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PSGR krishnammal college for Women,
• vitamin B12, vitamin B12 or vitamin B-12, also
called cobalamin, is a water-soluble vitamin with a
key role in the normal functioning of the brain and
nervous system, and for the formation of blood. It is
one of the eight B vitamins.
• It is normally involved in the metabolism of every
cell of the human body, especially affecting DNA
synthesis and regulation, but also fatty acid synthesis
and energy production.
• Vitamin B12 is important for the way the body
works, and people who don't have enough of it may
feel tired or have a lack of energy.
• Vitamin B12 helps in the production of healthy
red blood cells that carry oxygen around the
• Not having enough vitamin B12 is called
vitamin B12 deficiency anaemia.
• This condition makes the body produce larger
than normal red blood cells, described as
megaloblastic or macrocytic, which don't do
their job as well.
• Once diagnosed, vitamin B12 deficiency can
usually be treated successfully with B12
injections and sometimes with B12 tablets.
• Species from the following genera are known to synthesize
B12: Acetobacterium, Aerobacter, Agrobacterium, Alcaligenes,
Azotobacter, Bacillus, Clostridium, Corynebacterium,
Flavobacterium, Lactobacillus, Micromonospora,
Mycobacterium, Nocardia, Propionibacterium,
Protaminobacter, Proteus, Pseudomonas, Rhizobium,
Salmonella, Serratia, Streptomyces, Streptococcus and
• Industrial production of B12 is through fermentation of
selected microorganisms. Streptomyces griseus, a bacterium
once thought to be a yeast, was the commercial source of
vitamin B12 for many years. The species Pseudomonas
denitrificans and Propionibacterium shermanii are more
commonly used today
Methylcobalamin (shown) is a form of vitamin
B12. Physically it resembles the other forms of
vitamin B12, occurring as dark red crystals that
freely form cherry-colored transparent solutions
Minot and Murphy -reported -liver juice, in
Rickets and Smith -isolated from liver cells of
animals in 1942.
Rikes - Microbial source of Cyancobalamine
demonstrated in 1948. – Streptomyces griesus
Steps involved in Microbial Cyanocobalamine production
For Media preparation
Carbon source- as
1.Corn steep glucose
2. Beet molasses
• Nitrogen source-as
• 1.Ammonium phosphate
• 2. Ammonium hydroxide
Step-1 Formulation of medium
Medium+ Cobalt salt
Starter culture of
Propioni bacterium shermanii
Anaerobic fermentation for 3
Aerobic fermentation for 4
Acid treatment and heating
Released Pseudo-vitamin B12
Released Pseudo-vitamin B12
Addition of cyanide solution
Cyanocobalamine in liquid
Adsorption on IRC-50 resin
Elution with a phenolic
Cyanocobalamine in solvent
• Prepared medium is sterilized by autoclaving
• The sterilized medium is then used for fermentation.
3.Making Starter culture
The following microbes were suitable for Industrial fermentation of
2. Streptomyces olivaceous
4. Pseudomonas denitrificans
Mutant strains of--- Inoculum
Improved strain- produce 50,000
times more vitamin B12 than wild
strain .This is the great boon for
biotechnologists to produce vit B12.
• Most fermentations are batch processes
• Nutrients and the inoculum are added to the sterile fermenter and left
to get on with it!
• Anti-foaming agent may be added.
• Once the desired amount of product is present in the fermenter the
contents are drained off and the product is extracted.
• After emptying, the tank is cleaned & prepared for a new batch.
• Some products are made by a continuous
• Sterile medium is added to the fermentation
with a balancing withdrawal of broth for
• UPSTREAM PROCESSING
• Upstream processing encompasses any
technology that leads to the synthesis of a
product. Upstream includes the exploration,
development and production.
• DOWNSTREAM PROCESSING
The extraction and purification of a
biotechnological product from fermentation is
referred to as downstream processing
• It is a Batch fermentation
• Continuous fermentation also found to be effective.
• Sterilized medium is filled in stirred tank fermenter
1% of inoculum (Starter culture) is added in to the
• Anaerobic condition is maintained to encourage the
production of 5,6-dimethyl benzimidazole
cobalamide (DBC) by P.shermanii
• Anaerobic Fermentation is over sterile air is pumped
in to the fermenter.
• The culture is stirred well for proper aeration
• Aerobic fermentation is performed for 4 days.
• During this process some amount of DBC and pseudo
vitaminB12 (adeninyl cobalamine) are produced.
• DBC and pseudovitaminB12 (adeninyl cobalamine)
are immediate precursors of cyanocobalamine
Recovery of Cyanocobalamine
• Inside the microbial cells the cyanocobalamine exits in the
form of natural substances such as DBC and
• The cultured broth contains 10-23mg vitB12 per liter.
• It is harvested and centrifuged at high speed to get a
concentrated mass of cells.
• The cell mass is treated with a dilute acid and heat stock at
• During this treatment precursors of pseudovitaminB12 are
• Then it is treated with Cyanide solution to split the DBC and
pseudovitamin B12 .
• As a result cyanocobalamine (Vit B12) is released free in the
• Cyanocobalamine in the liquid is seperated by using an
adsorption column chromatography with IRC-50 resin. The
adsorbed cyanocobalamine is then eluted out of the column
using phenolic solvent.
• The solvent fraction is evaporated by exposing it to
• As result crystals of cyanocobalamine is let in the vessel. It is
stored for future use.
• It is a food preservative
• It is a co-factor
• It is a protective medicine