Production of Penicillin, Citric Acid, Vit B12, Glutamic Acid, Griseofluvin

Study of the Production of
Penicillin, Citric Acid, Vit B12,
Glutamic Acid, Griseofluvin
2 July 2021 Abhijit Debnath BP605T and Biotech Unit-1 1
CO1.1
Noida Institute of Engineering and Technology
(Pharmacy Institute) Greater Noida
Abhijit Debnath
Asst. Professor
NIET, Pharmacy
Institute
Unit: 5
Subject Name: Biotechnology
(BP605T)
Course Details
(B. Pharm 6th Sem)

Study of the
Production of
 Penicillin,
 Citric Acid,
 Vit B12,
 Glutamic Acid,
 Griseofluvin
CO1.1
Noida Institute of Engineering and Technology
(Pharmacy Institute) Greater Noida

 Penicillin is a secondary metabolite produced by certain bacteria, which is used an
antibiotic .
 ‘Antibiotic’ literally means ‘against life’ – but antibiotics only kill life that is harmful
to livingcreatures , i.e. bacteria.
 A bacterial infection is caused by millions of tiny bacteria that are trying to
survive and in multiply the body . An antibiotic attacksand kill these bacteria .
 Before the development of penicillin , many people suffered and died from
bacterial infections that are no longer considered dangerous today.
PENICILLIN CO5.2

PENICILLIUM CO5.2

 Opticallyactive.
 Soluble inwater.
 Acid resistant.
 Hydrolyzed by hot inorganic acid.
 Effective in treatment of respiratory track infection.
PENICILLIN PROPERTIES CO5.2

BIOSYNTHESIS OF PENICILLIN BY
PENICILLIUM CHRYSOGENUM CO5.2

NAM-N ACETYL
MURAMICACID
NAG-N ACETYL
GLUCOSAMINE
PENICILLIN MODE OF ACTION CO5.2

1.Natural penicillin's
2.Beta lactase
resistantpenicillin's
3.Aminopenicillins
penicillin G
Penicillin VK
Methicillin
nafcillin
Ampicillin
amoxicillin
PENICILLIN CLASSIFICATION CO5.2

4.carboxypenicillins
5.Ureidopenicillins
6.penicillin/inihibitor
combination
Carcenicillin
ticarcillin
Mezlocillin
piperacillin
Ampicillin/sulbactam
Ticarcillin/clavulanate
PENICILLIN CLASSIFICATION CO5.2

The industrial production of penicillin as broadly
classified in to two processes namely,
Upstream
process
Downstream
process
PRODUCTION OF PENICILLIN CO5.2

Upstream processing
 Upstream processing encompasses anytechnology that leads
to the synthesis of a product.
 Upstream includes
= the exploration
= development
= production

Downstream processing
 The extraction and purificationof a biotechnological product from
fermentation is referred to as downstream processing.

Culture methods
 The fungus can be cultured in two methods ,namely
Surface culture
method
Submerged culture
method

Surface culture method
 In surface culture method ,the fungus is cultured on the surface
of a liquid medium without agitation.
 Afteran appropriate incubation period ,the penicillin is extracted
from the medium.
 This is an old method.

Submerged culture method
 In submerged culture method ,the fungus is grow in a liquid
medium which is vigorously aerated and agitated.
 After an appropriate incubation period ,the penicillin is separated
from the medium.
 Today penicillin is produced by thesubmerged method .

medium
fermentation
centrifugation
filtration
Solvent extraction
precipitation
crystallization
PRODUCTION PROCESS OF PENICILLIN CO5.2

 Ph6.5
 Temperature 20-24c
 Oxygen
 Nitrogen (corn steep liquor8.5%)
 Glucose 1% (preferred for penicilliumnotatum)
 80% ethanol
 Phenyl aceticacid
 Probenecid
MEDIA FORMULATION OF PENICILLIN CO5.2

 Lactose 1%
 Calcium carbonate1%
 Sodium hydrogen phosphate0.4%
 Antifoaming agent : Vegetableoil
MEDIA FORMULATION OF PENICILLIN CO5.2

 121 degree celcius at 30 psi (pounds persquare inch).
 For high temperature short time for sterilization is used to
minimize thedegradation of certain components ofmedia.
HEAT STERILIZATION CO5.2

• Usually done by fed-batchmode
• High amount of glucose result in low yield of Penicillin.
• Temperature : 20 to 24c
• pH : 6.0 to 6.5 units
• Pressure : 1.02 atmosphere (higher than atmospheric pressure to
prevent contamination)
FERMENTATION CO5.2

•Sparging of air provided for providing
sufficient oxygen required for cellviability.
IMPELLER:
•Rotor used to increase the pressure
and flow offluid.
•Used to mix culturethroughout the medium
• Fungal cells arehardy
•Hence handled at rotation speed
around 200rpm
FERMENTORS
FERMENTATION CO5.2

o First done in lab by adding penicillium spores to the liquid
medium.
o After growth , inoculated into thefermentor.
o In some cases spores are directly inoculated into the fermentor.
Spore: produced during stresscondition
SEEDCULTURE CO5.2

