Vitamins

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Vitamins

  1. 1. Industrial production of Vitamins Gisha G P S3 Biotechnology 1
  2. 2. • Vitamin B12 • Vitamin B2 • Vitamin C • proVitamin A 2Industrial production of vitamins
  3. 3. introduction • Vitamins are defined as essential micronutrients that are required in trace quantity and are very important compounds in diet • Synthesized by prototropic microorganisms • Microbes excrete vitamins in excess of their metabolic needs under highly specific and artificial condition 3Industrial production of vitamins
  4. 4. Cyanocobalamin (Vitamin B 12) Industrial production of vitamins 4
  5. 5. • Cyanocobalamin, by definition vitamin B12, is the industrially produced stable cobalamin form which is not found in nature. • Vitamin B12 is obtained exclusively by fermentation process. • Important dietary component, requirement 0.001 mg/day. • Cyanocobalamin consist of a cobinamide linked to a nucleotide. • Cobinamide – cobalt linked to cyanide grp, surrounded by 4 reduced pyrrole ring. • Nucleotide – 5 , 6 – dimethyl benziminazole 5Industrial production of vitamins
  6. 6. 6Industrial production of vitamins
  7. 7. Micro –organisms in industrial production of vit. b12 • Streptomyces griseus , S. olivaceus , Bacillus megaterium , B. coagulans , Pseudomonas denitrificans , Propionibacterium freudenreichii , P. shermanii and a mixed fermentation of a Proteus spp and a Pseudomonas sp. 7Industrial production of vitamins
  8. 8. • Manufactured by submerged fermentation • Aeration and agitation of medium essential • Fermentation process completed in 3 to 5 days 8Industrial production of vitamins
  9. 9. Vit.B12 production using Streptomyces olivaceus NRRL B-1125 Industrial production of vitamins 9
  10. 10. Preparation of inoculum Industrial production of vitamins 10 • Pure slant culture of Streptomyces olivaceus NRRL B-1125 is inoculated and grown in 100 to 250 ml of inoculum medium. • Seeded flask are kept on shaker for incubation . • Flask cultures are used to inoculate large amount of inoculum media arranged in series of tank . • 2 or 3 successive transfers are made to obtain required amount of inoculum cultures. • Inoculum of production tank must be 5% of the volume of production medium
  11. 11. Industrial production of vitamins 11
  12. 12. Production medium • Consist of carbohydrate ,proteinaceous material , and source of cobalt and other salts . • Sterilization of medium batchwise or continuously . • Batch – medium heated at 250°F for 1 hr • Continuous – 330°F for 13 min by mixing with live steam. 12Industrial production of vitamins Components Amount ( %) Distillers solubles 4.0 Dextrose 0.5 to 1 CaCO3 0.5 COCl2.6H2O 1.5 to 10 p.p.m.
  13. 13. Temperature , pH , aeration and agitation • Temperature : 80°F • pH : At starting of process pH falls due to rapid consumption of sugar, then rises after 2 to 4 due to lysis of mycelium pH 5 is maintained with H2SO4 and reducing agent Na2SO4 . • Aeration and agitation : Optimum rate of aeration is 0.5 vol air/vol medium/min. Excess aeration cause foaming. 13Industrial production of vitamins
  14. 14. Antifoam agent , prevention of contamination • Antifoam agent : soya bean oil , corn oil, lard oil and silicones (sterilized before adding) . • Prevention of contamination : essential to maintain sterility , contamination results in reduced yields , equipments must be sterile and all transfers are carried out under aseptic conditions . 14Industrial production of vitamins
  15. 15. Yields • Yield of cobalamin are usually in the range of 1 to 2 mg. per litre in the fermented broth 15Industrial production of vitamins
  16. 16. Recovery • Cobalamin associated with mycelium- boiling mixture at pH 5 liberates the cobalamin quantitatively from mycelium. • Broth containing cobalamin is subjected to further work up depending on type of product to be produced 16Industrial production of vitamins
  17. 17. RecoveRy contd…. • Filtration - to remove mycelium. • Filtered broth treated with cyanide – (cobalamin to cyanocobalamin). • Adsorption chromatography , ion exchange chromatography – adsorbents : activated charcoal , bentonite , fuller’s earth . Industrial production of vitamins 17 Bentonite Fuller’s earth
