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.
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.
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.