OLIGOSACCHARIDE PREPARATION COUNTERACTING RESPIRATORY DISEASE EFFECT
[001] The present invention relates to an oligosaccharide preparation for treating, ameliorating, preventing and/or deferring respiratory disease, preferably infectious bronchitis, and/or effect(s) thereof.
[002] The global demand for products from poultry is continuously rising while at the same time farmers are facing persistent financial pressure. It is thus of outmost importance in poultry husbandry to ensure animal welfare, sustainability and animal health, while optimizing animal performance and product quality.
[003] Infectious diseases, in particular infectious diseases of the respiratory tract such as infectious bronchitis (IB) or Newcastle disease (ND) are known to occur in all regions and are among the leading causes for economic losses in poultry husbandry. Clinical signs of birds infected by infectious bronchitis virus (IBV) or Newcastle disease virus (NDV) include gasping, coughing, sneezing, damages of the respiratory epithelium, accumulation of mucus, tracheal rales, conjunctivitis, sinusitis, dyspnea, asphyxia etc. Concomitantly, feed consumption and weight gain, egg production and egg quality are reduced, feed conversion ratio (FCR) increased. In non-vaccinated flocks, morbidity is typically 100%, mortality is up to 60% depending on the specific virus strain infecting the flock.
[004] As a countermeasure in present poultry farming, birds are routinely vaccinated against NDV and IBV, for instance by spraying chicks with live-attenuated IBV. However, the taxon of IBV comprises several strains and new IBV types and variants emerge from mutation and recombination events. As a consequence, IBV vaccines as currently applied can only provide cross-protection against some of these variants, and IB outbreaks cannot be prevented entirely despite vaccination.
[005] In view of the prior art as outlined above, it is an objective of the present invention to provide means and methods for treating, ameliorating, preventing and/or deferring respiratory disease and the effect(s) associated and/or caused by said respiratory disease.
[006] Surprisingly, this objective is achieved by using an oligosaccharide preparation to prevent and/or defer respiratory disease, preferably infectious bronchitis, and/or effect(s) of said respiratory disease, preferably infectious bronchitis (i.e. infectious bronchitis caused by IBV); wherein the oligosaccharide preparation comprises at least n fractions of oligosaccharides each having a distinct degree of polymerization selected from 1 to n (DP1 to DPn fractions), wherein n is an integer greater than or equal to 2; and wherein each fraction comprises from at least about 0.5% to about 90%, e.g. from 1 % to 90%, or e.g. from about 0.5% to about 15%, of anhydrosubunit containing oligosaccharides by relative abundance as determined by mass spectrometry.
[007] In another aspect, the invention relates to a use of an oligosaccharide preparation to improve efficacy of a vaccination against respiratory disease, preferably infectious bronchitis; wherein the oligosaccharide preparation comprises at least n fractions of oligosaccharides each having a distinct degree of polymerization selected from 1 to n (DP1 to DPn fractions), wherein n is an integer greater than or equal to 2; and wherein each fraction comprises from at least about 0.5% to about 90%, e.g. from 1 % to 90%, or e.g. from about 0.5% to about 15%, of anhydrosubunit containing oligosaccharides by relative abundance as determined by mass spectrometry.
[008] In a further aspect, the invention relates to a use of an oligosaccharide preparation to reduce, prevent and/or defer mortality of a respiratory disease in a flock of poultry being challenged by the respiratory disease, preferably wherein the respiratory disease is infectious bronchitis (in particular IB as caused by IBV); wherein the oligosaccharide preparation comprises at least n fractions of oligosaccharides each having a distinct degree of polymerization selected from 1 to n (DP1 to DPn fractions), wherein n is an integer greater than or equal to 2; and wherein each fraction comprises from at least about 0.5% to about 90%, e.g. from 1% to 90%, or e.g. from about 0.5% to about 15%, of anhydro-subunit containing oligosaccharides by relative abundance as determined by mass spectrometry.
[009] In another aspect, the invention relates to a use of an oligosaccharide preparation to reduce, ameliorate, prevent and/or defer morbidity of a respiratory disease (in particular morbidity of a respiratory disease in a flock of poultry being challenged by or at risk of being challenged by the respiratory disease), preferably wherein the respiratory disease is infectious bronchitis (in particular IB as caused by IBV); wherein the oligosaccharide preparation comprises at least n fractions of oligosaccharides each having a distinct degree of polymerization selected from 1 to n (DP1 to DPn fractions), wherein n is an integer greater than or equal to 2; and wherein each fraction comprises from at least about 0.5% to about 90%, e.g. from 1 % to 90%, or e.g. from about 0.5% to about 15%, of anhydro-subunit containing oligosaccharides by relative abundance as determined by mass spectrometry.
[010] In another aspect, the invention relates to an oligosaccharide preparation for use in a method of treating, ameliorating, preventing and/or deferring respiratory disease, preferably infectious bronchitis (in particular IB as caused by IBV), and/or effect(s) of said respiratory disease; wherein the oligosaccharide preparation comprises at least n fractions of oligosaccharides each having a distinct degree of polymerization selected from 1 to n (DP1 to DPn fractions), wherein n is an integer greater than or equal to 2; and wherein each fraction comprises from at least about 0.5% to about 90%, e.g. from 1 % to 90%, or e.g. from about 0.5% to about 15%, of anhydro-subunit containing oligosaccharides by relative abundance as determined by mass spectrometry.
[011] In some embodiments, the invention relates to an oligosaccharide preparation for use in a method of treating, ameliorating, preventing and/or deferring respiratory disease, preferably infectious bronchitis, and/or effect(s) of said respiratory disease; wherein the oligosaccharide preparation comprises at least n fractions of oligosaccharides each having a distinct degree of polymerization selected from 1 to n (DP1 to DPn fractions), wherein n is an integer greater than or equal to 2; and wherein each fraction comprises from at least about 0.5% to about 90%, e.g. from 1% to 90%, or e.g. from about 0.5% to about 15%, of anhydro-subunit containing oligosaccharides by relative abundance as determined by mass spectrometry; and wherein the oligosaccharide preparation is administered to an individual (e.g. an animal, such as a bird, poultry, or chicken) having received a vaccination against the respiratory disease.
[012] In some embodiments, the oligosaccharide preparation is administered to an individual (e.g. an animal, such as a bird, poultry, or chicken) that will receive a vaccination against the respiratory disease.
[013] In some embodiments, the oligosaccharide preparation is administered to an individual (e.g. an animal, such as a bird, poultry, or chicken) that received at least a first vaccination against the respiratory disease, and which individual will receive at least a second vaccination against the respiratory disease.
[014] In some embodiments, the oligosaccharide preparation of the invention is comprised in a nutritional composition at an inclusion rate of more than 40 ppm; e.g. more than 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 600, 700, 800, 900, 1000 ppm etc.
[015] In some embodiments, n of the oligosaccharide preparation of the invention is at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 ,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57,
58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83,
84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100.
[016] In some embodiments, at least one fraction of the oligosaccharide preparation of the invention comprises less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydro-subunit containing oligosaccharides by relative abundance; and/or each fraction of the oligosaccharide preparation of the invention comprises greater than 0.2%, 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% anhydro-subunit containing oligosaccharides by relative abundance.
[017] In some embodiments, the oligosaccharide preparation of the invention has a weight average molecular weight from about 300 to 5000 g/mol, 500 to 5000 g/mol, 700 to 5000 g/mol, 500 to 2000 g/mol, 700 to 2000 g/mol, 700 to 1500 g/mol, 300 to 1500 g/mol, 300 to 2000 g/mol, 400 to 1300 g/mol, 400 to 1200 g/mol, 400 to 1100 g/mol, 500 to 1300 g/mol, 500 to 1200 g/mol, 500 to 1100 g/mol, 600 to 1300 g/mol, 600 to 1200 g/mol, or 600 to 1100 g/mol; and/or the oligosaccharide preparation of the invention has a number average molecular weight from about 1000 to 2000 g/mol, 1100 to 1900 g/mol, 1200 to 1800 g/mol, 1300 to 1700 g/mol, 1400 to 1600 g/mol, or 1450 to 1550 g/mol.