  18. 18. • Elution : water, water-acetone and solution of sodium cyanide or sodium thiocyanate . • further extraction – countercurrent distribution b/w cresol, amyl phenol or benzyl alcohol and water or single extraction into organic solvent (phenol) Industrial production of vitamins 18
  19. 19. RecoveRy contd…. • To aqueous concentrates , dissolve a Zn salt in a slight acidic solution & then rise the pH to bring about precipitation of ZnOH(impurities are removed) . • Chromatography on alumina & crystallization from methanol-acetone , ethanol-acetone, or acetone-water. Industrial production of vitamins 19
  20. 20. RecoveRy contd…. • To use as feed supplement , final fermented broth is evaporated to dryness. • Final broth contain 3% solids – in vacuo evaporation (15 to 20 % solid content). • Syrup – drum dried or spray dried.(contain 10 to 30 mg.lb. of cobalamin) Industrial production of vitamins 20
  21. 21. Vit.B12 production using Propionibacterium freudenreichii Industrial production of vitamins 21
  22. 22. • Reported to produce the highest yield s of Vit. B12 20 mg. per litre. • Production media : glucose , corn- steep , betaine , & cobalt Betaine -0.5 % Cobalt – 5µg./ml (excess cause reduced cobalamin formatn) • pH -7.5 • Temperature – 30°C Industrial production of vitamins 22
  23. 23. fermentation • 2 cycles : anaerobic 70 hrs ; aerobic 50 hrs. • Anaerobic cycle – formation of cobinamide (O2 inhibits the process). • Necessary to add 0.1% of 5, 6 – dimethyl benziminazole ( cannot readily synthesize ). • Aerobic cycle – nucleotide links with previously formed cobinamide . • Continuous culture – 2 tanks in series , retention period 60 hrs , yield 15µg./ml is obtained . Industrial production of vitamins 23
  24. 24. Vit.B12 production using other micro - organisms Industrial production of vitamins 24
  25. 25. Industrial production of vitamins 25 Species Medium Aeration Temp. (°C) Time (hr) Yield (mg./ltr) B. megaterium Molases , mineral salts, cobalt Aerobic 30 18 0.45 P. shermanii Glucose , corn- steep, ammonia , cobalt pH 7.0 Anaerobic(3 days) , aerobic (4 days) 30 150 23 B. coagulants Citric acid , triethanolamine , corn – steep , cobalt. Aerobic 55 18 6.0 P. denitrificans Oxalic acid , betaine , cobalt , mineral salts Aerobic ___ ___ 10
  26. 26. Industrial production of vitamins 26
  27. 27. Riboflavin (Vitamin B 2) Industrial production of vitamins 27
  28. 28. • Another important vitamin. • Require 0.07 – 0.1 mg./ 0.75 kg of body wt. in humans. • Recently, fermentation route has been widely used as it produces the vitamin in a single step, resulting in substantial cost savings. Industrial production of vitamins 28
  29. 29. Micro –organisms in industrial production of vit. b2 • Although bacteria (Clostridium sp.) and yeasts (Candida sp.) are good producers, two closely related ascomycete fungi, Eremothecium ashbyii and Ashbya gossypii, are considered the best riboflavin producers. • Ashbya gossypii produces 40 000 times more vitamin than it needs for its own growth. Industrial production of vitamins 29
  30. 30. Industrial production of vitamins 30 Eremothecium ashbyii Ashbya gossypii
  31. 31. Other micro -organisms producing Vit. B 2 • Gene amplification and substitution of wild type promoters and the regulatory regions with strong constitutive promoter from Bacillus subtilis phage SPO 1 have resulted in increased riboflavin production . The B. subtilis riboflavin-producing strain developed by Perkins et al. • Sybesma et al. developed Lactococcus lactis MG 1363 strain using both direct mutagenesis and metabolic engineering for simultaneous overproduction of both folate and riboflavin • Improved strains for the production of riboflavin were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes Industrial production of vitamins 31
  32. 32. Preparation of inoculum • Starts from slants or spores dried on sand. • After 1 or 2 stages, further propagation is carried on 1 or 2 tank inoculum stages. Industrial production of vitamins 32
