Fermentation of Penicillin | Antibiotics | Biotechnology

In this article we will discuss about the fermentation of penicillin.

Requirements:

1. Starter cultures on M₂ Agar.

2. M₂ agar (pH 7.0):

3. Seed medium:

4. Fermentation medium:

Dissolve 1.5 g of phenyl acetic acid in warm distilled water. Adjust pH to 7 and make up the volume to 100 ml. Add 1 ml of this to the medium.

5. Flasks.

6. Shaker.

Procedure:

1. Grow P. chrysogenum on M₂ agar for 120—144 hours at room temperature (28°-30°C).

2. Make a suspension of spores and inocu­late 10 ml of it on seed medium in Erlenmeyer flasks with 500 ml capacity.

3. Incubate at room temperature (28°— 30°C) on a shaker (240 rpm) for 48- 72 hours seed.

4. Inoculate this seed at the rate of 10% (V/V) to 45 ml of fermentation medium in 500 ml Erlenmeyer flasks (45 and medium + 5 ml seed).

5. Incubate at room temperature (28°- 30°C) for 120 hours on a rotary shaker (240 rpm).

6. Check the pH of the medium first be­fore inoculation (pH will be around 7.0- 7.5). Adjust it to 5.2 using 20% H₃PO₄.

7. After fermentation filter the broth and use the filtrate for chemical and biological as­says.

Chemical Assay— Iodimetric Method:

Principle:

Active penicillin molecule does not absorb iodine. Alkali inactivation product, Na penicillate does absorb I₂. Penicillin G inac­tivated with alkali absorbs nine atoms of I₂ per penicillin molecule. Residual I₂ is esti­mated with sodium thiosulphate.

Requirements:

1. Standard curve.

2. 1(N) NaOH (40 g NaOH in 1000 ml distilled water.).

3. I₂ solution 0.1 (N1).

12-12-7 g

KI-18.0 g

Grind I₂ and KI in a mortar and pestle. Add little water in it and remove the solution. Add, little more water and grind. Repeat the process till all the I₂ and KI are brought into solution. Make up the volume to 1000 ml and keep in brown bottle. Take 10 ml of this and make it upto 100 ml = N/58 I₂.

4. Sodium thiosulphate 0.1N-24.3g. Na₂S₂0₃ in 1000 ml distilled water + 1 ml chloroform as preservative.

5. Starch solution: lg starch powder—boil in 100 ml water and keep it at low tem­perature.

6. Methyl orange 0.04% in 20% ethanol.

7. Buffers for penicillin.

P0₄ buffer pH 6.0

KH₂P0₄-5.81g + lN NaOH-6.7ml made upto 1000 ml. Check pH on pH meter.

Acetate buffer pH 4.5

Na acetate-5.44 g + Glacial acetic acid – 2.4 ml made upto 1000 ml.

8. 1.2N HCl-100 ml conc. HCl 12(N) made upto 1000 ml.

9. Titration apparatus.

10. Test tubes.

11. Flasks.

Procedure:

1. For standard curve, dissolve the contents of Penicillin G from a vial (5,00,000 units) in 50 ml phosphate buffer (pH 6.0). One ml of this solution has 100,00 units of penicillin.

2. Prepare dilutions of penicillin G. as fol­lows:

1) Total units in the vial=500,000 units

2) Solution of this in 50 ml PO₄ buffer = 500,000/50 = 10,000 units/ml

3) 1 ml of dilution (2) + 9ml PO₄ buffer 10000/10 = 1000 units/ml (stock)

4) 1 ml of dilution (3) + 99 ml PO₄ buffer = 1000/100 = 10 units/ml (working stock)

5) 0.5 ml of dilution (4) + 9.5 ml PO₄ buffer = 5/10 = 0.5 units/ml (working stock)

6) 0.7 ml of dilution (4) + 9.3 ml PO₄ buffer = 7/10 = 0.7 units/ml

7) 1.0 ml of dilution (4) + 9.0 ml PO₄ buffer = 10/10 = 1.0 units/ml

8) 1.2 ml of dilution (4) + 8.8 ml PO₄ buffer = 12/10= 1.2 units/ml

9) 1.5 ml of dilution (4) + 8.5 ml PO₄ buffer = 15/10 =1-5 units/ml

10) 2.0 ml of dilution (4) + 8.0 ml PO₄ buffer = 20/10 = 2.0 units/ml

B_S = Blank standard.

E_S = Experimental standard.

B_T = Blank test (fermented broth).

E_T = Experimental test (fermented broth). 

Label these dilutions and use for chemical and biological assay.

Bioassay-Diffusion Method:

Requirements:

1. Test organism-Bacillus subtilis.

2. Fermented broth.

3. Standard graph of penicillin with diam­eter of zone of inhibition against units of penicillin.

4. Petri plates.

5. Bioassay medium butts.

6. Cork borer.

7. Glass marking pencil.

8. Incubator.

9. Base agar (any fungal medium).

Procedure:

1. Pour base agar into sterile Petri plates and allow it to solidify.

2. Inoculate B. subtilis into sterilised cooled (to 45°C), 10 ml medium in tubes, mix well by rolling it gently between the palms.

3. Pour it to the surface of the base agar, allow it to solidify.

4. With a sterile cork borer punch holes in the agar medium (3—4) in such a way that the zones of inhibition should not overlap.

5. Add different dilutions of standard peni­cillin along with fermented broth into the wells using a micropipette and label respective concentrations.

6. Keep the plates in a refrigerator (4°C) for an hour to allow even diffusion of the test solutions into the surrounding agar medium.

7. Incubate at 37°C for 24-48 hours and measure the diameter of the zones of in­hibition.

8. Prepare a graph with concentration of penicillin against the diameter of the zone of inhibition. From this find out the concentration of penicillin in the fer­mented broth.