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Title:
FEED COMPOSITION
Document Type and Number:
WIPO Patent Application WO/2021/046073
Kind Code:
A1
Abstract:
Provided herein, inter alia, are feed or feed additive compositions comprising chitin-glucan as well as methods for making and using the same for improving the performance of a subject with respect to feed conversion ratio (FCR), weight gain, feed efficiency, carcass quality, maintenance of a healthy guy microbiome, and/or decreased susceptibility to intestinal pathogens and diseases associated with the same.

Inventors:
GIBBS KIRSTY (GB)
LI WENTING (US)
Application Number:
PCT/US2020/048980
Publication Date:
March 11, 2021
Filing Date:
September 02, 2020
Export Citation:
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Assignee:
DUPONT NUTRITION BIOSCI APS (DK)
DUPONT NUTRITION & BIOSCIENCES USA 4 INC (US)
International Classes:
A23L33/135; A23K10/30; A23K20/163; A23K20/189; A23K20/195; A23K50/30; A23K50/75; A23L29/00; A23L29/269; A23L29/275; A23L31/15; A23L33/145; A61K31/722; A61K35/00; A61K35/741; A61K36/06; A61K47/69; C08B37/08
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Foreign References:
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Other References:
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Attorney, Agent or Firm:
SHOW, Matthew (US)
Download PDF:
Claims:
CLAIMS

We claim:

1. A feed additive composition or premix comprising chitin-glucan.

2. The feed additive composition or premix of claim 1, wherein chitin glucan comprises at least about 0.01% to 1% of feed additive composition or premix.

3. The feed additive composition or premix of claim 1 or claim 2, further comprising at least one direct fed microbial (DFM).

4. The feed additive composition or premix of claim 3, wherein the direct fed microbial is an antipathogen DFM.

5. The feed additive composition or premix of claim 3 or claim 4, wherein the DFM is a viable bacterium.

6. The feed additive composition or premix of any one of claims 3-5, wherein the composition comprises at least three DFMs.

7. The feed additive composition or premix of any one of claims 3-6, wherein the DFM is a strain which inhibits intestinal establishment of pathogenic microorganisms.

8. The feed additive composition or premix of claim 7, wherein the pathogenic microorganisms are Clostridium perjringens, E. coli, Salmonella spp., Campylobacter spp., protozoan parasites within the genus Eimeria or combinations thereof.

9. The feed additive composition or premix of any one of claims 3-8, wherein the DFMs are Bacillus strain 2084 Accession No. NRRL B-50013, Bacillus strain LSSAOl Accession No. NRRL B-50104 and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507.

10. The feed additive composition or premix of any one of claims 3-9, further comprising one or more of a phytase, a xylanase, a protease, a glucoamylase, and/or an amylase. 11. The feed additive composition or premix of claim 10, wherein the phytase is a 6-phytase or a 3 -phytase.

12. The feed additive composition or premix of claim 10, wherein the phytase is an E. coli phytase or a Buttiauxella phytase or a Citrobacter phytase or a Hqfnia phytase or an Aspergillus phytase or a Penicillium phytase or a Trichoderma phytase or a Hansenula phytase or a Peniphora phytase.

13. The feed additive composition or premix of any one of claims 3-12, wherein the DFM is present in the feed additive composition in a range from about 2.5x 103 CFU to about 6.7x 106 CFU.

14. The feed additive composition or premix of any one of claims 1-13, further comprising at least one essential oil.

15. The feed additive composition or premix of claim 14, wherein the essential oil is cinnamaldehyde and/or thymol.

16. The feed additive composition or premix of any one of claims 1-15, further comprising betaine.

17. A kit comprising the feed additive composition or premix of any one of claims 1-16 and instructions for administration.

18. A feed comprising the feed additive composition or premix of any one of claims 1-16.

19. The premix of any one of claims 1-16, comprising at least one mineral and/or at least one vitamin.

20. A method for improving one or more metrics in an animal comprising administering to the animal an effective amount of a feed additive composition comprising chitin-glucan, wherein said one or more metrics comprises of one or more of (a) improved feed conversion ratio (FCR); (b) improved weight gain; (c) improved feed efficiency; (d) reduced populations of pathogenic bacteria in the gastrointestinal tract; and/or (e) improved intestinal physiology and/or morphology compared to the performance of a subject that has not been administered a feed comprising chitin-glucan.

21. The method of claim 20, wherein improved intestinal physiology and/or morphology comprises one or more of (i) decreased gross morphological lesions; (ii) increased villus height; (iii) increased crypt depth; (iv) decreased immune cell infiltration; (v) increased ileal Lactobacillus sp. population; and/or (vi) increased cecal Butyricicoccus population.

22. The method of claim 20 or claim 21, wherein the chitin-glucan comprises from about 0.01% to about 1% of the diet of the animal.

23. The method of any one of claims 20-22, further comprising administering to the animal an effective amount of at least one direct fed microbial (DFM).

24. The method of claim 23, wherein the direct fed microbial is an antipathogen DFM.

25. The method of claim 23 or claim 24, wherein the DFM is a viable bacterium.

26. The method of any one of claims 23-25, comprising administering to the animal at least three DFMs.

27. The method of any one of claims 23-26, wherein the DFM is a strain which inhibits intestinal establishment of pathogenic microorganisms.

28. The method of claim 27, wherein the pathogenic microorganisms are Clostridium perfringens, E. coli, Salmonella spp., Campylobacter spp., protozoan parasites within the genus Eimeria or combinations thereof.

29. The method of any one of claims 23-28, wherein the DFMs are Bacillus strain 2084 Accession No. NRRL B-50013, Bacillus strain LSSAOl Accession No. NRRL B-50104 and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507.

30. The method of any one of claims 20-29, further comprising administering to the animal an effective amount of one or more of a phytase, a xylanase, a protease, a glucoamylase, and/or an amylase. 31. The method of claim 30, wherein the phytase is a 6-phytase or a 3-phytase.

32. The method of claim 31, wherein the phytase is an E. coli phytase or a Buttiauxella phytase or a Citrobacter phytase or a Hqfhia phytase or an Aspergillus phytase or a Penicillium phytase or a Trichoderma phytase or a Hansenula phytase or a Peniphora phytase.

33. The method of any one of claims 23-32, wherein the DFM is present in the feed additive composition in a range from about 2.5*103 CFU to about 6.7X106 CFU.

34. The method of any one of claims 20-33, further comprising administering an effective amount of at least one essential oil.

35. The method of claim 14, wherein the essential oil is cinnamaldehyde and/or thymol.

36. The method of any one of claims 20-35 further comprising administering an effective amount of betaine.

37. The method of any one of claims 20-36, wherein the animal has or is suspected to have necrotic enteritis or coccidiosis.

38. The method of any one of claims 20-37, wherein the animal is poultry or swine.

39. The method of claim 38, wherein the poultry is a broiler.

40. A method for treating or preventing necrotic enteritis in an animal in need thereof, comprising administering to the animal a feed additive composition comprising an effective amount of chitin-glucan.

41. The method of claim 40, wherein the chitin-glucan comprises from about 0.01% to about

1% of the diet of the animal.

42. The method of claim 40 or claim 41, further comprising administering to the animal an effective amount at least one direct fed microbial (DFM).

43. The method of claim 42, wherein the direct fed microbial is an antipathogen DFM.

44. The method of claim 42 or claim 43, wherein the DFM is a viable bacterium. 45. The method of any one of claims 42-44, comprising administering to the animal at least three DFMs.

46. The method of any one of claims 42-45, wherein the DFM is a strain which inhibits intestinal establishment of pathogenic microorganisms.

47. The method of claim 46, wherein the pathogenic microorganisms are Clostridium perfringens, E. coli, Salmonella spp., Campylobacter spp., protozoan parasites within the genus Eimeria or combinations thereof.

48. The method of any one of claims 42-47, wherein the DFMs are Bacillus strain 2084 Accession No. NRRL B-50013, Bacillus strain LSSAOl Accession No. NRRL B-50104 and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507.

49. The method of any one of claims 40-48, further comprising administering to the animal an effective amount of one or more of a phytase, a xylanase, a protease, a glucoamylase, and/or an amylase.

50. The method of claim 49, wherein the phytase is a 6-phytase or a 3-phytase.

51. The method of claim 50, wherein the phytase is an E. coli phytase or a Buttiauxella phytase or a Citrobacter phytase or a Hqfnia phytase or an Aspergillus phytase or a Penicillium phytase or a Trichoderma phytase or a Hansenula phytase or a Peniphora phytase.

52. The method of any one of claims 42-51, wherein the DFM is present in the feed additive composition in a range from about 2.5*103 CFTJ to about 6.7X106 CFU.

53. The method of any one of claims 40-52, further comprising administering an effective amount of at least one essential oil.

54. The method of claim 53, wherein the essential oil is cinnamaldehyde and/or thymol.

55. The method of any one of claims 40-55 further comprising administering an effective amount of betaine.

56. The method of any one of claims 40-55, wherein the animal is poultry or swine. 57. The method of claim 56, wherein the poultry is a broiler.

58. The method of any one of claims 40-57, wherein said administration a) increases the population of Firmicutes phylum bacteria in the ilium to a percentage that is higher than the population of Firmicutes phylum bacteria in a animal that is not administered a feed additive composition comprising an effective amount of chitin-glucan; and/or b) decreases the population of Bacteroidetes phylum bacteria in the ilium to a percentage that is lower than the population of Bacteroidetes phylum bacteria in an animal that is not administered a feed additive composition comprising an effective amount of chitin-glucan.

59. The method of any one of claims 40-58, wherein said administration results in a higher ratio of Firmicutes to Bacteroides bacteria in the ilium compared to the ratio of Firmicutes to Bacteroides bacteria in the ilium of an animal that is not administered a feed additive composition comprising an effective amount of chitin-glucan.

60. A method of preparing a feed additive composition comprising: admixing chitin-glucan and one or more ingredients selected from the group consisting of: a salt, polyol including sorbitol and glycerol, grain or a grain component, protein or at least a portion of a protein, sodium acetate, sodium acetate trihydrate, potassium sorbate, talc, polyvinyl alcohol (PVA), benzoate, sorbiate, 1,3-propane diol, glucose, parabens, sodium chloride, citrate, metabisulfite, formate or a combination thereof.

61. The method of claim 60, further comprising packaging the feed additive composition.

62. The method of claim 60 or claim 61, wherein the chitin-glucan is admixed with a composition comprising at least one protein or at least a portion of a protein.

63. The method of any one of claims 60-62, wherein the at least one protein or portion thereof is an animal protein or a vegetable protein, com, soybean meal, com dried distillers grains with solubles (cDDGS), wheat, wheat proteins, gluten, wheat by products, wheat bran, wheat dried distillers grains with solubles (wDDGS), com by products including com gluten meal, barley, oat, rye, triticale, full fat soy, animal by-product meals, an alcohol-soluble protein, a zein, a maize zein maize, a kafirin, a protein from oil seeds, or a combination thereof. 64. The method of claim 63, wherein the animal protein or vegetable protein is selected from the group consisting of one or more of a gliadin or an immunogenic fragment of a gliadin, a beta- casein, a beta-lactoglobulin, glycinin, beta-conglycinin, cruciferin, napin, hordeins, keratins, feather or hair meals, collagen, whey protein, fish protein, fish meals, meat protein, egg protein, soy protein and grain protein.

65. The method of claim 63, wherein the protein from oil seeds is selected from the group consisting of soybean seed proteins, sun flower seed proteins, rapeseed proteins, canola seed proteins and combinations thereof.

66. The method of any one of claims 60-65, wherein the chitin-glucan comprises from about 0.01% to about 1% of the feed additive composition.

67. The method of any one of claims 60-66, further comprising admixing at least one direct fed microbial (DFM).

68. The method of claim 67, wherein the direct fed microbial is an antipathogen DFM.

69. The method of claim 67 or claim 68, wherein the DFM is a viable bacterium.

70. The method of any one of claims 67-69, comprising admixing at least three DFMs.

71. The method of any one of claims 67-70, wherein the DFM is a strain which inhibits intestinal establishment of pathogenic microorganisms.

72. The method of claim 71, wherein the pathogenic microorganisms are Clostridium perfringens, E. coli, Salmonella spp., Campylobacter spp., protozoan parasites within the genus Eimeria, or combinations thereof.

73. The method of any one of claims 67-72, wherein the DFMs are Bacillus strain 2084 Accession No. NRRL B-50013, Bacillus strain LSSAOl Accession No. NRRL B-50104 and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507.

74. The method of any one of claims 60-73, further comprising admixing one or more of a phytase, a xylanase, a protease, a glucoamylase, and/or an amylase. 75. The method of claim 74, wherein the phytase is a 6-phytase or a 3-phytase.

76. The method of claim 75, wherein the phytase is an E. coli phytase or a Buttiauxella phytase or a Citrobacter phytase or a Hqfhia phytase or an Aspergillus phytase or a Penicillium phytase or a Trichoderma phytase or a Hansenula phytase or a Peniphora phytase.

77. The method of any one of claims 67-76, wherein the DFM is present in the feed additive composition in a range from about 2.5*103 CFU to about 6.7X106 CFU.

78. The method of any one of claims 60-77, further comprising admixing at least one essential oil.

79. The method of claim 78, wherein the essential oil is cinnamaldehyde and/or thymol.

80. The method of any one of claims 60-79 further comprising admixing betaine.

Description:
FEED COMPOSITION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/896,100, filed September 5, 2019, the disclosure of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] Provided herein, inter alia, are feed or feed additive compositions comprising chitin- glucan as well as methods for making and using the same for the improvement of a subject’s performance and the maintenance of a healthy gut microbiome.