[018] In some embodiments, the relative abundance of oligosaccharides in each of the n fractions of the oligosaccharide preparation of the invention decreases monotonically with its degree of polymerization.
[019] In some embodiments, the average DP of the oligosaccharide preparation of the invention is at least 2. In some embodiments the average DP of the oligosaccharide preparation of the invention is at least 3.
[020] In some embodiments, the average DP of the oligosaccharide preparation of the invention is less than 5.0.
[021] In some embodiments, the average DP of the oligosaccharide preparation of the invention is greater than or equal to 2, e.g. at least 3, or at least 4; and the average DP of the oligosaccharide preparation of the invention is less than 5.0.
[022] In some embodiments, the average DP of the oligosaccharide preparation of the invention is between 1 and 5; e.g. between 2 and 5, or between 2 and 4.94, or between 3 and 5, or between 3 and 3.94.
[023] In preferred embodiments, the average DP of the oligosaccharide preparation of the invention is determined based on the number average molecular weight distribution of the oligosaccharide preparation of the invention.
[024] The invention is further characterized by the following items:
[025] Item 1. Use of an oligosaccharide preparation to treat, ameliorate, prevent and/or defer respiratory disease, preferably infectious bronchitis (in particular infectious bronchitis caused by IBV), and/or effect(s) thereof (i.e. effects of, or effects caused by, or effects associated with said respiratory disease); wherein the oligosaccharide preparation comprises at least n fractions of oligosaccharides each having a distinct degree of polymerization selected from 1 to n (DP1 to DPn fractions), wherein n is an integer greater than or equal to 2; and wherein each fraction comprises from at least about 0.5% to about 90%, e.g. from 1 % to 90%, or e.g. from about 0.5% to about 15%, of anhydro-subunit containing oligosaccharides by relative abundance as determined by mass spectrometry.
[026] Item 2. Use of an oligosaccharide preparation to treat, ameliorate, prevent and/or defer respiratory disease, preferably infectious bronchitis (in particular infectious bronchitis caused by IBV), and/or effect(s) thereof (i.e. effects of, or effects caused by, or effects associated with said respiratory disease) in animals (preferably poultry, more preferably chicken) suffering from said respiratory disease, and/or at risk of suffering from said respiratory disease; wherein the oligosaccharide preparation comprises at least n fractions of oligosaccharides each having a distinct degree of polymerization selected from 1 to n (DP1 to DPn fractions), wherein n is an integer greater than or equal to 2; and wherein each fraction comprises from at least about 0.5% to about 90%, e.g. from 1% to 90%, or e.g. from about 0.5% to about 15%, of anhydro-subunit containing oligosaccharides by relative abundance as determined by mass spectrometry.
[027] Item 3. Use of an oligosaccharide preparation to improve efficacy, effectiveness and/or efficiency of a vaccination against respiratory disease, preferably infectious bronchitis (in particular infectious bronchitis caused by IBV); wherein the oligosaccharide preparation comprises at least n fractions of oligosaccharides each having a distinct degree of polymerization selected from 1 to n (DP1 to DPn fractions), wherein n is an integer greater than or equal to 2; and wherein each fraction comprises from at least about 0.5% to about 90%, e.g. from 1% to 90%, or e.g. from about 0.5% to about 15%, of anhydro-subunit containing oligosaccharides by relative abundance as determined by mass spectrometry.
[028] Item 4. Use of an oligosaccharide preparation to reduce, prevent and/or defer mortality in a flock of poultry being challenged by a respiratory disease, preferably infectious bronchitis (in particular infectious bronchitis caused by IBV); wherein the oligosaccharide preparation comprises at least n fractions of oligosaccharides each having a distinct degree of polymerization selected from 1 to n (DP1 to DPn fractions), wherein n is an integer greater than or equal to 2; and wherein each fraction comprises from at least about 0.5% to about 90%, e.g. from 1% to 90%, or e.g. from about 0.5% to about 15%, of anhydro-subunit containing oligosaccharides by relative abundance as determined by mass spectrometry. [029] Item 5. Use of an oligosaccharide preparation to reduce, ameliorate, prevent and/or defer morbidity of a respiratory disease, preferably infectious bronchitis (in particular infectious bronchitis caused by IBV); wherein the oligosaccharide preparation comprises at least n fractions of oligosaccharides each having a distinct degree of polymerization selected from 1 to n (DP1 to DPn fractions), wherein n is an integer greater than or equal to 2; and wherein each fraction comprises from at least about 0.5% to about 90%, e.g. from 1 % to 90%, or e.g. from about 0.5% to about 15%, of anhydro-subunit containing oligosaccharides by relative abundance as determined by mass spectrometry.
[030] Item 6. The use according to any one of the preceding items, wherein the oligosaccharide preparation is comprised in a nutritional composition at an inclusion rate of more than 40 ppm (e.g. more than 40 ppm, 50 ppm, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 ppm etc.).
[031] Item 7. The use according to any one of the preceding items, wherein n of the oligosaccharide preparation is at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20,
21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46,
47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72,
73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98,
99, or 100.
[032] Item 8. The use according to any one of the preceding items, wherein at least one fraction of the oligosaccharide preparation comprises less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydro-subunit containing oligosaccharides by relative abundance; and/or wherein each fraction of the oligosaccharide preparation comprises greater than 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% anhydro-subunit containing oligosaccharides by relative abundance.
[033] Item 9. The use according to any one of the preceding items, wherein the oligosaccharide preparation has a weight average molecular weight from about 300 to 5000 g/mol, e.g. from about 2000 to 2800 g/mol, 2100 to 2700 g/mol, 2200 to 2600 g/mol, 2300 to 2500 g/mol, or 2320 to
2420 g/mol; 500 to 5000 g/mol, 700 to 5000 g/mol, 500 to 2000 g/mol, 700 to 2000 g/mol, 700 to
1500 g/mol, 300 to 1500 g/mol, 300 to 2000 g/mol, 400 to 1300 g/mol, 400 to 1200 g/mol, 400 to
1100 g/mol, 500 to 1300 g/mol, 500 to 1200 g/mol, 500 to 1100 g/mol, 600 to 1300 g/mol, 600 to
1200 g/mol, or 600 to 1100 g/mol; and/or wherein the oligosaccharide preparation has a number average molecular weight from about 1000 to 2000 g/mol, 1100 to 1900 g/mol, 1200 to 1800 g/mol, 1300 to 1700 g/mol, 1400 to 1600 g/mol, or 1450 to 1550 g/mol. [034] Item 10. The use according to any one of the preceding items, wherein the relative abundance of oligosaccharides in each of the n fractions of the oligosaccharide preparation decreases monotonically with its degree of polymerization.
[035] Item 11 . The use according to any one of the preceding items, wherein the relative abundance of oligosaccharides in at least 5, 10, 20, or 30 DP fractions of the oligosaccharide preparation decreases monotonically with its degree of polymerization.
[036] Item 12. The use according to any one of the preceding items, wherein the oligosaccharide preparation comprises less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydro-subunit containing oligosaccharides by relative abundance.
[037] Item 13. The use according to any one of the preceding items, wherein each fraction of the oligosaccharide preparation comprises less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydro-subunit containing oligosaccharides by relative abundance.
[038] Item 14. The use according to any one of the preceding items, wherein at least one fraction of the oligosaccharide preparation comprises greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% anhydro-subunit containing oligosaccharides by relative abundance.
[039] Item 15. The use according to any one of the preceding items, wherein the oligosaccharide preparation comprises greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, or 25%, 30%, 40%, 50%, 60%, 70%, or 80% anhydro-subunit containing oligosaccharides by relative abundance.
[040] Item 16. The use according to any one of the preceding items, wherein each fraction of the oligosaccharide preparation comprises greater than 20%, 21%, 22%, 23%, 24%, or 25% anhydro-subunit containing oligosaccharides by relative abundance.
[041] Item 17. The use according to any one of the preceding items, wherein more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the anhydrosubunit containing oligosaccharides of the oligosaccharide preparation have only one anhydrosubunit.
