There are many steps to your question. You are not proposing something easy because there are so many diverse strains of those bacteria and their bacteriocin diversity can be immense. Also, they may produce microcins and phage, and the bacteriocins can be regulated by a number of different systems which require all sorts of environmental cues, including other bacteria.

The classic starting place is ammonium sulfate precipitation following filtration. Sephadex is also classically used.

In the end, however, research on nisin would best be served by using commercially available nisin. Some of the other gram positive bacteriocins can be purchased pure, and Sigma-Aldrich used to offer colicins as well.

U can simply check the antibacterial activity of the supernatant at neutral pH to confirm the bacteriocin, then grow it again in large (500-1000 ml) amount and precipitate the supernatant with ammonium sulfate, dissolve the PPT in PPB and dialyse it with 1 kda MWCO membrane against the same buffer for 12-24 h. The membrane will keep ur bacteriocin and u can use further then that crude bacteriocin for ur experiments, this is the simple protocol for bacteriocin, u can check more details in some published articles or books.

Try to do this: first you have to grow your producer strain onto the surface of a agar plate (in this case for lactic acid bacteria produce bacteriocin you can yous M17 agar plates, MRS agar plates, or Columbia agar plates supplemented with 5% whole human blood. or use any production media. after you make lawns of producer cultures grown onto the agar plates (maybe 80 plates) grown for at least 18 hours aerobically with 5% CO2. you transfer the whole culture (bacteria and agar everything) into centrifuge bottles and then transfer the bottles to -70C freezer for over night. after that you have to thaw the frozen cultures into the bottles by putting the bottles into a water bath 55C for one hour. centrifuge the cultures within the bottles 1000Xg and collect the bacteriocin containing supernatant and discard the agar debris. this liquid can be passed through XAD-16 column for concentration and purification. Please check my publication for more information. this method called freeze-thawed bacterial cultures and usually all bacteriocins of LAB can be recovered by this method.

Forgot to mention that make sure that the agar added to the plates should be no more than 0.8% to achieve successful freeze-thaw extraction.

Dear Paolo,

i suspect from your question that you are looking to extract a sample containing bacteriocin activity from various strains.You can either use the supernatant or transfer the cultures into liquid media or use a protocol similar to the above referred by the collegues. One parameter though that it might occur , would be the loss of activity (partial or total) due to interactions between the differents genres of bacteriocins (predator-prey relationships, one bacteriocin degrades another) that is why bacteriocins are preferably paired with lactic acid or thermal treatments at least in food. If you decide to go for transferring your cultures into liquid media (MRS broth foer example) or from supernatants , 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 Paolo,

I was in a similar situation as you are now (no liquid chromatography system in the lab), thus I tried various separation methods available.

By combining ammonium sulphate precipitation, solvent extraction and “manual” reverse-phase chromatography (on a plastic disposable C18 column), I have developed a method that worked fine for me (you can find my paper using this method here www.ncbi.nlm.nih.gov/pubmed/22739096 ).

Here it is:

Following cultivation of a producer, ammonium sulphate (50 g/L) was added to the liquid culture medium. After stirring for 3 h at 4 ◦C, the cells were collected by centrifugation (4225×g for 30 min at 4 ◦C) and were re-suspended in 70% isopropanol (adjusted to pH 2 with 1 M HCl). Following a second stirring step for 16 h at 4 ◦C, cell debris was removed by centrifugation (4500×g for 30 min at 4 ◦C). The supernatant was concentrated (Rotavapor®; BÜCHI, Flawil, Switzerland) and was subsequently re-ruspended in 0.01 M sodium phosphate buffer (pH 5.5). Bacteriocin was further purified by mixing with chloroform (1:1 v/v) and vortexing vigorously for 30 s. Following subsequent centrifugation (18 506×g for 40 min at 4 ◦C), the proteins at the chloroform–aqueous interface were recovered, re-suspended in bidistilled deionised water (bdH2O) and adjusted to pH 2 using 1 M HCl. The bacteriocin sample was loaded onto a disposable Sep-Pak® column containing 5 g of C18 gel (Waters, Milford, MA) pre-equilibrated with methanol. Step gradient elution was performed with 30% (v/v) ethanol and a series of isopropanol solutions with concentration increments of 10% (v/v). The collected fractions were concentrated in a Speed Vac (Savant Instruments, Farmingdale, NY) and were subsequently re-ruspended in 0.01 M sodium phosphate buffer (pH 5.5).

I hope it will work for you.

All best,

Milos

Dear Paolo,

Many of the colleagues earlier have given you excellent ideas. Ammonium sulphate precipitation, followed by solid phase extraction is the commonly followed route. Nevertheless, I might be interesting to use the method developed in this old paper to extract some/all bacteriocins by absorption/desorption to producer cells. You can either use pure cultures of strains which are known to produce these bacteriocins or alternatively you can find target strains affected by these bacteriocins (for example Listeria inocua) in the same protocol. Absorption/desorption of some bacteriocins on the surface of the producer (target?) cells is pH dependent. Bacteriocins tend to attach to producer cells at high pH and detach from them at low pH. The whole procedure is very easy and it does not require that much time. This method may not be a high yield method but it could be used for first purification.

Good luck,

Panos