BACKGROUND

[0003] Intestinal heath remains a significant challenge for swine and poultry production. For example, in chickens, Emeria and Clostridium perfringem cause intestinal lesions and can lead to the condition known as necrotic enteritis (NE). Impaired production efficiency and animal welfare as well as increased condemnation at processing plants are often associated with clinical and sub-clinical necrotic enteritis infections. In addition, producers that have suffered from necrotic enteritis infections for 2.5 years can experience an average of 33% profit loss (Lovland and Kaldhusdal, 2001, Avian Pathology, 30(1): 73-81).

[0004] Because pathogens such as Eimeria and C. perfringem are almost always present in the environment, prevention is more important than treatment. Control of NE infection has traditionally been accomplished by the prophylactic dietary inclusion of antibiotics or ionophores. However, use of antibiotics in the production of swine and poultry has been associated with several negative outcomes, including the development of antibiotic-resistant microorganisms. Accordingly, given the movement towards antibiotic-free production regimes in the industry, new non-antibiotic-based preventative feed additives are required to control infection, prevent disease, and continuously improve animal efficiency and welfare during production. [0005] The subject matter disclosed herein addresses these needs and provides additional benefits as well.

SUMMARY

[0006] Provided herein, inter alia, are feed or feed additive compositions comprising chitin- glucan as well as methods for making and using the same for the improvement of a subject’s performance and maintenance of a healthy gut microbiome.

[0007] Accordingly, in some aspects, provided herein is a feed additive composition or premix comprising chitin-glucan. In some embodiments, chitin glucan comprises at least about 0.01% to 1% of feed additive composition or premix. In some embodiments of any of the embodiments disclosed herein, the feed additive composition or premix further comprises at least one direct fed microbial (DFM). In some embodiments, the direct fed microbial is an antipathogen DFM.

In some embodiments of any of the embodiments disclosed herein, the DFM is a viable bacterium. In some embodiments of any of the embodiments disclosed herein, the composition comprises at least three DFMs. In some embodiments of any of the embodiments disclosed herein, the DFM is a strain which inhibits intestinal establishment of pathogenic microorganisms. In some embodiments, the pathogenic microorganisms are Clostridium perfringens, E. coli, Salmonella spp., Campylobacter spp., protozoan parasites within the genus Eimeria or combinations thereof. In some embodiments of any of the embodiments disclosed herein, the DFMs are Bacillus strain 2084 Accession No. NRRL B-50013, Bacillus strain LSSAOl Accession No. NRRL B-50104 and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507. In some embodiments of any of the embodiments disclosed herein, the feed additive composition or premix further comprises one or more of a phytase, a xylanase, a protease, a gluco amylase, and/or an amylase. In some embodiments, the phytase is a 6-phytase or a 3-phytase. In some embodiments, the phytase is an E. coli phytase or a Buttiauxella phytase or a Citrobacter phytase or a Hqfhia phytase or an Aspergillus phytase or a Penicillium phytase or a Trichoderma phytase or a Hansenula phytase or a Peniphora phytase. In some embodiments of any of the embodiments disclosed herein, the DFM is present in the feed additive composition in a range from about 2.5x 10 3 CFU to about 6.7X10 6 CFU. In some embodiments of any of the embodiments disclosed herein, the feed additive composition or premix further comprises at least one essential oil. In some embodiments, the essential oil is cinnamaldehyde and/or thymol. In some embodiments of any of the embodiments disclosed herein, the feed additive composition or premix further comprises betaine. In some embodiments of any of the embodiments disclosed herein, the premix comprises at least one mineral and/or at least one vitamin.

[0008] In other aspects, provided herein is a kit comprising any feed additive composition or premix disclosed herein and optional instructions for administration and/or a feed comprising any feed additive composition or premix disclosed herein.

[0009] In further aspects, provided herein is a method for improving one or more metrics in an animal comprising administering to the animal an effective amount of a feed additive composition comprising chitin-glucan, wherein said one or more metrics comprises of one or more of (a) improved feed conversion ratio (FCR); (b) improved weight gain; (c) improved feed efficiency; (d) reduced populations of pathogenic bacteria in the gastrointestinal tract; and/or (e) improved intestinal physiology and/or morphology compared to the performance of a subject that has not been administered a feed comprising chitin-glucan. In some embodiments, improved intestinal physiology and/or morphology comprises one or more of (i) decreased gross morphological lesions; (ii) increased villus height; (iii) increased crypt depth; (iv) decreased immune cell infiltration; (v) increased ileal Lactobacillus sp. population; and/or (vi) increased cecal Butyricicoccus population. In some embodiments of any of the embodiments disclosed herein, the chitin-glucan comprises from about 0.01% to about 1% of the diet of the animal. In some embodiments of any of the embodiments disclosed herein, the method further comprises administering to the animal an effective amount of at least one direct fed microbial (DFM). In some embodiments, the direct fed microbial is an antipathogen DFM. In some embodiments of any of the embodiments disclosed herein, the DFM is a viable bacterium. In some embodiments of any of the embodiments disclosed herein, the method comprises administering to the animal at least three DFMs. In some embodiments of any of the embodiments disclosed herein, the DFM is a strain which inhibits intestinal establishment of pathogenic microorganisms. In some embodiments, the pathogenic microorganisms are Clostridium perfringens, E. coli, Salmonella spp., Campylobacter spp., protozoan parasites within the genus Eimeria or combinations thereof. In some embodiments of any of the embodiments disclosed herein, the DFMs are Bacillus strain 2084 Accession No. NRRL B-50013, Bacillus strain LSSAOl Accession No. NRRL B-50104 and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507. In some embodiments of any of the embodiments disclosed herein, the method further comprises administering to the animal an effective amount of one or more of a phytase, a xylanase, a protease, a glucoamylase, and/or an amylase. In some embodiments, the phytase is a 6-phytase or a 3-phytase. In some embodiments, the phytase is an E. coli phytase or a Buttiauxella phytase or a Citrobacter phytase or a Hqfnia phytase or an Aspergillus phytase or a Penicillium phytase or a Trichoderma phytase or a Hansenula phytase or a Peniphora phytase. In some embodiments of any of the embodiments disclosed herein, the DFM is present in the feed additive composition in a range from about 2.5* 10 3 CFUJ to about 6.7x1o 6 CFU. In some embodiments of any of the embodiments disclosed herein, the method further comprises administering to the animal an effective amount of at least one essential oil. In some embodiments, the essential oil is cinnamaldehyde and/or thymol. In some embodiments of any of the embodiments disclosed herein, the method further comprises administering to the animal an effective amount of betaine. In some embodiments of any of the embodiments disclosed herein, the animal has or is suspected to have necrotic enteritis or coccidiosis. In some embodiments of any of the embodiments disclosed herein, the animal is poultry or swine. In some embodiments, the poultry is a broiler.

[0010] In yet other aspects, provided herein are methods for treating or preventing necrotic enteritis in an animal in need thereof, comprising administering to the animal a feed additive composition comprising an effective amount of chitin-glucan. In some embodiments, the chitin- glucan comprises from about 0.01% to about 1% of the diet of the animal. In some embodiments of any of the embodiments disclosed herein, the method further comprises administering to the animal an effective amount at least one direct fed microbial (DFM). In some embodiments, the direct fed microbial is an antipathogen DFM. In some embodiments of any of the embodiments disclosed herein, the DFM is a viable bacterium. In some embodiments of any of the embodiments disclosed herein, the method comprises administering to the animal at least three DFMs. In some embodiments of any of the embodiments disclosed herein, the DFM is a strain which inhibits intestinal establishment of pathogenic microorganisms. In some embodiments, the pathogenic microorganisms are Clostridium perfringens, K coli, Salmonella spp., Campylobacter spp, protozoan parasites within the genus Eimeria or combinations thereof. In some embodiments of any of the embodiments disclosed herein, the DFMs are Bacillus strain 2084 Accession No. NRRL B-50013, Bacillus strain LSSAOl Accession No. NRRL B-50104 and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507. In some embodiments of any of the embodiments disclosed herein, the method further comprises administering to the animal an effective amount of one or more of a phytase, a xylanase, a protease, a glucoamylase, and/or an amylase. In some embodiments, the phytase is a 6-phytase or a 3-phytase. In some embodiments, the phytase is an E. coli phytase or a Buttiauxella phytase or a Citrobacter phytase or a Hqfnia phytase or an Aspergillus phytase or a Penicillium phytase or a Trichoderma phytase or a Hansenula phytase or a Peniphora phytase. In some embodiments of any of the embodiments disclosed herein, the DFM is present in the feed additive composition in a range from about 2.5x 10 3 CPU to about 6.7x1o 6 CFU. In some embodiments of any of the embodiments disclosed herein, the method further comprises administering to the animal an effective amount of at least one essential oil. In some embodiments, the essential oil is cinnamaldehyde and/or thymol. In some embodiments of any of the embodiments disclosed herein, the method further comprises administering to the animal an effective amount of betaine. In some embodiments of any of the embodiments disclosed herein, the animal is poultry or swine. In some embodiments, the poultry is a broiler. In some embodiments of any of the embodiments disclosed herein, said administration a) increases the population of Firmicutes phylum bacteria in the ilium to a percentage that is higher than the population of Firmicutes phylum bacteria in a animal that is not administered a feed additive composition comprising an effective amount of chitin-glucan; and/or b) decreases the population of Bacteroidetes phylum bacteria in the ilium to a percentage that is lower than the population of Bacteroidetes phylum bacteria in an animal that is not administered a feed additive composition comprising an effective amount of chitin-glucan. In some embodiments of any of the embodiments disclosed herein, said administration results in a higher ratio of Firmicutes to Bacteroides bacteria in the ilium compared to the ratio of Firmicutes to Bacteroides bacteria in the ilium of an animal that is not administered a feed additive composition comprising an effective amount of chitin-glucan.

[0011] In other aspects, provided herein is a method for preparing a feed additive composition comprising: admixing chitin-glucan and one or more ingredients selected from the group consisting of: a salt, polyol including sorbitol and glycerol, grain or a grain component, protein or at least a portion of a protein, sodium acetate, sodium acetate trihydrate, potassium sorbate, talc, polyvinyl alcohol (PVA), benzoate, sorbiate, 1,3-propane diol, glucose, parabens, sodium chloride, citrate, metabisulfite, formate or a combination thereof. In some embodiments, the method further comprises packaging the feed additive composition. In some embodiments of any of the embodiments disclosed herein, wherein the chitin-glucan is admixed with a composition comprising at least one protein or at least a portion of a protein. In some embodiments of any of the embodiments disclosed herein, the at least one protein or portion thereof is an animal protein or a vegetable protein, com, soybean meal, com dried distillers grains with solubles (cDDGS), wheat, wheat proteins, gluten, wheat by products, wheat bran, wheat dried distillers grains with solubles (wDDGS), com by products including com gluten meal, barley, oat, rye, triticale, full fat soy, animal by-product meals, an alcohol-soluble protein, a zein, a maize zein maize, a kafirin, a protein from oil seeds, or a combination thereof. In some embodiments, the animal protein or vegetable protein is selected from the group consisting of one or more of a gliadin or an immunogenic fragment of a gliadin, a beta-casein, a beta- lactoglobulin, glycinin, beta-conglycinin, cmciferin, napin, hordeins, keratins, feather or hair meals, collagen, whey protein, fish protein, fish meals, meat protein, egg protein, soy protein and grain protein. In some embodiments, the protein from oil seeds is selected from the group consisting of soybean seed proteins, sun flower seed proteins, rapeseed proteins, canola seed proteins and combinations thereof. In some embodiments of any of the embodiments disclosed herein, the chitin-glucan comprises from about 0.01% to about 1% of the feed additive composition. In some embodiments of any of the embodiments disclosed herein, the method further comprises admixing at least one direct fed microbial (DFM). In some embodiments, the direct fed microbial is an antipathogen DFM. In some embodiments of any of the embodiments disclosed herein, the DFM is a viable bacterium. The method of any one of claims 67-69, comprising admixing at least three DFMs. In some embodiments of any of the embodiments disclosed herein, the DFM is a strain which inhibits intestinal establishment of pathogenic microorganisms. In some embodiments, the pathogenic microorganisms are Clostridium perfringens, E. coli, Salmonella spp., Campylobacter spp., protozoan parasites within the genus Eimeria, or combinations thereof. In some embodiments of any of the embodiments disclosed herein, the DFMs are Bacillus strain 2084 Accession No. NRRL B-50013, Bacillus strain LSSAOl Accession No. NRRL B-50104 and Bacillus strain 15A-P4 ATCC Accession No. PTA- 6507. In some embodiments of any of the embodiments disclosed herein, the method further comprises admixing one or more of a phytase, a xylanase, a protease, a glucoamylase, and/or an amylase. In some embodiments, the phytase is a 6-phytase or a 3 -phytase. In some embodiments, the phytase is an E. coli phytase or a Buttiauxella phytase or a Citrobacter phytase or a Hqfnia phytase or an Aspergillus phytase or a Penicillium phytase or a Trichoderma phytase or a Hansenula phytase or a Peniphora phytase. In some embodiments of any of the embodiments disclosed herein, the DFM is present in the feed additive composition in a range from about 2.5><10 3 CFU to about 6.7x10 6 CFU. In some embodiments of any of the embodiments disclosed herein, the method further comprises admixing at least one essential oil. In some embodiments, the essential oil is cinnamaldehyde and/or thymol. In some embodiments of any of the embodiments disclosed herein, the method further comprises admixing betaine.

[0012] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.

[0013] Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. The disclosure of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 is a bar graph depicting the frequency (% of birds recorded) of duodenum lesion scores of birds fed different level of chitin-glucan at 21 d of age.

[0015] FIG. 2 is a bar graph depicting ileal bacteria composition at phylum level of birds fed different level of chitin-glucan at 21 d of age.

[0016] FIG. 3 is a bar graph depicting ileal bacteria Firmicutes to Bacteroides ratio at phylum level of birds fed different level of chitin-glucan at 21 d of age.