[042] Item 18. The use according to any one of the preceding items, wherein the oligosaccharide preparation has a DP1 fraction content from 1 to 40 % by relative abundance. [043] Item 19. The use according to any one of the preceding items, wherein the oligosaccharide preparation has a DP2 fraction content from 1 to 35 % by relative abundance.
[044] Item 20. The use according to any one of the preceding items, wherein the oligosaccharide preparation has a DP3 fraction content from 1 to 30 % by relative abundance.
[045] Item 21 The use according to any one of the preceding items, wherein the oligosaccharide preparation has a DP4 fraction content from 0.1 to 20 % by relative abundance.
[046] Item 22. The use according to any one of the preceding items, wherein the oligosaccharide preparation has a DP5 fraction content from 0.1 to 15 % by relative abundance.
[047] Item 23. The use according to any one of the preceding items, wherein the ratio of DP2 fraction to DP1 fraction of the oligosaccharide preparation is 0.02-0.40 by relative abundance.
[048] Item 24. The use according to any one of the preceding items, wherein the ratio of DP3 fraction to DP2 fraction of the oligosaccharide preparation is 0.01-0.30 by relative abundance.
[049] Item 25. The use according to any one of the preceding items, wherein the aggregate content of DP1 and DP2 fractions in the oligosaccharide preparation is less than 50, 30, or 10% by relative abundance.
[050] Item 26. The use according to any one of the preceding items, wherein the oligosaccharide preparation comprises at least 103, 104, 105, 106 or 109 different oligosaccharide species.
[051] Item 27. The use according to any one of the preceding items, wherein two or more independent oligosaccharides of the oligosaccharide preparation comprise different anhydrosubunits.
[052] Item 28. The use according to any one of the preceding items, wherein the oligosaccharide preparation comprises one or more anhydro-subunits that are products of reversible thermal dehydration of monosaccharides.
[053] Item 29. The use according to any one of the preceding items, wherein the oligosaccharide preparation comprises one or more anhydro-glucose, anhydro-galactose, anhydro-mannose, anhydro-allose, anhydro-altrose, anhydro-gulose, anhydro-indose, anhydro-talose, anhydrofructose, anhydro-ribose, anhydro-arabinose, anhydro-rhamnose, anhydro-lyxose, or anhydroxylose subunits.
[054] Item 30. The use according to any one of the preceding items, wherein the oligosaccharide preparation comprises one or more anhydro-glucose, anhydro-galactose, anhydro-mannose, or anhydro-fructose subunits. [055] Item 31. The use according to any one of the preceding items, wherein the oligosaccharide preparation comprises one or more 1,6-anhydro-p-D-glucofuranose or 1,6-anhydro-p-D- glucopyranose subunits. In some embodiments, the oligosaccharide preparation comprises both 1,6-anhydro-p-D-glucofuranose and 1,6-anhydro-p-D-glucopyranose anhydro-subunits.
[056] Item 32. The use according to any one of the preceding items, wherein a ratio of 1,6- anhydro-p-D-glucofuranose to 1,6-anhydro-p-D-glucopyranose is from about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6, 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1 in the oligosaccharide preparation.
[057] Item 33. The use according to any one of the preceding items, wherein the ratio of 1,6- anhydro-p-D-glucofuranose to 1 ,6-anhydro-p-D-glucopyranose is about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:8, 1:9, or 1:10 within the oligosaccharide preparation.
[058] Item 34. The use according to any one of the preceding items, wherein the ratio of 1,6- anhydro-p-D-glucofuranose to 1,6-anhydro-p-D-glucopyranose is about 2:1 in the oligosaccharide preparation.
[059] Item 35. The use according to any one of the preceding items, wherein the ratio of 1,6- anhydro-p-D-glucofuranose to 1,6-anhydro-p-D-glucopyranose is about from 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6, 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1 in each fraction of the oligosaccharide preparation.
[060] Item 36. The use according to any one of the preceding items, wherein the ratio of 1,6- anhydro-p-D-glucofuranose to 1 ,6-anhydro-p-D-glucopyranose is about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:8, 1:9, or 1:10 in each fraction of the oligosaccharide preparation.
[061] Item 37. The use according to any one of the preceding items, wherein the ratio of 1,6- anhydro-p-D-glucofuranose to 1 ,6-anhydro-p-D-glucopyranose is about 2:1 in each fraction of the oligosaccharide preparation.
[062] Item 38. The use according to any one of the preceding items, wherein at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of anhydro-subunits in the oligosaccharide preparation are selected from a group consisting of 1,6-anhydro-p-D-glucofuranose and 1,6- anhydro-p-D-glucopyranose. [063] Item 39. The use according to any one of the preceding items, wherein the weight average molecular weight of the oligosaccharide preparation is about from 300 to 5000 g/mol, 500 to 5000 g/mol, 700 to 5000 g/mol, 500 to 2000 g/mol, 700 to 2000 g/mol, 700 to 1500 g/mol, 300 to 1500 g/mol, 300 to 2000 g/mol, 400 to 1300 g/mol, 400 to 1200 g/mol, 400 to 1100 g/mol, 500 to 1300 g/mol, 500 to 1200 g/mol, 500 to 1100 g/mol, 600 to 1300 g/mol, 600 to 1200 g/mol, or 600 to 1100 g/mol.
[064] Item 40. The use according to any one of the preceding items, wherein the number average molecular weight of the oligosaccharide preparation is about from 300 to 5000 g/mol, 500 to 5000 g/mol, 700 to 5000 g/mol, 500 to 2000 g/mol, 700 to 2000 g/mol, 700 to 1500 g/mol, 300 to 1500 g/mol, 300 to 2000 g/mol, 400 to 1000 g/mol, 400 to 900 g/mol, 400 to 800 g/mol, 500 to 900 g/mol, or 500 to 800 g/mol.
[065] Item 41 . The use according to any one of the preceding items, wherein the distribution of the degree of polymerization is determined and/or detected by MALDI-MS, GC-MS, LC-MS, SEC, HPLC and/or combination(s) thereof (e.g. MALDI-MS and SEC).
[066] Item 42. The use according to any one of the preceding items, wherein the degree of polymerization of the oligosaccharide preparation may be determined based on its molecular weight and molecular weight distribution.
[067] Item 43. The use according to any one of the preceding items, wherein the oligosaccharide preparation is comprised in the nutritional composition at a concentration of at least 50 g per ton of feed (e.g. at least 70 g, 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g per ton of feed); and/or wherein the oligosaccharide preparation comprised in the nutritional composition at an inclusion rate of at least 50 ppm (e.g. at least 50, 70, 100, 150, 200, 300, 400, 500 ppm); and/or wherein the oligosaccharide preparation comprised in the nutritional composition at a concentration of at least 50 ppm (e.g. at least 50, 70, 100, 150, 200, 300, 400, 500 ppm).
[068] Item 44. The use according to any one of the preceding items, wherein the oligosaccharide preparation is fed for at least one day, preferably for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,
14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39,
40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65,
66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 ,
92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 ,
132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150,
151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169,
170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, or 245 days, most preferably, the nutritional composition is fed continuously, i.e. uninterruptedly.
[069] Item 44. The use according to any one of the preceding items, wherein an average DP of the oligosaccharide preparation of the oligosaccharide preparation is at least 2, or at least 3, or at least 4.
[070] Item 44. The use according to any one of the preceding items, wherein an average DP of the oligosaccharide preparation is less than 5.0.
[071] Item 44. The use according to any one of the preceding items, wherein an average DP of the oligosaccharide preparation is greater than or equal to 2, e.g. at least 3, or at least 4; and/or wherein the average DP of the oligosaccharide preparation is less than 5.0.
[072] Item 44. The use according to any one of the preceding items, wherein the average DP of the oligosaccharide preparation is determined based on its number average molecular weight distribution.
