microbial sources of antibiotics.pptx

8/11/2019 Microbial sources of Antibiotics.pptx

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Microbial sources of Antibiotics…



Definition

"An antibiotic is the complex organic chemical substance

which is produced as the secondary metabolite by onemicro-organism and acts as a toxin against other micro-

organisms; either inhibiting their growth or killing them.“

Greek word… Anti – against; Bios – life.



Historical account

• Selman Waksman (1942) coined the term "antibiotics“

– Discovered streptomycin.

• Sir Alexander Fleming (1929) : Penicillin (Penicillium

notatum ). It was the first antibiotic to be produced

industrially

• Fleming, Chain and Florey shared the Nobel Prize in1945 for their contributions in the field of antibiotics.

E. Chain H. FloreyA. Fleming



Microbial source…

• Bacterial origin…

- Bacillin from B.subtilis - against G+ve & G-ve.

• Fungal origin… - Penicillin from P.nottatum, P.chrysogenum - against G+ve.

• From actinomycetes…

- Streptomycin from S.griseus - against G-ve & some G+ve.



Microbial sources of antibiotics



Mode of Action……

• Cell wall synthesis inhibitors.♦ Penicillins - against G+ve.

• Protein synthesis inhibitors.

♦ Aminoglycosides - against G-ve.

• Nucleic acid synthesis inhibitors.

♦ Quinolones - against G-ve > G+ve.

• Cell membrane disruptors.

♦ Polymyxin B - against G-ve only.

• Antimetabolites.

♦ Sulfonamides – broad spectrum.



Mode of action



Antibiotic Spectrum of Activity

• No antibiotic is effective against all

microbes



The process of antibiotic production

• Medium : The fermentation medium is prepared as per therequirement.

• Inoculum : The selected strain of the micro-organism iscultivated under aseptic conditions. This is used as the „seed‟or „inoculum‟.

• Fermentation : The medium and the inoculum are poured inthe sterilized fermenter tank. The pH, temperature, etc. of thefermenting medium are regulated as per the specificrequirements. The fermenting medium is constantly stirredand aerated. This increases the yield.

• Recovery of the antibiotic : After the process is over, thefermented medium is filtered to remove the microbe. Thefiltrate contains the antibiotic. It is recovered using variousmethods, e.g. precipitation, redissolving, filtration, etc.



PENICILLIN

• Inhibits transpeptidation reaction

during peptidoglycan synthesis.

• Has a cidal activity against G+ve.

Structure: β-lactam ring fused with

thiazolidine ring.

Produced by,

P.nottatum,

P.chrysogenum &

some of the

Aspergillus sps

6-APA



Types of Penicillin

Natural Penicillin

►No Precursors added

during Fermentation;

Eg: Penicillin G,

Penicillin V

Biosynthetic Penicillin

► Precursors added during

Fermentation.

Eg: Penicillin G, Penicillin V.

Semisynthetic Penicillin

►Prepared from

6- Aminopenicillanic acid →

Modification by Chemical

means. (Eg: acylation)

Eg: Ampicillin, Methicillin,

Oxocillin,Propicillin, etc,..

ADVANTAGES…..

- can be administerd orally.

- Some – Resistant to

penicillanase

- Can not be administed Orally.

- Resistant to penicillanase



PRODUCTION…

Strain selection… HIGH-YIELDING STRAINS.

↓

Achieved by “Sequential genetic selection”.

P.Chrysogenum NRRL 1951. B25 ( > 200 units/ml)

↓ X- Rays ↓ U.V

P.Chrysogenum, X-1612 P.Chrysogenum Stanford 25099

( > 500 units/ml)

↓ U.V

P.Chrysogenum, Q-176 ( > 761 units/ml)



The process…

• Inoculation medium…( Moyer & Coghill medium)

► Glycerol, Cane molasses, Corn-steep liquor, MgSO4, KH2PO4,

Peptone, NaCl, CuSo4,Fe-tartarate.

