PaPer 64 Ital. J. Food Sci., vol. 28 - 2016 - Keywords: D value, inactivation, ultrasound, Zygosaccharomyces rouxii - INACTIVATION OF ZYGOSACCHAROMYCES ROUXII USING POWER ULTRASOUND AT DIFFERENT TEMPERATURES, PH AND WATER ACTIVITY CONDITIONS S. KIRIMLI1 and B. KUNDUHOGLU2* 1Institute of Science, University of Eskisehir Osmangazi, 26480 Eskisehir, Turkey 2Department of Biology, Science and Arts Faculty, University of Eskisehir Osmangazi, 26480 Eskisehir, Turkey *Corresponding author: Tel. +90 222 2393750 ext. 2845, Fax +90 222 2393578, email: bkunduh@gmail.com AbstrAct In this study, the effect of ultrasound treatments (20 kHz) combined with mild temperatures (thermo-sonication) on the inactivation of Z. rouxii was examined. Additionally, the effect of pH (4 and 7) and water activity (aw 0.99 and 0.94) of the sonication medium on yeast inactivation was determined. the D (40-55) values at a thermo-sonication amplitude of 80% were shorter than that obtained at 40%. Using thermo-sonication, particularly at a low aw, was associated with a signif- icant synergistic effect for Z. rouxii inactivation (p<0.05). In most thermo-sonications at 50° and 55°c, the FDA requirement of a 5-log cycle reduction could be achieved (>5.7-log reductions in <0.2-0.2 min). Our findings show that sonication offers advantages in terms of reduced duration and temperature of pasteurization, without a reduction in structural and sensory quality partic- ularly for fruit juices. mailto:bkunduh%40gmail.com?subject= Ital. J. Food Sci., vol. 28 - 2016 65 INtrODUctION the yeast Zygosaccharomyces rouxii rep- resents a major cause of spoilage of foods and drinks that are packaged according to good manufacturing practices (GMP), including fruit juices, sauces, carbonated drinks, wine, salad dressings, and ketchups (JAMEs and strAt - FOrD, 2003; PItt and HOcKING, 1985; LOUrEI- rO and MALFEItO-FErrEIrA, 2003; FUGEL- sANG and EDWArDs, 2007; DEÁK, 2008). typi- cal physiological characteristics of Z. rouxii in- clude tolerance to low-acidity preservatives, ex- treme osmotolerance, and the ability to adapt to high glucose concentrations, low water ac- tivity (aw) and thermal treatment (EMMErIcH and rADLEr, 1983; JAMEs and strAtFOrD, 2003; MArtOrELL et al., 2007). thus, Z. rouxii is important to consider in examining spoilage during the processing of foods with low-acidi- ty and high-sugar content. the food industry most frequently uses tradi- tional pasteurization methods such as low tem- perature long time (LtLt) and high temperature short time (Htst) to achieve shelf-life stability for fruit juices and drinks due to these meth- ods’ effectiveness and low cost. However, these procedures are associated with the loss of vi- tamins and volatile aromatic substances (KÖr- MENDY, 2007; VAsANtHA rUPAsINGHE and LI JUAN YU, 2012). In addition to thermal pasteur- ization, other methods that are commonly uti- lized to prolong shelf-life include chemical pre- servatives such as potassium sorbate, sodium benzoate (VAsANtHA rUPAsINGHE and LI JUAN YU, 2012), citric acid and sulfur dioxide (WILEY, 1994; bAtEs et al., 2001). chemical preserva- tives used to prolong shelf-life may be associ- ated with adverse health consequences in hu- mans, depending on the characteristics of the consumer population and the frequency of con- sumption (IsMAN, 2000). Although thermal treatment is the most com- mon technique to inactivate microorganisms in food, there is an increased interest in the use of alternative food preservation methods as a re- sponse to consumer demand for food with con- served innate characteristics and no artificial preservatives (cOrbO et al., 2009; VAsANtHA rUPAsINGHE and LI JUAN YU, 2012; ALZAMO- rA et al., 2003). some of the non-thermal food preservation methods that may represent an al- ternative to thermal treatment include electric or magnetic fields, microwave radiation, ioniz- ing radiation, high-intensity light pulses and high-hydrostatic pressure (cOrbO et al., 2009; DI bENEDEttO et al., 2010). Additionally, pow- er