[0017] FIG. 4A is a bar graph depicting frequency (% of birds recorded) of duodenal lesion scores of birds fed chitin-glucan alone or in combination with Bacillus-based direct fed microbials at 21 d of age. FIG. 4B is a bar graph depicting frequency (% of birds recorded) of jejunal lesion scores of birds fed chitin-glucan alone or in combination with Bacillus-based direct fed microbials at 21 d of age. [0018] FIG. 5 depicts the structure of chitin and b (l,3)-glucan chains.

DETAILED DESCRIPTION

[0019] Chitin is a natural high molecular weight polymer widely found in nature, in fact the second major biopolymer after cellulose. Chitin is a polysaccharide whose structure is close to that of cellulose. It is the main component of insect and crustacean cuticule and is also part of the cell walls of some fungi and other organisms. Beta glucans are polysaccharides connected by beta glycosidic linkages that can be found in various organisms, such as yeast, mushrooms, fungi, cereal grains, and other sources. Chitin-glucan is a complex tridimensional biopolymer that combines two types of polysaccharide chains: covalently linked chitin and beta (1,3) glucan.

[0020] As will be further detailed below, the inventors of the instant application have surprisingly discovered that the addition of chitin-glucan to animal feed (for example, chicken and swine feed) improved one or more performance metrics such as, without limitation, feed conversion ratio (FCR), weight gain, “healthy” gut microbial populations, and/or feed efficiency. Surprisingly, it was also found that animals fed chitin-glucan did not develop intestinal lesions to the same extent when challenged with agents known to cause necrotic enteritis compared to challenged animals that were not fed chitin-glucan. The inventors further unexpectedly discovered that the protective effects of chitin-glucan with respect to preventing necrotic enteritis in necrotic enteritis-challenged animals were further potentiated when chitin-glucan was coadministered with a direct fed microbial (DFM) (a.k.a. a probiotic). In addition to decreased intestinal lesions, administration of chitin-glucan was associated with improved gut health and the maintenance of beneficial bacteria in the intestinal lumen compared to animals that were not fed chitin-glucan.

[0021] Accordingly, the inventors have discovered that animal feed or feed additive compositions supplemented with chitin-glucan co-polymers can promote growth and efficient utilization of feed as well as prevent and/or decrease the severity of intestinal conditions such as necrotic enteritis without the need for additional supplementation by antibiotics, which are known to contribute to unwanted outcomes such as antibiotic resistant microorganisms. L Definitions

[0022] As used herein, the term “chitin-glucan” refers to a vegetal biopolymer comprising two types of covalently linked polysaccharide chains: P(l,4)-poly-N-acetyl-D-glucosamine and b (l,3)-D-glucan. In some embodiments, chitin-glucan is extracted from the cell walls of a fungal cell, such as a yeast or the A. niger mycelium. In some embodiments, the ratio between chitin and b (l,3)-glucan in the chitin-glucan co-polymer can range from about 20:80 to 40:60 (w/w), such as any of about 20:80, 25:75, 30:70, 35:65, or 40:60, inclusive of all ratios in between these values. The structure of chitin and b (l,3)-glucan chains are illustrated in FIG. 5. In other embodiments, “chitin-glucan” can mean a pure copolymer which consists of links of N-acetyl-D- glucosamine units and, optionally, of a minor proportion of D-glucosamine units linked to one another by (l,6)-type linkages in the alpha conformation (chitin link), and of links of D-glucose units linked to one another by linkages of beta(l,3), beta(l,3)(l,6) or beta(l,3Xl,4) type, and preferably beta(l,3) type (beta-glucan links).

[0023] The term "chitin-glucan copolymers," as used herein, refers to copolymers obtained after extraction of fungal or yeast biomass but before enzymatic reaction by means of glucanase enzymes. The amount of chitin in said chitin-glucan copolymers is defined by the organism from which it is extracted. In a one embodiment, chitin-glucan copolymers derived from the mycelium of Aspergillus niger is used in the compositions and methods disclosed herein and comprise between about 30% and 50% (w/w) of chitin (such as any of about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% chitin) and between 50 to 70% (w/w) of beta-glucan (such as any of about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%). The terms "chitin" and "chitin polymers" are used herein as synonyms.

[0024] The terms “animal” and “subject” are used interchangeably herein and refer to nonruminant animals, i.e., mono-gastric animals. Examples of mono-gastric animals include, but are not limited to, pigs and swine, such as piglets, growing pigs, sows; and poultry. The term “poultry,” as used herein, means domesticated birds kept by humans for their eggs, their meat or their feathers. These birds are most typically members of the superorder Galloanserae, especially the order Galliformes which includes, without limitation, chickens, quails, ducks, geese, emus, ostriches, pheasant, and turkeys. In a further embodiment, the animal is a chicken, such as a broiler or a layer. In some embodiments, the subject or animal is not a human.

[0025] As used herein, “prevent,” “preventing,” “prevention” and grammatical variations thereof refers to a method of partially or completely delaying or precluding the onset or recurrence of a disorder or condition (such as necrotic enteritis) and/or one or more of its attendant symptoms or barring an animal from acquiring or reacquiring a disorder or condition or reducing an animal's risk of acquiring or reacquiring a disorder or condition or one or more of its attendant symptoms.

[0026] As used herein, the term “reducing” in relation to a particular trait, characteristic, feature, biological process, or phenomena refers to a decrease in the particular trait, characteristic, feature, biological process, or phenomena. The trait, characteristic, feature, biological process, or phenomena can be decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater than 100%.

[0027] As used herein “administer” or “administering” is meant the action of introducing one or more microbial strain, an exogenous feed enzyme and/or a strain and an exogenous feed enzyme to an animal, such as by feeding or by gavage.

[0028] As used herein, “effective amount” means a quantity of a substance (for example, chitin- glucan, a direct fed microbial (DFM), or an essential oil (EO)) to improve one or more metrics in an animal. Improvement in one or more metrics of an animal (such as, without limitation, any of improved feed conversion ratio (FCR); improved weight gain; improved feed efficiency; improved gut microbiome status (i.e. more healthy (“good”) bacterial and/or less unhealthy (“bad”) bacteria; and/or improved carcass quality can be measured as described herein or by other methods known in the art. An effective amount can be administered to the animal by providing ad libitum access to feed containing the substance (e.g., chitin-glucan). Additionally, substance (e.g., chitin-glucan) can also be administered in one or more doses.

[0029] As used herein, the term “feed” is used synonymously herein with “feedstuff.” Feed broadly refers to a material, liquid or solid, that is used for nourishing an animal, and for sustaining normal or accelerated growth of an animal including newborns or young and developing animals. The term includes a compound, preparation, mixture, or composition suitable for intake by an animal (such as, e.g., for poultry such as quail, ducks, turkeys, and chickens). In some embodiments, a feed or feed composition comprises a basal food composition and one or more feed additives or feed additive compositions.

[0030] The term "feed additive" as used herein refers to components included for purposes of fortifying basic feed with additional components to promote feed intake, treat or prevent disease, or alter metabolism. Feed additives include pre-mixes. As used herein, the term “feed additive” also refers to a substance which is added to a feed. Feed additives may be added to feed for a number of reasons. For instance, to enhance digestibility of the feed, to supplement the nutritional value of the feed, improve the immune defense of the recipient and/or to improve the shelf life of the feed. In some embodiments, the feed additive supplements the nutritional value of the feed and/or improves the immune defense of the recipient. In some embodiments, the feed additive is not for administration to a human.

[0031] A “premix,” as referred to herein, may be a composition composed of micro-ingredients such as, but not limited to, one or more of vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other essential ingredients. Premixes are usually compositions suitable for blending into commercial rations.

[0032] The term “performance” as used herein may be determined by the feed efficiency and/or weight gain of the animal and/or by the feed conversion ratio and/or by the digestibility of a nutrient in a feed (e.g, amino acid digestibility or phosphorus digestibility) and/or digestible energy or metabolizable energy in a feed and/or by nitrogen retention and/or by animals’ ability to avoid the negative effects of diseases or by the immune response of the subject. Performance characteristics may include but are not limited to: body weight; weight gain; mass; body fat percentage; height; body fat distribution; growth; growth rate; milk production; mineral absorption; mineral excretion, mineral retention; bone density; bone strength; feed conversion rate (FCR); average daily feed intake (ADFI); Average daily gain (ADG) retention and/or a secretion of any one or more of copper, sodium, phosphorous, nitrogen and calcium; amino acid retention or absorption; mineralization, bone mineralization carcass yield and carcass quality.

[0033] As used herein, “improving one or more metrics in an animal” refers to improvements on measurements relevant to the growth and/or health of an animal (such as a domesticated bird, for example, a chicken), measured by one or more of the following parameters: average daily weight gain (ADG), overall weight, mortality, feed conversion (which includes both feed:gain and gain:feed), feed intake, intestinal health status, decreased feed conversion ratio (FCR), gut barrier integrity (such as crypt depth), reduced pathogen infection (such as C. perfringens infection), reduced disease (such as necrotic enteritis) and/or reduced pathogen shedding in feces. “An improvement in a metric” or “improved metric” as used herein, refers to an improvement in at least one of the parameters listed under the metrics in an animal definition. The improvement in one or more metrics may be in respect to a control in which the feed for feed additive composition used does not comprise chitin-glucan.

[0034] As used herein, the term “feed efficiency” refers to the amount of weight gain in an animal that occurs when the animal is fed ad-libitum or a specified amount of food during a period of time. By “increased feed efficiency” it is meant that the use of a feed additive composition according the present invention in feed results in an increased weight gain per unit of feed intake compared with an animal fed without said feed additive composition being present.

[0035] As used herein, “feed conversion ratio” refers to a measure of a subject's efficiency in converting feed mass into increases of a desired output and is calculated by dividing the mass of the food eaten by the output for a specified period. For example, if an animal is raised for meat (e.g, beef), the output may be the mass gained by the animal. If an animal is raised for another intended purpose (e.g, milk production), the output will be different. The term “feed conversion ratio” may be used interchangeably with the terms “feed conversion rate” or “feed conversion efficiency.” By “lower feed conversion ratio” or “improved feed conversion ratio” it is meant that the use of a feed additive composition in feed results in a lower amount of feed being required to be fed to an animal to increase the weight of the animal by a specified amount compared to the amount of feed required to increase the weight of the animal by the same amount when the feed does not comprise said feed additive composition.

[0036] As used herein, "microorganism" or “microbe” refers to a bacterium, a fungus, a virus, a protozoan, and other microbes or microscopic organisms. [0037] The term “direct-fed microbial” (“DFM”) as used herein is source of live (viable) microorganisms that when applied in sufficient numbers can confer a benefit to the recipient thereof, i.e., a probiotic. A DFM can comprise one or more of such microorganisms such as bacterial strains. Categories of DFMs include Bacillus, Lactic Acid Bacteria and Yeasts. Thus, the term DFM encompasses one or more of the following: direct fed bacteria, direct fed yeast, direct fed yeast and combinations thereof. Bacilli are unique, gram-positive rods that form spores. These spores are very stable and can withstand environmental conditions such as heat, moisture and a range of pH. These spores germinate into active vegetative cells when ingested by an animal and can be used in meal and pelleted diets. Lactic Acid Bacteria are gram-positive cocci that produce lactic acid which are antagonistic to pathogens. Since Lactic Acid Bacteria appear to be somewhat heat-sensitive, they are not used in pelleted diets. Types of Lactic Acid Bacteria include Bifidobacterium, Lactobacillus and Streptococcus.

[0038] The terms “probiotic,” “probiotic culture,” and “DFM” are used interchangeably herein and define live microorganisms (including bacteria or yeasts, for example) which, when for example ingested or locally applied in sufficient numbers, beneficially affects the host organism, i.e. by conferring one or more demonstrable health benefits on the host organism such as a health, digestive, and/or performance benefit. Probiotics may improve the microbial balance in one or more mucosal surfaces. For example, the mucosal surface may be the intestine, the urinary tract, the respiratory tract or the skin. The term “probiotic” as used herein also encompasses live microorganisms that can stimulate the beneficial branches of the immune system and at the same time decrease the inflammatory reactions in a mucosal surface, for example the gut. Whilst there are no lower or upper limits for probiotic intake, it has been suggested that at least 10 6 -10 12 , for example at least 10 6 -10 10 , for example 10 8 -10 9 , cfu as a daily dose will be effective to achieve the beneficial health effects in a subject.

[0039] The term “CFU” as used herein means “colony forming units” and is a measure of viable cells in which a colony represents an aggregate of cells derived from a single progenitor cell.

[0040] As used herein the term “betaine” refers to trimethylglycine. The compound is also called trimethylammonioacetate, 1 -carboxy-N,N,N-trimethylmethaneaminium, inner salt and glycine betaine. It is a naturally occurring quaternary ammonium type compound having the formula

[0041] Betaine has a bipolar structure comprising a hydrophilic moiety (COO-) and a hydrophobic moiety (N+) capable of neutralizing both acid and alkaline solutions. In its pure form, betaine is a white crystalline compound that is readily soluble in water and lower alcohols. In the present invention betaine can be used, for example, as an anhydrous form, or as a hydrate or as an animal feed acceptable salt. In one embodiment, when betaine is present, it is present as the free zwitterion. In one embodiment, when betaine is present, it is present as anhydrous betaine. In one embodiment, when betaine is present, it is present as a monohydrate.

[0042] As used herein an “animal feed acceptable salt” means any non-toxic salt that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound or a derivative of a compound described herein. Acids commonly employed to form acceptable salts include inorganic acids such as hydrogen bisulfide, hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid, as well as organic acids such as para-toluenesulfonic, salicylic, tartaric, bitartaric, ascorbic, maleic, besylic, fumaric, gluconic, glucuronic, formic, glutamic, methanesulfonic, ethanesulfonic, benzenesulfonic, lactic, oxalic, para-bromophenylsulfonic, carbonic, succinic, citric, benzoic and acetic acid, and related inorganic and organic acids. Such animal feed acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fiimarate, maleate, butyne-l,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, di nitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephathalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, [beta]-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1 -sulfonate, naphthalene-2-sulfonate, mandelate and the like salts. Preferred animal feed acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid. Suitable cations for forming feed acceptable salts include ammonium, sodium, potassium, calcium, magnesium and aluminum cations, among others.