[073] Item 45. Oligosaccharide preparation for use in a method of treating, ameliorating, preventing and/or deferring respiratory disease, preferably infectious bronchitis (in particular infectious bronchitis caused by IBV), and/or effect(s) thereof (i.e. effects of, or effects caused by, or effects associated with said respiratory disease); wherein the oligosaccharide preparation comprises at least n fractions of oligosaccharides each having a distinct degree of polymerization selected from 1 to n (DP1 to DPn fractions), wherein n is an integer greater than or equal to 2; and wherein each fraction comprises from at least about 0.5% to about 90%, e.g. from 1% to 90%, or e.g. from about 0.5% to about 15%, of anhydro-subunit containing oligosaccharides by relative abundance as determined by mass spectrometry.
[074] Item 46. Oligosaccharide preparation for use in a method of treating, ameliorating, preventing and/or deferring respiratory disease, preferably infectious bronchitis (in particular infectious bronchitis caused by IBV), and/or effect(s) thereof (i.e. effects of, or effects caused by, or effects associated with said respiratory disease), in animals (preferably poultry, more preferably chicken) suffering from said respiratory disease, and/or at risk of suffering from said respiratory disease; wherein the oligosaccharide preparation comprises at least n fractions of oligosaccharides each having a distinct degree of polymerization selected from 1 to n (DP1 to DPn fractions), wherein n is an integer greater than or equal to 2; and wherein each fraction comprises from at least about 0.5% to about 90%, e.g. from 1 % to 90%, or e.g. from about 0.5% to about 15%, of anhydro-subunit containing oligosaccharides by relative abundance as determined by mass spectrometry.
[075] Item 47. The oligosaccharide preparation for use according to any one of items 45 or 46, wherein the oligosaccharide preparation is administered in addition to a vaccination against the respiratory disease.
[076] Item 48. The oligosaccharide preparation for use according to any one of items 45-47, wherein the oligosaccharide preparation is comprised in a nutritional composition at an inclusion rate of more than 40 ppm (e.g. more than 40 ppm, 50 ppm, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 ppm etc.).
[077] Item 49. The oligosaccharide preparation for use according to any one of items 45-48, wherein n of the oligosaccharide preparation is at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15,
16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 ,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67,
68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93,
94, 95, 96, 97, 98, 99, or 100.
[078] Item 50. The oligosaccharide preparation for use according to any one of items 45-49, wherein at least one fraction of the oligosaccharide preparation comprises less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydro-subunit containing oligosaccharides by relative abundance; and/or wherein each fraction of the oligosaccharide preparation comprises greater than 0.2%, 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% anhydro-subunit containing oligosaccharides by relative abundance.
[079] Item 51. The oligosaccharide preparation for use according to any one of items 45-50, wherein the oligosaccharide preparation has a weight average molecular weight from about 300 to 5000 g/mol, e.g. from about 2000 to 2800 g/mol, 2100 to 2700 g/mol, 2200 to 2600 g/mol, 2300 to 2500 g/mol, or 2320 to 2420 g/mol; 500 to 5000 g/mol, 700 to 5000 g/mol, 500 to 2000 g/mol, 700 to 2000 g/mol, 700 to 1500 g/mol, 300 to 1500 g/mol, 300 to 2000 g/mol, 400 to 1300 g/mol, 400 to 1200 g/mol, 400 to 1100 g/mol, 500 to 1300 g/mol, 500 to 1200 g/mol, 500 to 1100 g/mol, 600 to 1300 g/mol, 600 to 1200 g/mol, or 600 to 1100 g/mol; and/or wherein the oligosaccharide preparation has a number average molecular weight from about 1000 to 2000 g/mol, 1100 to 1900 g/mol, 1200 to 1800 g/mol, 1300 to 1700 g/mol, 1400 to 1600 g/mol, or 1450 to 1550 g/mol. [080] Item 52. The oligosaccharide preparation for use according to any one of items 45-51 , wherein the relative abundance of oligosaccharides in each of the n fractions of the oligosaccharide preparation decreases monotonically with its degree of polymerization.
[081] Item 53. The oligosaccharide preparation for use according to any one of items 45-52, wherein the relative abundance of oligosaccharides in at least 5, 10, 20, or 30 DP fractions of the oligosaccharide preparation decreases monotonically with its degree of polymerization.
[082] Item 54. The oligosaccharide preparation for use according to any one of items 45-53, wherein the oligosaccharide preparation comprises less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydro-subunit containing oligosaccharides by relative abundance.
[083] Item 55. The oligosaccharide preparation for use according to any one of items 45-54, wherein each fraction of the oligosaccharide preparation comprises less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydro-subunit containing oligosaccharides by relative abundance.
[084] Item 56. The oligosaccharide preparation for use according to any one of items 45-55, wherein at least one fraction of the oligosaccharide preparation comprises greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% anhydro-subunit containing oligosaccharides by relative abundance.
[085] Item 57. The oligosaccharide preparation for use according to any one of items 45-56, wherein the oligosaccharide preparation comprises greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, or 25%, 30%, 40%, 50%, 60%, 70%, or 80% anhydro-subunit containing oligosaccharides by relative abundance.
[086] Item 58. The oligosaccharide preparation for use according to any one of items 45-57, wherein each fraction of the oligosaccharide preparation comprises greater than 20%, 21%, 22%, 23%, 24%, or 25% anhydro-subunit containing oligosaccharides by relative abundance.
[087] Item 59. The oligosaccharide preparation for use according to any one of items 45-58, wherein more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the anhydro-subunit containing oligosaccharides of the oligosaccharide preparation have only one anhydro-subunit. [088] Item 60. The oligosaccharide preparation for use according to any one of items 45-59, wherein the oligosaccharide preparation has a DP1 fraction content from 1 to 40 % by relative abundance.
[089] Item 61. The oligosaccharide preparation for use according to any one of items 45-60, wherein the oligosaccharide preparation has a DP2 fraction content from 1 to 35 % by relative abundance.
[090] Item 62. The oligosaccharide preparation for use according to any one of items 45-61 , wherein the oligosaccharide preparation has a DP3 fraction content from 1 to 30 % by relative abundance.
[091] Item 63. The oligosaccharide preparation for use according to any one of items 45-62, wherein the oligosaccharide preparation has a DP4 fraction content from 0.1 to 20 % by relative abundance.
[092] Item 64. The oligosaccharide preparation for use according to any one of items 45-63, wherein the oligosaccharide preparation has a DP5 fraction content from 0.1 to 15 % by relative abundance.
[093] Item 65. The oligosaccharide preparation for use according to any one of items 45-64, wherein the ratio of DP2 fraction to DP1 fraction of the oligosaccharide preparation is 0.02-0.40 by relative abundance.
[094] Item 66. The oligosaccharide preparation for use according to any one of items 45-65, wherein the ratio of DP3 fraction to DP2 fraction of the oligosaccharide preparation is 0.01-0.30 by relative abundance.
[095] Item 67. The oligosaccharide preparation for use according to any one of items 45-66, wherein the aggregate content of DP1 and DP2 fractions in the oligosaccharide preparation is less than 50, 30, or 10% by relative abundance.
[096] Item 68. The oligosaccharide preparation for use according to any one of items 45-67, wherein the oligosaccharide preparation comprises at least 103, 104, 105, 106 or 109 different oligosaccharide species.
[097] Item 69. The oligosaccharide preparation for use according to any one of items 45-68, wherein two or more independent oligosaccharides of the oligosaccharide preparation comprise different anhydro-subunits. [098] Item 70. The oligosaccharide preparation for use according to any one of items 45-69, wherein the oligosaccharide preparation comprises one or more anhydro-subunits that are products of reversible thermal dehydration of monosaccharides.
[099] Item 71. The oligosaccharide preparation for use according to any one of items 45-70, wherein the oligosaccharide preparation comprises one or more anhydro-glucose, anhydrogalactose, anhydro-mannose, anhydro-allose, anhydro-altrose, anhydro-gulose, anhydro-indose, anhydro-talose, anhydro-fructose, anhydro-ribose, anhydro-arabinose, anhydro-rhamnose, anhydro-lyxose, or anhydro-xylose subunits.
[100] Item 72. The oligosaccharide preparation for use according to any one of items 45-71, wherein the oligosaccharide preparation comprises one or more anhydro-glucose, anhydrogalactose, anhydro-mannose, or anhydro-fructose subunits.
