Vitamins

Industrial production of Vitamins

Gisha G PS3 Biotechnology

1

• Vitamin B12• Vitamin B2• Vitamin C

• proVitamin A

2Industrial production of vitamins

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


Cyanocobalamin(Vitamin B 12)

Industrial production of vitamins 4

• 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


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.


• Manufactured by submerged fermentation

• Aeration and agitation of medium essential

• Fermentation process completed in 3 to 5 days


Vit.B12 production using Streptomyces olivaceus

NRRL B-1125


Preparation of inoculum


• 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

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.

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.


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 .


Yields

• Yield of cobalamin are usually in the range of 1 to 2 mg. per litre in the fermented broth


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


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 .


Bentonite Fuller’s earth


• 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)

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

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)


Vit.B12 production using Propionibacterium

freudenreichii


• 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


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 .


Vit.B12 production using

other micro - organisms



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

Riboflavin (Vitamin B 2)


• 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.


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.



Eremothecium ashbyii Ashbya gossypii

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


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.


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.


• 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


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

• 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)


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


recovery• 2 steps (for animal feed

supplements)i. Evaporation of the whole broth in

multiple effect evaporatorsii. Drying by drum-drier / spray drier


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.


Ascorbic acid (Vit c)


• 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


• 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.


• 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.



• 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

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

• 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

yield

• Analytical yields are about 95% – 98%.

• Recovery yields are about 85%.


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


ProVitamin a


• 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.


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.



Phycomyces blakesleeanus

Choanephora cucurbitarum

Blakeslea trispora - phase contrast image

• 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)


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.



Coriolus versicolor in submerged culture

Food-grade pigments from Streptomyces sp. isolated from the marine sponge Callyspongia diffusa


Carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae

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


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


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