[0043] As used herein, “essential oil” refers to the set of all the compounds that can be distilled or extracted from a plant from which the oil is derived and that contributes to the characteristic aroma of that plant. See e.g., H. McGee, On Food and Cooking, Charles Scribner's Sons, p. 154- 157 (1984). Non-limiting examples of essential oils include thymol and cinnamaldehyde.

[0044] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number can be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. For example, in connection with a numerical value, the term “about” refers to a range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in context.

[0045] As used herein, the singular terms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise.

[0046] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.

[0047] It is also noted that the term “consisting essentially of” as used herein refers to a composition wherein the component(s) after the term is in the presence of other known component(s) in a total amount that is less than 30% by weight of the total composition and do not contribute to or interferes with the actions or activities of the component(s).

[0048] It is further noted that the term "comprising,” as used herein, means including, but not limited to, the component(s) after the term “comprising.” The component(s) after the term “comprising” are required or mandatory, but the composition comprising the component(s) can further include other non-mandatory or optional component(s).

[0049] It is also noted that the term “consisting of,” as used herein, means including, and limited to, the component(s) after the term "consisting of.” The component(s) after the term “consisting of’ are therefore required or mandatory, and no other components) are present in the composition.

[0050] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0051] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0052] Other definitions of terms may appear throughout the specification.

P. Compositions

A. Chitin-glucan

[0053] Provided herein are premix, feed, and feed additive compositions comprising chitin- glucan. A chitin-glucan copolymer can be advantageously obtained according to the process described in International patent application publication no. WO 03/068824 and French patent application FR 0507066, the disclosures of each of which is incorporated by reference. This process is described in particular in application FR 0507066 on page 18, line 14 et seq. In some embodiments, Aspergillus niger is used as fungal source in this process.

[0054] The D-glucose-unit linkage and the proportion between the alpha(l,6)-chitin and beta- glucan chains can depend on the fungus and on the strain. For example, it has been shown that an Aspergillus niger mycelium contains the chitin-glucan copolymer with a ratio by mass of chitin to beta-glucan of between 30:70 and 60:40, with a D-glucose-unit linkage mainly of the beta(l,3) type (U.S. patent application publication no. 20100003292, incorporated by reference herein in its entirety).

[0055] The chitin-glucan copolymer is generally in the form of a white powder. It is essentially insoluble in aqueous and organic solvents irrespective of the temperature and the pH. It is hygroscopic, being generally capable of absorbing approximately 10 times its mass in water. This chitin-glucan powder can, for example, be produced by means of industrial processes in such a way as to obtain a product with a fine particle size according to the present invention.

[0056] The chitin-glucan co-polymer suitable for use in the compositions and methods disclosed herein can be purified, from a fungal source, such as from the mycelium of Ascomycete-type fungi (e.g. Aspergillus niger). The hydrolysates of the purified extracts, i.e. the copolymers of chitin and beta-glucan of lower molecular mass, can also be used in the feed or feed additive compositions disclosed herein. The terms “chitin-glucan copolymer” or “chitin-glucan” also can, in some embodiments, include all the compounds obtained starting from chitin-glucan, by physical or chemical modification of the copolymer, according to a physical, chemical or enzymatic process, insofar as the properties of the chitin-glucan copolymer remain equivalent for the applications envisaged and insofar as the copolymers are insoluble in water and/or soluble in an organic solvent.

[0057] The availability and the quality in particular of Aspergillus niger, which is a co-product of the industrial production of citric acid for the food and pharmaceutical industry, make it an ideal starting material of choice for uses in the animal feed industry. Other fungal sources containing the chitin and beta-glucan polysaccharides can also be used, for instance Basidiomycetes , in particular the fungi Lentimla edodes (shiitake) and Agaricus bisporus.

[0058] As used herein, the phrase “polysaccharides of fimgal origin,” refers to the purified extracts of fungal cell walls composed predominantly of chitin and beta-glucan polysaccharides, in the form of copolymers, and derivatives thereof. The purified extracts can comprise a chitin- glucan content of greater than 70% by mass relative to the total mass of the extract, such as greater than 80%, such as greater than 85% and such as greater than 90%. [0059] It is generally accepted that fungal cell wall polysaccharides can be separated into two groups according to their solubility in an alkaline medium, and that the cell wall backbone is insoluble. It is also known that the insoluble fraction consists of chitin and of beta-glucan polymers, in variable proportions depending on the species, that the beta-glucan units are linked by linkages of variable structure, and that the bond between the chitin and beta-glucan links is stable, as shown, for example, by Siestma & Wessels for Saccharomyces cerevisiae Zygomycete), Neurospora crassa (Ascomycete), Aspergillus nidulcms (Ascomycete) and Coprims cinereus (Basidiomycete) (Siestma J H & Wessels J G. (1981), J. Gen. Microbiol. 125:209, incorporated herein by reference). It is known that the chitin and beta-glucan links of the insoluble fraction of Aspergillus niger are linked to one another covalently, as mentioned, for example, by Stagg C M and Feather M S ( Biochim . Biophys. (1973) Acta 320:64, incorporated herein by reference). Methods for determining the nature of the covalent linkage between the chitin and beta-glucan links have been described, for example, by Fontaine et al., for Aspergillus fumigatus (Fontaine T et al., (2000), J. Bio. Chem. 275:27594, incorporated herein by reference), and by Kollar et al., for the yeast Saccharomyces cerevisiae (Kollar R, et al. (1995), J. Biol. Chem. 270: 1170, incorporated herein by reference).

[0060] The fungal extract according to some embodiments of the present invention can be obtained from the mycelium cell wall of fungi of various groups, including the Zygomycete group, the Basidiomycete group, the Ascomycete group (of which Aspergillus niger is part) and the Deuteromycete group, and/or a mixture thereof. Said source of fungi should be chosen so as to allow the extraction of a polysaccharide as defined above and hereinafter. There exist sources of fungi which comprise beta-glucans, but these units are soluble in water in particular, or comprise no or few chains of chitin structure, and therefore do not make it possible to obtain the polysaccharide used in the compositions and methods disclosed herein. The present invention covers all fungi that make it possible to obtain the chitin-glucan polymer defined in the present application.

[0061] The ratio of the chitin to the beta-glucan is between 95:5 and 5:95, such as between 70:30 and 10:90 (m/m). The chitin part of the chitin-glucan copolymer can be composed of at least 85% of N-acetyl-D-glucosamine units and at most 15% of D-glucosamine units, such as of at least 90% of N-acetyl-D-glucosamine units and at most 10% of D-glucosamine units. [0062] Commercially available sources of chitin-glucan can be obtained from Kitozyme, LLC (Herstal, Belgium).

B. Enzymes

[0063] In one embodiment, the disclosure relates chitin-glucan containing feed or feed additive compositions further containing at least one enzyme. Suitable enzymes for use in accordance with the methods disclosed herein include, without limitation, glucoamylases, xylanases, amylases, phytases, beta-glucanases, and proteases.

1. Glucoamylases

[0064] Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is an enzyme, which catalyzes the release of D-glucose from the non-reducing ends of starch or related oligo- and poly -saccharide molecules. Glucoamylases are produced by several filamentous fungi and yeast.

[0065] In one embodiment, provided herein are chitin-glucan-containing feed or feed additive compositions including one or more glucoamylase. The glucoamylase may be any commercially available glucoamylase. Suitably the glucoamylase may be an 1,4-alpha-D-glucan glucohydrolase (EC 3.2.1.3). All E.C. enzyme classifications referred to herein relate to the classifications provided in Enzyme Nomenclature — Recommendations (1992) of the nomenclature committee of the International Union of Biochemistry and Molecular Biology — ISBN 0-12-226164-3, which is incorporated herein

[0066] Glucoamylases have been used successfully in commercial applications for many years. Additionally, various mutations have been introduced in fungal glucoamylases, for example, Trichoderma reesei glucoamylase (TrGA), to enhance thermal stability and specific activity. See, e.g., WO 2008/045489; WO 2009/048487; WO 2009/048488; and U.S. Pat. No. 8,058,033. Glucoamylase activity can be assessed using any means known in the art, including those described in the Examples section, infra.

[0067] A glucoamylase may be derived from any suitable source, e.g., derived from a microorganism or a plant. Glucoamylases can be from fungal or bacterial origin, selected from the group consisting of Aspergillus glucoamylases, in for example, Aspergillus niger G1 or G2 glucoamylase (Boel et al., 1984, EMBO J. 3(5): 1097-1102), or variants thereof, such as those disclosed in WO 92/00381, WO 00/04136 and WO 01/04273 (from Novozymes, Denmark); the A. awamori glucoamylase disclosed in WO 84/02921, Aspergillus oryzae glucoamylase (Hata et al., 1991 ,Agric. Biol. Chem. 55(4): 941-949), or variants or fragments thereof. Other Aspergillus glucoamylase variants include variants with enhanced thermal stability: G137A and G139A (Chen et al., 1996, Prot. Eng. 9: 499-505); D257E and D293E/Q (Chen et al., 1995, Prot. Eng. 8: 575-582); N182 (Chen et al., 1994, Biochem. J. 301: 275-281); disulphide bonds, A246C (Fierobe et al., 1996, Biochemistry 35: 8698-8704; and introduction of Pro residues in positions A435 and S436 (Li et al., 1997, Protein Eng. 10: 1199-1204.

[0068] Other glucoamylases include Athelia rolfsii (previously denoted Corticium rolfsi) glucoamylase (see U.S. Pat. No. 4,727,026 and Nagasaka et al., 1998, Appl Microbiol. Biotechnol. 50: 323-330), Talaromyces glucoamylases, in particular derived from Talaromyces duponti, Talaromyces emersonii (WO 99/28448), Talaromyces leycettams (U.S. Pat. No. Re. 32,153), and Talaromyces thermophilus (U.S. Pat. No. 4,587,215).

[0069] Bacterial glucoamylases include glucoamylases from Clostridium , in particular C. thermoamylolyticum (EP 135138) and C. thermohydrosulfiiricum (WO86/01831), Trametes cingulata, Pachykytospora papyracea, and Leucopaxillus giganteus, all disclosed in WO 2006/069289; or Peniophora rufomarginata disclosed in WO2007/124285 or PCT/US2007/066618; or a mixture thereof. A hybrid glucoamylase may be used in the present invention. Examples of hybrid glucoamylases are disclosed in WO 2005/045018. Specific examples include the hybrid glucoamylase disclosed in Tables 1 and 4 of Example 1 (which hybrids are hereby incorporated by reference).

[0070] The glucoamylase may have a high degree of sequence identity to any of above mentioned glucoamylases, i.e., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature enzymes sequences mentioned above.

[0071] Commercially available glucoamylase compositions include AMG 200L; AMG 300L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL, SPIRIZYME™ B4U, SPIRIZYME ULTRA, SPIRIZYME™ EXCEL and AMG™ E (from Novozymes A/S, Denmark); OPTIDEX™ 300, GC480™ and GC147™ (from Genencor Int, USA); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™ and G990 ZR (from Genencor Int.).

2. Xylanases

[0072] Xylanase is the name given to a class of enzymes that degrade the linear polysaccharide b-l,4-xylan into xylose, thus breaking down hemicellulose, one of the major components of plant cell walls. Xylanases, e.g., endo-P-xylanases (EC 3.2.1.8) hydrolyze the xylan backbone chain. In one embodiment, provided herein are chitin-glucan-containing feed or feed additive compositions comprising and one or more xylanase.

[0073] In another embodiment, provided herein are feed or feed additive compositions including one or more xylanase. In one embodiment, the xylanase may be any commercially available xylanase. Suitably the xylanase may be an endo-l,4-P-d-xylanase (classified as E.G. 3.2.1.8) or a ILb-xylosidase (classified as E.G. 3.2.1.37). All E.C. enzyme classifications referred to herein relate to the classifications provided in Enzyme Nomenclature — Recommendations (1992) of the nomenclature committee of the International Union of Biochemistry and Molecular Biology — ISBN 0-12-226164-3, which is incorporated herein

[0074] In another embodiment, the xylanase may be a xylanase from Bacillus, Trichodermna, Therinomyces, Aspergillus and Penicillium. In still another embodiment, the xylanase may be the xylanase in Axtra XAP® or Avizyme 1502®, both commercially available products from Danisco A/S. In one embodiment, the xylanase may be a mixture of two or more xylanases. In still another embodiment, the xylanase is an endo-l,4-P-xylanase or a 1,4-P-xylosidase. In yet another embodiment, the xylanase is from an organism selected from the group consisting of: Bacillus, Trichoderma, Thermomyces, Aspergillus, Penicillium, and Humicola. In yet another embodiment, the xylanase may be one or more of the xylanases or one or more of the commercial products recited in Table 13.

Table 13: Representative commercial xylanases [0075] In one embodiment, the disclosure relates to a feed or feed additive composition comprising one or more xylanase. In one embodiment, the composition comprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, and greater than 750 xylanase units/g of composition.

[0076] In one embodiment, the composition comprises 500-1000, 1000-1500, 1500-2000, 2000- 2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000- 6500, 6500-7000, 7000-7500, 7500-8000, and greater than 8000 xylanase units/g composition.

[0077] It will be understood that one xylanase unit (XU) is the amount of enzyme that releases 0.5 |imol of reducing sugar equivalents (as xylose by the Dinitrosalicylic acid (DNS) assay- reducing sugar method) from an oat-spelt-xylan substrate per min at pH 5.3 and 50° C. (Bailey, et al., Journal of Biotechnology, Volume 23, (3), May 1992, 257-270).