[101] Item 73. The oligosaccharide preparation for use according to any one of items 45-72, wherein the oligosaccharide preparation comprises one or more 1,6-anhydro-p-D-glucofuranose or 1,6-anhydro-p-D-glucopyranose subunits. In some embodiments, the oligosaccharide preparation comprises both 1,6-anhydro-p-D-glucofuranose and 1,6-anhydro-p-D-glucopyranose anhydro-subunits.
[102] Item 74. The oligosaccharide preparation for use according to any one of items 45-73, wherein a ratio of 1,6-anhydro-p-D-glucofuranose to 1,6-anhydro-p-D-glucopyranose is from about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6, 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1 in the oligosaccharide preparation.
[103] Item 75. The oligosaccharide preparation for use according to any one of items 45-74, wherein the ratio of 1,6-anhydro-p-D-glucofuranose to 1,6-anhydro-p-D-glucopyranose is about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:8, 1:9, or 1:10 within the oligosaccharide preparation.
[104] Item 76. The oligosaccharide preparation for use according to any one of items 45-75, wherein the ratio of 1,6-anhydro-p-D-glucofuranose to 1,6-anhydro-p-D-glucopyranose is about 2:1 in the oligosaccharide preparation.
[105] Item 77. The oligosaccharide preparation for use according to any one of items 45-76, wherein the ratio of 1,6-anhydro-p-D-glucofuranose to 1,6-anhydro-p-D-glucopyranose is about from 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6, 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1 in each fraction of the oligosaccharide preparation. [106] Item 78. The oligosaccharide preparation for use according to any one of items 45-77, wherein the ratio of 1 ,6-anhydro-p-D-glucofuranose to 1 ,6-anhydro-p-D-glucopyranose is about 10:1 , 9:1 , 8:1 , 7:1 , 6:1 , 5:1 , 4:1 , 3:1 , 2:1 , 1 :1 , 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :8, 1 :9, or 1 :10 in each fraction of the oligosaccharide preparation.
[107] Item 79. The oligosaccharide preparation for use according to any one of items 45-78, wherein the ratio of 1 ,6-anhydro-p-D-glucofuranose to 1 ,6-anhydro-p-D-glucopyranose is about 2:1 in each fraction of the oligosaccharide preparation.
[108] Item 80. The oligosaccharide preparation for use according to any one of items 45-79, wherein at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of anhydrosubunits in the oligosaccharide preparation are selected from a group consisting of 1 ,6-anhydro- P-D-glucofuranose and 1 ,6-anhydro-p-D-glucopyranose.
[109] Item 81. The oligosaccharide preparation for use according to any one of items 45-80, wherein the weight average molecular weight of the oligosaccharide preparation is about from
300 to 5000 g/mol, 500 to 5000 g/mol, 700 to 5000 g/mol, 500 to 2000 g/mol, 700 to 2000 g/mol,
700 to 1500 g/mol, 300 to 1500 g/mol, 300 to 2000 g/mol, 400 to 1300 g/mol, 400 to 1200 g/mol,
400 to 1100 g/mol, 500 to 1300 g/mol, 500 to 1200 g/mol, 500 to 1100 g/mol, 600 to 1300 g/mol,
600 to 1200 g/mol, or 600 to 1100 g/mol.
[110] Item 82. The oligosaccharide preparation for use according to any one of items 45-81 , wherein the number average molecular weight of the oligosaccharide preparation is about from 300 to 5000 g/mol, 500 to 5000 g/mol, 700 to 5000 g/mol, 500 to 2000 g/mol, 700 to 2000 g/mol, 700 to 1500 g/mol, 300 to 1500 g/mol, 300 to 2000 g/mol, 400 to 1000 g/mol, 400 to 900 g/mol, 400 to 800 g/mol, 500 to 900 g/mol, or 500 to 800 g/mol.
[111] Item 83. The oligosaccharide preparation for use according to any one of items 45-82, wherein the distribution of the degree of polymerization is determined and/or detected by MALDI- MS, GC-MS, LC-MS, SEC, HPLC and/or combination(s) thereof (e.g. MALDI-MS and SEC).
[112] Item 84. The oligosaccharide preparation for use according to any one of items 45-83, wherein the degree of polymerization of the oligosaccharide preparation may be determined based on its molecular weight and molecular weight distribution.
[113] Item 85. The oligosaccharide preparation for use according to any one of items 45-84, wherein the oligosaccharide preparation is comprised in the nutritional composition at a concentration of at least 50 g per ton of feed (e.g. at least 70 g, 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g per ton of feed); and/or wherein the oligosaccharide preparation comprised in the nutritional composition at an inclusion rate of at least 50 ppm (e.g. at least 50, 70, 100, 150, 200, 300, 400, 500 ppm); and/or wherein the oligosaccharide preparation comprised in the nutritional composition at a concentration of at least 50 ppm (e.g. at least 50, 70, 100, 150, 200, 300, 400, 500 ppm).
[114] Item 86. The oligosaccharide preparation for use according to any one of items 45-85, wherein the oligosaccharide preparation is fed for at least one day, preferably for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125,
126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144,
145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163,
164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182,
183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 ,
202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220,
221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239,
240, 241 , 242, 243, 244, or 245 days, most preferably, the nutritional composition is fed continuously, i.e. uninterruptedly.
[115] Item 87. The oligosaccharide preparation for use according to any one of items 45-86, wherein an average DP of the oligosaccharide preparation is at least 2, or at least 3, or at least 4.
[116] Item 88. The oligosaccharide preparation for use according to any one of items 45-87, wherein an average DP of the oligosaccharide preparation is less than 5.0.
[117] Item 89. The oligosaccharide preparation for use according to any one of items 45-88, wherein an average DP of the oligosaccharide preparation is greater than or equal to 2, e.g. at least 3, or at least 4; and/or wherein the average DP of the oligosaccharide preparation is less than 5.0.
[118] Item 90. The oligosaccharide preparation for use according to any one of items 45-89, wherein the average DP of the oligosaccharide preparation is determined based on its number average molecular weight distribution.
[119] It is also considered that the oligosaccharide preparation according to the present invention may be provided in the form of a powderous formulation comprising at least 20% (w/w) of the oligosaccharide preparation referred to herein; at least 25% (wt/wt) of a silica-based adsorbate (e.g. diatomaceous earth, amorphous precipitated silica) having an average particle size D of less than or equal to 3000 pm (e.g. 100-500, 200-500, 200-300 pm etc.); and optionally 0-25% (wt/wt) of water and/or an auxiliary substance; wherein the % are based on the total weight of the powderous formulation. For instance, such a powderous formulation may comprise 30-70% (wt/wt) of the oligosaccharide preparation as referred to herein; 30-70% (wt/wt) of a silica based adsorbate (e.g. having an average particle size of at least 50 pm); and 0-21% (wt/wt) of water; wherein the % are based on the total weight of the powderous formulation. In some embodiments the oligosaccharide preparation is formulated as described in any one of Examples 22-26 and 33 of WO 2020/097458.