ultrasound (Us) is a promising novel technol- ogy that minimizes the need for treatment, in- creases food quality, and conserves the charac- teristics and sensory qualities of the food. Power Us is defined as the use of pressure waves be- tween 20 and 100 kHz. the lethal effect of ultra- sonic processing on microorganisms is achieved through the conversion of electrical energy to ul- trasonic sound waves via the ultrasonic trans- ducer and through the formation and collapse of vast numbers of small bubbles in each second during the propagation of ultrasonic waves with- in liquids. the quick formation and collapse of these bubbles (cavitation) creates very high local temperatures (5500°c) and pressures (50 MPa), which cause disruption of the cell wall and dam- age to the cell membrane and DNA (JIrANEK et al., 2008; MANVELL, 1997; KNOrr et al., 2004; O’DONNELL et al., 2010; cÁrcEL et al., 2012; LEIGHtON, 1998; sOrIA and VILLAMIEL, 2010). the duration and temperature of the procedure, the composition and volume of the liquid, and the form and dimensions of the microorganism are among the determinants of the antimicro- bial efficiency of ultrasonic processes (bEVILAc- QUA et al., 2013). the possible areas of use for microbial inac- tivation by power Us have been relatively well studied in the food industry. It has been report- ed that to achieve the FDA-required 5-log re- duction in microorganisms, sonication should be used in combination with mild heat treat- ment and/or pressure (FDA, 2001; WALK- LING-rIbEIrO et al., 2009; bAUMANN et al., 2005; D’AMIcO et al., 2006; UGArtE-rOMErO et al., 2006; sALLEH-MAcK and rObErts, 2007; tIWArI et al., 2009). Many studies have report- ed the synergistic effect of the combination of non-thermal technologies and heat treatment on microbial inactivation (GUYOt et al., 2007; LEE et al., 2009; LEIstNEr and GOrrIs, 1995; rAsO et al., 1998; rEDDY et al., 2006; rOss et al., 2003). However, to our knowledge, there are no studies examining the combined effect of heat, pH and aw on Z. rouxii inactivation using Us. therefore, the aim of this research was to evaluate the effect of Us with heat (thermo-son- ication) on the inactivation of Z. rouxii at dif- ferent pH and aw conditions. For this purpose, citrate buffer was chosen as the model medi- um, and the effect of thermo-sonication on Z. rouxii was tested under different pH and aw conditions. thus, the optimum procedural pa- rameters defined for Z. rouxii inactivation may be utilized as a model for the Us-assisted pas- teurization of real fruit juices and other drinks at mild temperature conditions. MAtErIALs AND MEtHODs Maintenance of test strain Zygosaccharomyces rouxii (NrrL Y-229) was obtained as a lyophilized culture from the Ars culture collection (Northern regional research Laboratory, United states Department of Agri- culture, Midwest Area-National center for Agri- cultural Utilization research Microbial Genom- 66 Ital. J. Food Sci., vol. 28 - 2016 ics & bioprocessing research Unit 1815 N Uni- versity street, Peoria, IL 61604). the culture tube was opened aseptically, the contents were transferred to a 2% sabouraud Dextrose broth (sDb, Merck, Germany), and the mixture was incubated for 48-72 h at 30°c. the stock cul- tures were then grown on sabouraud Dextrose Agar (sDA, Merck, Germany) slants and stored at 4°c until use. Preparation of yeast culture for inactivation studies Z. rouxii subcultures were prepared by inoc- ulating a test tube that contained 5 ml of ster- ile sDb with one single colony from a culture plate. the tubes were then incubated at 30°c for 48 h. Erlenmeyer flasks (250 mL) containing 50 mL of sDb were inoculated with this subcul- ture. the flasks were incubated under agitation (130 rpm). the broth cultures were transferred to sterile centrifuge tubes, and pellets were ob- tained at 5500 rpm for 10 min. the pellets were then washed with saline water (0.85% Nacl) and resuspended in the same medium. Z. rouxii sus- pensions prepared in this way were used to in- oculate sonication