionic, and butyric acids and salts thereof, and lactic acid or salts thereof

11. The method of any one of claims 3-10, wherein the amount of the bacterial composition and/or the MMO administered in step (a) is different than the amount of the bacterial composition and/or the MMO administered in step (c).

12. The method of any one of claims 3-11, wherein the periodicity and/or duration of the administration of the bacterial composition and/or the MMO administered in step (a) is different than the periodicity and/or duration of the bacterial composition and/or the MMO administered in step (c).

35. The method of any one of claims 2-34, wherein the mammal is receiving MMO at a dose representing over 25% of the total dietary fiber.

36. The method of any one of claims 2-35, wherein the mammal is receiving MMO at a dose representing over 40% of the total dietary fiber.

37. The method of any one of claims 2-36, wherein the mammal is receiving MMO at a dose representing over 50% of the total dietary fiber.

38. The method of any one of claims 2-37, wherein the MMO is administered to said mammal prior to the administration of the bacterial composition.

39. The method of any one of claims 2-38, wherein the MMO is administered to said mammal after the administration of the bacterial composition.

40. The method of any one of claims 2-38, wherein the MMO is administered to said mammal contemporaneously with the administration of the bacterial composition.

41. The method of any one of claims 2-38, wherein the MMO consists of carbohydrate polymers found in mammalian milk which are not metabolized by any combination of digestive enzymes expressed from mammalian genes.

42. The method of any one of claims 2-41, wherein the MMO comprises fucosyllactose, lacto-N-fucopentose, lactodifucotetrose, sialyllactose, disialyllactone-N-tetrose, 2′

-fucosyllactose, 3′

-sialyllactoseamin, 3′

-fucosyllactose, 3′

-sialyl-3-fucosyllactose, 3′

-sialyllactose, 6′

-sialyllactosamine, 6′

-sialyllactose, difucosyllactose, lacto-N-fucosylpentose I, lacto-N-fucosylpentose II, lacto-N-fucosylpentose III, lacto-N-fucosylpentose V, sialyllacto-N-tetraose, their derivatives, or combinations thereof.

43. The method of any one of claims 2-42, wherein the MMO is administered in a food composition.

44. The method of claim 43, wherein the food composition comprises mammalian milk, mammalian milk derived product, and/or mammalian donor milk.

45. The method of claim 43, wherein the food composition comprises infant formula, a milk replacer, an enteral nutrition product, and/or a meal replacer for a mammal, preferably for a human.

46. The method of any one of claims 43-45, wherein the food composition is sufficient to sustain growth of the mammal.

47. The method of any one of claims 2-46, wherein the bacterial composition comprises bacteria that are capable of consuming MMO by the internalization of that MMO within the bacterial cell.

48. The method of any one of claims 2-47, wherein the bacterial composition comprises bacteria that can grow in an anaerobic culture whose sole carbon source is MMO.

49. The method of any one of claims 2-48, wherein at least one species of the bacterial composition comprises bacteria capable of and/or activated for colonization of the colon.

50. The method of any one of claims 2-49, wherein the bacterial composition comprises bacteria capable of and/or activated for colonization of the colon, and wherein the bacteria expresses genes for a transporter that internalizes one or more MMOs intact.

51. The method of any one of claims 2-50, wherein the bacterial composition comprises bacteria of a genus selected from the group consisting of Bifidobacteria, Lactobacillus, and Pediococcus.

52. The method of claim 51, wherein the bacteria are B. adolescentis, B. animalis, B. animalis subsp. animalis, B. animalis subsp. lactis, B. bifidum, B. breve, B. catenulatum, B. longum, B. longum subsp. infantis, B. longum subsp. longum, B. pseudocatanulatum, B. pseudolongum, L. acidophilus, L. antri, L. brevis, L. casei, L. coleohominis, L. crispatus, L. curvatus, L. fermenturn, L. gasseri, L. johnsonii, L. mucosae, L. pentosus, L. plantarum, L. reuteri, L. rhamnosus, L. sakei, L. salivarius, P. acidilactici, P. argentinicus, P. claussenii, P. pentosaceus, P. stilesii L. paracasei, L. kisonensis., L. paralimentarius, L. perolens, L. apis, L. ghanensis, L. dextrinicus, L. shenzenensis, L. harbinensis, P. parvulus, or P. lolii.