3. Amylases

[0078] Amylase is a class of enzymes capable of hydrolysing starch to shorter-chain oligosaccharides, such as maltose. The glucose moiety can then be more easily transferred from maltose to a monoglyceride or glycosylmonoglyceride than from the original starch molecule. The term amylase includes a-amylases (KG. 3.2.1.1), G4-forming amylases (E.G. 3.2.1.60), b- amylases (E.G. 3.2.1.2) and g-amylases (E.C. 3.2.1.3). Amylases may be of bacterial or fimgal origin, or chemically modified or protein engineered mutants. In another embodiment, provided herein are chitin-glucan-containing feed or feed additive compositions including one or more amylase.

[0079] In one embodiment, the amylase may be a mixture of two or more amylases. In another embodiment, the amylase may be an amylase, e.g. an a-amylase, from Bacillus licheniformis and an amylase, e.g. an a-amylase, from Bacillus amyloliquefaciens. In one embodiment, the a- amylase may be the a-amylase in Axtra XAP® or Avizyme 1502®, both commercially available products from Danisco A/S. In yet another embodiment, the amylase may be a pepsin resistant a-amylase, such as a pepsin resistant Trichoderma (such as Trichoderma reesei) alpha amylase. A suitably pepsin resistant a-amylase is taught in UK application number 101 1513.7 (which is incorporated herein by reference) and PCT/IB2011/053018 (which is incorporated herein by reference).

[0080] In one embodiment, the amylase for use in the present invention may be one or more of the amylases in one or more of the commercial products recited in Table 14.

Table 14: Representative commercial amylases

[0081] II will be understood that one amylase unit (AU) is the amount of enzyme that releases 1 mmol of glucosidic linkages from a water insoluble cross-linked starch polymer substrate per min at pH 6.5 and 37° C. (this may be referred to herein as the assay for determining 1 AU).

[0082] In one embodiment, the disclosure relates to a feed or feed additive composition comprising one or more amylase. In one embodiment, the composition comprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, and greater than 750 amylase units/g composition.

[0083] In one embodiment, the composition comprises 500-1000, 1000-1500, 1500-2000, 2000- 2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000- 6500, 6500-7000, 7000-7500, 7500-8000, 8000-8500, 8500-9000, 9000-9500, 9500-10000, 10000-11000, 11000-12000, 12000-13000, 13000-14000, 14000-15000 and greater than 15000 amylase units/g composition.

4. Proteases

[0084] The term protease as used herein is synonymous with peptidase or proteinase. The protease may be a subtilisin (E.G. 3.4.21.62) or a bacillolysin (E.G. 3.4.24.28) or an alkaline serine protease (E.G. 3.4.21.x) or a keratinase (E.G. 3.4.X.X). In one embodiment, the protease is a subtilisin. Suitable proteases include those of animal, vegetable or microbial origin. Chemically modified or protein engineered mutants are also suitable. The protease may be a serine protease or a metalloprotease. e.g., an alkaline microbial protease or a trypsin-like protease. In another embodiment, provided herein are chitin-glucan-containing feed or feed additive compositions including one or more protease.

[0085] Examples of alkaline proteases are subtilisins, especially those derived from Bacillus sp., e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309 (see, e.g., U.S. Pat. No. 6,287,841), subtilisin 147, and subtilisin 168 (see, e.g., WO 89/06279). Examples of trypsin-like proteases are trypsin {e.g, of porcine or bovine origin), and Fusarium proteases (see, e.g, WO 89/06270 and WO 94/25583). Examples of useful proteases also include but are not limited to the variants described in WO 92/19729 and WO 98/20115.

[0086] In another embodiment, the protease may be one or more of the proteases in one or more of the commercial products recited in Table 15.

Table 15: Representative commercial proteases

[0087] In one embodiment, the protease is selected from the group consisting of subtilisin, a bacillolysin, an alkine serine protease, a keratinase, and a Nocardiopsis protease.

[0088] It will be understood that one protease unit (PU) is the amount of enzyme that liberates from the substrate (0.6% casein solution) one microgram of phenolic compound (expressed as tyrosine equivalents) in one minute at pH 7.5 (40 mM NazPOVlactic acid buffer) and 40° C. This may be referred to as the assay for determining 1 PU.

[0089] In one embodiment, the disclosure relates to a feed or feed additive composition comprising one or more protease. In another embodiment, the disclosure relates to a feed or feed additive composition comprising one or more xylanase and protease. In still another embodiment, the disclosure relates to a feed or feed additive composition comprising one or more amylase and protease. In yet another embodiment, the disclosure relates to a feed or feed additive composition comprising one or more xylanase, amylase and protease.

[0090] In one embodiment, the composition comprises 10-50, 50-100, 100-150, 150-200, 200- 250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700- 750, and greater than 750 protease units/g composition. [0091] In one embodiment, the composition comprises 500-1000, 1000-1500, 1500-2000, 2000- 2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000- 6500, 6500-7000, 7000-7500, 7500-8000, 8000-8500, 8500-9000, 9000-9500, 9500-10000, 10000-11000, 11000-12000, 12000-13000, 13000-14000, 14000-15000 and greater than 15000 protease units/g composition.

5. Phytases

[0092] In another embodiment, provided herein are chitin-glucan-containing feed or feed additive compositions including one or more phytase. The phytase for use in the present invention may be classified a 6-phytase (classified as E.C. 3.1.3.26) or a 3-phytase (classified as E.C. 3.1.3.8). In one embodiment, the phytase for use in the present invention may be one or more of the phytases in one or more of the commercial products below in Table 16:

Table 16: Representative commercial phytases [0093] In one embodiment the phytase is a Citrobacter phytase derived from e.g. Citrobacter freundii , In some embodiments, C. freundii NCIMB 41247 and variants thereof e.g. as disclosed in W02006/038062 (incorporated herein by reference) and W02006/038128 (incorporated herein by reference), Citrobacter braakii YH-15 as disclosed in WO 2004/085638, Citrobacter braakii ATCC 51113 as disclosed in W02006/037328 (incorporated herein by reference), as well as variants thereof e.g. as disclosed in W02007/112739 (incorporated herein by reference) and WO2011/117396 (incorporated herein by reference), Citrobacter amalonaticus, In some embodiments, Citrobacter amalonaticus ATCC 25405 or Citrobacter amalonaticus ATCC 25407 as disclosed in W02006037327 (incorporated herein by referenced, Citrobacter gillenii,

In some embodiments, Citrobacter gillenii DSM 13694 as disclosed in W02006037327 (incorporated herein by reference), or Citrobacter intermedius, Citrobacter koseri, Citrobacter murliniae, Citrobacter rodentium, Citrobacter sedlakii, Citrobacter werkmanii, Citrobacter youngae, Citrobacter species polypeptides or variants thereof.

[0094] In some embodiments, the phytase is an E. coli phytase marketed under the name

Phyzyme XP™ Danisco A/S. Alternatively, the phytase may be a Buttiauxella phytase, e.g. a Buttiauxella agrestis phytase, for example, the phytase enzymes taught in WO 2006/043178, WO 2008/097619, WO2009/129489, W02008/092901, PCT/US2009/41011 or

PCT/IB2010/051804, all of which are incorporated herein by reference.

[0095] In one embodiment, the phytase may be a phytase from Hqfhia, e.g. from Hqfhia alvei, such as the phytase enzyme(s) taught in US2008263688, which reference is incorporated herein by reference. In one embodiment, the phytase may be a phytase from Aspergillus, e.g. from Apergillus orzyae. In one embodiment, the phytase may be a phytase from Penicillium, e.g. from Penicillium fimiculosum.

[0096] In some embodiments, the phytase is present in the feed or feed-additive compositions in range of about 200 FTU/kg to about 1000 FTU/kg feed. In some embodiments, about 300 FTU/kg feed to about 750 FTU/kg feed. In some embodiments, about 400 FTU/kg feed to about 500 FTU/kg feed. In one embodiment, the phytase is present in the feedstuff at more than about 200 FTU/kg feed, suitably more than about 300 FTU/kg feed, suitably more than about 400 FTU/kg feed. In one embodiment, the phytase is present in the feedstuff at less than about 1000 FTU/kg feed, suitably less than about 750 FTU/kg feed. In some embodiments, the phytase is present in the feed additive composition in range of about 40 FTU/g to about 40,000 FTU/g composition; about 80 FTU/g composition to about 20,000 FTU/g composition; about 100 FTU/g composition to about 10,000 FTU/g composition; and about 200 FTU/g composition to about 10,000 FTU/g composition. In one embodiment, the phytase is present in the feed additive composition at more than about 40 FTU/g composition, suitably more than about 60 FTU/g composition, suitably more than about 100 FTU/g composition, suitably more than about 150 FTU/g composition, suitably more than about 200 FTU/g composition. In one embodiment, the phytase is present in the feed additive composition at less than about 40,000 FTU/g composition, suitably less than about 20,000 FTU/g composition, suitably less than about 15,000 FTU/g composition, suitably less than about 10,000 FTU/g composition.

[0097] It will be understood that as used herein 1 FTU (phytase unit) is defined as the amount of enzyme required to release 1 mmiol of inorganic orthophosphate from a substrate in one minute under the reaction conditions defined in the ISO 2009 phytase assay — A standard assay for determining phytase activity and 1 FTU can be found at International Standard ISO/DIS 30024: 1-17, 2009. In one embodiment, the enzyme is classified using the E.C. classification above, and the E.C. classification designates an enzyme having that activity when tested in the assay taught herein for determining 1 FTU.

C. Direct Fed Microbials (DFMs)

[0098] In one embodiment, a DFM can be included in the chitin-glucan-containing formulations disclosed herein and, optionally, may be formulated as a liquid, a dry powder or a granule. In one embodiment, the DFMs and chitin-glucan can be formulated as a single mixture. In another embodiment, the DFMs and chitin-glucan can be formulated as separate mixtures. In still another embodiment, separate mixtures of DFMs and chitin-glucan can be administered at the same time or at different times. In still another embodiment, separate mixtures of DFMs and chitin-glucan can be administered simultaneously or sequentially. In yet another embodiment, a first mixture comprising DFMs can be administered followed by a second mixture comprising chitin-glucan. In still another embodiment, a first mixture comprising chitin-glucan can be administered followed by a second mixture comprising DFMs.

[0099] The dry powder or granules may be prepared by means known to those skilled in the art, such as, in top-spray fluid bed coater, in a buttom spray Wurster or by drum granulation (e.g. High sheer granulation), extrusion, pan coating or in a microingredients mixer.

[0100] In another embodiment, the chitin-glucan, enzymes, and/or DFMs may be coated, for example encapsulated. Suitably the chitin-glucan, enzymes, and/or DFMs may be formulated within the same coating or encapsulated within the same capsule. Alternatively, one or more of the enzymes and/or DFMs may be formulated within the same coating or encapsulated within the same capsule while the chitin-glucan can be formulated in a separate coating.

[0101] In some embodiments, such as where the DFM is capable of producing endospores, the DFM may be provided without any coating. In such circumstances, the DFM endospores may be simply admixed with the chitin-glucan co-polymer. The chitin-glucan, enzymes, and/or DFMs may be encapsulated as mixtures (i.e. comprising one or more, two or more, three or more or all) or they may be encapsulated separately, e.g. singly.

[0102] At least one DFM may comprise at least one viable microorganism such as a viable bacterial strain or a viable yeast or a viable fungi. In some embodiments, the DFM comprises at least one viable bacteria. It is possible that the DFM may be a spore forming bacterial strain and hence the term DFM may be comprised of or contain spores, e.g. bacterial spores. Thus, the term “viable microorganism” as used herein may include microbial spores, such as endospores or conidia. Alternatively, the DFM in the feed additive composition described herein may not comprise of or may not contain microbial spores, e.g. endospores or conidia. The microorganism may be a naturally-occurring microorganism or it may be a transformed microorganism.

[0103] A DFM as described herein may comprise microorganisms from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus , Bacillus, Pediococcus, Enterococcus, Leuconostoc, Camobacterium, Propionibacterium, Bifidobacterium, Clostridium and Megasphaera and combinations thereof. In some embodiments, the DFM comprises one or more bacterial strains selected from the following Bacillus spp: Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus pumilis and Bacillus amyloliquefaciens.

[0104] The genus ‘Bacillus”, as used herein, includes all species within the genus “Bacillus,” as known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis,

B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciem, B. clausii,

B. halodurans, B. megaterium, B. coagulans, B. circulans, B. gibsonii, B. pumilis and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as Bacillus stearothermophilus, which is now named “ Geobacillus stearothermophilus, or Bacillus pofymyxa, which is now “ Paenibacillus potymyxa" The production of resistant endospores under stressful environmental conditions is considered the defining feature of the genus Bacillus, although this characteristic also applies to the recently named Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.

[0105] In another aspect, the DFM may be further combined with the following Lactococcus spp: Lactococcus cremoris and Lactococcus lactis and combinations thereof. The DFM may be further combined with the following Lactobacillus spp: Lactobacillus buchneri, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus kefiri, Lactobacillus bifldus, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus bulgaricus, Lactobacillus sakei, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus farciminis, Lactobacillus lactis, Lactobacillus delbreuckii, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus farciminis, Lactobacillus rhamnosus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii and Lactobacillus jensenii, and combinations of any thereof.

[0106] In still another aspect, the DFM may be further combined with the following Bifidobacteria spp: Bifidobacterium lactis, Bifidobacterium bifidium, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium adolescentis, and Bifidobacterium angulatum, and combinations of any thereof.

[0107] There can be mentioned bacteria of the following species: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus pumilis, Enterococcus , Enterococcus spp, and Pediococcus spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus lactis, Bifidobacterium bifidum, Bacillus subtilis, Propionibacterium thoenii, Lactobacillus farciminis, Lactobacillus rhamnosus, Megasphaera elsdenii, Clostridium butyricum, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Bacillus cereus, Lactobacillus salivarius ssp. Salivarius, Propionibacteria sp and combinations thereof.

