Large scale production of streptomycin.pptx

Large scale production of streptomycin.pptx

1. LARGE SCALE PRODUCTION OF ANTIBIOTICS-STREPTOMYCIN 1

2. Aminoglycoside antibiotics Antibiotics composed of one or more amino sugars and an aminocyclitol (6 carbon ring) are referred as aminoglycosides. These complex compounds are exclusively the products of various species of soil actinomycetes in the genera Streptomyces and Micromonospora . Streptomycin is obtained from Streptomyces griseus and dihydrostreptomycin is isolated from Streptomycin humidus . 2

3. streptomycin It is an antibiotic drug. Discovered by Schatz, Bugie and Wakman in 1944. Belongs to a class of drugs called aminoglycosides and is the first antibiotic remedy for tuberculosis caused by Mycobacterium tuberculosis. Streptomycin is the broad spectrum aminoglycoside antibiotic which works actively against gram-negative bacteria. Commercially available as Hypochloride with a chemical formula C21H39N7O12.3HCL . It is derived from actinomycetes, Streptomyces griseus. It cause kidney damage and deafness as side effects. 3

4. Streptomycin cont … Streptomycin is active against a large number of bacteria found among gram-negative, gram-positive and acid fast groups. Has little activity against anaerobic bacteria, fungi, protozoa and viruses. Streptomycin was found to be soluble in water and insoluble in organic solvents such as ether, chloroform and acetone. Useful in the control of plant diseases caused by bacteria. DISADVANTAGE Neurotoxicity due to which hearing impairment and balance maintenance is lost due to prolonged streptomycin treatment at high dosage. It’s reduction to dihydrostreptomycin results in decreased toxicity and for this reason only dihydrostreptomycin is being produced. 4

5. PHYSICAL AND CHEMICAL PROPERTIES Physical state - solid Form - Hygroscopic powder Odour - Faint amine like odour Taste - Slightly bitter Solubility - Soluble in water Nature - Hygroscopic Molecular weight - 581.58 g/ mol Ph - Polar organic base On decomposition - Emit fumes of nitrogen oxide 5

6. structure It is made of 3 basic structural units 1. Streptidine (a diguanidino compound) 2. Strptose (a aldose sugar) 3. Streptoscamine (N-methyl L-glucosamine) Both guanido groups of streptidine are essential for antibiotic activity and removal of one group reduces antibiotic activity up to 90%. 6

7. Mode of action of streptomycin Streptomycin is a protein synthesis inhibitor. It binds to small 16s rRNA of the 30s subunit of bacterial ribosome and interfere with the binding of formyl- methionine –tRNA to the 30s subunit. This leads to codon misreading, eventual inhibition of protein synthesis and ultimately death of microbial cells. Speculation on this mechanism indicates that binding of molecule to 30s subunit interferes with 50s subunit association with mRNA strand. This results in an unstable ribosomal m-RNA complex, leading to frame-shift mutation and defective protein synthesis leading to cell death. 7

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9. Organism required for streptomycin production Streptomyces griseus is an actinomycete used for the preparation of streptomycin. Krinsky was the first scientist isolated S. griseus during time of first world war from the Russian soil. S. griseus is commonly present in soil and it is gram-negative bacteria. It produces grey mycelium during sporulation and produces grey yellow reverse pigment when it grows in the form of colony. Streptococcus griseus secretes toxin valinomycin , which cause apoptosis in natural killer cells and mitochondrial swelling in peripheral lymphocytes. Due to presence of valinomycin, excessive use of streptomycin can cause neurotoxicity like hearing loss, dizziness etc. 9

10. LARGE SCALE Production of streptomycin ANTIBIOTIC Growth of Streptomyces griseus Maintain the hymns of Streptomyces griseus spores in the stock soil culture. Then, inoculate the inoculum of S. griseus into agar media at 28 degree Celsius. After that, spores of S. griseus sporulate and build up the mycelial network in a flask or seed banks. Sporulation of S. griseus occurs well in a liquid culture media and forms endospores in low nutrient availability. Growth of S. griseus requires a PH range of 5-11. Duration- varies between 5-7 days. 10

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12. b. Preparation of medium Medium is a nutritive substance in which cultures are grown for scientific purposes. Culture medium for streptomycin consists of- 1. Carbon source- starch, dextrin, glucose, glycerol and other economically available material. 2. Nitrogen source- natural agricultural by-products, soybean meal, corn steep liquor, cotton seed flour, casein hydrolyte or yeast and it’s extract. Inorganic N salts like ammonium sulphate and ammonium nitrates are also used. 3. Animal oils, vegetable oils and mineral oils are also used. 12

13. Hockenhull nutrient medium Glucose - 2.5% Soy meal - 4% Sodium chloride - 0.25% Distillers dried soluble - 0.5% pH - 7.6-8.0 13

14. Preparation of medium cont … Carbon source- monosaccharide like glucose provides best carbon source that helps in building up of streptomycin. Sugars like fructose, maltose, starch etc can also be used as carbon source but oligosaccharides and polysaccharides give low yield. 2. Nitrogen source- contents like soy meal, meat extract, ammonium salts. 3. Mineral source- magnesium, calcium, potassium. 4. Growth stimulating sources- sources like L-naphthalene acetic acid and phenylacetic acid . 5. Compounds like proline, oils, fatty acids, antioxidants are also used to accelerate streptomycin production. 14

