Thank you very much for your kind suggestion. According to your suggestion if I use  antifungal antibiotics,  I am not sure, whether will it interrupt the growth of E. coli O104/ influence VBNC state  of that bacteria inoculated into orange juice or not, so if you have any idea,please kindly give me.@Hani Anton

You can use sorbate type food preseratives if you want to have a real life scenario.

Hey

Did you filtered the juice before the inocule with E. coli? That would prevent any other type of contamination. 

Also you can supplement it with some fungicide, like cyclohexamide - that wouldn't affect the bacterial growth, since it only affects fungi. 

But the best option is to filter the juice and then make the uncoil under aseptic conditions. 

Best regards

Dear Santos filtering is not a better option as the orange juice is thick and contains several suspended constituents. Nahar you can use food preservatives (as we use packaged fruit juices which contain preservatives) or anti fungal agents as suggested by   Antoun, Ozturk, and Santos. I don't think that these compounds will interfere with bacterial growth due to their mechanism of action. But you should get a minimum concentration of anti fungal by optimization to minimize this risk. 

Thanks all for giving  me some valuable suggestions.

@ Santosh Kumar and Santos.  I would like to inform you that actually  in my experiment I used orange juice that was bought from supermarket and it already had some preservatives. I used 2 brands of juice , one is rich in fiber for which I centrifuged it at 1200 rpm for 5 min and inoculated the target bacteria and kept at refrigeration temperature and sampling was done and cfu was counted spreading on LB agar. After certain time, around 23 days,   when target bacteria were no detectable in LB agar, Then I go for revival of target bacteria using special media. After growing in  that media I spread  it in LB agar and found,  more fungus growth  than my target bacteria. In this case what can I do? could you please help me to give some suggestions?

Azium,

If you are applying some treatment after the inoculation to inactivate e.coli, the addition of any antifungal, or the use of a selective media will affect your results. Some cell will be sublethaly injured, and those cell in a media enriched with an antifungal, or the use a selective media could be an stressfull condition, making it unable to growth, and those sublethaly injured cells will afect your results. This cells in a real scenario can be the ones that cause an intoxication. 

So if you are preparing the orange juice so you may prepare it in sterile conditions (desinfection of the fruit, then the prepration in a cold room 5ºC, and all the instruments you use might be sterilized) this would reduce the initial contamination, the pasteurize at 85ºC per 3 minutes... also E. coli O104 and O157 growth at 37ºC so it would not affect your plate counts. 

In a real scenario, there exist an a positive relationship (comensalism) between yeast & moulds and bacterias, due the low pH of the juice the first in growth are yeast and moulds, so as an effect of their growth the pH rise and the medium pH gets perfect for the growth of bacteria, with out this it would be very difficult for bacteria to growth, and as far as i know there is just one bacteria available to growth in the juice characteristics, Alicyclobacillus acidoterrestris.

You can use "natamycin". It's a food grade anti-fungai. It's effective on yeast and fungi and you won't need more than 40 ppm of it. 10 ppm is usually effective in most foods.

Thanks all.

@Juan-Pablo. I am sorry that  according to your opinion, whether yeast and mold is necessary to grow in my experimental juice to help growing of my target bacteria or not, I am not clear, could you please clarify it?. By the way I would like to inform you that I used Orange that was bought from supermarket and fully sterilized.

Another question, In my experiment, I inoculated bacteria in 100 ml of Orange juice  in a flask and every 3 days interval, I removed 500 microlitre of juice by hand stirring clockwise  10 times and anticlockwise 10 times.Now my question is that whether hand shaking is enough for homogenizing the bacteria for dilution or not? and another question- I cannot understand that when transferring sample, every time shaking the flask containing juice will affect the counting cfu as shaking change the environment everytime, If problem. What can I do ,could you please give me some suggestions?

Hello, you may treat the juice with pasteurization before inoculating with E. Coli, of course the inoculation have to be done under aseptic conditions. this would prevent your juice from fungi contamination for a while. another solution you may use antifungal in this juice but you have to be sure that it is not affecting E. coli.

have you tried Millipore filtering the juice, simple and you are not changing anything. You can then add your E. coli

Thanks all for suggestions.

Hi. I think you Should use antifungal agents such as nystatin in order for preventing the fungal growth.

Just add a few micrograms of the antibiotic - ACTIDIONE to your orange juice along with the strains of E. coli.