The document discusses the production of the antifungal antibiotic griseofulvin. It describes the fermentation process using the fungus Penicillium griseofulvum to produce griseofulvin. The process involves growing the fungus in a liquid medium, extracting and purifying the griseofulvin through various steps like filtration, precipitation, and crystallization to obtain the final product. Griseofulvin is then used to treat fungal infections like ringworm.

1. S H I K H A K U M A R I
M . T E C H - I
B I O C H E M I C A L E N G I N E E R I N G
Production of Antifungal
Antibiotics

2. Introduction
 Antifungal antibiotics are used to treat fungal
infections, which are most commonly found on the
skin, hair and nails.
 They come as creams, sprays, solutions, shampoos,
tablets designed to go into the vagina (pessaries),
medicines to take by mouth, and injections.
 The length of treatment depends on what type of
fungal infection you have, how severe it is and if you
have any other health problems - for example,
problems with your immune system.

3. Site of Action of Antifungal Drugs
Griseofulvin
Inhibits mitotic
spindle formation

4. Classification of Antifungal Drugs
1- Antifungal Antibiotics :
 Griseofulvin
 Polyene macrolide : Amphotericin- B & Nystatin
2- Synthetic :
 Azoles :
 A) Imidazoles : Ketoconazole , Miconazole
 B) Triazoles : Fluconazole , Itraconazole

5. THE COST OF RESEARCH AND DEVELOPMENT
 Candidate compounds have to pass tests for:
Toxicity
Allergic effects
Mutagenicity
Carcinogenicity
 The estimated cost of a new drug is between $100
million to $500 million.
 Development can take as long as 5-10 years.

6. Manufacturers Of Antifungal Antibiotics
 Versicor and Biosearch Italia, US based company
 PURE SOURCE, FLORIDA
 Avni Chlorochem Pvt. Ltd., Vadodara
 Alichem Pvt. Ltd., Nilanga
 Remedica Products, Europe

7. ORIGIN, CHEMICAL NATURE, AND ACTIVITY OF ANTIFUNGAL
ANTIBIOTICS
Antibiotic Producing
organism
Chemical
nature
Active upon
Clavicin Aspergillus
clavatus
Unsaturated
ketone
Bacteria and
fungi
Gliotoxin Trichoderma Contains sulfur
and
nitrogen
Bacteria and
fungi
Trichothecin Trichothecium
roseum
Unsaturated
ketone
Fungi
Viridin Triclhoderma
viride
Contains carbon,
hydrogen,
oxygen
Fungi
Eumycin Bacillus subtilis Alcohol -soluble Bacteria and
fungi
Pyocyanin Pseudomonas a-ketophenazine Bacteria and

8. Griseofulvin
 Griseofulvin is an antifungal antibiotic first isolated from a
Penicillium species in 1939.
 It was discovered by Harold Rastrick and his team.
 It is a secondary metabolite produced 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.
 Griseofulvin binds microtubule proteins, inhibit cell wall
synthesis ,inhibits nuclear division.
 Induces apoptosis in human tumor cell lines.

9. Production Of Griseofulvin
• Fermentation has been widely used for the production
of a wide variety of substances that are highly
beneficial to individuals and industry.
• Over the years, fermentation techniques have gained
immense importance due to their economic and
environmental advantages.
• Ancient techniques have been further modified and
refined to maximize productivity.
• This has also involved the development of new
machinery and processes.
• Two broad fermentation techniques have emerged as a
result of this rapid development: Submerged
Fermentation and Solid State Fermentation .

10. Preparation of media
 Medium
 Czapek Dox Medium
 Chemicals
 Glucose 5%
 NaNO3 0.2%
 KH2PO4 0.1%
 Mg2SO4. 7H2O 0.05%

11. Steps involved in the manufacturing process
Fermentation
Pretreatment Of
fermentation broth
Filtration
Extraction
Decolorization
Isolation and
Separation
Precipitation and
Purification

12. Schematic Representation

13. Submerged-State Fermentation
 The pH of Czapek-Dox medium adjusted between 6.0-7.2.
 Medium dispensed in the fermenter .
 Fresh sample of mycelial suspension of fungus Penicillium
griseofulvum obtained.
 Solution autoclaved for 200 minutes at 120°C at 15lbs
pressure and fermented for 14 days at 24°C.
 Broth heated above 60°C for 20-30minutes
 Period of heating may be short, 5-10 minutes at 80°C
having been found to provide a satisfactory increase in
filtration rate.

16. Pre treatment of fermentation broth
 The broth is heated above 60°C for 20-
30minutes.
 After heating, sufficient coagulation of material
occurs to produce a valuable improvement in
separation characteristics of the broth.