[0108] A direct-fed microbial described herein comprising one or more bacterial strains may be of the same type (genus, species and strain) or may comprise a mixture of genera, species and/or strains. Alternatively, a DFM may be combined with one or more of the products or the microorganisms contained in those products disclosed in WO2012110778 and summarized as follows: Bacillus subtilis strain 2084 Accession No. NRRLB-50013, Bacillus subtilis strain LSSAOl Accession No. NRRL B-50104, and Bacillus subtilis strain 15A-P4 ATCC Accession No. PTA-6507 (from Enviva Pro® (formerly known as Avicorr®); Bacillus subtilis Strain C3102 (from Calsporin®); Bacillus subtilis Strain PB6 (from Clostat®); Bacillus pumilis (8G- 134); Enterococcus NCIMB 10415 (SF68) (from Cylactin®); Bacillus subtilis Strain C3102 (from Gallipro® & GalliproMax®); Bacillus licheniformis (from Gallipro®Tect®); Enterococcus and Pediococcus (from Poultry star®); Lactobacillus, Bifidobacterium and/or Enterococcus from Protexin®); Bacillus subtilis strain QST 713 (from Proflora®); Bacillus amyloliquefaciens CECT-5940 (from Ecobiol® & Ecobiol® Plus); Enterococcus faecium SF68 (from Fortiflora®); Bacillus subtilis and Bacillus licheniformis (from BioPlus2B®); Lactic acid bacteria 7 Enterococcus faecium (from Lactiferm®); Bacillus strain (from CSI®); Saccharomyces cerevisiae (from Yea-Sacc®); Enterococcus (from Biomin IMB52®); Pediococcus acidilactici, Enterococcus, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Lactobacillus salivarius ssp. salivarius (from Biomin C5®); Lactobacillus farciminis (from Biacton®); Enterococcus (from Oralin E1707®); Enterococcus (2 strains), Lactococcus lactis DSM 1103 (from Probios-pioneer PDFM®); Lactobacillus rhamnosus and Lactobacillus farciminis (from Sorbiflore®); Bacillus subtilis (from Animavit®); Enterococcus (from Bonvital®); Saccharomyces cerevisiae (from Levucell SB 20®); Saccharomyces cerevisiae (from Levucell SC 0 & SC10® ME); Pediococcus acidilacti (from Bactocell); Saccharomyces cerevisiae (from ActiSaf® (formerly BioSaf®)); Saccharomyces cerevisiae NCYC Sc47 (from Actisaf® SC47); Clostridium butyricum (from Miya-Gold®); Enterococcus (from Fecinor and Fecinor Plus®); Saccharomyces cerevisiae NCYC R-625 (from InteSwine®); Saccharomyces cerevisia (from BioSprint®); Enterococcus and Lactobacillus rhamnosus (from Provita®); Bacillus subtilis and Aspergillus oryzae (from PepSoyGen-C®); Bacillus cereus (from Toyocerin®); Bacillus cereus var. toyoi NCIMB 40112/CNCM 1-1012 (from TOYOCERIN®), or other DFMs such as Bacillus licheniformis and Bacillus subtilis (from BioPlus® YC) and Bacillus subtilis (from GalliPro®).

[0109] The DFM may be combined with Enviva® PRO which is commercially available from Danisco A/S. Enviva Pro® is a combination of Bacillus strain 2084 Accession No. NRRL B-50013, Bacillus strain LSSAOl Accession No. NRRL B-50104 and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507 (as taught in US 7,754,469 B - incorporated herein by reference). Preferably, the DFM described herein comprises microorganisms which are generally recognized as safe (GRAS) and, preferably are GRAS-approved. A person of ordinary skill in the art will readily be aware of specific species and/or strains of microorganisms from within the genera described herein which are used in the food and/or agricultural industries and which are generally considered suitable for animal consumption.

[0110] In some embodiments, it is important that the DFM be heat tolerant, i.e. is thermotolerant. This is particularly the case when the feed is pelleted. Therefore, in another embodiment, the DFM may be a thermotolerant microorganism, such as a thermotolerant bacteria-,including for example Bacillus spp.

[0111] In other aspects, it may be desirable that the DFM comprises a spore producing bacteria, such as Bacilli, e.g. Bacillus spp. Bacilli are able to form stable endospores when conditions for growth are unfavorable and are very resistant to heat, pH, moisture and disinfectants.

[0112] The DFM described herein may decrease or prevent intestinal establishment of pathogenic microorganism (such as Clostridium perfringens and/or E coli and/or Salmonella spp and/or Campylobacter spp.). In other words, the DFM may be antipathogenic. The term “antipathogenic” as used herein means the DFM counters an effect (negative effect) of a pathogen.

[0113] As described above, the DFM may be any suitable DFM. For example, the following assay “DFM ASSAY” may be used to determine the suitability of a microorganism to be a DFM. The DFM assay as used herein is explained in more detail in US2009/0280090. For avoidance of doubt, the DFM selected as an inhibitory strain (or an antipathogenic DFM) in accordance with the “DFM ASSAY” taught herein is a suitable DFM for use in accordance with the present disclosure, i.e. in the feed additive composition according to the present disclosure. Tubes were seeded each with a representative pathogen (e.g., bacteria) from a representative cluster. Supernatant from a potential DFM, grown aerobically or anaerobically, is added to the seeded tubes (except for the control to which no supernatant is added) and incubated. After incubation, the optical density (OD) of the control and supernatant treated tubes was measured for each pathogen. Colonies of (potential DFM) strains that produced a lowered OD compared with the control (which did not contain any supernatant) can then be classified as an inhibitory strain (or an antipathogenic DFM). Thus, The DFM assay as used herein is explained in more detail in US2009/0280090. In some embodiments, a representative pathogen used in this DFM assay can be one (or more) of the following: Clostridium, such as Clostridium perfringens and/or Clostridium difficile, and/or E coli and/or Salmonella spp and/or Campylobacter spp. In one preferred embodiment, the assay is conducted with one or more of Clostridium perfringens and/or Clostridium difficile and/or E. coli, preferably Clostridium perfringens and/or Clostridium difficile, more preferably Clostridium perfringens.

[0114] Antipathogenic DFMs include one or more of the following bacteria and are described in W02013029013.: Bacillus subtilis strain 3BP5 Accession No. NRRL B-50510,

Bacillus amyloliquefaciens strain 918 ATCC Accession No. NRRL B-50508, and Bacillus amyloliquefaciens strain 1013 ATCC Accession No. NRRL B-50509.

[0115] DFMs may be prepared as culture(s) and carriers) (where used) and can be added to a ribbon or paddle mixer and mixed for about 15 minutes, although the timing can be increased or decreased. The components are blended such that a uniform mixture of the cultures and carriers result. The final product is preferably a dry, flowable powder. The DFM(s) comprising one or more bacterial strains can then be added to animal feed or a feed premix, added to an animal's water, or administered in other ways known in the art (preferably simultaneously with the enzymes described herein. Inclusion of the individual strains in the DFM mixture can be in proportions varying from 1% to 99% and, preferably, from 25% to 75% Suitable dosages of the DFM in animal feed may range from about lxl 0 3 CFU/g feed to about lxl 0 10 CFU/g feed, suitably between about lxl 0 4 CFU/g feed to about lxl 0 8 CFU/g feed, suitably between about 7.5xl0 4 CFU/g feed to about 1x10 7 CFU/g feed. In another aspect, the DFM may be dosed in feedstuff at more than about 1x10 3 CFU/g feed, suitably more than about 1x10 4 CFU/g feed, suitably more than about 5x10 4 CFU/g feed, or suitably more than about 1x10 5 CFU/g feed.

[0116] The DFM may be dosed in a feed additive composition from about lxl 0 3 CFU/g composition to about 1x10 13 CFU/g composition, such as 1x10 5 CFU/g composition to about 1x10 13 CFU/g composition, such as between about 1x10 6 CFU/g composition to about 1x10 12 CFU/g composition, and such as between about 3.75x10 7 CFU/g composition to about 1x10 11 CFU/g composition. In another aspect, the DFM may be dosed in a feed additive composition at more than about 1x10 5 CFU/g composition, such as more than about 1x10 6 CFU/g composition, and such as more than about 3.75xl0 7 CFU/g composition. In one embodiment, the DFM is dosed in the feed additive composition at more than about 2x10 s CFU/g composition, such as more than about 2x10 6 CFU/g composition, suitably more than about 3.75xl0 7 CFU/g composition.

D. Feed Additive Compositions

[0117] In one embodiment, provided herein are feed additive compositions comprising chitin-glucan co-polymers disclosed herein. The chitin-glucan can comprise at least about 0.01% to 1% (such as any of about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, or 1%, inclusive of all percentages falling in between these values) of the feed additive composition.

[0118] In one embodiment, the feed additive composition may be used in the form of solid or liquid preparations or alternatives thereof. Examples of solid preparations include powders, pastes, boluses, capsules, ovules, pills, pellets, tablets, dusts, and granules which may be wettable, spray-dried or freeze-dried. Examples of liquid preparations include, but are not limited to, aqueous, organic or aqueous-organic solutions, suspensions and emulsions.

[0119] In another embodiment, the feed additive composition can be used in a solid form. In one embodiment, the solid form is a pelleted form. In solid form, the feed additive composition may also contain one or more of: excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine; disintegrants such as starch (In some embodiments, com, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates; granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia; lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

[0120] Examples of nutritionally acceptable carriers for use in preparing the forms include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

[0121] In one embodiment, the feed additive composition is formulated to a dry powder or granules as described in W02007/044968 (referred to as TPT granules) or WO 1997/016076 or WO 1992/012645 (each of which is incorporated herein by reference).

[0122] In one embodiment, the feed additive composition may be formulated to a granule feed composition comprising: one or more of chitin-glucan and/or enzymes disclosed herein. In one embodiment, the active agent of the granule retains activity after processing. In one embodiment, the active agent of the granule retains an activity level after processing selected from the group consisting of: 50-60% activity, 60-70% activity, 70-80% activity, 80-85% activity, 85-90% activity, and 90-95% activity.

[0123] In yet another embodiment, the granule may be produced using a feed pelleting process and the feed pretreatment process may be conducted between 70° C and 95° C for up to several minutes, such as between 85° C and 95° C. In another embodiment, the granule may be produced using a steam-heated pelleting process that may be conducted between 85° C and 95° C for up to several minutes.

[0124] In one embodiment, the granule may have a moisture barrier coating selected from polymers and gums and the moisture hydrating material may be an inorganic salt. The moisture hydrating coating may be between 25% and 45% w/w of the granule and the moisture barrier coating may be between 2% and 20% w/w of the granule.

[0125] In one embodiment, the active agent retains activity after conditions selected from one or more of: (a) a feed pelleting process; (b) a steam-heated feed pretreatment process; (c) storage; (d) storage as an ingredient in an unpelleted mixture; and (e) storage as an ingredient in a feed base mix or a feed premix comprising at least one compound selected from trace minerals, organic acids, reducing sugars, vitamins, choline chloride, and compounds which result in an acidic or a basic feed base mix or feed premix.

[0126] In some embodiments, the feed additive compositions may be diluted using a diluent, such as starch powder, lime stone or the like. In another embodiment, the feed additive composition may be formulated by applying, e.g. spraying, a substance (such as an enzyme, DFM, or chitin-glucan) onto a carrier substrate, such as ground wheat for example.

[0127] In one embodiment, the feed additive composition may be formulated as a premix. By way of example only, the premix may comprise one or more feed components, such as one or more minerals and/or one or more vitamins.

[0128] In one embodiment, the chitin-glucan-containing feed additive composition may be formulated with at least one physiologically acceptable carrier selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a wheat component, sucrose, starch, NaaSO^ Talc, PVA, sorbitol, benzoate, sorbiate, glycerol, sucrose, propylene glycol, 1,3- propane diol, glucose, parabens, sodium chloride, citrate, acetate, phosphate, calcium, metabisulfite, formate and mixtures thereof.

[0129] In another embodiment, the feed additive composition can be delivered as an aqueous suspension and/or an elixir. The feed additive composition may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, propylene glycol and glycerin, and combinations thereof.

E. Feedstuffs

[0130] In another embodiment, provided herein are feed additive compositions containing any of the chitin-glucan-containing compositions disclosed herein that may be used as a feed or in the preparation of a feed. The feed may be in the form of a solution or as a solid depending on the use and/or the mode of application and/or the mode of administration. When used as a feed or in the preparation of a feed, such as functional feed, the feed additive composition may be used in conjunction with one or more of the following: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, a nutritionally active ingredient. The chitin-glucan can comprise at least about 0.01% to 1% (such as any of about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, or 1%, inclusive of all percentages falling in between these values) of the feed.

[0131] In one embodiment, the feed additive composition disclosed herein is admixed with a feed component to form a feedstuff. In one embodiment, the feed may be a fodder, or a premix thereof, a compound feed, or a premix thereof. In one embodiment, the feed additive composition disclosed herein may be admixed with a compound feed, a compound feed component or a premix of a compound feed or to a fodder, a fodder component, or a premix of a fodder.

[0132] In one embodiment, fodder may be obtained from one or more of the plants selected from: alfalfa (lucerne), barley, birdsfoot trefoil, brassicas, Chau moellier, kale, rapeseed (canola), rutabaga (swede), turnip, clover, alsike clover, red clover, subterranean clover, white clover, grass, false oat grass, fescue, Bermuda grass, brome, heath grass, meadow grasses (from naturally mixed grassland swards, orchard grass, rye grass, Timothy-grass, com (maize), millet, oats, sorghum, soybeans, trees (pollard tree shoots for tree-hay), wheat, and legumes.

[0133] Compound feeds can be complete feeds that provide all the daily required nutrients, concentrates that provide a part of the ration (protein, energy) or supplements that only provide additional micronutrients, such as minerals and vitamins. The main ingredients used in compound feed are the feed grains, which include com, soybeans, sorghum, oats, and barley.