  33. 33. Production medium • Fermentor 10,000 to 1,00,000 gals range. • Production medium designed according to type of micro- organism. • Ashbya gossypii : sources - palm oil ,corn steep liquor, glucose, molasses , whey, collagen , soya oil , glycine. • Stahmann et al. reported riboflavin yields in excess of 15 g/L of culture broth in a sterile aerobic submerged fermentation of Ashbya gossypii with a nutrient medium containing molasses or plant oil as major carbon source. Industrial production of vitamins 33
  34. 34. • Ertrk et al. studied fermentative production of riboflavin by Ashbya gossypii in a medium containing whey. • The quantities of riboflavin produced by Ashbya gossypii in whey with different supplements Industrial production of vitamins 34 Supplement Quantity of riboflavin (mg./L) Bran 389.5 Glycine + peptone 120 Sucrose 87.5 Glycine 78.3 Yeast extract 68.4 Peptone 23.2 Soyabean oil 17.5
  35. 35. • For Eremothecium ashbyii - still slops from alcohol industry with skim milk , soya bean meal or casein (protein source) , maltose / sucrose/ glucose(carbohydrate source). • low cost organic wastes as flavinogenic factors and the various concentrations at which they induced flavinogenecity resulting in higher yields of riboflavin (Kalingan and Liao, 2002). • Organic wastes like beef extract, hog casings, blood meal or fish meal supported the production of riboflavin from Eremothecium ashbyii NRRL 1363. • Recent studies with wild type of E. ashbyii have yielded 3.3 g/L of riboflavin using molasses and peanut seed cake as carbon and nitrogen source, respectively (Kalingan, A. E. and M. R. V. Krishnan ,1997) Industrial production of vitamins 35
  36. 36. conditions • pH : 6 to 7.5 • Temperature : 26 to 28 °C • Fermentation : submerged aerated fermentation • Fermentation time : 96 to 120 hrs • Aeration & agitation required. • Yield : 3 to 6 g or more / litre Industrial production of vitamins 36
  37. 37. recovery • 2 steps (for animal feed supplements) i. Evaporation of the whole broth in multiple effect evaporators ii. Drying by drum-drier / spray drier Industrial production of vitamins 37
  38. 38. RecoveRy contd… • For drug & fine food use i. Heating of fermentation broth at 120°C for 1 hr. ii. Filtration. iii. Precipitation by adding dithionite (hydrosulfite). iv. Other purification steps – crystallization. Industrial production of vitamins 38
  39. 39. Industrial production of vitamins 39
  40. 40. Ascorbic acid (Vit c) Industrial production of vitamins 40
  41. 41. • L-ascorbic acid is an important metabolite for most living organisms. • Strong reducing agent and plays a part in cellular oxidation – reduction reactions. • Daily intake : 60mg for persons aged 15 & above 50mg for age group b/w 11 to 14 45mg for children under 11 Industrial production of vitamins 41
  42. 42. • L-ascorbic acid finds its use mainly in food industry, being a vitamin as well as an antioxidant. • Approximately 50 % of synthetic ascorbic acid is used in vitamin supplements and pharmaceutical preparations. Industrial production of vitamins 42
  43. 43. • At present the majority of commercially manufactured L-ascorbic acid is synthesized via Reichstein process using D-glucose as a starting material. • Reichstein process involves six chemical steps and one fermentation step for oxidation of D-sorbitol to L-sorbose . • Bacterium used for this bioconversion is Acetobacter suboxydans. • Very high level of aeration is required. Industrial production of vitamins 43
  44. 44. Industrial production of vitamins 44
  45. 45. Industrial production of vitamins 45 • With higher sorbitol concentration fermentation is slower in the beginning. • Production medium is inoculated with culture of Acetobacter suboxydans (3 % of total medium volume) • Complete fermentation cycle takes 25 – 30 hrs. Ingredients Amount D- Sorbitol 200 -250 g. Corn –steep liquor 5 ml. Tap water 1 ltr. Antifoam as required
  46. 46. Fermentor (for vit.c production) • Vertical fermentors , ( 5,000 to 10,000 gal. capacity) • Designed for contain large volumes of air . • Fermentors are equipped with a turbo mixer type agitator & baffles along the side walls to ensure maximal turbulence of the solution. • Air inlet underneath the agitator. Industrial production of vitamins 46
