Effect of ultrasound on the survival of Saccharomyces cerevisiae: influence of temperature, pH and amplitude

Innovative Food Science & Emerging Technologies 2 Ž2001. 31᎐39

Effect of ultrasound on the survival of Saccharomyces cere¨isiae: influence of temperature, pH and amplitude b , S.M. Alzamoraa,2 S. Guerrero a,U,1, A. Lopez-Malo ´ a

b

Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Uni¨ ersidad de Buenos Aires, Ciudad Uni¨ ersitaria, 1428 Buenos Aires, Argentina Departamento de Ingenierıa y Alimentos, Uni¨ ersidad de las Americas, Puebla, Ex Hacienda Santa Catarina Martir, ´ Quımica ´ ´ ´ Puebla 72820, Mexico ´ Accepted 23 December 2000

Abstract The resistance of Saccharomyces cere¨ isiae cells to the action of ultrasound Ž20 kHz, wave amplitude in the range 71᎐110 ␮m. was analyzed at 35, 45 and 55⬚C in Sabouraud broth at pH 3.0 and 5.6. The inactivation rate where a first-order kinetic was observed exhibited D values between 0.5 and 31 min. The resistance of the yeast decreased as ultrasonic wave amplitude increased, with the z values for this effect ranging between 128 and 323 ␮m. In the pH range investigated, the reduction of pH did not affect ultrasound yeast sensitivity except for experiments performed at 71.4 ␮m wave amplitude and 45⬚C. At moderate temperatures, decimal reduction time values were reduced by the simultaneous effect of ultrasound but at 55⬚C, no advantages were observed by adding sonication. Structural studies performed in cells sonicated at 45⬚C and 95.2 ␮m of wave amplitude indicated the treatment provoked puncturing of cell walls with leakage of content as well as damage at subcellular level. However, when ultrasound was applied at 55⬚C, no structural differences were appreciated between sonicated cells and only heat-treated cells. 䊚 2001 Elsevier Science Ltd. All rights reserved. Keywords: Saccharomyces cere¨ isiae; Ultrasound; Inactivation; pH; Amplitude; Temperature Industrial rele¨ ance: The potential of ultrasound in food science and technology is still underutilized and more intensive technology transfer i.e. from the medical field should take place. This paper reveals the potential of combining ultrasound at specific amplitudes and moderate heat for inactivation of yeast and induction of cell leakage.

1. Introduction Damage in preserved products caused by heatinduced degradation of sensory and nutritional properties has focussed the interest in new non-thermal preservation methods that provoke less food quality degradation ŽGould, 1995.. Under this concept, the employment of ultrasonic waves represents an alternaU

Corresponding author. Tel.: q54-11-45763366. E-mail address: [email protected] ŽS. Guerrero.. 1 Member of Consejo Nacional de Investigaciones Cientıficas y ´ Tecnicas. ´ 2 Member of Consejo Nacional de Investigaciones Cientıficas y ´ Tecnicas. ´

tive for the development of minimally processed foods. This emerging preservation factor in combination with heat or another hurdle could enhance the desired inactivation effect. In particular, the pH has received special attention as a preservation factor in foods like fruits because they can tolerate significant reductions of this parameter without flavor impairment. The lethal effect of ultrasound has been attributed to the ca¨ itation phenomenon ŽRaso, Pagan, Condon & Sala, 1998.. Pressure waves created by mechanical vibrations of low frequency form millions of microscopic bubbles or cavities containing gas and vapor, which expand during the negative pressure excursion and implode violently during the positive excursion with the release of large amounts of energy ŽMason, 1990.. This

1466-8564r01r$ - see front matter 䊚 2001 Elsevier Science Ltd. All rights reserved. PII: S 1 4 6 6 - 8 5 6 4 Ž 0 1 . 0 0 0 2 0 - 0

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S. Guerrero et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 31᎐39

implosion Žknown as ca¨ itation. is accompanied by a number of mechanical effects. One of these is the formation of microjets of solvent which impact on the solid surface causing pitting and erosion ŽHagenson & Doraiswamy, 1998.. High-intensity or power ultrasound has been used to facilitate the microbial decontamination of various types of food and other surfaces, ultrasound appearing to be particularly effective when used in combination with other decontamination techniques, such as heating, extremes of pH, chlorination and hydrogen peroxide ŽAhmed & Russell, 1975; Lillard, 1993; McClements, 1995.. Recently, the combined effects of ultrasound and simultaneously applied heat treatment Žthermo-ultrasonication ., static pressure Žmanosonication. and heat treatment under pressure Žmanothermosonication. for inactivation of pathogenic microorganisms were also investigated ŽRaso et al., 1998.. Resistance of different species to ultrasound differs widely. Sporulated microorganisms are much more resistant than vegetative ones and fungi are more resistant in general than vegetative bacteria ŽAlliger, 1975.. There is little information about the effect of ultrasound conditions on yeast inactivation. The objective of this research was to study the lethality of the combined effect of ultrasound, pH Ž3.0 and 5.6. and mild heat treatment Ž45 and 55⬚C. on Saccharomyces cere¨ isiae viable cells suspended in laboratory media. The effect of the amplitude of the sonication waves was also evaluated.