17. Filtration
 Drum covered with diatomaceous earth matter and allowed
to rotate under vacuum with half immersed in the slurry tank.
 Small amount of coagulation agent added to broth and
pumped into the slurry tank.
 As drum rotates in the slurry tank under vacuum thin layer of
coagulated particles adhere to drum.
 The layer thickens to form cake.
 As the cake portion in the drum comes to the upper region
which is not immersed in the liquid it is washed with water
and dewatered immediately by blowing air over it.
 Then before the dried portion is again immersed into the
liquid it is cut off from drum by knife.

20. Extraction
 Griseofulvin is extracted in the cold acetone when it is
used as an extraction agent.
 The extractions with the cold acetone may be carried out
with the efficiencies between 75-96% or even upto
99.5%.
 The quantity of the solvent used in the extraction at large
scale production should be kept minimum.
 The volume of acetone should be 3-5 times of the
mycelial left.

22. Decolorization
 The color of the extract can be improved by the addition
of calcium hydroxide usually 2.5-50 g/liter preferably 5-30
g/liter.
 The pH of the extract should be above 10.
 It can be neutralize by the removal of lime or by using
mineral acid.

23. Isolation and Separation
 The impurities or waxy substances are removed by
washing the extract with a solvent in which extract is
immiscible and also griseofulvin is insoluble.
 Hydrocarbon solvents, generally aliphatic hydrocarbons
such as hexane or petroleum containing a high portion of
hexane are in general suitable for this step.

24. Precipitation and Purification
 Griseofulvin can be precipitated from the solvent extract in various
ways. One of the method is using the liquid solvent in which
griseofulvin is substantially insoluble. Griseofulvin non-solvent is
preferably water.
 The alkaline water is more effective for the removal of colored
impurities present in the crystals of the griseofulvin.
 Water is made alkaline with ammonia or an alkali metal carbonate
or alkali metal hydroxide. The suitable pH is about 8.5.
 The purity of the precipitate is generally improved by washing with
a solvent for the small quantities of impurities remaining. The
suitable washing media are dry or wet acetone, a lower alkanol for
example methanol or butanol. Marked purification is obtained with
the use of methanol for this step.

26. •Submerged fermentation utilizes free flowing liquid substrates,
such as molasses and broths.
•The bioactive compounds are secreted into the fermentation
broth.
•The substrates are utilized quite rapidly ; hence need to be
constantly replaced/supplemented with nutrients.
•This fermentation technique is best suited for microorganisms
such as bacteria that require high moisture content.
•An additional advantage of this technique is that purification of
products is easier.
•Submerged Fermentation is primarily used in the extraction of
secondary metabolites that need to be used in liquid form.
Submerged Fermentation (SmF)

28. Solid State Fermentation
 Solid state Fermentation utilizes solid substrates like bran,
bagasse, and paper-pulp.
 The main advantage of using these substrates is that nutrient-
rich waste materials can be easily recycled as substrates.
 In this fermentation technique, the substrates are utilized very
slowly and steadily, so the same substrate can be used for long
fermentation periods.
 Hence, this technique supports controlled release of nutrients.
 SSF is best suited for fermentation techniques involving fungi
and microorganisms that require less moisture content.
 However, it cannot be used in fermentation processes
involving organisms that require high water activity, such as
bacteria. (Babu and Satyanarayana, 1996).

29. Uses
 Anti-inflammatory properties
 Inhibits keratolytic action
 It is used in the treatment of
 Ringworm of the Scalp
 Ringworm of the Body
 Ringworm of beard
 Fungal Disease of the Nails
 Ringworm of Groin Area

30. Diseases Being Treated

31. Side-effects
The most common side-effects are
•Nausea
•Vomiting
•Diarrhoea
•Flatulence, cracking at the side of the mouth
•Soreness
•Blackening of the tongue and thirst
•Headache
•Peripheral neuritis
•Mental confusion
•Fatigue
•Blurred vision
•Increases alcohol intoxication

32. References
 Hazen et al., U.S. Pat. No. 2,797,183, supra
 Dutcher et al., Antibiotics Annual, l953l954, pages
191-194, Medical Encyclopedia, Inc., New York, NY.
 U.S. Pat. No. 2,786,781 to Vandeputte et al.
 http://patient.info/health/antifungal-medicines
 http://www.life-worldwide.org/fungal-
diseases/griseofulvin

33. References
 US patents
 US Patent 3069329A Published on Dec 18, 1962 published by
Dorey Michael John, Ivor L S Mitchell, David W Rule, Walker Cecile
 US Patent 3616247 Published on Nov 4, 1968 published by
Harold George Hemming, Malcolm Lehan, David Giles
 US Patent 2986496 Published on May 30, 1961 published by
Alan Rhodes, Bracknell And Derek L. Fletcher
 US Patent 3152150 Published on Oct 6, 1964 published by
Wilson Alan, Arthur P Best, Arthur R Lockwood, Alan H Raper, Gordon C
Sayer, Richards John Willis