vessels at a final concentra- tion of 108 cFU/mL. Preparation of citrate buffer All sonication and control group treatments in this study were applied in citrate buffer me- dium. citrate buffer was prepared as two stock solutions (stock solution A: 0.1 M citric acid, c 6 H 8 O 7 .H 2 O reagent, carlo Erba, Italy; and stock solution b: 0.2 M di basic sodium phosphate, Na 2 HPO 4 .2H 2 O, Merck, Germany). the final pH of the citrate buffer was measured using a pH meter (WtW InoLab 730, Germany). Water activity (a w ) of the citrate buffer was ad- justed to a w 0.94 with glycerol (Merck, Germa- ny). the aw values of the citrate buffer medium were measured at room temperature (23-25°c) with an AquaLab water activity meter (Decagon Devices, Inc., UsA). Combined treatments (Thermo-sonication treatments; TS-T) sonication was performed with a Vc-750 Watt Us generator and a Vibracell® WcX 750 (sonics and Materials, ct, UsA) model ultrasonic processor at a frequency of 20 kHz (maximum 124 μm amplitude). A solid sonication probe (13 mm in diameter) was used in all treatments. Lev- els of 40% (49.6 μm amplitude) and 80% ultra- sonic power (99.2 μm amplitude) were applied in each case. Most of the sonication treatments were applied for 20 min. A 100 ml sterile wa- ter-jacketed vessel (Part No. 830-00010, son- ics and Materials, ct, UsA) was used to hold the citrate buffer. the temperature of the citrate buffer in the vessel was controlled by a refriger- ated circulating water bath (Polyscience-9102, IL, UsA). the temperature of the medium in the vessel was monitored during the sonication pro- cess using the digital thermometer (sonics and Materials, ct, UsA) of the ultrasonic processor. the vessels and probes were sterilized at 121°c for 15 min before and after each experiment. the preparation of the sonication vessels and the sonication process are described below. Ad- ditionally, the experimental design of the com- bined treatments (ts-t at different medium con- ditions) and thermal treatments alone (t-t: con- trol group treatments, at the same medium con- ditions) are summarized in table 1. (1) A total of 99 mL of citrate buffer was placed in a water-jacketed vessel. (2) A sonication probe was immersed in the center of the vessel. (3) the sonication procedure produces heat in a liquid medium; thus, to fix the temperature of the citrate buffer in the vessel at the target treat- ment temperature (40, 45, 50 or 55°c) during the sonication process, the temperature of the circulating water bath was adjusted to 7-10°c less than the target temperature. then sonica- tion was started. (4) Immediately after reaching the target tem- perature, 1 mL of yeast suspension was add- ed to produce a final concentration of 108 cFU/ ml in the citrate buffer in the sonication vessel. (5) At the beginning and during the treat- ment, 1 ml samples of citrate buffer samples were collected from the vessel and serially di- luted in sterile saline water (1:10). If necessary, the sampling intervals and treatment times were adjusted (e.g., in the case of high temperature table 1 - summary of the experimental design with ther- mo-sonication (ts-t) and thermal treatments (t-t) at dif- ferent pH and aw levels. Variables Treatment Sonication pH aw Treatments Temperatures Levels TS-T T-T - 4 0,99 + 0,94 + 7 0,99 + 0,94 + 40% 4 0,99 + 0,94 + 7 0,99 + 0,94 + 80% 4 0,99 + 0,94 + 7 0,99 + 0,94 + -: no sonication. 40°C, 45°C, 50°C and 55°C Ital. J. Food Sci., vol. 28 - 2016 67 levels). survival was determined using the drop- plate and spread-plate techniques. Aliquots of 0.02 ml (for drop-plate technique) or 0.1 mL (for spread-plate technique) were taken from the di- lutions and plated on sDA. the plates were in- cubated at 30°c for 48 h, and counts of survi- vors in treated samples were conducted. All ex- periments were repeated at least two times. Thermal Treatments (T-T) alone the survival and growth of Z. rouxii was also determined in citrate buffer at different tem- peratures (40, 45, 50 or 55°c) and under differ- ent