## 3 The Influence of Ultrasound on the Physical and Chemical Properties of Juices
Freshly squeezed orange juice was sonicated for 1, 10, 20 and 30 min at a frequency of 24 kHz to evaluate its effect on selected microorganisms. Ultrasound was generated continuously using a UP200H processor from Hielscher Ultrasound Technology and an S3 probe (Hielscher). Overheating of the samples during the ultrasound treatment was prevented by circulating ice water through the treatment chamber. The number of aerobic mesophilic bacteria was significantly reduced when the prepared product was sonicated for 30 min, while 20 min of sonication was sufficient to eliminate yeasts and moulds. The results of this study indicate that the applied process conducted at a tempera  
Figure 3: Mechanisms responsible for the effect of microbial inactivation of sonication. ture of 43-45 \({}^{\circ}\)C can be used to process orange juice and leads to an improvement in its microbiological safety. Adekunte et al. [56] used a 1500 W ultrasonic processor (VC 1500, Sonics and Materials Inc., Newtown, USA) for the sonication of tomato juice. Samples whose processing temperature ranged from 32 to 45 \({}^{\circ}\)C were processed at a constant frequency of 20 kHz. The performed tests showed the inactivation of yeast (_Pichia fermentans_) by 5 log, and the level of their reduction depended on the applied amplitude and duration of the process, which were 61 \(\upmu\)m and 7.5 min, respectively. The yeast inactivation observed may result from the aforementioned combination of physical and chemical mechanisms occurring during cavitation. The formation of free radicals and H2O2 during sonication helps to inactivate microbes. Nevertheless, the physical effects of the process may not be effective enough to inactivate yeast cells that are difficult to disrupt through micro-jet action. Rather, ultrasound may lead to rupture of yeast cells and subsequent release of protein [57]. Satisfactory research results were obtained by Bhat et al. [58] when freshly squeezed kasturi lime fruit juice (_Citrus microcarpa_) was sonicated for 30 and 60 min at 20 \({}^{\circ}\)C and 25 kHz (using an ELMA(r) cleaning bath, model T700h W/Acc,ingen, Germany) for microbiological evaluation of the product via analysis of total plate counts, yeast and mould. Sonication of juice samples for 60 min showed a significant reduction in most microorganisms compared to 30 min treatment of samples and control (untreated) samples along with the achievement of final product safety and quality standards through 5-log reductions, which is the mandatory level set by the FDA for fruit and vegetable juices. In the work of Khandpur and Gogate [59], the effectiveness of ultrasound in the processing of various fruit and vegetable juices obtained from oranges, sweet limes, carrots and spinach was assessed in terms of the growth of microorganisms and changes in quality parameters during storage. Ultrasonic treatment with a constant frequency (20 kHz) for 15 min at a temperature below 30 \({}^{\circ}\)C was adopted as the optimal process conditions. The results of the authors' research unequivocally confirmed the usefulness of sonication to maintain the microbiological safety of beverages characterized by an extended shelf life and excellent quality parameters compared to untreated (control) and thermally treated juices. The analyses of Choo et al. [60] indicate that regardless of the method of processing used for noni fruit juice (_Morinda citrifolia_ L.), i.e., ultrasonic exposure for 60 min at a constant temperature of 30 \({}^{\circ}\)C and frequency of 37 kHz or pasteurization at 90 \({}^{\circ}\)C for 60 s, the level of microbes may be within the acceptable range. No yeasts or moulds were detected during 8 weeks of refrigerated storage (4 \({}^{\circ}\)C), while the total number of aerobic mesophilic bacteria remained below 10\({}^{4}\) CFU/mL, indicating that the juice was edible.