Have you confirmed that the inhibition is not due to an acidification of the media by secreted lactate?

Firstly, thank you very much for your answer. I performed Bradford Analysis for the supernatant which I obtained. Normally there is casein in MRS Broth, I used MRS Broth as a blank. The protein ratios in the supernatant were higher than the protein in the blank. Also one of the pathogens that I use for antimicrobial activity is E.coli. I know that E.coli can also survive at low pH. And the lactic acid bacteria which I use also had a good antimicrobial activity on E.coli. Therefore I think that the antimicrobial effect is due to protein. Do I also need to verify with another method? What do you recommend?

Hi, in my previous laboratory before we performed an agar well diffusion assay for checking whether the strains produce bacteriocin-like we neutralize the cell-free supernatant first (NCFS). Then, if it has shown clear inhibition, we confirmed it by digestion of NCFS with proteinase K and doing agar well diffusion again, if it abolished the activity, we can suspect that the strain(s) produce bacteriocin-like substance before checking the protein band by SDS-PAGE or Native PAGE. Good luck.

I've to repeat this part, you're right. Thank you very much for your answer, I'll try.

Dear Esra,

There is no "best method" for isolation of bacteriocins from LABs. The following factors affect on isolation.

1.Kind of LAB

2.Kind of bacteriocin (peptide or glycopeptide)

3.Molecular weight of bacteriocin. This affects on stacking and resolving gels percent and type of PAGE. Maybe Tris-Tricine-SDS-PAGE is required.

4.Thermal stability of bacteriocin

5.pH stability of bacteriocin

6.Presence of protease in broth medium

7.Time of harvesting

From one hand, the most important point is optimization of Percentage (%) saturation concentration of (NH4)2SO4 in MRS broth separately for each bacteriocin. On the other hand, in what percentage do you choose stocking and resolving gel in SDS-PAGE? For regular, low molecular or very low molecular weight?

Meanwhile, don't forget that precipitation with ammonium sulfate is the first step of separation and not all of it.

Firstly, thank you very much for your answer. You can be sure that this information will be very useful to me. I don't know the molecular weight of bacteriocin and so I performed different percentages of SDS-PAGE gels (12,16,20%). I think I need to do the characterization of the bacteriocin first. Then I'll to return to the SDS-PAGE and ammonium sulfate precipitation steps. Thanks again for your ideas. I'll try.

Hello, i think first of all you need characterization of the bacterio

cin. The modified agar-well diffusion method is most frequently used for antibacterial activity assay to screen for potentially bacteriocin producing strains and to elementarily characterize their bacteriocins and the relatives.

Hello, thank you very much for your answer. You're right, I'll try the characterization tests.

@Esra Bedir, @Heru Pramono, @Zhanylbubu Mamatova,@Say-yed Hesameddin Tafreshi

please scientists, could you explain to me how to estimate the bacteriocin activity (antimicrobial activity) after each purification step of the obtained fraction?

Best regards

Dear Makhlouf Fatima Zohra,

The measurement of bacteriocins is a quantitative and precise task and not an estimate. For a correct biological measurement (bioassay), you need the indicator strain, the right culture medium, and the most importantly, the right measurement method. The measurement method may be different based on the type of bacteriocin. You can find the appropriate method for the bacteriocins you are studying them, by searching in the databases. Don't forget that you have to set up the method anyway.

Dear Say-yed Hesameddin Tafreshi

Thank you for your answer and your help

By searching in the databases, I found the appropriate method for the bacteriocins that I am studing, but what is not clear in this method is the way of quantification and expression of the bacteriocin activity after each purification step (antibacterial activity)

Dear Makhlouf Fatima Zohra,

You have two options.

Option 1: You work with a known bacteriocin. In this case, you must compare your results and inhibition zone diameters with standard concentration of the bacteriocin (such as nisin).

Option 2: You work with a unknown bacteriocin. In this case, at first you must define "Arbitrary Unit" activity of the bacteriocin and then report the results. For example, each 2 mm of inhibition zone diameter equals to 100 Arbitrary Unit (AU) of the bacteriocin biological activity. Now, you can compare your results in different purification steps. Of course, protein assay must be done in each step for Specific Activity and Purification Fold measurements. I hope that I've answered your question.

Dear Say-yed Hesameddin Tafreshi

Thank you so much for your help and your answer.

Just one last question, how can I deduce the relationship between the diameter and AU : 1AU=? mm, or it's arbitrary I get to decide my self?

Dear Makhlouf Fatima Zohra,

Exactly correct. You must define the "Arbitrary Unit" according to your inhibition zone diameters. There is no standard for definition of "Arbitrary Unit". Of course, there is logic in the definition. For example, when the inhibition diameters are in the range between 5 to 20 mm, you can define each mm equals to 10 "Arbitrary Unit". Why? Because, your upper limit (20 mm) equals to 200 AU and in the next steps of purification, you will not encounter the problem of large numbers.

Please consider two point.

1. You should avoid using large numbers. For instance, 25000 AU/mL for biological activity of the culture medium is not suitable.

2. Principally, inhibition diameters have logarithmic relationship. Therefor, 10 or 100 are a good idea for "Arbitrary Unit" definition.

Finally, decision is yours and you get to decide yourself.

Dear Say-yed Hesameddin Tafreshi

Thank you so much for your insights and your help

بارك الله فيك وجزاك خيرا

Kind regards

Dear Makhlouf Fatima Zohra,

God willing, you will be successful in your work.

There are several methods available for bacteriocin isolation from lactic acid bacteria (LAB). The choice of method depends on factors such as the nature of the bacteriocin, the LAB strain, and the specific goals of the isolation process. Here are a few commonly used methods:

It is important to note that each method has its advantages and limitations, and the optimal approach may vary depending on the specific requirements of the study. Top of Form

Dear Saif Wahid,

Thank you very much for your answer. I'm currently working on the precipitation method with ammonium sulfate. This method has many disadvantages therefore I may try the other methods you mentioned. Thanks again for your help.