Since I am doing a project of bioactive compounds from bacteria and fungi I would like to produce high amounts of all types of vitamins and other active compounds
You need an assay for the compound -- like a mutant of E. coli that can't make that compound
Then you could ask in a mixture of cells, are there any organisms excreting that compound. You could take the E. coli mutant and mark it with a metabolic dye so that when it is metabolizing it will be fluorescent.
If the bulk collection gives you any activity, you can separate the cells and repeat the test
Of course, if you can grow the bacteria or fungi, you can just add the mutant cells to the mixture and see if there is enhanced growth around any colonies that are growing.
Just a few thoughts
Ken Nealson
I do not work with bacteria, but I believe that the same methods can be used used with protozoarios. I identified vitamin K, vitamin E and carotenoides using methods of HPLC. These vitamins must be extracted of the bacteria with hexane or with other solvents. These are the works: Intraerythrocytic stages of Plasmodium falciparum biosynthesize vitamin E. Sussmann RA, Angeli CB, Peres VJ, Kimura EA, Katzin AM. FEBS Lett. 2011 Dec 15;585(24):3985-91.
Intraerythrocytic stages of Plasmodium falciparum biosynthesize menaquinone. Tonhosolo R, Gabriel HB, Matsumura MY, Cabral FJ, Yamamoto MM, D'Alexandri FL, Sussmann RA, Belmonte R, Peres VJ, Crick DC, Wunderlich G, Kimura EA, Katzin AM. FEBS Lett. 2010 Dec 1;584(23):4761-8.
Carotenoid biosynthesis in intraerythrocytic stages of Plasmodium falciparum. Tonhosolo R, D'Alexandri FL, de Rosso VV, Gazarini ML, Matsumura MY, Peres VJ, Merino EF, Carlton JM, Wunderlich G, Mercadante AZ, Kimura EA, Katzin AM.J Biol Chem. 2009 Apr 10;284(15):9974-85.
adel mahasneh
by microbiological assay technique.you could use some Lactobacilli ex. L plantarum which is known not to synthesize any vitamins and assay the culture filtrate of your suspected bacterial strain.
A suggestion when trying to find a needle in a haystack. Look for vitamin precursors that the bacteria requires to produce the target vitamin, label it with a fluorescent dye for instance, allow an incubation period and then examine bacterial cells either by epifluorescent microscopy or by flow cytometry. For essential vitamins you can grow the bacterial soup on vitamin deficient media, subculture them onto the same media and then further tests on those that grow.
Look for the symbiotic growth of bacteria one which is producing vitamin and one which is not producing but still requires that vitamin.. isolation of such relationship will help.
First you need a quantitative assay for the presence of the desired compound(s). HPLC, gas spectrometry, antibody binding, photospectroscopy, whatever works best. Then test your starting pools of bacteria for the compound(s). Then you'll need to start dividing your bacterial population into fractions. Test each fraction for the presence of the compound. Remove those fractions that lack the desired compound. Repeat until you have isolated a pure culture of bacteria that produce the compound(s) you want.
The simplest way will be the use of vitamin auxotrophs. Auxotroph is an organism that needs a known ingredient (like vitamin in this case) to be added in the medium for its growth. Auxotrophs for many vitamins are available in the literature.
After growing a pool of bacteria you want to test in minimal medium (medium without a particular vitamin) in a Petri plate, layer an auxotroph in minimal medium. The growth of auxotroph indicate the secretion of a particular vitamin.
i am also interested to identify LAB which have ability to produce vitamins? please suggest some direct methods?
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