• Fermentation medium…

► Corn-steep liquor, Lactose, Glucose, CaCO3, KH2PO4,

Phenyl acetic acid – precursor.

• PH→6.5

• Submerged production.

• Aerobic process.

• Growth Phase → 40 hrs.

• Doubling time → 6 hrs.



Production strains storage…

Production strains are geneticallyunstable.

To store,…at dormancy…

Spore suspension + inert solidsupport → desiccated.

Spore suspension → lyophilized inapp.media.

Spore suspension → stored underliquid nitrogen.

In very strict ASEPTIC CONDITION.

Stored spore suspension.

↓ 2 – 3 times culturing n solid

media/broth.

↓

Flask culture.

↓

Smaller Fermentor

(0.5 – 1 m3)

↓

Little bigger one

( 10 – 20 m3)

↓ Production vessel

Inoculum preparation…



The DSP…. 3 stages…

Penicillin – Extracellular – present in the medium.

Removal of myceliumRotary vacuum

Filtration filter

Filtrate →

subjected toCOUNTERCURRENT SOLVENT EXTRACTION.

( PODBIELNIAK EXTRACTOR )

i. Extraction of the penicillin into an organic

solvent.(amyl or butyl acetate / methyl isobutyl

ketone). pH → 2 - 2.5.

ii. Extraction from the organic solvent into an

aqueous buffer. pH → 7 – 7.5

iii. Extraction from aqueous buffer into organic

solvent.

iv. Extraction of the solvent to obtain the ↓

Penicillin salt

Obtained penicillin salt solution.

↓

Charcoal treatment to remove

Pyrogens

↓

Filter sterilization.

Seitz filter → remove bacteria.

↓

Crystallization.

For Parental use For Oral use

• Powder / • Tabletted.

suspension.



STREPTOMYCIN… An Aminoglycoside…

• Discovered by American

biochemists Selman Waksman,

Albert Schatz, and Elizabeth

Bugie in 1943.

• Consists - an N-methyl-a -L-

glucosamine ring, an a -L-

streptose & a streptidine ring,

linked together by glycosidic

bonds.

• Binds to 30S ribosomal subunit

& inhibits protein synthesis.

• Cidal against G-ve.

• Treatment of Tuberculosis,

Plague.

• Produced by S.griseus



Streptomycin production …

• Submerged culture Method …

• Inoculation medium….Same as penicillin.

• Production medium…

► Glucose / Fructose / Mannitol, Peptone / Meat extract / Soyextract, Mg++, Ca++, k+, etc,..

• Precursors: Proline, Phenyl acetic acid

• Antioxidant: Sodium sulphite.

• Temperature → 28•c; PH range → 7.6 – 8.0

• High agitation and aeration are needed.

• Process lasts for about 10 days.



The process…

Spores are inoculated into a

medium - Obtain high mycelial

biomass

↓

Introduction into aninoculum tank.

↓

production tank.



The Phases….

I. Mycelial growth phase

- Rapid growth producing mycelial biomass.

- Large requirement for O2, Glc, N & P .

- PH up to 8.0 – due to the productiono of NH3.

- little production of streptomycin.

II. Streptomycin production phase

- Stretomycin production & No new mycelialgrowth.

- NH3 is utilized.

- PH ↓↓ to 7.0.

- Glc & O2 required in large quantity.III. Autolysis of mycelium

- Carbohydrates become depleted.

- Streptomycin production ceases .

- Mycelium undergoes Autolysis

- PH ; No O2 requirement; Antibiotic Recovery



The DSP…

Streptomycin – Extracellular – present in the medium.

Culture.

↓ FILTRATION

Filtrate.

↓

Adsorbtion onto activated charcoal.

↓

Elution with acid alcohol.

↓ Precipitation with acetone.

↓

Further purification

by the use of column chromatography.

microbial sources of antibiotics.pptx

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