2. Materials and methods 2.1. Microorganism and inocula preparation

was also used. The pH of the Sabouroud broth was measured with a glass electrode on a Methrom pHmeter E632 ŽHerisam, Switzerland.. 2.3. Thermal and ultrasonic treatments Treatments were carried out in a 150-ml double-wall cylindrical vessel Ždiameter, 6.3 cm; height, 7.6 cm. connected to a thermostatically-controlled water bath ŽHAAKE, Model Rotovisco RV12, Germany. whose temperature was fixed to attain 35.0, 45.0 and 55.0⬚C in the culture medium. Ninety-five milliliters of Sabouraud Broth with or without pH adjustment were poured into the vessel. Temperature of the culture medium was continuously monitored by a thermocouple to ensure a constant pre-determined temperature value "0.3⬚C. Ultrasound ŽVibracell 䊛 VCX600, Sonic Materials Inc., Chicago. at 20 kHz and 71.4 Ž60%., 83.3 Ž70%., 95.2 Ž80%. or 107.10 Ž90%. micrometers of wave amplitude was applied to the medium with an immersed 13-mm diameter probe. The equipment had automatic amplitude compensation to ensure uniform probe amplitude regardless of the varying loading conditions and line voltage fluctuations. The probe was previously calibrated following the steps suggested by the manufacturer. After 2 or 3 min of sonication the desired temperature was reached and it was maintained constant throughout the experiment by recycling water 10⬚ below the selected value. Five milliliters of the culture was inoculated into the vessel. The average initial yeast number was 5 = 10 6 CFUrml of the system. Due to a lot of bubbles generated by the cavitation process, the systems were always highly mixed since the beginning of the experiments. During the ultrasonic treatment, 1-ml samples were taken out at preset intervals, cooled in cold water and immediately transferred to the dilution bottles.

Saccharomyces cere¨ isiae KE 162 obtained from the culture collection of the laboratory of the Universidad ŽPuebla, Mexico. was employed. The de las Americas ´ initial inoculum was prepared by transferring a loopful of a stock culture maintained on Sabouraud agar slants to 20 ml of Sabouraud broth contained in 50-ml Erlenmeyer flasks. The organism was grown at 27⬚C Ž"1⬚C. until it reached the stationary phase Žf 24 h.. The yeast growth was also checked by measuring the absorbance Ž A 660nm s 1.0. in order to obtain a similar inoculum level in all experiments Ž( 1 = 10 8 cellsrml.. Microbiological media were purchased from Merck ŽArgentina.. Quımica ´

Yeast survivors were plated on to Sabouraud Agar by spread plating with 0.1 ml of sample suspension serially diluted Ž1:10. with sterile 0.1% wrw peptoned water. Plates of three serial dilutions were incubated at 27⬚C for 3 days. Two plates were used for each dilution. Survival curves were obtained by plotting Log (number of sur¨ i¨ orsr initial number) vs. time of treatment. Each condition was assayed in duplicate and the average was reported

2.2. Laboratory media

2.5. Determination of the decimal reduction time

The treatment medium consisted of Sabouraud Broth acidified to pH 3.0 with citric acid ŽAnedra Quımica, ´ Argentina.. The medium was sterilized at 121⬚C for 15 min. Sabouraud Broth without pH adjustment ŽpH 5.6.