[0134] In one embodiment, a feedstuff as disclosed herein may comprise one or more feed materials selected from the group comprising cereals, such as small grains (e.g, wheat, barley, rye, oats and combinations thereof) and/or large grains such as maize or sorghum; by products from cereals, such as com gluten meal, Distillers Dried Grain Solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citms pulp; protein obtained from sources such as soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; oils and fats obtained from vegetable and animal sources; and minerals and vitamins.

[0135] In yet another embodiment, a feedstuff may comprise at least one high fiber feed material and/or at least one by-product of the at least one high fiber feed material to provide a high fiber feedstuff. Examples of high fiber feed materials include: wheat, barley, rye, oats, by products from cereals, such as com gluten meal, Distillers Dried Grain Solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp. Some protein sources may also be regarded as high fiber: protein obtained from sources such as sunflower, lupin, fava beans and cotton

[0136] In still another embodiment, the feed may be one or more of the following: a compound feed and premix, including pellets, nuts or (cattle) cake; a crop or crop residue: com, soybeans, sorghum, oats, barley, com stover, copra, straw, chaff, sugar beet waste; fish meal; freshly cut grass and other forage plants; meat and bone meal; molasses; oil cake and press cake; oligosaccharides; conserved forage plants: hay and silage; seaweed; seeds and grains, either whole or prepared by crushing, milling etc.; sprouted grains and legumes; yeast extract.

[0137] In one embodiment, the feed additive composition of disclosed herein is admixed with the product (e.g. feedstuff). Alternatively, the feed additive composition may be included in the emulsion or raw ingredients of a feedstuff. In another embodiment, the feed additive composition is made available on or to the surface of a product to be affected/treated. In still another embodiment, the feed additive compositions disclosed herein may be applied, interspersed, coated and/or impregnated to a product (e.g. feedstuff or raw ingredients of a feedstuff) with a controlled amount of chitin-glucan.

IP. Methods

A. Methods for Improving Performance Metrics in an Animal

[0138] Further provided herein are methods for increasing one or more performance metrics of an animal. In another embodiment, the disclosure relates to methods of increasing performance metrics of a bird. In still another embodiment, the disclosure relates to methods of increasing performance metrics of poultry, including but not limited to broilers, chickens and turkeys. In another embodiment, the disclosure relates to methods of increasing performance metrics of a pig.

[0139] In yet another embodiment, the disclosure relates to a method comprising administering to an animal a composition comprising chitin glucan and, optionally, exogenous feed enzymes, direct fed microbials (such as any of the DFMs described herein), essential oils, or betaine. In still another embodiment, the disclosure relates to a method comprising administering to an animal an effective amount of a composition comprising chitin-glucan and optional exogenous feed enzymes to increase performance of the animal. This effective amount can be administered to the animal in one or more doses. In one embodiment, the animal is poultry, such as a broiler. In still another embodiment, the animal is a pig, such as a piglet, growing pig, or sow.

[0140] In another embodiment, the disclosure relates to a method comprising administering to an animal (such as a domesticated bird or a pig) an effective amount of a composition comprising chitin-glucan (such as any of the chitin-glucan-containing feed or feed additive compositions disclosed herein) and optionally exogenous feed enzymes, direct fed microbials (such as any of the DFMs described herein), essential oils, and/or betaine to increase average daily feed intake. In some embodiments, the average daily feed intake increases by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, or 110%, inclusive of all values falling in between these percentages, relative to animals who are not administered one or more of the chitin-glucan-containing compositions disclosed herein. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is a feed or feedstuff. Chitin-glucan can comprise at least about 0.01% to 1% (such as any of about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, or 1%, inclusive of all percentages falling in between these values) of the feed additive composition, feed, or feedstuff.

[0141] In another embodiment, the disclosure relates to a method comprising administering to an animal (such as a domesticated bird or a pig) an effective amount of a composition comprising chitin-glucan (such as any of the chitin-glucan-containing feed or feed additive compositions disclosed herein) and optionally exogenous feed enzymes, direct fed microbials (such as any of the DFMs described herein), essential oils, and/or betaine to increase average daily weight gain. In some embodiments, the average daily weight gain increases by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, or 110%, inclusive of all values falling in between these percentages, relative to animals who are not administered one or more of the chitin-glucan-containing compositions disclosed herein. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is a feed or feedstuff. Chitin-glucan can comprise at least about 0.01% to 1% (such as any of about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, or 1%, inclusive of all percentages falling in between these values) of the feed additive composition, feed, or feedstuff.

[0142] In another embodiment, the disclosure relates to a method comprising administering to an animal (such as a domesticated bird or a pig) an effective amount of a composition comprising chitin-glucan (such as any of the chitin-glucan-containing feed or feed additive compositions disclosed herein) and optionally exogenous feed enzymes, direct fed microbials (such as any of the DFMs described herein), essential oils, and/or betaine to increase total weight gain. In some embodiments, total weight gain increases by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, or 110%,, inclusive of all values falling in between these percentages, relative to animals who are not administered one or more of the chitin-glucan-containing compositions disclosed herein. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is a feed or feedstuff. Chitin-glucan can comprise at least about 0.01% to 1% (such as any of about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, or 1%, inclusive of all percentages falling in between these values) of the feed additive composition, feed, or feedstuff.

[0143] In another embodiment, the disclosure relates to a method comprising administering to an animal (such as a domesticated bird or a pig) an effective amount of a composition comprising chitin-glucan (such as any of the chitin-glucan-containing feed or feed additive compositions disclosed herein) and optionally exogenous feed enzymes, direct fed microbials (such as any of the DFMs described herein), essential oils, and/or betaine to increase feed conversion, which can be measured by either feed:gain or gain:feed. In some embodiments, feed conversion increases by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, or 110%, inclusive of all values falling in between these percentages, relative to animals who are not administered one or more of the chitin-glucan-containing compositions disclosed herein. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is a feed or feedstuff. Chitin-glucan can comprise at least about 0.01% to 1% (such as any of about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, or 1%, inclusive of all percentages falling in between these values) of the feed additive composition, feed, or feedstuff.

[0144] In another embodiment, the disclosure relates to a method comprising administering to an animal (such as a domesticated bird or a pig) an effective amount of a composition comprising chitin-glucan (such as any of the chitin-glucan-containing feed or feed additive compositions disclosed herein) and optionally exogenous feed enzymes, direct fed microbials (such as any of the DFMs described herein), essential oils, and/or betaine to increase feed efficiency. In some embodiments, feed efficiency increases by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, or 110%, inclusive of all values falling in between these percentages, relative to animals who are not administered one or more of the chitin-glucan-containing compositions disclosed herein. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is a feed or feedstuff. Chitin-glucan can comprise at least about 0.01% to 1% (such as any of about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, or 1%, inclusive of all percentages falling in between these values) of the feed additive composition, feed, or feedstuff.

[0145] In another embodiment, the disclosure relates to a method comprising administering to an animal (such as a domesticated bird or a pig) an effective amount of a composition comprising chitin-glucan (such as any of the chitin-glucan-containing feed or feed additive compositions disclosed herein) and optionally exogenous feed enzymes, essential oils, and/or betaine to decrease mortality. In some embodiments, mortality decreases by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, inclusive of all values falling in between these percentages, relative to animals who are not administered one or more of the chitin-glucan-containing compositions disclosed herein. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is a feed or feedstuff.

[0146] In another embodiment, the disclosure relates to a method comprising administering to an animal (such as a domesticated bird or a pig) an effective amount of a composition comprising chitin-glucan (such as any of the chitin-glucan-containing feed or feed additive compositions disclosed herein) and optionally exogenous feed enzymes, direct fed microbials (such as any of the DFMs described herein), essential oils, and/or betaine to decrease feed conversion ratio (FCR). In some embodiments, FCR decreases by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, inclusive of all values falling in between these percentages, relative to animals who are not administered one or more of the chitin-glucan-containing compositions disclosed herein. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is a feed or feedstuff. Chitin-glucan can comprise at least about 0.01% to 1% (such as any of about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, or 1%, inclusive of all percentages falling in between these values) of the feed additive composition, feed, or feedstuff.

[0147] In another embodiment, the disclosure relates to a method comprising administering to an animal (such as a domesticated bird or a pig) an effective amount of a composition comprising chitin-glucan (such as any of the chitin-glucan-containing feed or feed additive compositions disclosed herein) and optionally exogenous feed enzymes, direct fed microbials (such as any of the DFMs described herein), essential oils, and/or betaine to improve one or more of gut barrier integrity, intestinal physiology, and/or intestinal morphology. In some embodiments, gut barrier integrity increases by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, or 110%, inclusive of all values falling in between these percentages, relative to animals who are not administered one or more of the chitin-glucan-containing compositions disclosed herein. “Gut barrier integrity” can refer to, without limitation, epithelial damage and epithelial permeability which is characterized by a shortening of villi, a lengthening of crypts and an infiltration of inflammatory cells (such as, without limitation, CD3+ cells). The latter damage and inflammation markers can also be associated with a “severe” macroscopic appearance of the gut - compared to a “normal” appearance - when evaluated using a scoring system such as the one described by Teirlynck et al. (2011). In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is a feed or feedstuff. Chitin-glucan can comprise at least about 0.01% to 1% (such as any of about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, or 1%, inclusive of all percentages falling in between these values) of the feed additive composition, feed, or feedstuff.

[0148] In another embodiment, the disclosure relates to a method comprising administering to an animal (such as a domesticated bird or a pig) an effective amount of a composition comprising chitin-glucan (such as any of the chitin-glucan-containing feed or feed additive compositions disclosed herein) and optionally exogenous feed enzymes, direct fed microbials (such as any of the DFMs described herein), essential oils, and/or betaine to decrease or prevent pathogen shedding in the feces (such as, without limitation, shedding of Clostridium perfringens, an Eimeria sp., Campylobacter jejuni, a Salmonela sp., and/or Escherichia coli). In some embodiments, pathogen shedding in the feces decreases by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, inclusive of all values falling in between these percentages, relative to animals who are not administered one or more of the chitin-glucan-containing compositions disclosed herein. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is a feed or feedstuff. Chitin-glucan can comprise at least about 0.01% to 1% (such as any of about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, or 1%, inclusive of all percentages falling in between these values) of the feed additive composition, feed, or feedstuff.

[0149] In another embodiment, the disclosure relates to a method comprising administering to an animal (such as a domesticated bird or a pig) an effective amount of a composition comprising chitin-glucan (such as any of the chitin-glucan-containing feed or feed additive compositions disclosed herein) and optionally exogenous feed enzymes, direct fed microbials (such as any of the DFMs described herein), essential oils, and/or betaine to influence the pattern and/or population of the gut microflora. In some embodiments, the phrase “influence the pattern and/or population of the gut microflora” refers to the preferential promotion of the growth of “beneficial” gut microorganisms such as, without limitation, bacteria from the phylum Firmicutes (which includes, without limitation, bacteria from the class Bacilli which, in turn includes, without limitation, bacteria from the orders Bacillales and Lactobacillales). In other embodiments, the phrase “influence the pattern and/or population of the gut microflora” refers to the reduction in the growth of “harmful” gut microorganisms such as, without limitation, bacteria from the phylum Proteobacteria (which includes, without limitation, bacteria from the class Epsilonproteobacteria and Gammaproteobacteria which, in turn includes, without limitation, bacteria from the orders Campylobacterales, and Enterobacteriales).

[0150] In some embodiments, administration of chitin-glucan-containing feed or feed additive compositions increases the population of beneficial gut microorganisms (eg., bacteria from the phylum Firmicutes) by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,

11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, or 110%, inclusive of all values falling in between these percentages, relative to animals who are not administered one or more of the chitin-glucan-containing compositions disclosed herein. In other embodiments, administration of chitin-glucan-containing feed or feed additive compositions decreases the population of harmfiil gut microorganisms (eg., bacteria from the phylum Proteobacteria) by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, inclusive of all values falling in between these percentages, relative to animals who are not administered one or more of the chitin-glucan-containing compositions disclosed herein. The increase in beneficial gut microorganisms and the decrease in harmfiil microorganisms can occur simultaneously. Additionally, the increase in beneficial gut microorganisms and the decrease in harmful microorganisms can occur in one or more of the duodenum, the jejunum, and/or the ileum. Chitin-glucan can comprise at least about 0.01% to 1% (such as any of about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, or 1%, inclusive of all percentages falling in between these values) of the feed additive composition, feed, or feedstuff.

[0151] The composition comprising chitin-glucan may be administered to the animal in one of many ways. For example, the composition can be administered in a solid form as a veterinary pharmaceutical, may be distributed in an excipient. In other embodiments, the composition can be administered as a drench, formulated with a liquid oil phase, incorporating the chitin-glucan. In further embodiments, the composition can be administered as a paste. In some embodiments, water, and directly fed to the animal, may be physically mixed with feed material in a dry form, or the composition may be formed into a solution and thereafter sprayed onto feed material. The method of administration of the compositions disclosed herein to the animal is considered to be within the skill of the artisan.

[0152] When used in combination with a feed material, the feed material can include com, soybean meal, byproducts like distillers dried grains with solubles (DDGS), and vitamin/mineral supplement. Other feed materials can also be used.

[0153] Thus, in at least some embodiments, the effective amount of the composition comprising chitin-glucan is administered to an animal by supplementing a feed intended for the animal. As used herein, “supplementing,” refers to the action of incorporating the effective amount of chitin-glucan described herein directly into the feed intended for the animal. Thus, the animal, when feeding, ingests the chitin-glucan co-polymer described herein.

B. Methods for treating or preventing intestinal disease

[0154] In another embodiment, the disclosure relates to a method comprising administering to an animal (such as a domesticated bird or pig) an effective amount of a composition comprising chitin-glucan (such as any of the chitin-glucan-containing feed or feed additive compositions disclosed herein) and optionally exogenous feed enzymes, direct fed microbials (such as any of the DFMs described herein), essential oils, and/or betaine to decrease or prevent pathogen populations in the gastrointestinal tract (such as, without limitation, infection by Clostridium perfringens, an Eimeria sp., Campylobacter jejuni, a Salmonela sp., and/or Escherichia coli). In some embodiments, pathogen populations decrease by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, inclusive of all values falling in between these percentages, relative to animals who are not administered one or more of the chitin-glucan-containing compositions disclosed herein. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is a feed or feedstuff. Chitin-glucan can comprise at least about 0.01% to 1% (such as any of about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, or 1%, inclusive of all percentages felling in between these values) of the feed additive composition, feed, or feedstuff.

