PRODUCTION OF VITAMIN A

By
Suman Rai
Dept of Microbiology
Central Campus of Technology
 The fat soluble vitamin A, as such is present only in
foods of animal origin.
 However, its provitamins carotenes are found in
plants.
 Retinol, retinal and retinoic acid are regarded as
vitamers of vitamin A.
1. Retinol (vitamin A alcohol) :
 lt is a primary alcohol containing β-ionone ring. The
side chain has two isoprenoid units, four double bonds
and one hydroxyl group.
 Retinol is present in animal tissues as retinyl ester
with long chain fatty acids.
2. Retinal (vitamin A aldehyde) :
 This is an aldehyde form obtained by the
oxidation of retinol. Retinal and retinol are
interconvertible.
 Previously, the name retinine was used for
retinal.
3. Retinoic acid (vitamin A acid) :
 This is produced by the oxidation of retinal.
However, retinoic acid cannot give rise to the
formation of retinal or retinol.
4. p-Carotene (provitamin A) :
 This is found in plant foods.
 lt is cleaved in the intestine to produce two
moles of retinal.
 ln humans, this conversion is inefficient, hence
p-carotene possessesa bout one-sixth vitamin
A activity compared to that of retinol.
 Carotenoids are found in animals and plant
tissues.
 However, originate extensively in plant or
microbes.
 β-carotene (provitamin A) is converted into
vitamin A in the intestinal mucous membrane
and store in the liver as palmitate ester.
 It (β-carotene) is primarily use as a food
coloring agent such as lycopene or
xanthophylls (do not contain provitamin A).
 Carotenoids are highly unsaturated isoprene derivatives.
 Naturally occurring carotenoids are tetraterpenoids
consisting of 8 isoprene residues.
 When cultures of both sexual forms are mixed, a
significant increase in carotene production occur by
microbial fermentation.
 Only compounds with the β-ionone strucrure (ring str)
are effective as provitamin A.
 Thus, 2 molcules of Vit A can be formed from β-
carotene.
 Only 1 molecule of Vit A can form from α & γ carotene
since they have 1 β-ionone ring.
 Production process for β-carotene (Ninet and Renaut,
1979) is describe below;
 Production process for β-carotene
Blakeslea trispora B. trispora Inoculum storage: Spores in sterile soil.
NRRL 2456 (+) NRRL 2457 (-)

Culture on Culture on
agar slant agar slant Incubation: 168 hr/27OC

Preculture Preculture 2L Erlenmeyer flask wth 400 ml Medium A.

Incubation: 48 hr/26OC, shaker

Mix Preculture
170 L fermenter with 120 L mediumA.
Inoculum: 400ml of each preculture.
Production culture Incubation: 40hr/26OC; stirring 170 rpm;
aeration 1.1vvm
800 L fermenter with 320 L medium B.
sterilization 55 min, 122OC. Inoculum: 32L of
Mix Preculture. Incubation: 185 hr/26 OC;
stirring 210 rpm; aeration 1.3vvm
Medium A (g/L): Corn steep liqour-70; Corn
starch-50; KH2PO4-0.5; MnSO4.H2O-0.1;
Medium B(g/L) : Distiller’s soluble -70; Corn
starch-60; Soybean meal-30; Cottonseedoil-30;
Antioxidant-0.35; MnSO4.H2O-0.2;
ThiaminHCl-0.5; Isoniazid-0.6; Kerosene-20ml;
Tap water; pH-6.3.
 Isoniazid and Kerosene are sterilized separately.
After 48 hr, add 1g/L β-ionone and 5 ml/L
kerosene are added; glucose feeding (Total
addition 42g/L) until the end of the fermentation.
 when cultures of Blakeslea trispora of both
sexual forms (+ &- strains) are mixed, a
significant increase in carotene production in
the (-) strain is achieved .
 Production is induced by trisporic acids, a
mixture of closely related substances.
 Trisporic acids are not precursors of β-carotene
but rather they act as (+) gamones (sexual
hormones).
 They are derived biosynthetically from β-
carotene.
 The production of β-carotene is carried out by
sub-merged fermentation using a mixed culture
of (+) & (-)strains.
 The propertion of two strains need not be equal
and since it is the (-)strains which produces the
β-carotene.
 Another activator of β-carotene is isoniazid,
particularly in combination with β-ionone.
 β-ionone alone is toxic to the production
organism, but in the presence of plant oils it
promotes carotene production.
 β-ionones themseves are not incorporated into
carotene but affect the synthesis of various of
the enzymes involved in the biosynthetic
pathway.
 They can be replaced by terpenes or
cyclohexanone and their trimethyl derivatives.
 The addition of purified kerosene to the
medium doubles the yield by increasing the
solubility of the hydrophobic substrate.
 Antioxidant is added for the fermentation
process due to the low solubility of β-carotene.
 Product Recovery
 The mycelium is removed from the mixture.
 It is then dehydrated with methanol.
 IT is extracted with methylene chloride (75-
92% yield).
 The crude product is further purified by
chromatography separation.
 The agar is ground and washed continuously
with alcohol for 72 hours, followed by ether
for the same length of time.
 The peptone was washed with ether only.
 Cultures were incubated at room temperature
in total darkness.
 The organism used was an unidentified
diphtheroid, Corynebacterium sp.