Decimal reduction time ŽD. value was calculated as the time required to reduce the number of viable cells by one log cycle or to kill 90% of population at a given temperature, pH, and sonic wave amplitude. Determi-

2.4. Enumeration procedure

S. Guerrero et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 31᎐39

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nation coefficients adjusted by the degree of freedom 2 . of the error Ž R adj were calculated. An analysis of variance was performed to determine significance differences among D values due to the variables: pH value ŽpH., wave amplitude ŽWA. and temperature ŽT. and their interactions. In all cases significance was expressed by the calculation of the Fisher statistic ŽF. at a 5% significance level. Additional information was obtained by performing multiple range analysis calculating significant differences among D values due to each factor. z a values were defined as the number of micrometers needed to reduce the D value by one log cycle as it is expressed by the following equation: D1 s 10 D2

A 2 y A1 za

where Di : D value corresponding to wave amplitude A i Ž i: 1,2.. Microsoft 䊛 Excel and Statgraphics Plus 7.0 䊛 packages were used for data analysis. 2.6. Transmission electron microscopy Ten milliliters of sonicated Ž20 min, 95.2 ␮m, 45⬚C and 2.5 min, 95 ␮m, 55⬚C. or heated Ž3 min, 55⬚C. Sabouraud Broth ŽpH 5.6. inoculated with Saccharomyces cere¨ isiae were centrifuged at 4000 = g for 1 min at 4⬚C and the supernatant discarded. The yeast cells were resuspended in 2 ml of 2.5% Žwrv. glutaraldehyde in 0.1 M phosphate buffer, pH 7.1, for 2 h at 4⬚C; then cells were washed with 0.1 M phosphate buffer and post-fixed with KMnO4 Ž2% wrw. in 0.1 M phosphate buffer for 17 h at 4⬚C, washed well with distilled water, dehydrated with alcohol series and embedded in Epon 812 ŽShell Chemical Company, USA., during 48 h ŽBolondi, Gaggino & Monesiglio, 1995.. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined with a microscope JEOL model JEM 1200 EX II at 80 kV ŽJEOL, Japan.. Cells suspended in Sabouraud Broth and maintained at 45⬚C for 20 min were fixed in a similar manner and served as a control.

3. Results and discussion S. cere¨ isiae survival curves during ultrasonic treatments at 35, 45 and 55⬚C Žwave amplitudes ranging between 71.4 and 107.1 ␮m. at pH 5.6 and 3.0 are shown in Figs. 1 and 2, respectively. At 55⬚C, in the first four log 10 cycles of inactivation, a first-order kinetics was observed at both pH values while inactivation at 35 and 45⬚C followed first-order kinetics throughout the whole assayed sonication time. Resistance to ultrasound in those ranges was expressed as decimal reduc-

Fig. 1. Survival curves of Saccharomyces cere¨ isiae during ultrasonic treatments in Sabouroud broth at pH 5.6. Wave amplitude: 71.4 Ž⽧.; 83.3 ŽB.; 95.2 Ž'.; and 107.1 Ž䢇. micrometers ŽpH 5.6.: Ža. 35⬚C; Žb. 45⬚C; and Žc. 55⬚C. Error bar: S.D. Predicted values using first order model, ᎏ. ŽNo: initial number of yeast cells and N: survival yeast count, CFUrml..

tion time ŽD value. according to the logarithmic death rate model. Table 1 lists this inactivation parameter for S. cere¨ isiae cells under combined conditions of temperature, pH and sonication. Table 2 summarizes the results of the analysis of

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S. Guerrero et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 31᎐39

Fig. 2. Survival curves of Saccharomyces cere¨ isiae during ultrasonic treatments in Sabouroud broth at pH 3.0. Wave amplitude: 71.4 Ž⽧.; 83.3 ŽB.; 95.2 Ž'.; and 107.1 Ž䢇. micrometers ŽpH 3.0.: Ža. 35⬚C; Žb. 45⬚C; and Žc. 55⬚C. Error bar: S.D. Predicted values using first order model,ᎏ. ŽNo: initial number of yeast cells and N: survival yeast count, CFUrml..

variance as the significance of main effects ŽWA, T and pH. and interactions ŽWA᎐T, WA᎐pH and pH᎐T. on