 To remove biomass such as fungus,other impurities from themedium.
 Phosphoric acid is added pH become8.5
 Thus pH is maintained at 6.0 to6.5.
 Rotary vacuum filter is used for large scale production.
 This can leads to the loss of penicillin activity.
ROTARY VACCUM FILTER
FILTERATION CO5.2

 AMYL ACETATE or BUTYL ACETATE is added to dissolve
penicillin infiltrate.
 Now, penicillin is present in the form of solution.
 Othersolids areconsidered as wastes.
ADDITION OF SOLVENTS CO5.2

 Tubular bowl or chamber bowl centrifuge is used.
 Toseparate solid waste from liquid component which contains the
penicillin.
 Supernatent is transferred to downstreamprocess.
CENTRIFUGAL EXTRACTION CO5.2

PENICILLIN + ACETATE SOLUTION
Mixed with
1.Phosphate buffer
2.Chloroform solution
3.Again phosphatebuffer
4.Ether solution
EXTRACTIONCO5.2

ETHER SOLUTION
CONTAINING PENICILLIN
Mixed with SODIUM BICARBONATE
Penicillin sodiumsalt
BASKET CENTRIFUGATION
Basket centrifuge
EXTRACTIONCO5.2

 Toremove the moisture present in the penicillin salt.
 Hot gas is pumped from the base of the chamber.
 Powdered salt is contained in a vaccum chamber.
 Results in dried form ofpenicillin.
FLUID BED DRYING CO5.2

• Stored in containers in driedenvironment.
• Then packaged into
• Liquid penicillin
• Penicillin in pills
STORAGE CO5.2

starterculture
(penicillium)
 Medium
(corn steepliquor lactose
Yeast extract pHbuffers
minerals )
batch fermenter
(10 times in 6 days to remove 30% culture add 30%
fresh medium )
PROCESS CO5.2

rotating filter
filtrate fungal cells
Dissolve in butylacetate animal feed
Potassium ions added to
Precipitate salt ofpenicillin
Wash, filter and dry 99.55%
purepenicillin
PROCESS CO5.2

 Have excellent tissue penetration.
 Bactericidal against sensitivestrains.
 Relatively nontoxic.
 Efficacious in the treatment ofinfections.
 Inexpensive in comparision with other antibiotics.
 Newer penicillin’s are resistant to stomach acid , such as penicillin V
or a broader spectrum ,such as ampicillin andamoxicillin.
ADVANTAGES CO5.2

 Acid liability – most of these drugs are destroyed by gastric acid.
 Lack of activityagainst most gram negative organisms.
 Short duration ofaction.
 Many patients experience GIupset.
 Painful if given intramuscularly.
DISADVANTAGES CO5.2

Large number of micro-organisms including bacteria, fungi and yeasts have been
employed to produce citric acid.
The main advantages of using this micro-organisms are:
a) Its easy of handling
b) Its ability to ferment a variety of cheap raw materials
c) High yields
MICRO-ORGANISMS USED FOR CITRIC ACID PRODUCTION CO5.2

Fungi:
A. spergillus nagger
A. aculeatus
A. awamori
A. carbonarius
A. wentii
A. foetidus
Penicillium janthinelum
Bacteria:
Bacillus licheniformis
Arthrobacter paraffinens
Corynebacterium species
Yeasts:
Saccahromicopsis lipolytica
Candida tropicalis
C. oleophila
C. guilliermondii
C. parapsilosis
C. citroformans
Hansenula anamosa
MICRO-ORGANISMS CO5.2

 Fermentation is the most economical and widely used ay for
synthesis citric acid production.
 The industrial citric acid production can be carried in three different
ways:
 surface fermentation
 submerged fermentation
 solid state fermentation
CITRIC ACID PRODUCTION CO5.2

 Surface fermentation using Aspergillus niger may be done on rice bran as
is the case in Japan, or in liquid solution in flat aluminium or stainless
steel pans.
 Special strains of Aspergillus Niger which can produce citric acid despite
the high content of trace metals in rice bran are used.
SURFACE FERMENTATION CO5.2

• In this case the strains are inoculated of about 15cm depth in
fermentation tank.
• The culture is enhanced by giving aeration using air bubbles.
• And its allowed to grow for about 5 to 14 days at 27 to 33 degree Celsius.
• The citric acid produced in the fermentation tank and it is purified.
SUBMERGED FERMENTATION CO5.2

 It is simplest method for citric acid production.
 Citric acid production reached a maximum(88g/kg dry matter)when fermentation
as carried out with cassava having initial moisture of 62% at 26degree Celsius for
120 hours.
 Solid state fermentation is also known as koji process, was first developed in Japan.
SOLID STATE FERMENTATION CO5.2

 The biomass is separated by filtration.
 The liquid is transferred to recovery process
 Separation of citric acid from the liquid precipitation.
 Calcium hydroxide is added to obtain calcium citrate.
SEPARATION CO5.2

Tetra
hydrate
Wash the
precipitate
Dissolve it with
dilute sulfuric acid,
yield citric acid and
calcium sulfate
precipitate
Bleach and
crystallization
Anhydrous or
mono hydrate
citric acid
SEPARATION PROCESS: CO5.2

