Well try to use your stock culture and repeat the experiment. Also you should know which growth phase the organism is active and from this phase make the bioassay again. Also in some cases the repeated subculturing of the organism can make spontaneous mutations , leading to a phenomenon like that

Thanks Gamal

I was trying to stick to the original cultures but there was a finite amount . Most of the inhibitory activity occurred  after 7-9 days at a lower temperature. 

I suggest that you go back to your original culture and try to measure antibiotic activity at different physical factors, nutritional factors, and growth phase points

Thanks Yehia

restreak on agar, pick sporulating healthy single colonies. Test fermentation 10 of them. I suspect serially subcultured cultures contain a lot of bld and non producing strains.

Best to keep spores prepared from single isolates.

I agree with the comments given above. In addition, you need to consider your methods for maintaining your stock culture. By the way, how did you maintain your stock culture?