the D values. Wave amplitude ŽWA. and temperature ŽT. were the most significant variables Ž ␣ F 0.05. influencing S. cere¨ isiae inactivation during ultrasonic treatment. Non-significant interactions between WA, T and pH were observed. Multiple range analysis showed significant differences Ž ␣ F 0.05. between D values determined at 35, 45 and 55⬚C. No significant differences Ž ␣ - 0.05. were observed between D values obtained at pH 3.0 and pH 5.6 except for D value obtained at 45⬚C, 71.4 ␮m Ž60%. amplitude and pH 3.0, which was significantly smaller than the one obtained at the same conditions and pH 5.6. D values for ultrasonic treatment ranged between 0.6 and 30.9 min and the determination coefficients were highly significant ŽTable 1.. S. cere¨ isiae inactivation due to thermal treatment alone at both pH values is exhibited in Fig. 3. The D55 values corresponding to only heat treatment at both pH values were similar Ž; 1.2" 0.5 min. to those shown in Table 1 for sonicated systems at the same temperature. The D45 values estimated for the systems with pH 3.0 and 5.6 and without sonication were 45 " 1 min and 98 " 2 min, respectively. At 35⬚C the microorganism did not lose viability during the assayed time Ž30 min. as this temperature is in the optimal temperature range for S. cere¨ isiae growth. At the lowest temperature, the only effect of ultrasound resulted in decreased values of S. cere¨ isiae counts during the whole treatment. The D 35 values were between 19 and 31 min depending on the applied wave amplitude and system pH. When the temperature was increased to 45⬚C, D values for the sonication treatment decreased by up to approximately 60% as compared with the corresponding values at 35⬚C, depending on the wave amplitude. At 55⬚C, the inactivation was similar for both thermoultrasound and only heat treatment. The simultaneous introduction of ultrasound led to an evident increased heat sensitivity of the yeast at 45⬚C for both pH values but when the temperature of the medium increased to 55⬚C, additional effect by ultrasonic waves was not observed. This fact could be probably the result of an increased thermal effect that would masque the effect of sonication, andror a decrease of the violence of implosion due to the increased vapor pressure and reduced viscosity at higher temperatures ŽMason, 1990; Sala, Burgos, Condon, ´ Lopez ´ & Raso, 1995.. These results confirmed previous ones obtained in our laboratory with the same yeast in the temperature range 45᎐55⬚C at pH 5.6 ŽLopez-Malo, Guerrero & Alzamora, 1999.. Futher´ more, the effectiveness of the combined heatrultrasonication treatment and its decrease as temperature of treatment increases has already been reported by different authors for vegetative bacteria cells, including various bacilli, Staphylococcus aureus, thermoduric streptococci and Salmonella typhimurium ŽGarcıa, ´ Bur-

S. Guerrero et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 31᎐39

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Table 1 D values Žmin. and their confidence intervals for Saccharomyces cere¨ isiae inactivation by ultrasonic treatment in Sabouroud broth. Influence of pH, temperature and wave amplitude Wave amplitude Ž␮m.

pH

Temperature 35⬚C

45⬚C 2

D

R

adj

55⬚C 2

D

R

adj

D

R2adj

71.4

5.6 3.0

29.1" 3.2 30.9" 4.1

0.970 0.959

26.3" 1.7 17.7" 1.1

0.988 0.987

4.3" 0.5 0.6" 0.1

0.988 0.970

83.3

5.6 3.0

26.8" 1.3 24.6" 2.1

0.990 0.980

18.1" 0.5 16.9" 1.1

0.997 0.989

2.1" 0.2 1.0" 0.1

0.977 0.989

95.2

5.6 3.0

21.2" 0.9 22.9" 2.1

0.993 0.981

15.1" 0.8 14.3" 0.8

0.990 0.992

1.9" 0.2 1.7" 0.2

0.986 0.991

107.1

5.6 3.0

21.4" 1.6 19.5" 3.4

0.988 0.981

14.1" 0.7 14.7" 0.7

0.994 0.993

1.3" 0.2 1.7 " 0.2

0.980 0.985

gos, Sanz & Ordonez, 1989; Mason, Paniwnyk & ´˜ Lorimer, 1996; Sala et al., 1995.. However, Ciccolini, Taillandier, Wilhem, Delmas and Strehaiano Ž1997. studied the survival of S. cere¨ isiae suspended in water at 45, 50 and 55⬚C at different ultrasonic powers, and found that application of ultrasonic waves at non-lethal temperature Ž45⬚C. did not display a deactivation action while synergy between ultrasound and heat was confirmed at the higher temperatures. This behaviour is not in agreement with our results. The effect observed was very slight because of the short exposure times Ž10 min. to ultrasonication Žas compared with the sonication time needed to inactivate S. cere¨ isiae reported in Table 1., the inoculum preparation and the yeast strain assayed.

At 35 and 45⬚C, an increase in the amplitude of the ultrasound wave generated decreasing D values until a given value Žthat was dependent on temperature and pH. was reached. Further increases in amplitude values did not result in an increased inactivation. This effect was not evident at 55⬚C ŽTable 1.. As can be seen in Fig. 4, the resistance of S. cere¨ isiae decreased exponentially when the wave amplitude increased linearly at 35 and 45⬚C. Decimal reduction time dependence on amplitude exhibited z a values significantly Ž ␣ - 0.05. different for each assayed condition ŽTable 3.. At the highest amplitude assayed, titanium particles from the tip could be observed in the medium. Scarce information is found in the literature on the influence of wave amplitude on microorganism inactivation. It has been reported that the intensity of ultra-

Table 2 Analysis of variance showing the effect of ultrasonic treatment variables as significance of the main effects and interactions on D value of Saccharomyces cere¨ isiaea Source

Degree of freedom Žd.f..