medium conditions (pH 4 and 7 and aw 0.99 and 0.94) without sonication. treatments were performed in a shaking water bath (Memmert, Germany). the T-T process is described below and given in table 1. (i) A total of 99 ml of citrate buffer was placed in a flask. (ii) to reach the target temperatures (40°, 45°, 50° and, 55°c), 99 mL of citrate buffer in flasks was pre-heated in a shaking water bath. the temperature of the citrate buffer in the flasks was monitored using a digital thermometer. (iii) the citrate buffer reached the target tem- perature level. (iv) One milliliter of yeast suspension was add- ed to achieve a final concentration of 108 cFU/ ml in the citrate buffer. this step corresponded to the beginning of the treatment time. (v) During the treatment, 1 ml samples of the citrate buffer were collected from the flasks and serially diluted in saline water (1:10). the sam- pling intervals were 0, 1, 2, 4, 8, 12, 24 and 48 h. Viability counts were conducted as described above. As shown in table 1, 48 different (32 ts-t + 16 t-t) treatment conditions were studied to de- termine yeast inactivation, and each treatment was repeated in parallel at least two times. Determination of D values In this study, the inactivation of Z. rouxii was described using the first-order inactivation ki- netic model. the D values were directly calcu- lated from the k values (the slope of the inacti- vation curve) and the r2 values. First-Order Kinetic Model: Where; No= initial cell number (cFU/mL), t = treatment time (min), N = number of the surviving cells (cFU/mL) af- ter t minutes of treatment, k = slope of inactivation curve (min-1), k´ = log of slope of inactivation curve (min-1), and D = decimal reduction time, or the time required for a 1-log cycle reduction in the microbial pop- ulation. Data were fitted to this model with a linear re- gression using the Microsoft Excel program. Ad- ditionally, log reductions (log cFU/mL) for each process were calculated using data of the initial and final yeast numbers in the vessel. Viability of yeast cells in treated samples during storage In this step of the study, we determined the growth of the survivors during storage at differ- ent temperatures (4° and 25°c). two samples (10 mL) were taken from each treatment, with 5-log cycle reductions achieved; they were asep- tically transferred into 10 mL double-strength glass bottles containing sDb and stored at 4° and 25°c for 60 d in the dark. During storage, 1 ml aliquots were taken predetermined intervals from each bottle and were then transferred into sDb; the tubes were incubated at 30°c for 3-5 days, and yeast growth was checked. the sam- pling intervals were 1, 7, 15, 30, 45 and 60 d. Statistical analysis Variance analysis was used to determine the effect of inactivation factors on D values. the plate-count data were logarithmically trans- formed for statistical analysis. the results (log 10 cFU/ml) were subjected to an analysis of vari- ance (sPss Ver. 11.5, chicago, Il, UsA). For all experiments, a p value ≤0.05 was considered to indicate statistical significance. rEsULts AND DIscUssION In the present study, the inactivating effect of ultrasound waves (20 kHz) on Z. rouxii was in- vestigated in a model medium (citrate buffer). A total of 48 different experiments were performed to determine the effect of heat (40, 45, 50 and 55°c), pH (4 and 7), and aw (0.99 and 0.94) on ultrasonic inactivation (40 and 80% amplitude) of Z. rouxii. During the sonication procedure, pe- riodical sampling from the sonication chamber was conducted to determine the number of via- ble cells of Z. rouxii (cFU/ml). A first-degree ki- netics reaction was used to establish inactivation plots for Z. rouxii that were subsequently utilized to estimate the “D values” based on slope and r2. Additionally, yeast reduction was determined based on a comparison of the cell numbers be- fore and after the procedure. the difference in D values, as defined by the ts-t and t-t pro- cesses, were assessed using variance analysis. 