Here is a list of top three antibiotics:- 1. Penicillins 2. Tetracyclines and Chloramphenicol 3. Streptomycin.
1. Penicillins:
Penicillins, a group of several penicillin, differ from one another in the side chain attached to its amino group. Most of these penicillins are 6-aminopenicillanic acid derivatives and all have β-lactam ring which is responsible for the antibiotic activity (Fig. 20.16).
Pencillin acts against Gram- positive bacteria and inhibit their cell wall synthesis. These are non-toxic to mammals except for certain allergic reactions. More than 100 penicillins have been synthesized so far.
Penicillium species required the production medium which contains lactose (1%), calcium carbonate (1%), corn steep liquor 8.5%, glucose (1%), sodium hydrogen phosphate (0.4%) and phenyl acetic acid (0.5g). The pH is kept between 5 and 6 and temperature for incubation is 23- 25°C. Aeration and agitation are necessary.
(i) Fermentation:
Penicillin is produced by fed-batch culture using Penicillium chrysogenum Q-176, a fungus that can be grown in stirred fermenters. The inoculum under aerobic condition (seed) can be produced when there is glucose in sufficient amount in the medium.
If a particular penicillin is produced, specific precursor (substance added prior or simultaneously with the fermentation which are incorporated without any major change in the molecules) is added in the medium for e.g. phenyl acetic acid or its derivatives such as ethanol amide to get penicillin G (Fig. 20.17). The antifoam agents such as vegetable oil (com or soybean oil) is added to the medium before sterilization.
The spore suspension is inoculated in flasks, each containing 15 g barley seeds. These flasks are vacuum dried, to which sterilized quartz is added. The preparation of inoculum takes place on barley seeds. The flask containing 15 g barley seeds are to be mixed with mother culture, and incubated at 25°C for 7 days.
The spores developed on barley seeds are suspended in distilled water to make spore suspension. After testing the antibiotic activity, the seeds containing flasks are ready for seeding in fermenter.
Three phases of growth can be differentiated during cultivation of Penicillium chrysogenum:
(a) First Phase:
In this phase, growth of mycelium occurs; yield of antibiotic is quite low. Lactic acid present in corn steep liquor is utilized at a maximum rate by the microorganism. Lactose is used slowly. Ammonia is liberated into the medium resulting into rise in pH.
(b) Second Phase:
There was intense synthesis of penicillin in this phase, due to rapid consumption of lactose and the ammonium nitrogen (NH3N). The mycelial mass increases; the pH remain unchanged.
(c) Third Phase:
The concentration of antibiotic decreases in the medium. The autolysis of mycelium starts liberation of ammonia and slight rise in pH.
(ii) Recovery:
When the fermentation cycle (7 days) is completed, the whole batch is harvested for recovery. Its activity disappears on evaporation to dryness, hydrolysed to penicilloic acid. Penicillin has tendency that it remains in aqueous phase at normal pH and in solvent phase at acidic pH.
This property of penicillin is used in recovery of potassium penicillin from natural solutions. Once the fermentation is completed the broth is separated from fungal mycelium and processed by absorption, precipitation and crystallization to yield the final product (Fig. 20.18). This basic product can then be modified by chemical procedures to yield a variety of semisynthetic penicillins such as ampicillin, amoxycillin, etc.
(iii) Units of Antibiotics:
The potency of antibiotics is normally expressed in terms of units/ ml of solution or in 1 mg (U/mg). In most of the cases, the antibiotic vial shows the quantity expressed in terms of weight as well as activity for example 1 mg of the penicillin G (benzyl penicillin) is equivalent to 1665 units.
Production of Semisynthetic Penicillins:
The main objective of producing semisynthetic penicillin is to generate compounds with improved properties e.g. acid stability, resistance to enzymic degradation, broader spectrum of activity, etc.
In such cases, side chain is removed to form 6-amino penicillanic acid (6-APA) via immobilization in a coloumn of penicillin acylase. For example. Penicillin G is converted to 6-APA and phenylacetic acid. The 6-APA is then chemically acylated to produce a semisynthetic penicillin.
2. Tetracyclines and Chloramphenicol:
Due to the broad antibiotic activity of the tetracyclines (against Gram-positive and Gram-negative bacteria, rickettsia, and some large viruses), these compounds are widely used in medicine. The tetracyclines are also valuable for relatively low toxicity.
There are three important actinomycetes namely, Streptomyces aureofaciens, S. ramosus and Nocardia sulphurea which form tetracyclines. Several semisynthetic preparations are obtained from the other tetracyclines such as 6-di-methyl tetracycline, 7-chloro-tetracycline, 7-chloro-6-de-methyl chloro-tetracycline, 7-bro-mo-tetracycline, 5-oxytetracycline including de-oxycycline and metacycline by chemical modification of the oxytetracyclines. (Fig. 20.19)
Streptomyces aureofaciens produces aureomycin as observed by Duggar (1948, 1949). The name of the antibiotic was corresponded to the name of the species aureofaciens and secondly, due to the golden colour of its crystals. Now this antibiotic is named as chlorotetracyclines due to its chemical nature. (Fig. 20.20)
(i) Media Composition:
Following medium is normally used for the production of chlorotetracyclins:
Sugar – 3%
Corn steep liquor – 1%
CaCO3 – 1%
(NH4)2SO4 – 0.2%
NH4Cl – 0.1%
The pH is kept at 6-7. The conditions of the medium for the growth and biosynthesis of chlorotetracyclines are important. The yield of antibiotic depends upon pH, age of the inoculum, and composition of the medium. Aeration of the culture is also very important for the biosynthesis of antibiotic.
