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Ultrasonication is a non-thermal method of food preservation that has the advantage of inactivating microbes in food without causing the common side-effects associated with conventional heat treatments, such as nutrient and flavour loss. In this work high intensity ultrasound was used to investigate inactivation Enterobacteriae count in raw milk. Raw milk with 4% of milk fat was treated with ultrasonic probe that was 12 mm in diameter and with 20 kHz frequency immerged in milk directly. For ultrasounds treatment, three parameters varied according to the statistical experimental design. Centre composite design was used to optimize and design experimental parameters: temperature (20, 40 and 60 °C), amplitude (120, 90 and 60 μm) and time (6, 9 and 12 minutes). All analyses were performed immediately after sonication and after 3 and 5 days of storage in refrigeration at 4 °C. The facts that substantially affect the inactivation of microorganisms using ultrasound are the amplitude of the ultrasonic waves, the exposure/contact time with the microorganisms, and the temperature of treatment. The achieved results indicate significant inactivation of microorganisms under longer period of treatments with ultrasonic probe particularly in combination with higher temperature and amplitude. Output optimal value of Enterobacteriae count has been defined by Statgraphics where lowest Enterobacteriae count (1.06151 log CFU mL-1) was as follows for specific ultrasound parameters: amplitude of 120 μm, treatment time for 12 min and temperature of 60 °C., Ultrazvuk kao netermalna metoda procesiranja hrane ima svoje prednosti pri inaktivaciji mikroorganizama u hrani u odnosu na klasične metode procesiranja hrane budući da uzrokuje manje negativnih učinaka npr. gubitak važnih nutrijenata te aroma. U ovom radu ispitivana je inaktivacija Enterobakterija u mlijeku primjenom ultrazvuka visokog intenziteta. Sirovo mlijeko sadržaja masti 4 %, tretirano je ultrazvučnom sondom promjera 12 mm i frekvencije 20 kHz. Eksperiment je planiran i proveden koristeći kompjuterski program STATGRAFICS (Centre composite design - CCD). Ultrazvučna obrada provedena je prateći tri parametra - temperaturu (20, 40 i 60 °C), amplitudu (120, 90 i 60 μm) te vrijeme procesiranja (6, 9 i 12 minuta). Sve analize provedene su neposredno nakon sonificiranja te nakon 3 odnosno 5 dana skladištenja uzoraka u hladnjaku na temperaturi od 4 °C. Inaktivirajući učinak ultrazvuka na mikroorganizme (Enterobakterije) ovisio je o primijenjenoj amplitudi ultrazvuka, vremenu i temperaturi procesiranja mlijeka. Dobiveni rezultati pokazuju značajan inaktivirajući učinak ultrazvuka kod dužeg perioda obrade ultrazvučnom sondom u kombinaciji sa povišenom temperaturom i amplitudom od 120 μm. Optimalna vrijednost (najmanja) Enterobakterija (1,06151 log cfu mL-1) u mlijeku određena je Statgraphics-om pri sljedećim uvjetima obrade ultrazvukom: amplituda 120 μm, vrijeme obrade 12 minuta i temperatura od 60 °C.