Introduction to Fermentation, and production of penicillin and penicillin G

Industrial production of penicillin by using fermentation techniques.

1. FERMENTATION PHARMACEUTICAL PROCESS CHEMISTRY PRESENTED BY Mr. Darshan N U B Pharm ( M Pharm) M Pharmacy II Semester Dept. of Pharmaceutical Chemistry SACCP PRESENTING TO Mr. Purushotham K N M Pharm ( Ph.D ) ASST. Professor Dept. of Pharmaceutical Chemistry SACCP 1

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3. Products of Fermentation 3

4. 4 Wine Beer Vinegar Kimchi Pepperoni Sauerkraut Yogurts cheese

5. Importance of Fermentation It allows the cells to get two ATP gain from one molecule of glucose, even without oxygen Fermentation takes away the end products of glycolysis Fermentation is important to the Baking industry Fermentation is important in wineries and Breweries because yeast uses fermentation to produce alcohol Fermentation is important in muscles because it allows the muscle to keep getting a little energy from glucose even when the oxygen supply cannot keep up with the demand 5

6. Fermentation is carried out in an equipment called as fermentor . Fermentor can be defined as a vessel in which sterile nutrient media and pure culture of microorganism are mixed and fermentation process is carried out under aseptic and optimum condition. The fermenter provides sterile environment , an optimum condition that are important for micro-organism to grow. 6

7. Aerobic Fermentation Aerobic fermentation is  a metabolic process by which cells metabolize sugars via fermentation in the presence of oxygen and occurs through the repression of normal respiratory metabolism . Anaerobic Fermentation Anaerobic fermentation  occurs in the fermentation vessel once the oxygen is discharged and replaced with N 2 , CO 2 , or another by-product of the fermentation process. Anaerobic fermentation is usually a slower process. 7

8. PENCILLIN Penicillin refers to a group of beta-lactam antibiotics used in the treatment of bacterial infections caused by susceptible, usually Gram-positive organism. The chemical structure of penicillin was determined by Dorothy Crowfoot Hodgkin in the early 1940 s, enabling synthethic production. 8

9. The structure of penicillin composed of 4-membered beta lactam ring with thiazolidine nucleus. It contains three chiral carbon atoms ( C3,C5,C6). 6-Amino penicillanic acid (PAP) is the basic skeleton structure of all penicillins. 9 6-(APA)

10. General Structure of Penicillin 10 Generic Name Chemical Name R Group Penicillin G Benzyl penicillin Penicillin V Phenoxy-methyl penicillin Penicillin X P-Hydroxy benzyl penicillin Penicillin F 2-pentenyl penicillin Penicillin K n-Heptyl Penicillin CH 3 (CH 2 ) 7 -

11. Production of Penicillin P. Chrysogenum is high yielding strain and therefore most widely used as production strain Inoculum Preparation: Purpose is to develop a pure inoculum in an adequate amount. To do so various sequential steps are necessary like: 1.A starter culture is needed for inoculation. 2.After getting growth on solid media, one or two growth stages should allowed in shaken flask cultures to create a suspension, which can be transferred to seed tanks for further growth. 3.After about 24-28 hours, the content of the seed tanks is transferred to the primary fermentation tanks. 11

12. 4. All the bio parameters like temperature, pH , aeration, agitation etc. Should be properly maintained Bio parameters pH : near 6.5 Temperature : 26 to 28 C Aeration : a continuous stream of sterilized air is pumped into it. Agitation : have baffles which allow constant agitation (200 rpm) 12

13. Nutrient Media Fermentation broth contains all necessary elements required for the proliferation of the microorganism. Generally, it contains a carbon source, nitrogen source, mineral source. Precursors and antifoam agents. Carbon Source Lactose in a concentation of 6 %. Other carbohydrates like glucose & sucrose. Complex as well as cheap source like molasses, or soya meal can also be used which are made up of lactose and glucose sugars. 13

14. Ni trogen Source Ammonium salts such as ammonium sulphate, ammonium acetate, ammonium lactate or ammonia gas are used for this reason Sometime corn steep liquor may be used. Mineral Source These elements include phosphorus, sulphur, magnesium, zinc, iron, and copper which generally added in the form of water soluble salts. Precursors Various types of precursors are added into production medium to produce specific type of penicillin 14

15. For example, if phenyl acetic acid is provided then only Penicillin-G will be produced but if hydroxy phenyl acetic acid is provided then then penicillin –X will be produced. Phenoxy acetic acid is provided as precursor for Penicillin V production When corn steep liquor is provided as nitrogen source, it also provides Phenyl acetic acid derivatives; therefore it is widely used in the production of penicillin-G 15

16. Anti-foam agents Anti-foaming agents such as lard oil, octadecanol and silicones are used to prevent foaming during fermentation 16

17. Penicillin is commercially produced in the industry by culturing the fungus penicillium notatum or penicillin chrysoenum. The fungus can be cultured in two methods: Surface culture method Submerged culture method 17

18. Surface culture method: Fungus is cultured on the surface of the liquid media without agitation After appropriate an incubation period the penicillin is extracted from the media. Submerged culture method: Fungus is cultured in a liquid media which is vigorously aerated and agitated. After appropriate an incubation period the penicillin is extracted from the media. Now day, penicillin is produced by this method 18

19. 19 Media Formulation • Lactose: 1% • Calcium Carbonate: 1% • Cornsteep Liquor: 8.5% Corn steep liquor is a by-product of corn wet-milling. A viscous concentrate of corn solubles which contains amino acids, vitamins and minerals, it is an important constituent of some growth media. • Glucose: 1% • Phenyl acetic acid: 0.5g • Sodium hydrogen phosphate: 0.4% • Antifoaming Agent: Vegetable oil

20. 20 Biosynthesis of Penicillin

21. Master stock of P. chrysogenum Sporulation (flask culture) Spore suspension Germination /seed tank Production fermenter Filtration Filtrate 21 Flow diagram for Penicillin G Production

22. Filtrate pot. bicarbonate/acetate Rich solvent Aqueous extraction Rich solvent solution Active carbon treatment Azeotropic distillation Solvent wash Crystalline mass Drying /Blending Potassium penicillin G 22

23. Semi Synthetic Penicillins Semi-synthetic penicillins such as Ampicillin, Methicillin, oxocillin , propicillin are prepared by chemical degradation of 6-Aminopenicillanic acid. Few of semi-synthetic penicillins are known and some of them have proved to be definitely superior to Penicillin G in clinical application 23

24. 24 Ampicillin Amoxicillin Methicillin Nafacillin Oxacillin

25. Reference Pharmaceutical Microbiology by N.K. Jain Medicinal Chemistry volume II by ILANGO and P. Valentina Wikipedia Date 28/07/2022 25

26. THANK YOU 26