53. The method of claim 51 or 52, wherein the bacteria are B. longum.

54. The method of claim 53, wherein the bacteria are B. longum subsp. infantis.

55. The method of any one of claims 1-54, wherein the mammal is a human.

56. The method of any one of claims 1-55, wherein the mammal is a non-human mammal.

57. The method of claim 56, wherein the non-human mammal is a buffalo, camel, cat, cow, dog, goat, guinea pig, hamster, horse, pig, rabbit, sheep, monkey, mouse, or rat.

58. The method of claim 56 or 57, wherein the non-human mammal is a mammal grown for human consumption.

59. The method of claim 56 or 57, wherein the non-human mammal is a companion or performance animal.

60. The method of any one of claims 1-59, wherein the mammal is an infant.

61. The method of claim 60, wherein the infant is a pre-term infant or a term infant.

62. The method of claim 60 or 61, wherein the infant is an infant born by C-section.

63. The method of any one of claims 60-62, wherein the infant is a dysbiotic infant.

64. The method of claim 63, wherein the dysbiotic infant has (a) a watery stool, (b) Clostridium difficile levels of greater than 10⁶ cfu/g feces, greater than 10⁷ cfu/g feces, or greater than 10⁸ cfu/g feces, (c) Enterobacteriaceaes at levels of greater than 10⁶, greater than 10⁷, or greater than 10⁸ cfu/g feces, and/or (d) a stool pH of 5.5 or above, 6.0 or above, or 6.5 or above.

3. A method of maintaining the health of the mammal, said method comprising the step of:

a) treating a mammal by;

i. administering a bacterial composition comprising bacteria capable of colonization of the colon;

ii. administering MMO;

or

iii. both (i) and (ii); and

further comprising the steps of;

b) monitoring the mammal by;

i. obtaining a fecal sample from the mammal;

ii. determining the level of a dysbiotic indicator in the sample;

iii. identifying a dysbiotic state in the mammal based on the level and/or content of the dysbiotic indicator in the sample; and

c) administering the bacterial composition and/or the MMO in response to the identified dysbiotic state.

13. A method of increasing and/or maintaining a level of short chain fatty acids (SCFA) or organic acids in the colon of a mammal, comprising:

a) administering a bacterial composition comprising bacteria capable of colonization of the colon; and

b) administering MMO;

wherein the bacteria and the MMO are administered in respective amounts sufficient to maintain a level of short chain fatty acids (SCFA) or organic acids in the feces of said mammal.

14. The method of claim 13, wherein the SCFA comprises one or more of acetic, propionic, and butyric acids and salts thereof, and lactic acid or salts thereof

15. The method of claim 14, wherein acetic acid makes up at least 30% of the SCFA.

16. The method of any one of claims 13-15, wherein said level of SCFA or organic acids is the level indicative of a healthy microbiome.

21. The method of claim 13-20, wherein the bacterial composition is administered for a period and in an amount sufficient to maintain the species capable of consuming MMO by the internalization of that MMO within the bacterial cell at a level of at least 10⁶ CFU/g feces, preferably 10⁸ CFU/g feces.

17. A method of reducing the antibiotic resistance gene load of at least one gene in a mammal'"'"'s gut comprising:

a) administering a bacterial composition comprising at least one species capable of consuming MMO by the internalization of that MMO within the bacterial cell, and;

b) administering an MMO at a dose representing over 10% of the total dietary fiber.

18. The method of claim 17, wherein the antibiotic resistance gene load of said at least one gene is reduced by greater than 25%, 50%, 75% 80%, or 85%.

