In order to determine the antimicrobial activity of several lactic acid bacteria (Lactobacillus spp.), I want to separate bacteriocins from the culture broth. Can anybody suggest the most effective method or protocol?
Alternatively, if your bacteriocin was not precipitated by ammonium sulfate, you can separate the bacteriocin substance from culture broth by using Amberlite XAD-16. See below for details of the method I used.
1. Harvested for cell-free-supernatant (CFS) from culture broth by centrifugation (8000 rpm for 15 min at 4°C).
2. Heat the CFS at 80°C for 20 min to destroy proteolytic enzyme and some living cells.
3. Pour the CFS into Amberlite XAD-16 (Sigma-Aldrich, Steinheim, Germany) and soak with gentle shaking for 3-5 hrs. The bacteriocin will be adsorbed with the Amberlite XAD-16 resin at this step.
4. Filtrate for the adsorbed resin and elute the bacteriocin from the resin by using 70% isopropanol (JT Baker, Pennsylvania, United State), pH 2.
5. Adjust pH of the eluent to 5.5 and evaporate at 60 hPa, 42°C to get rid of isopropanol.
6. Test for the antimicrobial activity of the partial purified bacteriocin.
If you have some more question, please feel free to let me know.
A very common approach is to use ammonium sulfate precipitation.
Here is an example protocol:
100ml cultures were grown overnight and then centrifuged at 7000 × g for 10 min.
2. The supernatants were then removed and cooled at 4°C, and 30g of ammonium
sulfate was then added to each solution.
3. The solutions were then incubated at 4°C for 45 min with moderate shaking.
4. Afterwards the solutions were centrifuged at 20 000 × g for 30 min. at 4°C.
5. The supernatants were then decanted and the pellets were dissolved in 2ml dH2O for a final concentration of 50x.
6. The solutions were then incubated for 10 min at 70°C to kill any remaining bacterial
cells.
7. The peptide concentrates were then stored at -20°C.
I think you have to use salt precipitation using Ammonium sulfate then use dialysis to separate the salted out protein from Ammonium sulfate.
Alternatively, if your bacteriocin was not precipitated by ammonium sulfate, you can separate the bacteriocin substance from culture broth by using Amberlite XAD-16. See below for details of the method I used.
1. Harvested for cell-free-supernatant (CFS) from culture broth by centrifugation (8000 rpm for 15 min at 4°C).
2. Heat the CFS at 80°C for 20 min to destroy proteolytic enzyme and some living cells.
3. Pour the CFS into Amberlite XAD-16 (Sigma-Aldrich, Steinheim, Germany) and soak with gentle shaking for 3-5 hrs. The bacteriocin will be adsorbed with the Amberlite XAD-16 resin at this step.
4. Filtrate for the adsorbed resin and elute the bacteriocin from the resin by using 70% isopropanol (JT Baker, Pennsylvania, United State), pH 2.
5. Adjust pH of the eluent to 5.5 and evaporate at 60 hPa, 42°C to get rid of isopropanol.
6. Test for the antimicrobial activity of the partial purified bacteriocin.
If you have some more question, please feel free to let me know.
Dear Marimunthu,
there are several ways of separating bacteriocins from fermented broths including gel filtration, nanofiltration,which I personally think is the most effective, high pressure liquid chromatography (HPLC) , ammonium sulfate precipitation. Each method has different volumetric yields but also different yield in potency and activity, a good relevant publication is the following,Parada JL, Caron CR, Medeiros ABP, Soccol CR. Bacteriocins from lactic acid bacteria: purification, properties and use as biopreservatives. Braz Arch Bio Tech 2007; 50: 512-54.http://www.scielo.br/scielo.php?pid=S1516-89132007000300018&script=sci_arttext&tlng=es . I have done extensive work on bacteriocin recovery produced of lactic acid bacteria , especially of lactococcoi and lactobacilli for example Separation of lactobacilli bacteriocins from fermented broths using membranes see details on my profile https://www.researchgate.net/profile/Myrto-Panagiota_Zacharof/?ev=hdr_xprf, or follow the link to the final publication http://www.sciencedirect.com/science/article/pii/S1359511313002158.
Best wishes, Myrto
Dear Anandharaj,
For determination of primary antimicrobial activity of bacteriocins, separation is not necessary. But consider following factors for isolation and purification of bacteriocins.
1. Heat stability or heat sensitivity of bacteriocins
2. Resistance of bacteriocins to pH changes
3. Complexity of the bacteriocins (is bacteriocin in combination with carbohydrates or lipids?).
4. Effects of solvents and salts on antimicrobial activity of bacteriocins (solvents and salts used in many different isolation and purification methods.
Enhancement of novel extracellular bacteriocin production by media optimization using LAB isolate from
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