[120] Methods for manufacturing oligosaccharide preparations according to the invention are described in WO 2020/097458, WO 2016/007778, in particular in the Examples described therein, in particular in any one of Examples 1-7, 16-18 of WO 2020/097458 A1 ; in the methods described in paragraph [317], and/or in any one of Examples 73-77, 80-89, 97-99, 101-110 of WO 2016/007778 A1 ; preferably oligosaccharide preparations according to the invention are manufactured as described in Example 16 of WO 2020/097458 A1. In particular, oligosaccharide preparations according to the invention are characterized by the step of heating an aqueous composition comprising one or more feed sugars and a catalyst to a temperature and for a time sufficient to induce polymerization. In some embodiments, the feed sugar(s) is/are glucose, mannose, galactose, lactose, fructose, sucrose, xylose, arabinose, glucosamine, N- acetylglucosamine, N-acetylgalactosamine, glucuronic acid, galacturonic acid, fucose, rhamnose, chinovose or any combination thereof; glucose and mannose, glucose and galactose, glucose and lactose, glucose and fructose, glucose and arabinose, glucose and sucrose, glucose and xylose, glucose and galactose and arabinose etc. In some embodiments, the catalyst is any one of (+)-camphor-10-sulfonic acid; 2-pyridinesulfonic acid; 3-pyridinesulfonic acid; 8-hydroxy-5- quinolinesulfonic acid hydrate; a-hydroxy-2-pyridinemethanesulfonic acid; (P)-camphor-IO- sulfonic acid; butylphosphonic acid; diphenylphosphinic acid; hexylphosphonic acid; methylphosphonic acid; phenylphosphinic acid; phenylphosphonic acid; tert-butylphosphonic acid; SS)-VAPOL hydrogenphosphate; 6-quinolinesulfonic acid, 3-(1 -pyridinio)-1 - propanesulfonate; 2-(2-pyridinyl)ethanesulfonic acid; 3-(2-pyridyl)-5,6-diphenyl- 1 ,2,4-triazine- p,p'-disulfonic acid monosodium salt hydrate; 1 ,1'-binaphthyl-2,2'-diyl-hydrogenphosphate; bis(4- methoxyphenyl)phosphinic acid; phenyl(3,5-xylyl)phosphinic acid; L-cysteic acid monohydrate; poly(styrene sulfonic acid -co- divinylbenzene); lysine; ethanedisulfonic acid; ethanesulfonic acid; isethionic acid; homocysteic acid; HEPBS (N-(2-Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)); HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); 2-hydroxy-3- morpholinopropanesulfonic acid; 2-(N-morpholino)ethanesulfonic acid; methanesulfonic acid; methaniazide; naphthalene-1 -sulfonic acid; naphthalene-2-sulfonic acid; perfluorobutanesulfonic acid; 6-sulfoquinovose; triflic acid; 2-aminoethanesulfonic acid; benzoic acid; chloroacetic acid; trifluoroacetic acid; caproic acid; enanthic acid; caprylic acid; pelargonic acid; lauric acid; palmitic acid; stearic acid; arachidic acid; aspartic acid; glutamic acid; serine; threonine; glutamine; cysteine; glycine; proline; alanine; valine; isoleucine; leucine; methionine; phenylalanine; tyrosine; or tryptophan. In some embodiments, the feed sugar is glucose and the catalyst is (+)-camphor- 10-sulfonic acid, (P)-camphor-IO-sulfonic acid, butylphosphonic acid, HEPBS, naphthalene-1 - sulfonic acid, naphthalene-2-sulfonic acid or palmitic acid. In some embodiments, the feed sugars are glucose and mannose, and the catalyst is ethanedisulfonic acid, ethanesulfonic acid, triflic acid or lauric acid. In some embodiments, the feed sugar is galactose, and the catalyst is 2-(N- morpholino)ethanesulfonic acid, methanesulfonic acid or methaniazide.
[121] Merely for the sake of clarity, the term “oligosaccharide preparation” may refer to a preparation that comprises one or more oligosaccharides.
[122] As used herein, an “oligosaccharide” or “oligomer” may refer to a monosaccharide unit or a compound containing two or more monosaccharide subunits, which are linked by glycosidic bonds to form the oligosaccharide. An “oligosaccharide” may also refer to an anhydromonosaccharide unit or a compound containing two or more monosaccharide subunits, where at least one monosaccharide unit is replaced by an anhydro-subunit. An “oligosaccharide” may be optionally functionalized. As used herein, the term “oligosaccharide” encompasses all species of the oligosaccharide, wherein each of the monosaccharide subunits in the oligosaccharide is independently and optionally functionalized and/or replaced with its corresponding anhydromonosaccharide subunit.
[123] An “anhydro-subunit” may be a product of reversible thermal dehydration of a monosaccharide or monosaccharide subunit, or a sugar caramelization product. For example, an “anhydro-subunit” may be an anhydro-monosaccharide such as anhydro-glucose. As another example, an “anhydro-subunit” may be linked with one or more regular monosaccharide subunits and/or with one or more anhydro-monosaccharide subunits via glycosidic linkage(s).
[124] An oligosaccharide may be characterized to contain two or more monosaccharide subunits linked by glycosidic bonds. In this regard, a “gluco-oligosaccharide” may refer to a single glucose molecule, or to a compound containing two or more glucose monosaccharide subunits linked by glycosidic bonds. A “gluco-oligosaccharide” may also refer to an anhydro-glucose or a compound containing two or more glucose monosaccharide subunits linked by glycosidic bonds, wherein at least one monosaccharide subunit is replaced by an anhydro-glucose subunit. Similarly, a “galacto-oligosaccharide” may refer to a galactose or a compound containing two or more galactose monosaccharide subunits linked by glycosidic bonds. A “galacto-oligosaccharide” may also refer to an anhydro-galactose or a compound containing two or more galactose monosaccharide subunits linked by glycosidic bonds, wherein at least one monosaccharide subunit is replaced by an anhydro-galactose subunit. Analogously, a gluco-galactose- oligosaccharide may be a compound comprising one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic bonds. A glucogalactose oligosaccharide may also refer to a compound, wherein at least one of the monosaccharide subunits is replaced with its respective anhydro-monosaccharide subunit. A gluco-galacto-xylo-oligosaccharide may refer to a compound produced by the condensation reaction of glucose, galactose, and xylose. An oligosaccharide preparation comprising gluco- galacto-xylo-oligosaccharides may comprise gluco-galactose-oligosaccharides, gluco-xylo- oligosaccharides, galacto-xylo-oligosaccharides, gluco-galacto-xylo-oligosaccharides, and compounds containing one or more glucose monosaccharide subunits, one or more xylose monosaccharide subunits, and one or more galactose monosaccharide subunits linked by glycosidic bonds, as well as anhydro-monosaccharide subunit(s) thereof.
[125] As used herein, the term “monosaccharide unit” and “monosaccharide subunit” may be used interchangeably, unless suggested otherwise. A “monosaccharide subunit” may refer to a monosaccharide monomer in an oligosaccharide. For an oligosaccharide having a degree of polymerization (DP) of 1 , the oligosaccharide may be referred to as a monosaccharide subunit or monosaccharide. For an oligosaccharide having a degree of polymerization higher than 1 , its monosaccharide subunits are linked via glycosidic bonds.
[126] As used herein, the term “regular monosaccharide” may refer to a monosaccharide that does not contain an anhydro-subunit. The term “regular disaccharide” may refer to a disaccharide that does not contain an anhydro-subunit. Accordingly, the term “regular subunit” may refer to a subunit that is not an anhydro-subunit.
[127] The term “relative abundance” or “abundance” as used herein, may refer to the abundance of a species in terms of how common or rare the species exists. For example, a DP1 fraction (i.e. a fraction having a DP of 1) comprising 10% anhydro-subunit containing oligosaccharides by relative abundance may refer to a plurality of DP1 oligosaccharides, wherein 10%, by number, of the DP1 oligosaccharides are anhydro-monosaccharides.
[128] Degree of Polymerization (DP) distribution: A distribution of the degree of polymerization of the oligosaccharide preparation may be determined by any suitable analytical method and instrumentation, including but not limited to end group method, osmotic pressure (osmometry), ultracentrifugation, viscosity measurements, light scattering method, size exclusion chromatography (SEC), SEC-MALLS, field flow fractionation (FFF), asymmetric flow field flow fractionation (A4F), high-performance liquid chromatography (HPLC), and mass spectrometry (MS). For example, the distribution of the degree of polymerization may be determined and/or detected by mass spectrometry, such as MALDI-MS, LC-MS, or GC-MS. For another example, the distribution of the degree of polymerization may be determined and/or detected by SEC, such as gel permeation chromatography (GPC). As yet another example, the distribution of the degree of polymerization may be determined and/or detected by HPLC, FFF, orA4F. In another example, the degree of polymerization of the oligosaccharide preparation may be determined based on its molecular weight and molecular weight distribution (for a more detailed description see WO 2020/097458).