68 Ital. J. Food Sci., vol. 28 - 2016 Furthermore, the growth pattern of sublethally injured yeasts following ts-t and t-t process- es were evaluated under different storage con- ditions (at 4° and 25°c for 60 d). Inactivation of Z. rouxii at 40°C An overall assessment of the results of all combined procedures at 40°c showed a small- er D value at 80% amplitude (0.94 aw and 0.99 aw; pH = 4 and pH = 7) than at 40% amplitude (p<0.05) (Fig. 1a). A generally reduced microbio- logical resistance to heat occurs in an acidic en- vironment. However, in our study, the D 40 val- ues in the combined and thermal procedures at pH= 4 were statistically significantly higher than those at pH=7 (p=0.012). the aw of the medium also had an impact on Z. rouxii inactivation. the D 40 values estimated at 0.94 aw in all combined and thermal proce- dures were higher than those estimated at 0.99 w (p<0.05). thus, a low aw was considered to give Z. rouxii a higher resistance to heat and sonication. similar to this study, ALVArEZ et al. (2003) observed a 30-fold increase in the ther- mal decimal reduction time for Salmonella en- teriditis by decreasing aw from 1 to 0.96, where- as only a two-fold increase was observed with mano-sonication, and a synergistic lethal effect with the combined use of heat and ultrasound was observed. In our combined treatment procedures at 40°c, the reduction in Z. rouxii for the 40% and 80% amplitude levels was 0.4-1.6 log cFU/mL and 0.8-3.6 log cFU/ml, respectively (Fig. 1b). In a study by bEVILAcQUA et al. (2013), ultra- sound was used to determine the reduction in several spoiling yeasts, including Z. rouxii, in fruit juices; similar to our observations, there was a maximum reduction of 1.7 log cFU/mL Z. rouxii in orange juice after sonication (40°c, 20 kHz, amplitude 60%, time 4 min, pulse 2 s). According to the Hurdle concept, if the effect obtained via the combined use of two different inactivation factors is greater than the sum of the separate use of these methods, then a syner- gistic interaction is said to occur (LEIstNEr and GOrrIs, 1995). In the present study, treatment with a pH= 4 or 7 at 0.94 aw, with the combined use of ultrasound (40% and 80%) and heat, re- sulted in a significant synergistic interaction, al- though the D 40 value was higher than that ob- served with an aw of 0.99. In control treatments performed at the same temperature, sonication at 0.94 (pH 4 and 7) and 0.99 aw (pH 4 and 7), the reduction in D 40 values was, respectively 1/8-1/16 and 1/32-1/128 (Fig. 1a). Inactivation of Z. rouxii at 45°C the D 45 values estimated for combined treat- ments at 0.94 aw were greater than those ob- served with 0.99 aw; however, the D 45 val- ues were lower than those obtained at 40°c (Fig. 2a). Overall, our results suggest that in- creased treatment temperatures resulted in in- creased yeast inactivation. Additionally, all son- ications at 80% amplitude (0.94 aw and 0.99 aw; pH 4 and 7) had D values smaller than those found at 40% (p<0.05). the reduction in Z. rouxii for the 40% and 80% amplitude levels was 0.5-2.0 log cFU/mL and 1.1-3.9 log cFU/ mL, respectively (Fig. 2b). In treatments at 0.99 aw (pH 4 and 7), a synergistic interaction for Z. rouxii inactiva- tion was observed with the combined use of heat and ultrasound (40% and 80% amplitude). While synergy was present at 0.94 aw and pH values of 4 and 7 (40% and 80%), the D 45 val- ue was greater than that observed at 0.99 aw. compared with control treatments at the same temperature and pH, the reductions in D 45 ob- tained with the combined treatments at 0.94 and 0.99 aw were from 1/8-1/32 and 1/64- 1/128, respectively (Fig. 2a). LOPEZ-MALO et al. (2005) assessed the sonication inactivation (20 kHz, 90 µm) of Z. bailii in 2% sabouraud Glu- cose broth with a pH of 3.5 and at three dif- ferent aw (0.99, 0.97 and 0.95) and tempera- tures (45, 50 and 55°c) levels. consistent with our findings, the D value at 45°c obtained with thermal treatment (tt) was significantly great- er than that obtained with thermoultrasonica- tion (tUt) (p<0.05). these authors found that at 45°c and at 0.99, 0.97, and 0.95 aw, the D value was reduced from 15.4 to 7.4, 26.8 to 8.6 and 43.5 to 12.9, respectively, with tt and tUt. Additionally, along with the reduc- tion in aw, an increase in the D values was ob- served. Furthermore, a lower aw was associat- ed with a greater synergistic effect in tUt. In the present study, the average D 45 values of Z. rouxii for 0.99 and 0.94 aw at 45°c t -t (pH=4) were 98.66 and 140.1 min, respectively. In con- trast, treatment ts-t under the same condi- tions (80% amplitude: 99.2 µm) resulted in D 45 values of 0.58 and 4.33 min at 0.99 and 0.94 aw, respectively. Inactivation of Z. rouxii at 50°C In our sonication treatments, the minimum possible sampling interval from the sonication vessel was 20 s. therefore, in some combined treatments, especially those conducted with high temperatures and high aw values (i.e., aw=0.99; 50 and 55°c), samples were taken af- ter the first 20 s, and there were typically no vi- able yeast cells (for this reason, some D values in Figs. 3a and 4a are shown as <0.2 min). Ad- ditionally, yeast reductions are shown as >5.7- log cFU/mL because the maximum yeast reduc- tion was determined as 5.7-log cFU/mL in this study (Figs. 3b and 4b). similar to our results obtained at 45°c, the estimated D 50 for Z. rouxii at 50°c and 0.94 Ital. J. Food Sci., vol. 28 - 2016 69 Fig. 1 - D 40 values of Z. rouxii obtained from the ts-t and t-t (A) and reductions of Z. rouxii after ts-t and t-t at 40°c (b). bA b A Fig. 2 - D 45 values of Z. rouxii obtained from the ts-t and t-t (A) and reductions of Z. rouxii after ts-t and t-t at 45°c (b). Fig. 3 - D 50 values of Z. rouxii obtained from the ts-t and t-t (A) and reductions of Z. rouxii after ts-t and t-t at 50°c (b). bA 70 Ital. J. Food Sci., vol. 28 - 2016 Fig. 4 - D 55 values of Z. rouxii obtained from the ts-t and t-t (A) and reductions of Z. rouxii after ts-t and t-t at 55°c (b). bA aw was greater than that observed at 0.99 aw (Fig. 3a). Z. rouxii showed a greater resistance to combined treatments at a pH of 4 than at a pH of 7, most likely because Z. rouxii is a yeast with good adaptation to lower pH values. the maximum D 50 at 0.99 aw and pH 4 was 0.8 minutes, whereas the D 50 value at pH 7 was <0.2 minutes. In treatments at 0.99 aw and pH 4, the combined use of ultrasound (40% and 80%) provided a significant synergistic interac- tion for Z. rouxii inactivation. However, no such synergy could be observed at 0.99 aw and pH 7 for the combined treatment. A synergistic ef- fect could be observed at 0.94 aw, with pH val- ues of 4 and 7, with sonication (40% and 80%), and with D 50 values greater than that observed with 0.99 aw. compared with controls under the same temperature conditions, the reduction in D 50 in sonications of 0.94 aw (pH=4 and 7) and 0.99 aw (pH = 4 and 7) was 1/4-1/16 and 1/3- 1/6, respectively. And the reduction in Z. rouxii for the 40% and 80% amplitude levels was 1.1- >5.7 log cFU/ml and 3.6->5.7 log cFU/ml, re- spectively (Fig. 3b). Inactivation of Z. rouxii at 55°C compared with control treatments, the D 55 values obtained with the combined treatments at 55°c and 0.99 aw suggested that the use of ultrasound did not result in a significant dif- ferences (p>0.05) in yeast inactivation and that heat was the primary determinant of inactiva- tion. In all combined treatments (at 0.94 aw), the D 55 values for Z. rouxii were determined to be 0.2 minutes; at 0.99 aw, the D 55 values were deter- mined to be <0.2 minutes (Fig. 4a). compared with controls at the same temperature levels, the reduction in D 55 at sonications at 0.94 aw and pH levels of 4 and 7 was 1/8. In a similar study by GUErrErO et al. (2001), the inacti- vation of S. cerevisiae was examined at differ- ent amplitude levels (20 kHz, 71.4 and 107.10 µm), pH values (3 or 5.6), and temperatures (35°, 45°, and 55°c) in sabouraud broth. In line with our findings, the D value at 55°c was lower that obtained at other temperature levels (i.e., 