19. A method of decreasing the frequency of bowel movements in a mammal comprising:

administering to the mammal a bacterial composition comprising at least one species capable of consuming MMO by the internalization of that MMO within the bacterial cell, wherein the mammal is receiving MMO at a dose representing over 10% of the total dietary fiber.

20. The method of claim 19, wherein the firmness of the stool composition of the mammal is increased.

22. A method of altering the stool composition in a mammal comprising:

administering a bacterial composition comprising at least one species capable of consuming MMO by the internalization of that MMO within the bacterial cell to the mammal, wherein the mammal is receiving MMO at a dose representing over 10% of the total dietary fiber.

23. The method of claim 22, wherein the level of LPS in the stool composition is decreased as compared to the stool composition of a dysbiotic mammal.

24. The method of any one of claim 22 or 23, wherein the level of pathogenic bacteria in the stool composition is decreased as compared to the stool composition of a mammal not receiving the bacterial composition.

25. The method of any one of claims 22-24, wherein the pH of the stool composition is decreased as compared to the stool composition of a mammal not receiving the bacterial composition.

26. The method of any one of claims 22-25, wherein the level of inflammatory cytokines, especially IL-8 and/or IL-10, in feces is reduced relative to the level of the same cytokine for a dysbiotic mammal.

27. A method of reducing the levels of LPS and/or pathogenic bacteria in the gut of a mammal comprising:

a) administering a bacterial composition comprising at least one species capable of consuming MMO by the internalization of that MMO within the bacterial cell, and;

b) administering an MMO at a dose sufficient to maintain colonization of the colon of the mammal by bacteria in the bacterial composition of step (a).

28. The method of claim 27, wherein the method is a method of reducing the levels of LPS in the gut of a mammal.

29. The method of claim 27, wherein the method is a method of reducing the levels of pathogenic bacteria of a mammal.

30. The method of any one of claims 27-29, wherein the bacterial composition is provided to the mammal contemporaneously or within 2 hr of the addition of the MMO composition.

31. The method of any one of claims 27-31, wherein the pathogenic bacteria is Salmonella, E. coli, Enterobacteria, Clostridium, Klebsiella, or combinations thereof.

32. A method of reducing the risk of presenting metabolic disorders selected from the group consisting of Juvenile Diabetes (Type I), obesity, asthma, atopy, Celiac'"'"'s Disease, food allergies, autism, and combinations thereof, comprising:

a) administering a bacterial composition comprising at least one species capable of consuming MMO by the internalization of that MMO within the bacterial cell, and;

b) administering an MMO at a dose sufficient to maintain colonization of the colon of the mammal by bacteria in the bacterial composition of step (a),

wherein the risk is reduced as compared to a dysbiotic infant.

33. The method of claim 32, wherein the bacterial composition and MMO are administered daily for a period of from at least 10, at least 20, at least 30, at least 60, at least 90, at least 120, at least 150, or at least 180 days.

34. The method of claim 32 or 33, wherein the risk is reduced by 20, 30, 40, 50, 60, 70, 80, or 90%.

65. A method of obtaining a bacterial monoculture comprising a bacterial strain which can grow on mammalian milk oligosaccharides (MMO) as a sole carbon source, the method comprising the following steps:

a) obtaining a sample containing living microorganisms from fecal material of a nursing infant mammal that is not dysbiotic;

b) inoculating a culture medium whose sole carbon source is MMO with the sample from step (a);

c) incubating the inoculated culture anaerobically; and

d) recovering a pure bacterial strain from the incubated culture of step (c).

66. The method of claim 65, further comprising the step of:

exposing the sample from step (a) to mutagenesis prior to the inoculating step (b).

67. The method of claim 66, wherein the bacterial strain comprises bacteria of a genus selected from the group consisting of Bifidobacteria, Lactobacillus, and Pediococcus.