Inoculum preparation in seed lab of fermentation industries:-
Today many industries including pharmaceutical industries use fermentation process for manufacturing various products like drugs, semi-synthetic medicinal products, beverages, chemicals, etc. All such fermentation process needs pure cultures of microorganisms which are capable of utilizing substrates with the help of their enzymes. Enzymes are proteins; it does not mean all proteins are enzymes. Enzymes have active sites on which the substrates are utilized and fermentation process is done along with production of many byproducts which also has commercial values.
Inoculums are pure cultures of required microorganisms for fermentation process. Today pharmaceutical industries manufacture drugs using various microorganisms and these product ranges from anti-TB drugs like Rifampicin, anti-cholesterol drugs like Lovastatin and many other semi-synthetic drugs. These are also used in beverages manufacturing industries. Lyophilized cultures of such microorganisms are preserved at -180°C such that they can be enriched as and when required for manufacturing drugs. Lyophilized cultures are freeze dried form of microorganisms which are vacuum dried at -40C for more than 12 hours and with these ampoules are aseptically flame sealed and further stored in -180C.
As and when required, these are removed from deep freezer and are sub cultured on to the selective media using aseptic techniques. The lyophilized cultures are opened aseptically under clean rooms like class 100 environments and suspension of these cultures is made. This is later spread on to media using spread plate technique and incubated at favorable condition depending upon the requirements of organisms. The visible colonies appear on the media containing Petri-plates and this is later isolated using various colony morphological studies.
Few selected colonies area sub-cultures onto slants having selective media depending upon the specific organism under test. After the growth is completed and incubation period is completed, these are tested for its purity and antibiotic producing properties. The cultures from the selected slants are tested for their ability to produce specified products like antibodies and also for the most important part that is for its purity. Purity is specifically more important as this can be the first step to control contamination in actual manufacturing at large scale. Laboratory scale fermentation is done for testing the ability of microorganism under test to product the drugs, antibiotic, etc. All media as used during actual fermentation in large fermenters is added in same proportion in glass level fermentation test that is at laboratory level.
The few best organisms which were initially selected for sub-culturing are testing for selecting the best isolate among them so that It can be further isolated and used for actual fermentation. The ability of culture to produce the specified antibiotic (or specified product) is tested by means of using various purification & analytical methods like chromatography, spectro-photometry. The antibiotic titer is calculated and the best isolate is selected who's giving highest titer of antibiotic production.
This best culture is further studied for its purity again before taking it for actual production. Enrichment of the culture is most important and need to be performed under stringent aseptic conditions. The enrichment is done by providing all required carbon sources, nitrogen sources, minerals, salts for osmotic balancing of organism and required water quantity. The purity in enrichment is most critical part as there is chances that other non-required organisms can contaminate the whole process including the actual manufacturing at large scale and which may lead to severe commercial losses. Even the best selected cultures has to compete with the contaminants for its enrichment and this affects its properties and ability of producing higher product for which it has been selected. Avoiding contamination is a skill of microbiologist and which comes with experience and practice. Even with support from facility & environment like clean rooms, laminar flow class 100 areas, temperature, relative humidity, there is a chance that error can happen in aseptic techniques of microbiologists. If care is taken, this is not a problem. Once the enriched culture passes the purity test, it is used for actual production at large scale as inoculums. These inoculums act as seed for large scale fermentation process.
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