• Purification is a simple form of getting a pure citric acid followed by two simple techniques.
• Precipitation
• Filtration
PURIFICATION CO5.2

A . niger CA 16 and 79/20 Substrate cut , dried and powdered
Grown in PDA agar slant Mixed with water at different concentration
A. Niger spores 7days old culture Filtration @ sterilization
inoculation with 1 10 spores/25mLf
FILTERATION Filtrate for citric acid
CELL BIOMASS
CITRIC ACID PRODUCTION CO5.2

Nitrogen source
pH
Aeration
Trace elements
Temperature
FACTORS AFFECTING CITRIC ACID PRODUCTION CO5.2

99% of world production microbial processes
surface or submerged culture.
70% of total production of 1.5 million tons per year is used in food and beverage
industry as on acidifier or antioxidant to preserve or enhance the flavors and
aromas of fruit juices, ice cream and marmalades.
20% used pharmaceutical industry as anti oxidant
to preserve vitamins, effervescent, pH corrector, blood preservative, or in the
form of iron citrate.
INDUSTRIAL PRODUCTION OF CITRIC ACID CO5.2

 Chemical industry remaining 10% softening and treatment of textile.
 Also used in the detergent industry as a Phosphate substitute,
because of less entropic effect hardening of cement
TABLETS, OINTMENTS AND COSMETIC PREPARATIONS CO5.2

Food & drink:
Preservative and flavoring agent
Emulsifying agent in ice-cream.
Household cleaner:
Kitchen
Bathroom sprays.
Cosmetics:
Shampoos
Body wash
WASH CLEANERS:
Nail polish
Hand soap and other cosmetic products
APPLICATIONS/USES OF CITRIC ACID CO5.2

 A vitamin is an organic compound and a vital nutrient that an organism
requires in limited amounts.
 They are of great value in the growth and metabolism ofthe living cells.
 Vitamins are obtained with food, but a few are obtained by other
means ; humans can produce some vitamins from
precursors they consume while certain microorganism produce
vitamins too.
 Thirteen vitamins are universally recognized at present, vitamins
are classified by their biological and chemical activity.
 Vitamins can be classified as “Fat soluble vitamins” and “Water
soluble vitamins”
VITAMIN CO5.2

VITAMIN CO5.2

 Vitamin B12, also called Cobalamin, is a water-
soluble vitamin that has a key role in the normal functioning of the brain and nervous system,
and the formation of red blood cells.
 It is involved in the metabolism of every cell of the human body, especially affecting DNA
synthesis, fatty
acid and amino acid metabolism.
 It is synthesized only by microorganisms and not by animals (including humans) and plants.
 People with B12 deficiency may eventually develop
Pernicious anemia.
 It is the largest and most structurally complicated vitamin and can be produced industrially
only through bacterial fermentation-synthesis.
VITAMIN B12 CO5.2

VITAMIN B12 CO5.2

• Glutamic acid was discovered by Ritthausen in 1866.
• In 1908 professor Ikeda found that the material enhancing the taste of sea weed
(tangle) soup came from a sodium salt of glutamic acid (monosodium glutamate).
• Since then monosodium glutamate has been widely used in both the food industry and
by the general public as a flavor-enhancing agent.
• The demand of glutamic acid has increased rapidly. Before 1956 glutamic acid was mainly
obtained by the hydrolysis of plant proteins .
GLUTAMIC ACID CO5.2

• Glutamic acid is Important in brain metabolism hence various analogues of glutamic
acid are used in treating various neuropathic diseases.
• Since 1908, monosodium glutamate has been used as a flavor enhancing agent both by the
food industry and the general public.
• Poly glutamic acid (PGA) is a naturally occurring anionic polymer that is
biodegradable, edible, and non-toxic towards human and environment .
• It is a good candidate for various industrial applications including thickener, bitterness
reliving agent , Cryoprotectant. Curable biological adhesive heavy metals absorbers .
APPLICATION OF GLUTAMIC ACID CO5.2

Griseofulvin is an antifungal antibiotic first isolated from a Penicillium species in 1939. It
is a secondary metabolite produce by the fungus Penicillium griseofulvum. The
compound is insoluble in water, and slightly soluble in ethanol, methanol, acetone,
benzene, CHCl3, ethyl acetate, and acetic acid.
GRISOFULVIN- INTRODUCTION CO5.2

 Ringworm of the Beard
 Ringworm of Scalp
 Fungal Disease of the Nails
 Ringworm of Groin Area
 Athlete's Foot
 Ringworm of the Body.
APPLICATION OF GRISOFULVIN CO5.2

• Fermentation
• Pre treatment of fermentation broth
• Filtration
• Extraction
• Decolorization
• Isolation and separation
• Precipitation and purification
STEPS INVOLVED IN THE MANUFACTURING PROCESS GRISOFULVIN

Production of Penicillin, Citric Acid, Vit B12, Glutamic Acid, Griseofluvin

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Production of Penicillin, Citric Acid, Vit B12, Glutamic Acid, Griseofluvin