MEDIA AND FERMENTATION METHODS FOR PRODUCING POLYSACCHARIDES IN BACTERIAL CELL CULTURE

1 . A polysaccharide-producing bacterial cell culture medium comprising a vegetable hydrolysate, a yeast extract, and a carbon source.

2 . The medium of claim 1 , wherein the vegetable hydrolysate is a soy hydrolysate.

3 . The medium of claim 2 , wherein the soy hydrolysate is selected from the group consisting of HYPEP 1510 (Kerry Group Services Ltd.), HYPEP 4601 (Kerry Group Services Ltd.), HYPEP 5603 (Kerry Group Services Ltd.), HY-SOY (Kerry Group Services Ltd.), AMI-SOY (Kerry Group Services Ltd.), N-Z-SOY (Kerry Group Services Ltd.), N-Z-SOY BL4 (Kerry Group Services Ltd.), N-Z-SOY BL7 (Kerry Group Services Ltd.), SHEFTONE D (Kerry Group Services Ltd.), SE50M, SE50MK, soy peptone, BACTO soytone (Difco Laboratories Inc.), NUTRISOY 2207 (ADM), NUTRISOY (ADM), NUTRISOY flour (ADM), and soybean meal.

4 . The medium of claim 3 , wherein the soy hydrolysate is HYPEP 1510 (Kerry Group Services Ltd.).

5 . The medium of any one of claims 1 - 4 , wherein the concentration of the vegetable hydrolysate is between about 5 g/L and about 75 g/L.

6 . The medium of claim 5 , wherein the concentration of the vegetable hydrolysate is between about 10 g/L and about 50 g/L.

7 . The medium of claim 6 , wherein the concentration of the vegetable hydrolysate is about 28 g/L.

8 . The medium of any one of claims 1 - 7 , wherein the yeast extract is a yeast autolysate, an ultrafiltered yeast extract, or a synthetic yeast extract.

9 . The medium of claim 8 , wherein the yeast extract is an ultrafiltered yeast extract.

10 . The medium of claim 9 , wherein the ultrafiltered yeast extract is AMBERFERM 5902 (Sensient Technologies Corp.), BD DIFCO (BD Biosciences), HYPEP YE (Kerry Group Services Ltd.), ULTRAPEP YE (Kerry Group Services Ltd.), HY-YEST 412 (Kerry Group Services Ltd.), HY-YEST 441 (Kerry Group Services Ltd.), HY-YEST 444 (Kerry Group Services Ltd.), HY-YEST 455 (Kerry Group Services Ltd.), or HY-YEST 504 (Kerry Group Services Ltd.).

11 . The medium of any one of claims 1 - 10 , wherein the concentration of yeast extract is between about 1 g/L to about 50 g/L.

12 . The medium of claim 11 , wherein the concentration of yeast extract is between about 5 g/L to about 25 g/L.

13 . The medium of claim 12 , wherein the concentration of yeast extract is about 10 g/L.

14 . The medium of any one of claims 1 - 13 , wherein the carbon source is selected from the group consisting of glucose, dextrose, mannitol, lactose, sucrose, fructose, galactose, raffinose, xylose, and mannose.

15 . The medium of claim 14 , wherein the carbon source is glucose.

16 . The medium of any one of claims 1 - 15 , wherein the concentration of the carbon source is between about 25 g/L to about 100 g/L.

17 . The medium of claim 16 , wherein the concentration of the carbon source is between about 50 g/L to about 90 g/L.

18 . The medium of claim 17 , wherein the concentration of the carbon source is about 80 g/L.

19 . The medium of any one of claims 1 - 18 , wherein the medium comprises soy hydrolysate, an ultrafiltered yeast extract, and glucose.

20 . The medium of any one of claims 1 - 19 , wherein the medium further comprises a phosphate-containing ingredient.

21 . The medium of claim 20 , wherein the phosphate-containing ingredient is Na 2 HPO 4 , K 2 HPO 4 , or KH 2 PO 4 .

22 . The medium of any one of claims 1 - 21 , wherein the medium further comprises at least one amino acid, vitamin, nucleoside, or inorganic salt.

23 . A polysaccharide-producing bacterial cell culture medium having a total amino acid concentration greater than about 50 mM.

24 . The medium of claim 23 , wherein the medium comprises a total glycine concentration of between about 1.5 mM and about 60.0 mM.

25 . The medium of claim 24 , wherein the total glycine concentration is between about 5.0 mM and about 15.0 mM.

26 . The medium of claim 25 , wherein the total glycine concentration is about 7.5 mM.

27 . The medium of any one of claims 23 - 26 , wherein the medium comprises a total arginine concentration of between about 1.0 mM and about 30.0 mM.