15. c . fermentation Sterilize all the above contents and fed into the stirred tank fermentor . Then, transfer the bacterial inoculum of about 4-5% to the medium. Maintain a temperature between a range of 25-30 degree Celsius and pH between 7.6-8 inside the fermentor . The production of streptomycin requires continuous fermentation and growth of Streptomyces griseus requires continuous aeration and agitation. Fermentation process takes 4-10 days. 15

16. Fermentation cont … Fermentation process involves three consecutive phases: a. First phase b. Second phase c. Third phase a. First phase During first phase, S. griseus grows by utilizing soya meal and releases ammonia through proteolytic activity. pH rises upto 8 during this phase due to release of ammonia in to the medium. S. griseus slowly utilizes carbon source present in soya meal and glucose and thereby produce streptomycin at a slower rate and rapid growth with production of mycelial biomass. Takes about 24-48 hours. 16

17. Fermentation cont … b . Second phase After utilization of soy meal and glucose, ammonia is secreted and streptomycin is synthesized rapidly. Streptomycin starts accumulating in the medium. In this phase, incubation period lasts for 24 hours to 6-7 days. pH remains constant between 7.6 and 8. There is little or no production of mycelium. Glucose and oxygen are required in large quantity during this phase. 17

18. Fermentation cont … c. Third phase It is the final phase of fermentation where concentration of glucose depletes from medium that results in cessation of streptomycin production and bacterial cells begin to lyse. Requirement of oxygen decreases and contents of medium including sugar get exhausted. As a result, medium’s pH increases by deposition of ammonia after cell lysis. Before cell lysis, harvest the fermentative medium for recovery and purification. 18

19. Recovery and purification of streptomycin Recovery of streptomycin is done in following steps: Filtration Separate the bacterial biomass from the fermentative broth through Oliver’s pre-coated pressure filter. After filtration, remaining liquid is first acidified, filtered and then neutralized. 2. Adsorption Mix the activated carbon with clear broth obtained after filtration in a series of three adsorption tanks. Separate streptomycin from broth to remove impurities. 3. Broth is fed to pressure filter which releases out the spent broth. Wash the absorbate with dilute alcohol to remove impurities. 19

20. 4. Elution Elute the streptomycin through exchange method by adding dilute hydrochloric acid. The impurities like metallic salts are removed from solution which contaminate streptomycin. 5. Concentration Concentrate remaining solution at 60 degree Celsius under vaccum by series of three single-phase evaporators. 6. Crystallization Firstly, dissolve the streptomycin in methanol and then filter it again . Treat the filtrate with acetone to obtain a precipitate. Rewash the precipitate with acetone and later dry it under vaccum . It is purified further by dissolving in methanol and streptomycin in pure form is extracted as calcium chloride complex. 20

21. Final steps 1. Dissolve the crystalline streptomycin in the solution containing pyrogen free water. 2. Pass the solution through special biological filter like Seitz filter to eliminate microorganisms that can contaminate the streptomycin. 3. Dry the filtrate under vacumn to form a dried cake. 4. Crush the dried cake into a powdered form by employing high vacumn sublimation using sterile steel balls. 5. After that, weigh the powdered streptomycin into sterile vials. The whole process needs to be carried out in the air-conditioned room which provides filtered or stale air through ultraviolet lamps. 6. Finally, pack the powdered streptomycin in the rooms having 10% relative humidity. To avoid contamination, we can use special microbial filters, triethylene glycol as a disinfecting agent and UV lamps. All these factors maintain sterile condition for the production of streptomycin. 21

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23. uses Streptomycin is very effective against gram-negative bacteria, thus treat infections and diseases caused by gram-negative bacteria. Widely used in the treatment of severe disease like tuberculosis. Used in conjugation with antimycobacterial drugs to treat pulmonary infections caused by Mycobacterium avium complex. Works as an alternative of Penicillin G to treat rat-bite fever caused by streptobacillus moniliformis for those who are allergic to penicillin. Products like streptomycin-B and dihydroxystreptomycin is obtained from streptomycin. Patients suffering from endocarditis caused by Enterococcus can be treated with streptomycin if they are sensitive to gentamicin. Tularemia infections, plague brucellosis etc can be treated with streptomycin. 23

24. Uses cont … Streptomycin is used as pesticide that inhibits growth of bacteria, fungi, algae etc. Also used at time of cell culture to prevent undesired microorganisms growth. Streptomycin sulphate is also used in purification of biomolecules like protein and nucleic acids. 24

25. R eference Procópio , R. E. D. L., Silva, I. R. D., Martins, M. K., Azevedo , J. L. D., & Araújo , J. M. D. (2012). Antibiotics produced by Streptomyces. Brazilian Journal of Infectious Diseases, 16, 466-471. https://patents.google.com/patent/US2504067A/en https://biologyreader.com/production-of-streptomycin.html https://thesciencenotes.com/streptomycin-production-chemical-structure-medical-applications/ 25