When the culture is grown in submerged conditions continuous aeration is required to ensure the high yield of chlorotetracyclines. Chlorotetracycline is isolated from the culture fluid (after its separation from the mycelium) by extraction, precipitation or adsorption. It is poorly soluble in common organic solvents but is soluble in water and insoluble in ether.
Chlorotetracyline is more valuable medicinal preparation than streptomycin or penicillin. It is used to treat bacterial pneumonia, brucellosis, tularemia, pertusses, scarlet fever, anthrax, etc. It is also used against rickettsiosis and also against some viral diseases.
Oxytetracyclines (tetrramycin) is formed by S. rimosus. The species name is indicating due to fissure like appearance with slightly elevated edges on the agar-surface.
S. griseoflavus, S. armilatus, S. aureofaciens var. oxytetracyclini also produce oxytetracyline.
It requires following contents for its growth medium:
Com steep liquor – 0.5%
Starch – 3.0%
Ammonium sulphate – 0.4%
Sodium chloride – 0.5%
Calcium carbonate – 0.5%
In 24-28 hours of cultivation, submerged spores of streptomycete develop and then the secondary mycelium grows. This process requires aeration and there is liberation of protein N into the mycelium.
The ammonium salt and nitrates favour the biosynthesis of oxytetracylines, starch, glucose, maltose, galactose, glycerol etc. are also used as carbon source. Lactose and saccharose are not at all used by Streptomyces species. The consumption of carbohydrates increases, the pH of the medium decreases, and the biosynthesis of the antibiotic slows down.
When tetracyclines are taken in low concentration, these act bacteriostatically and only become bacteriocidal when the concentration increases. The chlorotetracyclines and oxytetracyclines inhibit phosphorylation processes by preventing the inclusion of phosphorus into nucleic acids.
The synthesis of proteins is essentially stopped while that of nucleic acid continues and sometimes is stimulated as well. Chloramphenicol in low concentration inhibits the protein synthesis.
The antibiotic prevents assimilation of amino acids but inhibits the formation of polypeptide chain. It also inhibits selectively the growth of many bacteria but fails to produce an appreciable effect on the growth of yeasts, fungi, protozoa, or animal cells in the same concentration.
3. Streptomycin:
It is effective against tuberculosis causing organism, Mycobacterium tuberculosis and Gram-negative bacteria. The prolonged use of streptomycin in mass can produce neurotoxic effects and loss in hearing. Since its discovery by Schatz, Burgie and Waksman (1944) most of the strains of S. griseus are genetically improved.
(i) Structure of Streptomycin:
The commercial available streptomycin is basically hydrochloride of streptomycin (C21H39N7O12.3HCl) with calcium chloride. During the production of streptomycin, mannosidostreptomycin or hydroxystreptomycin is also produced in the early fermentation. This salt is not economical and is easily converted to streptomycin by the action of S. griseus (Fig. 20.21). No precursor is reported to increase the yield.
(ii) Media Composition:
Following is the composition of medium (lit-1):
Soybean meal – 10g
Glucose – 10g
Peptone – 5g
Meat extract – 5g
Sodium chloride – 5g
The pH is kept at 7.6-8.0 before sterilization and after inoculation the culture is incubated at 28°C.
(iii) Production:
Streptomycin produced by S. griseus, an actinomycete that can be grown in stirred fermentation due to strong requirement of high aeration and agitation. Continuous fermentation process is required to produce streptomycin.
The spores can be produced on medium which provide enough sporulated growth to initiate liquid culture of mycelium. The optimum fermentation temperature is approximately 28°C and the whole process completes within 5-7 days.
There are three main steps in the production process of streptomycin:
(a) First Phase:
Growth of mycelium occurs, the proteolytic activity of S.griseus releases ammonia from the soybean meal, the carbon from soybean meal utilizes and induce growth but glucose is utilized at a minimum rate. The yield of streptomycin produced is low. The pH rises.
(b) Second phase:
The streptomycin synthesized at a rapid rate in this phase, due to rapid utilization of ammonia and glucose. The total incubation period lasts from 24 h to 6 – 7 days. No mycelial growth occurs in this phase. The pH remains from 7.6 to 8.
(c) Third Phase:
The sugar depletes from the medium resulting into cease of streptomycin production. The cells lyse, releasing ammonia resulting into raised pH. Before lysis, fermentative material is harvested for recovery of streptomycin.
(iv) Recovery:
After filteration, the broth is treated with activated carbon and then streptomycin is eluted with dilute acid. The eluted streptomycin is then precipitated by solvents, filtered, and dried before further purification. In another process, culture filtrate is acidified, filtered and neutralized.
It is passed through cation exchange column to absorb streptomycin. Later it is dissolved in methanol, after filteration acetone is added to yield streptomycin. About 1200 µg/ml yield is obtained. The yield is not affected by contamination except due to actinophages as found in penicillin production.
(v) Unit of Antibiotic:
One unit of streptomycin activity, however, is equivalent to the microgram of free base.