[129] Anhydro-subunit level: An oligosaccharide preparation may comprise n distinct fractions, wherein each fraction has a distinct DP, and wherein n is any integer, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 etc. In some embodiments, each of the n fractions of oligosaccharides of the oligosaccharide preparation as described herein independently comprises an anhydro-subunit level. For instance, in some embodiments, the DP1 fraction comprises 10% anhydro-subunit containing oligosaccharides by relative abundance, and the DP2 fraction comprises 15% anhydro-subunit containing oligosaccharides by relative abundance. For another example, in some embodiments, DP1 , DP2, and DP3 fraction each comprises 5%, 10%, and 2% anhydro-subunit containing oligosaccharides by relative abundance, respectively. In other embodiments, two or more fractions of oligosaccharides may comprise similar level of anhydro-subunit containing oligosaccharides. For example, in some embodiments, the DP1 and DP3 fraction each comprises about 5 % anhydro-subunit containing oligosaccharides by relative abundance.
[130] The level of anhydro-subunits may be determined by any suitable analytical methods, such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, HPLC, FFF, A4F, or any combination thereof. In some embodiments, the level of anhydro-subunits is determined, at least in part, by mass spectrometry such as MALDI-MS. In some embodiments, the level of anhydro-subunits may be determined, at least in part, by NMR. In some embodiments, the level of anhydro-subunits may be determined, at least in part, by HPLC. For example, in some embodiments, the level of anhydro-subunits may be determined by MALDI-MS, as illustrated in more detail in WO 2020/097458.
[131] Glycosidic Linkages: In some embodiments, the oligosaccharide preparation described herein comprise a variety of glycosidic linkages. The type and distribution of the glycosidic linkages may depend on the source and manufacturing method of the oligosaccharide preparation. In some embodiments, the type and distribution of various glycosidic linkages may be determined and/or detected by any suitable methods known in the art such as NMR. For example, in some embodiments, the glycosidic linkages are determined and/or detected by proton NMR, carbon NMR, 2D NMR such as 2D JRES, HSQC, HMBC, DOSY, COSY, ECOSY, TOCSY, NOESY, or ROESY, or any combination thereof. In some embodiments, the glycosidic linkages are determined and/or detected, at least in part, by proton NMR. In some embodiments, the glycosidic linkages are determined and/or detected, at least in part, by carbon NMR. In some embodiments, the glycosidic linkages are determined and/or detected, at least in part, by 2D HSQC NMR.
[132] In some embodiments, an oligosaccharide preparation may comprise one or more a-(1 ,2) glycosidic linkages, a-(1 ,3) glycosidic linkages, a-(1 ,4) glycosidic linkages, a-(1 ,6) glycosidic linkages, |3-(1 ,2) glycosidic linkages, |3-(1 ,3) glycosidic linkages, |3-(1 ,4) glycosidic linkages, - (1 ,6) glycosidic linkages, a-(1 ,1)-a glycosidic linkages, a-(1 ,1)-p glycosidic linkages, p-(1 ,1)-p glycosidic linkages, or any combination thereof.
[133] In some embodiments, the oligosaccharide preparations have a glycosidic bond type distribution of about from 0 to 60 mol%, 5 to 55 mol%, 5 to 50 mol%, 5 to 45 mol%, 5 to 40 mol%, 5 to 35 mol%, 5 to 30 mol%, 5 to 25 mol%, 10 to 60 mol%, 10 to 55 mol%, 10 to 50 mol%, 10 to 45 mol%, 10 to 40 mol%, 10 to 35 mol%, 15 to 60 mol%, 15 to 55 mol%, 15 to 50 mol%, 15 to 45 mol%, 15 to 40 mol%, 15 to 35 mol%, 20 to 60 mol%, 20 to 55 mol%, 20 to 50 mol%, 20 to 45 mol%, 20 to 40 mol%, 20 to 35 mol%, 25 to 60 mol%, 25 to 55 mol%, 25 to 50 mol%, 25 to 45 mol%, 25 to 40 mol%, or 25 to 35 mol% of a-(1 ,6) glycosidic linkages.
[134] Molecular Weight: The molecular weight and molecular weight distribution of the oligosaccharide preparation may be determined by any suitable analytical means and instrumentation, such as end group method, osmotic pressure (osmometry), ultracentrifugation, viscosity measurements, light scattering method, SEC, SEC-MALLS, FFF, A4F, HPLC, and mass spectrometry. In some embodiments, the molecular weight and molecular weight distribution are determined by mass spectrometry, such as MALDI-MS, LC-MS, or GC-MS. In some embodiments, the molecular weight and molecular weight distribution are determined by size exclusion chromatography (SEC), such as gel permeation chromatography (GPC). In other embodiments, the molecular weight and molecular weight distribution are determined by HPLC. In some embodiments, the molecular weight and molecular weight distribution are determined by MALDI-MS.
[135] In some embodiments, the weight average molecular weight of the oligosaccharide preparation is about from 100 to 10000 g/mol, 200 to 8000 g/mol, 300 to 5000 g/mol, 500 to 5000 g/mol, 700 to 5000 g/mol, 900 to 5000 g/mol, 1100 to 5000 g/mol, 1300 to 5000 g/mol, 1500 to 5000 g/mol, 1700 to 5000 g/mol, 300 to 4500 g/mol, 500 to 4500 g/mol, 700 to 4500 g/mol, 900 to 4500 g/mol, 1100 to 4500 g/mol, 1300 to 4500 g/mol, 1500 to 4500 g/mol, 1700 to 4500 g/mol, 1900 to 4500 g/mol, 300 to 4000 g/mol, 500 to 4000 g/mol, 700 to 4000 g/mol, 900 to 4000 g/mol, 1100 to 4000 g/mol, 1300 to 4000 g/mol, 1500 to 4000 g/mol, 1700 to 4000 g/mol, 1900 to 4000 g/mol, 300 to 3000 g/mol, 500 to 3000 g/mol, 700 to 3000 g/mol, 900 to 3000 g/mol, 1100 to 3000 g/mol, 1300 to 3000 g/mol, 1500 to 3000 g/mol, 1700 to 3000 g/mol, 1900 to 3000 g/mol, 2100 to 3000 g/mol, 300 to 2500 g/mol, 500 to 2500 g/mol, 700 to 2500 g/mol, 900 to 2500 g/mol, 1100 to 2500 g/mol, 1300 to 2500 g/mol, 1500 to 2500 g/mol, 1700 to 2500 g/mol, 1900 to 2500 g/mol, 2100 to 2500 g/mol, 300 to 1500 g/mol, 500 to 1500 g/mol, 700 to 1500 g/mol, 900 to 1500 g/mol, 1100 to 1500 g/mol, 1300 to 1500 g/mol, 2000-2800 g/mol, 2100-2700 g/mol, 2200-2600 g/mol, 2300-2500 g/mol, or 2320-2420 g/mol. In some embodiments, the weight average molecular weight of the oligosaccharide preparation is about from 2000 to 2800 g/mol, 2100 to 2700 g/mol, 2200 to 2600 g/mol, 2300 to 2500 g/mol, or 2320 to 2420 g/mol.
[136] Types of Oligosaccharides: In some embodiments, the species of oligosaccharides present in an oligosaccharide preparation referred to herein may depend on the type of the one or more feed sugars. For example, in some embodiments, the oligosaccharide preparations comprise a gluco-oligosaccharide when the feed sugars comprise glucose. For example, in some embodiments, the oligosaccharide preparations comprise a galacto-oligosaccharide when the feed sugars comprise galactose. For another example, in some embodiments, the oligosaccharide preparations comprise gluco-galacto-oligosaccharides when the feed sugars comprise galactose and glucose.
[137] In some embodiments, the oligosaccharide preparations comprise one or more species of monosaccharide subunits. In some embodiments, the oligosaccharide preparation may comprise oligosaccharides with 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different species of monosaccharides subunits.