[0155] In further embodiments, provided herein is a method for treating or preventing necrotic enteritis in an animal (such as a domesticated bird) in need thereof, comprising administering to the animal a feed additive composition comprising an effective amount of chitin-glucan glucan (such as any of the chitin-glucan-containing feed or feed additive compositions disclosed herein) and optionally exogenous feed enzymes, direct fed microbials (such as any of the DFMs described herein), essential oils, and/or betaine. In some embodiments, disease rates (as measured by, for example, intestinal lesion score, weight loss, or intestinal histology) decrease by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, inclusive of all values falling in between these percentages, relative to animals who are not administered one or more of the chitin-glucan-containing compositions disclosed herein. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is a feed or feedstuff. Chitin-glucan can comprise at least about 0.01% to 1% (such as any of about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, or 1%, inclusive of all percentages falling in between these values) of the feed additive composition, feed, or feedstuff.

[0156] The invention can be further understood by reference to the following examples, which are provided by way of illustration and are not meant to be limiting.

EXAMPLES

Example 1: Efficacy of chitin-glucan in animals

[0157] This example describes the effects of dietary chitin-glucan for improving one or more performance metrics in poultry.

Materials and Methods

[0158] Chitin-glucan extracted from the cell walls of the A. mger mycelium was obtained from a commercially available source (Kitozyme). Newly hatched male Ross 708 broiler chickens were housed in floor pens mixed with fresh and used litter with no exposure to coccidiosis previously. There were 39 birds per pen to allow approximately 1.0 ft 2 per bird. Birds were allocated to the pen using a complete randomized block design and fed one of the 3 experimental diets containing 0%, 0.01%, or 0.06% chitin-glucan were fed to animals from 0 to 42 day of age.

[0159] At 14 d of age, birds from all treatments were orally inoculated with 10X Advent ® Vaccine. At 21 d of age, respective intestinal lesion scoring at infection sites from 6 birds per pen (2 below, 2 above and 2 average pen weight) were evaluated, small intestine villa height and crypt depth determined on those birds. Three birds with average pen weight from each pen were euthanized and small intestine and ceca digesta were collected for microbial community analysis.

[0160] 16S Amplicon sequencing analysis: The 16S Amplicon data from Hlumina Miseq sequencing were processed by an in-house pipeline as described below. Paired-end reads were first merged by Flash (Mogoc et al. 2011) with parameters “-0 -M 800”. The forward and reverse primers were removed from the merged reads, and reads with overall quality score less than 20 were discarded by RDP Initial Process tool [Fish et al. 2013] with parameters “-F 2 -R 1 -m 220 -x 270 -n 0”. The average length of the trimmed reads is 253 bp. The trimmed reads were assigned to bacterial and archaeal taxonomy by RDP Classifier with training set no. 16 with bootstrap cutoff of 50% (Wang et al. 2007). Any sample with less than 5,000 sequences was excluded from downstream analysis. The reads passed the above quality processing steps were clustered at 99% by CD-HIT (Li et al., 2006) to obtain Operational Taxonomic Units (OTU). To focus on relatively high abundant OTUs, only OTUs with abundance above 0.1% in at least one sample were kept. The representative sequence from each OTU was assigned to the closest species by RDP pairwise alignment tool (Fish et al. 2013) against a vetted 16S reference database containing mostly 16S genes from type strains and public genomes.

Results

[0161] Performance results for broiler chickens at 42 days of age are shown in Table 1.

Table 1: Performance results of broiler chickens fed various level of chitin-glucan at 42 d of age.

Birds fed higher doses of chitin-glucan exhibited on average increased body weight, livability, growth rate, and European poultry efficiency factor while also having decreased feed conversion ratio.

[0162] As shown in Table 2, in animals challenged with Advent ® Vaccine, those that were fed chitin-glucan showed on average higher body weight gain and growth rate as well as decreased feed conversion ratio.

Table 2: Post challenge performance (14 to 21 d of age) results of broiler chickens fed various level of chitin-glucan.

[0163] Morphological and histological results from the challenged birds are shown in Table 3. As shown, treatment groups that were fed chitin-glucan exhibited decreased duodenal lesion score (FIG. 1), increased villus height and crypt depth, as well as decreased villus to crypt ratio. Table 3: Duodenum pathological results of broiler chickens fed various level of chitin- glucan at 21 d of age 1 .

1 6 birds were sampled for each treatment, total birds sampled were 48 per treatment

2 Evaluated at 3-points scale, with 0 = no lesion, 1 = minor lesion, 2 = moderate lesion and 3 = sever lesion

3 Calculated as: villus height/crypt depth

[0164] The ileum is the key site for nutrient absorption and also susceptible to health challenges. Changes in a host’s microbial community will inevitably impact the nutrient availability and consequently host’s nutritional status. Increasing the ratio of FirmicutesiBacteroidetes has been suggested to be positively related to heavier body weight in broiler chicken (Salaheen et al., 2017). As shown in FIG. 2 and FIG. 3, chickens fed chitin-glucan exhibited positive alterations in the ratio of FirmicutesiBacteroidetes in the ileum.

[0165] In conclusion, this example demonstrated that feeding chitin-glucan to broiler chickens can positively alter the microbiome of poultry as well as enhance growth performance and intestinal morphology.

Example 2: Chitin-glucan in combination with a Bacillus-bused direct fed microbials

[0166] The aim of this Example is to determine the efficacy of chitin-glucan for improving one or more performance metrics in poultry in combination with direct fed microbials (DFMs). A total of three trials were conducted at separate locations.

Materials and Methods

[0167] Trial 1: Newly hatched male Cobb 500 broiler chickens were housed in floor pens mixed with fresh and used litter with no exposure to cocddiosis previously. There were 26 birds per pen to allow approximately 0.9 ft 2 per bird. Birds were allocated to the pen using a complete randomized block design and fed one of the 2 experimental diets (with 8 replications per treatment) containing 0 or 0.01% chitin-glucan in combination with Bacillus-based direct fed microbials were fed to animals from 0 to 42 day of age at 1.5x 10 s CFU/kg of feed. Both diets contained Bacillus-based direct fed microbials in the background.

[0168] Trial 2: Newly hatched male Ross 308 broiler chickens were housed in floor pens mixed with fresh and used litter with no exposure to cocddiosis previously. There were 27 birds per pen to allow approximately 0.9 ft 2 per bird. Birds were allocated to the pen using a complete randomized block design and fed one of the 2 experimental diets (with 8 replications per treatment) containing 0 or 0.01% chitin-glucan in combination with Bacillus-based direct fed microbials were fed to animals from 0 to 42 day of age at 1.5x10 s CFU/kg of feed. From days 17 to 20, Clostridium perfringens were orally inoculated (3-6 x 10 5 cfii per broiler) to all treatments. On day 18, chickens received attenuated coccidiosis vaccine Paracox 5 (lOx recommended dose) via drinking water.

[0169] Trial 3: Newly hatched male Ross 308 broiler chickens were housed in floor pens mixed with fresh and used litter with no exposure to coccidiosis previously. There were 40 birds per pen to allow approximately 1.8 ft 2 per bird. Birds were allocated to the pen using a complete randomized block design and fed one of the 2 experimental diets (with 7 replications per treatment) containing 0 or 0.01% chitin-glucan alone or in combination with Bacillus-based direct fed microbials were fed to animals from 0 to 42 day of age at 1.5 c 10 s CFU/kg of feed. At 14 d of age, birds from all treatments were orally inoculated with 10X Advent ® Vaccine. At 21 d of age, respective intestinal lesion scoring at infection sites from 4 birds per pen (1 below, 1 above and 2 average pen weight) were evaluated.

Results

[0170] Performance results for broiler chickens at 42 days of age are shown in Table 4 for Trial

1.

Table 4: Performance results of broiler chickens fed chitin-glucan on top of Bacillus- based direct fed microbials at 42 d of age-Trial 1.

[0171] A diet supplemented with 0.01% chitin glucan that was supplemented with Bacillus- based direct fed microbials resulted in, on average, slightly higher body weight gain, feed intake, livability, growth rate, and European poultry efficiency factor as well as decreased FCR.

[0172] As shown in Table 5, the combination of chitin-glucan and Bacillus-based DFMs in Trial 2 resulted in higher average body weight gain, livability, growth rate, and European poultry efficiency factor as well as decreased FCR compared to birds that were fed neither chitin-glucan for DFMs. Additionally, the combination of chitin-glucan and DFMs showed improvements for body weight gain, growth rate, FCR, and European poultry efficiency factor over administration of chitin-glucan alone.

Table 5: Performance results of broiler chickens fed chitin-glucan in combination with Bacillus-based direct fed microbials at 42 d of age-Trial 2. [0173] Further, when Trial 2 birds were challenged with C. perjringem, those fed a combination of chitin-glucan and Bacillus-based DFMs exhibited improved body weight gain, growth rate, FCR, and European poultry efficiency factor compared to challenged birds that were fed neither chitin-glucan nor DFMs (Table 6).

Table 6: Post challenge performance (14 to 21 d of age) results of broiler chickens fed chitin-glucan in combination with Bacillus-based direct fed microbials at 42 d of age-Trial 2.

[0174] As shown in Table 7, the combination of chitin-glucan and Bacillus-based DFMs in Trial 3 resulted in higher average body weight gain, livability, growth rate, and European poultry efficiency factor as well as decreased FCR compared to birds that were fed neither chitin-glucan for DFMs. Additionally, the combination of chitin-glucan and DFMs showed improvements for body weight gain, growth rate, FCR, and European poultry efficiency factor over administration of chitin-glucan alone.

Table 7: Performance results of broiler chickens fed chitin-glucan alone or in combination with Bacillus-based direct fed microbials at 42 d of age-Trial 3.

1 European Poultry Efficiency Factor = (Live weight (kg) x Livability (%) x 100) / (Age x Feed conversion ratio).

[0175] Additionally, when Trial 3 birds were challenged with Advent ® Vaccine, those fed a combination of chitin-glucan and Bacillus-based DFMs exhibited improved body weight gain, growth rate, FCR, and European poultry efficiency factor compared to challenged birds that were fed neither chitin-glucan nor DFMs (Table 8).

Table 8: Post challenge performance (14 to 21 d of age) results of broiler chickens fed chitin-glucan alone or in combination with Bacillus-based direct fed microbials at 42 d of age- Trial 3.

1 European ! 1 oultry Efficiency Factor = (Live weight (kg) x Livability (%) x 100) / (Age x Feed conversion ratio).

[0176] Morphological analysis of intestinal pathology revealed that challenged birds from Trial 3 exhibited markedly less frequency of small intestine lesion scores when they were fed chitin- glucan alone or chitin-glucan in combination with Bacillus-based DFMs (Table 9; FIG. 4A and

4B).

Table 9: Intestine pathological results of broiler chickens fed chitin-glucan alone or in combination with Bacillus-based direct fed microbials at 42 d of age-Trial 3 1 .

1 Three birds were sampled for each treatment, total birds sampled were 48 per treatment. Lesion score evaluated at 3-points scale, with 0 = no lesion, 1 = minor lesion, 2 = moderate lesion and 3 = severe lesion

[0177] In conclusion, this Example demonstrated that dietary-supplemented chitin-glucan reduced the severity of small intestine lesions, with an even more beneficial result ws observed with the inclusion of Bacillus-based direct fed microbial.

Example 3: Ex vivo fermentation of chitin-glucan in a porcine model of intestinal digestion

[0178] This Example employs an ex vivo model to assess the effect of chitin-glucan on digestion in swine.

Materials and Methods

[0179] A total of 8 pigs (>30kg) were fed with barley-based commercial diet (Pekoni 90 Rae; Suomen Rehu) for at least two weeks. After the feeding period, 6 pigs were sacrificed and their intestinal tract digesta sampled in distal ileum, caecum and proximal colon. The two remaining pigs were sacrificed in the morning of the simulation day and the distal colon digesta was collected and applied as a bacterial inoculum in the fermentation.

[0180] Intestinal digesta recovered from pigs fed with the commercial barley-based diet were pooled to prepare the authentic growth medium (combination of liquid and solid phases) for the ex vivo laboratory simulation. The medium is thus truly authentic, and, consequently, provides significant benefits as compared to the use of artificial and synthetic growth media commonly used in in vitro models (5 replicates/treatment).

[0181] Inoculum (1.5%), substrate (solid and liquid phase), reduced anaerobic buffer and chitin- glucan were added in the simulation vessels in an anaerobic chamber. The total volume of the simulation vessels was 15 ml. The vessels were sealed with thick butyl rubber stoppers, transferred to 37°C and continuously mixed in a gyratory shaker at 100 r.p.m. The simulation was performed in a random order to avoid any potential systematic shifts. Incubation was continued for 18 hours during which the vessels were sampled for the analysis of gas production, pH and volatile fatty acids (VFA) at various timepoints.

Results

[0182] As shown in Table 10, cumulative gas production increased significantly in the 0.05% and 0.1% treatment groups over the course of 18 hours of fermentation. Additionally, fermentation in the 0.05% and 0.1% treatment groups was associated with decreased pH (Table 11) and increased butyric acid production (Table 12). Benefits of butyric acid production have been extensively evaluated. In short, it is the major metabolite for colonic epithelial cells, which are instrumental in nutrient absorption including water, sodium, and chloride. It also possesses antimicrobial activity for pathogenic bacteria control.

[0183] In conclusion, this Example demonstrated chitin-glucan can be fermented by the intestinal bacteria and stimulate butyric acid production.