  47. 47. • In commercial operations , high velocity jets & 30 lb. head pressure , used for maximal rate of fermentation , air to solution ratio is 1:3 • Require provision for cooling the fermentor along with medium , cooling achieved by circulating cold water either through coils or through jackets. Industrial production of vitamins 47
  48. 48. yield • Analytical yields are about 95% – 98%. • Recovery yields are about 85%. Industrial production of vitamins 48
  49. 49. Other microbes involved in ascorbic acid production • Glucanobacter oxydans , Acetobacter melanogenus , Pseudomonas albosesamae , Bacterium gluconicum , Brevibacterium ketosoreductum • Saccharomyces cerevisiae and Zygosaccharomyces bailii accumulate L- ascorbic acid intracellularly when incubated with L-galactose. • Skatrud and Huss described the method for the efficient production of L-ascorbic acid in algae ,Chlorella pyrenoidosa ATCC 53170 Industrial production of vitamins 49
  50. 50. ProVitamin a Industrial production of vitamins 50
  51. 51. • β-carotene (provitamin A) , occurs naturally as a component of green plants. • β-carotene after ingestion, is converted into vitamin A in the animal body. Industrial production of vitamins 51
  52. 52. Micro –organisms in industrial production • Members of Choanephoraceae family of Phycomycetes. • Extensive studies on Phycomyces blakesleeanus , Choanephora cucurbitarum , and Blakeslea trispora . • Production of β-carotene by wild-type Phycomyces blakesleeanus can be stimulated by light, chemicals, regulatory mutations, and sexual interaction between mycelia of opposite sex. Industrial production of vitamins 52
  53. 53. Industrial production of vitamins 53 Phycomyces blakesleeanus Choanephora cucurbitarum Blakeslea trispora - phase contrast image
  54. 54. • Barnett , Lilly , and Krause (1956) discovered increased yields of β- carotene results with Choanephora cucurbitarum if both plus and minus mating types are grown together . • Similar phenomenon with Blakeslea trispora (Hesseltine and Anderson , 1957) Industrial production of vitamins 54
  55. 55. Fermentation medium • Includes β – ionone, non ionic detergent , vegetable oil , fats , fatty acids , waxes, or white grease. • β – ionone is regarded as a steering factor activates enzyme responsible for β- carotene formation. • Inoculum of each mating type is grown separately , & apprx. 5% of each is added to fermentation medium. • β – ionone is added 48 hrs after inoculation. Industrial production of vitamins 55
  56. 56. Industrial production of vitamins 56 Coriolus versicolor in submerged culture Food-grade pigments from Streptomyces sp. isolated from the marine sponge Callyspongia diffusa
  57. 57. Industrial production of vitamins 57 Carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae
  58. 58. India based biotech companies in vitamin manufacturing • The manufacturing set up of Fermenta Biotech Limited located at Kullu in Himachal Pradesh and Dahej in Gujarat are modern facilities that cater to worldwide supplies of various grades of Vitamin D3 products and other specialty APIs. • Jubilant Life Sciences Limited, headquarters in Noida, New Delhi , an integrated pharmaceutical and life sciences company, is the largest Custom Research and Manufacturing Services (CRAMS) player and a leading Drug Discovery and Development Solution (DDDS) provider out of India. Globally No. 2 in Niacin & Niacinamide / Vitamin B3-Nutrition Ingredients Industrial production of vitamins 58
  59. 59. Reference • Biotechnological Production of Vitamins , S.A. SURVASE et al.: Production of Vitamins, Food Technol. Biotechnol. 44 (3) 381–396 (2006) • Microbial Production of Riboflavin Using Riboflavin Overproducers, Ashbya gossypii, Bacillus subtilis, and Candida famate: An Overview ,Seong Han Lim1, Jong Soo Choi2, and Enoch Y. Park1* , Biotechnol. Bioprocess Eng. 2001, 6: 75-88 • Carotene-Super producing Strains of Phycomyces , F. J. Murillo et.al, Applied and Environmental Microbiology, Nov. 1978, p. 639-642 • High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous • Industrial Microbiology by A H Patel • Industrial Microbiology by Casida 59Industrial production of vitamins
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