28 . The medium of claim 27 , wherein the total arginine concentration is between about 1.0 mM and about 20.0 mM.

29 . The medium of claim 28 , wherein the total arginine concentration is about 4.0 mM.

30 . The medium of any one of claims 23 - 29 , wherein the medium comprises a total cysteine concentration of between about 0.1 mM and about 5.0 mM.

31 . The medium of claim 30 , wherein the total cysteine concentration is between about 0.1 mM and about 3.5 mM.

32 . The medium of claim 31 , wherein the total cysteine concentration is about 0.4 mM.

33 . The medium of any one of claims 23 - 32 , wherein the medium comprises a total serine concentration of between about 5.0 mM and about 75.0 mM.

34 . The medium of claim 33 , wherein the total serine concentration is between about 5.0 mM and about 15.0 mM.

35 . The medium of claim 34 , wherein the total serine concentration is about 7.5 mM, or about 10 mM.

36 . The medium of any one of claims 23 - 35 , wherein the medium comprises a total glutamine concentration of between about 1.0 mM and about 30.0 mM.

37 . The medium of claim 36 , wherein the total glutamine concentration is between about 1.0 mM and about 20.0 mM.

38 . The medium of claim 37 , wherein the total glutamine concentration is about 4.0 mM.

39 . The medium of any one of claims 23 - 38 , wherein the medium comprises a total concentration of tyrosine of between about 0.1 mM and about 5.0 mM.

40 . The medium of claim 39 , wherein the total tyrosine concentration is between about 1.0 mM and about 3.5 mM.

41 . The medium of claim 40 , wherein the total tyrosine concentration is about 2.9 mM or about 3.0 mM.

42 . The medium of any one of claims 23 - 41 , wherein the medium comprises a total concentration of asparagine of between about 5.0 mM and about 50.0 mM.

43 . The medium of claim 42 , wherein the total asparagine concentration is between about 10.0 mM and about 30.0 mM.

44 . The medium of claim 43 , wherein the total asparagine concentration is about 20.0 mM.

45 . The medium of any one of claims 23 - 41 , wherein the medium does not contain asparagine.

46 . The medium of any one of claims 23 - 45 , wherein the medium further comprises a potassium salt.

47 . The medium of claim 46 , wherein the potassium salt is potassium chloride or potassium sulfate.

48 . The medium of claim 46 or claim 47 , wherein the total concentration of potassium salt is between about 0.1 g/L and about 25 g/L.

49 . The medium of claim 48 , wherein the total potassium salt concentration is between about 0.2 g/L and about 1.25 g/L.

50 . The medium of claim 49 , wherein the total potassium salt concentration is about 0.9 g/L.

51 . The medium of any one of claims 23 - 50 , wherein the medium further comprises a carbon source.

52 . The medium of claim 51 , wherein the carbon sources is selected from the group consisting of glucose, dextrose, mannitol, lactose, sucrose, fructose, galactose, raffinose, xylose, and mannose.

53 . The medium of claim 52 , wherein the carbon sources is glucose.

54 . The medium of any one of claims 51 - 53 , wherein medium comprises a total concentration of the carbon source of between about 25 g/L and about 100 g/L.

55 . The medium of claim 54 , wherein the total concentration of the carbon source is between about 25 g/L and about 80 g/L.

56 . The medium of claim 55 , wherein the total concentration of the carbon source is about 50 g/L.

57 . The medium of any one of claims 23 - 56 , wherein the medium further comprises sodium bicarbonate.

58 . The medium of claim 57 , wherein the medium comprises a concentration of sodium bicarbonate of between about 0.1 g/L and about 20 g/L.

59 . The medium of claim 58 , wherein the concentration of sodium bicarbonate is between about 0.5 g/L and about 1.0 g/L.

60 . The medium of claim 59 , wherein the concentration of sodium bicarbonate is about 0.84 g/L.

61 . The medium of any one of claims 23 - 60 , wherein the medium further comprises a yeast extract.

62 . The medium of claim 61 , wherein the yeast extract is selected from the group consisting of a yeast autolysate, an ultrafiltered yeast extract, and a synthetic yeast extract.

63 . The medium of claim 62 , wherein the yeast extract is an ultrafiltered yeast extract.

64 . The medium of claim 63 , wherein the ultrafiltered yeast extract is AMBERFERM 5902 (Sensient Technologies Corp.), BD DIFCO (BD Biosciences), HYPEP YE (Kerry Group Services Ltd.), ULTRAPEP YE (Kerry Group Services Ltd.), HY-YEST 412 (Kerry Group Services Ltd.), HY-YEST 441 (Kerry Group Services Ltd.), HY-YEST 444 (Kerry Group Services Ltd.), HY-YEST 455 (Kerry Group Services Ltd.), or HY-YEST 504 (Kerry Group Services Ltd.).