[138] Method of Manufacturing Oligosaccharide Preparations: The method of manufacturing an oligosaccharide preparation according to the invention is described in detail in WO 2020/097458 comprising heating an agueous composition comprising one or more feed sugars and a catalyst to a temperature and for a time sufficient to induce polymerization, wherein the catalyst is selected from the group consisting of: (+)-camphor-10-sulfonic acid; 2-pyridinesulfonic acid; 3- pyridinesulfonic acid; 8-hydroxy-5-guinolinesulfonic acid hydrate; a-hydroxy-2- pyridinemethanesulfonic acid; (P)-camphor-IO-sulfonic acid; butylphosphonic acid; diphenylphosphinic acid; hexylphosphonic acid; methylphosphonic acid; phenylphosphinic acid; phenylphosphonic acid; tert-butylphosphonic acid; SS)-VAPOL hydrogenphosphate; 6- quinolinesulfonic acid, 3-(1-pyridinio)-1 -propanesulfonate; 2-(2-pyridinyl)ethanesulfonic acid; 3- (2-pyridyl)-5,6-diphenyl-1 ,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate; 1,1'- binaphthyl-2,2'-diyl-hydrogenphosphate; bis(4-methoxyphenyl)phosphinic acid; phenyl(3,5- xylyl)phosphinic acid; L-cysteic acid monohydrate; poly(styrene sulfonic acid -co- divinylbenzene); lysine; Ethanedisulfonic acid; Ethanesulfonic acid; Isethionic acid; Homocysteic acid; HEPBS (N-(2-Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)); HEPES (4-(2- hydroxyethyl)-1 -piperazineethanesulfonic acid); 2-Hydroxy-3-morpholinopropanesulfonic acid; 2- (N-morpholino)ethanesulfonic acid; Methanesulfonic acid; Methaniazide; Naphthalene-1 -sulfonic acid; Naphthalene-2-sulfonic acid; Perfluorobutanesulfonic acid; 6-sulfoquinovose; Triflic acid; 2- aminoethanesulfonic acid; Benzoic acid; Chloroacetic acid; Trifluoroacetic acid; Caproic acid; Enanthic acid; Caprylic acid; Pelargonic acid; Lauric acid; Pamitic acid; Stearic acid; Arachidic acid; Aspartic acid; Glutamic acid; Serine; Threonine; Glutamine; Cysteine; Glycine; Proline; Alanine; Valine; Isoleucine; Leucine; Methionine; Phenylalanine; Tyrosine; Tryptophan.
[139] In some embodiments, the polymerization of the feed sugars is achieved by a step-growth polymerization. In some embodiments, the polymerization of the feed sugars is achieved by polycondensation.
[140] Feed Sugar: The one or more feed sugars used in the methods of manufacturing oligosaccharide preparations described herein may comprise one or more types of sugars. In some embodiments, the one or more feed sugars comprise monosaccharides, disaccharides, trisaccharides, tetrasaccharides, or any mixtures thereof.
[141] In some embodiments, the one or more feed sugars comprise glucose. In some embodiments, the one or more feed sugars comprise glucose and galactose. In some embodiments, the one or more feed sugars comprise glucose, xylose, and galactose. In some embodiments, the one or more feed sugars comprise glucose and mannose. In some embodiments, the one or more feed sugars comprise glucose and fructose. In some embodiments, the one or more feed sugars comprise glucose, fructose, and galactose. In some embodiments, the one or more feed sugars comprise glucose, galactose, and mannose.
[142] As used herein, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an agent” includes a plurality of such agents, and reference to “the oligosaccharide” includes reference to one or more oligosaccharides (or to a plurality of oligosaccharides) and equivalents thereof known to those skilled in the art, and so forth.
[143] When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range, in some instances, will vary between 1 % and 15% of the stated number or numerical range.
[144] In the following, the present invention is further described by non-limiting examples.
Examples
[145] The present invention as disclosed herein is not limited to specific embodiments, figures, methodology, examples, protocols etc. described herein, but solely limited by the claims.
Example 1
[146] For a broiler feeding trial, 32,600 male Ross 308 day-old broilers were fed with either a control diet or with a test diet. 10,800 broilers received the control diet, 21 ,800 broilers received the test diet. The control diet was a basal diet for broilers (corn, soybean meal). The test diet contained 0.9 kg/MT of an oligosaccharide preparation as described herein in addition to the basal diet. In detail, the oligosaccharide preparation comprised more than two DP fractions, and had an average DP of less than 5, as determined based on its number average molecular weight distribution. Each of the DP fractions of the oligosaccharide preparation contained approximately 0.5%-15%, of anhydro-subunit containing oligosaccharides by relative abundance as determined by mass spectrometry. Feeding was performed according to common recommendations for the breed; no in-feed antibiotic growth promoting substance or ionophores.
[147] All birds were vaccinated against Newcastle disease, infectious bronchitis disease, NB & IBQX. Feed and water were provided ad libitum. Starter (d0-10d) crumble, grower (d11-d22) pellets, finisher (d 23-d35) pellets. Total trial duration was 38 days.
[148] Despite vaccination, IBV infected the birds, causing an outbreak in the birds receiving the control diet on day 20. Surprisingly, in birds receiving the test diet, the disease broke out on day 29. Also, FCR and body weight gain were significantly improved in the test birds compared to the control birds (1.58 compared to 1.69; and 2615 g compared to 2534 g, respectively). Moreover, mortality was significantly lower in birds receiving the test diet compared to birds receiving the control diet. In particular, the mortality from d23-d35 was 14.48% in the control group and 2.965% in the test group. In comparison, the mortality from d0-d10 was not significantly different between the two groups of birds. Therefore, the oligosaccharide preparation was found to make the birds more resilient and less impacted by the disease, and to delay and defer the IB outbreak.
Example 2
[149] For a separate feeding trial with layer chicken, 24 replicates of eight Bovans white hens were fed either a control diet (comprising a phytase and a non-starch polysaccharide xylanase), or a first test diet (control diet additionally comprising 450 ppm of the oligosaccharide preparation of the invention and as described in Example 1), or a second test diet (control diet additionally comprising 900 ppm of the oligosaccharide preparation of the invention and as described in Example 1). All birds were vaccinated against IBV and NDV.
[150] The trial was started at an age of the birds of 22 weeks and continued to an age of 60 weeks. Housing conditions, feed consumption, egg production and egg weight, haugh unit, yolk color, eggshell breaking strength, fertilizer moisture, and mortality were monitored weekly.
[151] In terms of egg production, egg mass, and FCR, birds receiving any one of the test diets performed superior over birds receiving the control diet throughout the trial. At week 26 of age, IB and ND broke out. At week 36 of age, another ND outbreak was recorded. In agreement with the finding of Example 1 , disease outbreak affected those birds less that were receiving any one of the test diets, compared to birds of the control group.
[152] In weeks 22-25 of age, mean hen-day egg production (HDEP) was 1.39% and 4.13% higher in birds receiving 450 ppm and 900 ppm of the oligosaccharide preparation, respectively, compared to birds of the control group. This improved productivity was even more pronounced in weeks 26-30, i.e. at the time of disease outbreak, where HDEP was 7.66% and 11 .34% higher in birds receiving 450 ppm and 900 ppm of the oligosaccharide preparation, respectively, compared to birds receiving the control diet. Also in terms of mean egg mass, birds receiving either of the test diets outperformed the control birds by at least 2.2% higher mean egg mass (g/d). Again, this improvement was particularly prominent at weeks 26-30, where birds receiving 450 ppm or 900 ppm of the oligosaccharide preparation of the invention produced 12.2% or 11.9% higher mean egg mass, respectively, than birds receiving the control diet. Also with respect to mean FCR, test birds were 1-1 .5% lower (i.e. more efficient) than control birds in weeks 22-26 of age; 9.9-11 .2% lower in weeks 26-30; 2.8-6.3% lower in weeks 30-34; 1.6-4.9% lower in weeks 34-38 etc. Moreover, mean eggshell breaking strength was found to be approximately 1 % improved by eggs produced by test birds during the weeks prior to disease outbreak, and approximately 3-11% improved compared to control birds in weeks during or after disease outbreak. Notably, pC>2 and pCO2 as blood biomarkers indicated that birds of the test groups were subjected to less severe respiratory stress than birds of the control group upon disease outbreak.