INTRODUCTION
The aim of the trial, carried out at the Minnesota University (USA) in 2018, was to determine if natural preservative (cimenol ring) at different concentrations (1000, 700, 400 and 200 ppm) could inactivate Clostridium perfringens after contact times of 10 minutes, 20 minutes, 60 minutes, and 24 hours.

METHODOLOGY
1.    The following concentrations of natural preservative (cimenol ring) in sterile RO water were prepared: 2000, 1400, 800 and 400 ppm.
2.    1 ml from all 4 vials were removed and placed in 4 new separate vials. 1 extra vial was prepared as control with only 1 ml of sterile RO water.
3.    To all 5 vials, 1ml overnight broth culture of Clostridium perfringens was added. Final preservative dilutions were: 1000, 700, 400 and 200 ppm, respectively.
4.    All vials were incubated at room temperature (25ºC) under anaerobic conditions. 
5.    After various time points (10 min, 20 min, 60 min and 24 hours) 200 μl from each vial were removed and serial 10-fold dilutions (100 to 10-7) were prepared in PBS as a diluent.
6.    Later, 100 μl of each sample dilution were transferred into separate sterile Petri dishes containing TSA (trypticase soy agar medium). 
7.    Plates were incubated at 37ºC for 24 hours under anaerobic conditions.
8.    Inoculated plates were examined and number of bacterial colonies was counted.
9.    Bacterial titers in all vials were calculated and recorded in a table.

RESULTS

The upper chart shows that 1000 ppm dilution at 24 hours was the only one that reached >99,99% of inactivation of CFU. Lower concentrations achieved 98,45% and 70% inactivation, at 700 and 400 ppm respectively, and also after 24 hours.

CONCLUSIONS
This natural preservative with cimenol ring is effective against C. perfringens, and its efficacy is related to the dose and time of contact with the pathogen.

Cimenol ring is commercially available under the name of Alquermold Natural, manufactured by Biovet S.A.