35° and 45°c) (p<0.05), whereas sonica- tion, amplitude, and medium pH were not as- sociated with a change in that reduction. How- ever, in our study, the combined treatment with 0.94 aw resulted in an increased yeast inacti- vation (p<0.05), regardless of the pH and am- plitude, and was associated with a synergistic effect. the D 55 values obtained for all sonica- tion procedures at 80% were lower than those obtained at 40%, although the differences were not statistically significant (p>0.05). the reduction in Z. rouxii for the 40% and 80% amplitude levels was 4.7->5.7 log cFU/mL and 5.6->5.7 log cFU/mL, respectively (Fig. 4b). Viability of yeast cells in treated samples during storage the growth during storage of sublethally in- jured yeast after combined treatments was tested. samples were taken from treatments in which the 5-log cycle yeast reductions had been achieved (Figs. 3b and 4b) and stored for 60 d under different storage temperatures (4° and 25°c). As a result, none of the samples ex- hibited yeast growth during storage. this find- ings suggests that thermo-sonication is associ- ated with irreversible cell damage. In a study by MArX et al. (2011) examining the effect of con- tinuous and pulsed thermo-sonication (20 kHz frequency, at 60°c, 100% amplitude, for 30 min) on S. cerevisiae inactivation, the structural dam- age occurring in yeast cells after treatment was examined using scanning electron microsco- py. they observed more broken cells using con- tinuous rather than pulsed thermo-sonication treatments; however, they did not find any via- ble cells in their samples. Ital. J. Food Sci., vol. 28 - 2016 71 cONcLUsIONs compared with controls, all thermo-sonica- tion procedures at 40, 45, 50 and 55°c resulted in a significant decrease in the D values (p=0.00) for Z. rouxii in our study. this finding shows a decreased resistance of Z. rouxii cells to heat to- gether with the use of Us. the amplitude of the ultrasound waves was effective in the reduction of yeast cells, with lower D values obtained at the 80% amplitude than at 40%. the use of Us, particularly in medium with a low aw, resulted in significant synergistic effects for Z. rouxii inactivation. However, thermo-soni- cations performed at low aw (0.94) were associ- ated with a more prolonged D value and a less marked reduction. Additionally, low aw was as- sociated with the relative protection of yeast cells against thermo-sonication, particularly at low- er temperatures. Furthermore, as the sonication temperatures increased, the effects of amplitude, medium pH and aw on yeast reduction tended to weaken. In- creased sonication temperatures (50° and 55°c) resulted in significant yeast inactivation (>5.7-log reductions). In most of the combined treatments at 50° and 55°c, the FDA requirement of a min- imum of 5-log cycle reduction (within <0.2-0.2 min) could be met. However, although heat was the primary determinant of the yeast inactivation in combined treatments with high aw (55°c), the synergistic effect of Us was more prominent than at 0.94 aw. the absence of yeast growth at 60 d that was observed in the samples obtained from the sonication chamber after combined treat- ments indicates that thermosonication was as- sociated with irreversible yeast damage. the findings of this study indicate that Us combined with mild heat treatments (50° and 55°c) has the potential to inactivate Z. rouxii in fruit juices and beverages as an alternative to traditional pasteurization methods. 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Paper Received August 21, 2014 Accepted April 15, 2015 http://www.sciencedirect.com/science/journal/09242244/21/7 http://www.sciencedirect.com/science/journal/13504177/14/3 http://www.sciencedirect.com/science/journal/09242244 http://www.sciencedirect.com/science/journal/09242244 http://www.sciencedirect.com/science/journal/09242244/20/3 http://www.sciencedirect.com/science/journal/09242244/20/3 http://cdn.intechopen.com/pdfs-wm/28909.pdf http://cdn.intechopen.com/pdfs-wm/28909.pdf