65 . The medium of any one of claims 61 - 64 , wherein the concentration of yeast extract is between about 1 g/L to about 50 g/L.

66 . The medium of claim 65 , wherein the concentration of yeast extract is between about 5 g/L to about 25 g/L.

67 . The medium of claim 66 , wherein the concentration of yeast extract is about 10 g/L.

68 . The medium of any one of claims 23 - 67 , wherein the medium comprises at least about 50 mM of amino acids, a potassium salt, a carbon source, and optionally, a yeast extract.

69 . The medium of claim 68 , wherein the medium comprises at least about 50 mM of amino acids, between about 5.0 mM and about 15.0 mM of glycine, between about 0.2 g/L and about 1.25 g/L of a potassium salt, between about 25 g/L and about 80 g/L of a carbon source, and between about 5 g/L to about 25 g/L of a yeast extract.

70 . The medium of claim 69 , wherein the medium comprises at least about 60 mM of amino acids, about 7.5 mM of glycine, about 0.9 g/L of potassium chloride, 50 g/L of glucose, and about 10 g/L of an ultrafiltered yeast extract.

71 . A method of cultivating a polysaccharide-producing bacteria comprising a) adding a medium of any one of claims 1 - 70 to a bioreactor, b) seeding the medium with a polysaccharide-producing bacteria, and c) cultivating the bacteria by fermentation, wherein said cultivation comprises the addition of a nutrient at a constant rate to the medium.

72 . The cultivation method of claim 71 , wherein the nutrient is a carbon source.

73 . The cultivation method of claim 72 , wherein the carbon source is glucose.

74 . The cultivation method of any one of claims 71 - 73 , wherein the cultivated bacteria have a cell density of at least 9.0.

75 . The cultivation method of any one of claims 71 - 74 , wherein the cultivated bacteria have a polysaccharide concentration of at least about 250 mg/L.

76 . The cultivation method of any one of claims 71 - 75 , wherein the polysaccharide-producing bacteria is selected from the group consisting of Streptococcus agalactiae, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria meningitidis, Escherichia coli, Salmonella typhi, Haemophilus influenzae, Klebsiella pneumoniae, Enterococcus faecium , and Enterococcus faecalis.

77 . A method of cultivating a polysaccharide-producing bacteria comprising a) adding a medium of any one of claims 1 - 70 to a bioreactor, b) seeding the medium with a polysaccharide-producing bacteria, and c) cultivating the bacteria by perfusion, wherein the cultivation comprises (i) removing spent medium from the culture, (ii) adding fresh medium, and (iii) retaining the bacteria.

78 . The cultivation method of claim 77 , wherein the rate of perfusion is between about 0.07 VVH to about 2.00 VVH.

79 . The cultivation method of claim 78 , wherein the rate of perfusion is between about 0.67 VVH to about 1.33 VVH.

80 . The cultivation method of claim 79 , wherein the rate of perfusion is about 1.20 VVH.

81 . The cultivation method of claim 77 , wherein the rate of perfusion is varied.

82 . The cultivation method of claim 81 , wherein the perfusion starts at a first rate and the rate is increased to a second rate.

83 . The cultivation method of claim 81 , wherein the perfusion starts at a first rate and the rate is decreased to a second rate.

84 . The cultivation method of any one of claims 77 - 83 , wherein the duration of perfusion is between about 1 hour and about 15 hours.

85 . The cultivation method of claim 84 , wherein the duration of perfusion is between about 1 hour and about 10 hours.

86 . The cultivation method of claim 85 , wherein the duration of perfusion is about 7 hours.

87 . The cultivation method of any one of claims 77 - 86 , wherein the cell growth of the cultivated bacteria is at least 2-fold greater than the cell growth in a batch fermentation system.

88 . The cultivation method of any one of claims 77 - 87 , wherein the cultivated bacteria have reached a cell density of at least 20.0.

89 . The cultivation method of any one of claims 77 - 88 , wherein the cultivated bacteria have reached a polysaccharide concentration of at least about 600 mg/L.

90 . The cultivation method of any one of claims 77 - 89 , wherein wherein the polysaccharide-producing bacteria is selected from the group consisting of Streptococcus agalactiae, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria meningitidis, Escherichia coli, Salmonella typhi, Haemophilus influenzae, Klebsiella pneumoniae, Enterococcus faecium , and Enterococcus faecalis.