Mean square sum ŽMS.

Fisher statistic ŽF. 7.2b 0.4 ŽNS. 212.1b

Main effects WA pH T

3 1 2

39.4 2.3 1157.6

Interactions WA-pH WA-T pH-T

3 6 2

4.1 11.2 10.4

6 23

5.5 111.7

Residual Total a

0.75 ŽNS. 2.0 ŽNS. 1.9 ŽNS.

Abbre¨ iations: WA, wave amplitude; T, temperature; pH, pH value; NS, non-significant. b Significant at 5% level.

Fig. 3. Survival curves of Saccharomyces cere¨ isiae during heat treatment in laboratory media at 45⬚C, pH 5.6 ŽI.; 55⬚C, pH 5.6 Ž'.; 45⬚C, pH 3.0 Ž⽧. and 55⬚C, pH 3.0 Ž䢇.. Error bar: S.D. Ž N: survival yeast count, CFUrml..

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S. Guerrero et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 31᎐39

Fig. 4. Relationship between D value and the wave amplitude of the ultrasonic treatment at 35⬚C Ž⽧. and 45⬚C ŽB.; Ža. pH 5.6 and Žb. pH 3.0. Error bar: S.D.

sound effect is directly related to the amplitude: when ultrasound amplitude increases, the zone undergoing cavitation increases, leading to more inactivation. Raso et al. Ž1998. and Pagan, ´ Manas, ˜ Raso and Condon ´ Ž1999. observed that the higher the amplitude of ultrasonic waves in the range 21᎐150 ␮m, the greater inactivation of S. faecium, L. monocytogenes, Y. enterocolitica, S. enteritidis and A. hydrophila in manothermosonication treatments, a linear relationship being between amplitude of sonication and log D. The effect of pH on ultrasound effectiveness reported in the literature seem to be contradictory: some authors found no influence of pH on resistance, while a higher ultrasound sensitivity at acidic pH values was reported by others ŽSala et al., 1995.. In our case, we only found a significant difference when WAs 71.4 ␮m and T s 45⬚C were used. This could be attributed to the enhanced heat inactivation of the yeast at pH 3.0 and the less pronounced effect of sonication at low amplitude. The effect of pH on thermal inactivation at moderate temperatures has been already mentioned by Agab and Collins Ž1992a,b.. They found that pH reduction effect on thermal inactivation of the yeast was noticed at temperatures - 55⬚C probably because at the highest temperature the death cell mechanism mainly involved cell disruption by the lethal effect of heat. Fig. 5 shows the transmission electron microscopy ŽTEM. micrographs obtained to observe structural

changes in S. cere¨ isiae cells treated with ultrasound at 45⬚C . Sonication produced several modifications in the cellular structure of the yeast, from cytological disruption of organelles ŽFig. 5E,F,I,K,L. till puncturing of the cell wall and breakage of plasma membrane ŽFig. 5B,C,H,J,L.. Many cells showed wall rupture or fragmentation. These observations indicated that pitting and erosion of cell surface is not the only mechanism responsible of S. cere¨ isiae damage since the inner structure of the cell seems to be strikingly affected. Ciccolini et al. Ž1997. suggested that yeast cells could contain cavitation nuclei and sonication could cause an internal cavitation as well as internal microstreaming, modifying the cellular structure. He observed under light microscopy that sonicated S. cere¨ isiae cells Ž45⬚C and 50, 100 or 180 W. were intact but reacted differently to vital staining with methylene blue if compared to untreated cells. Alliger Ž1975. also mentioned that disruption of subcellular particles during sonication was often faster than disruption of cell walls and that ultrasound rapidly disrupted mitochondria with fine membrane fragmentation. Baker’s yeasts in the process of being disintegrated by ultrasound were observed by these authors using light microscopy. First the cells distorted and swelled slightly; then puncturing of the wall occurred above the middle and the content emptied. When the whole cells were observed, they appeared undamaged but they did not grow, indicating damage at the subcellular level that could not be visualized with light microscopy. Kinsloe, Ackerman and Reid Ž1954. showed electron photomicrographs of S. cere¨ isiae exposed to a 9-kHz magnetostriction oscillator where fragmented cells, some in which the end had been broken, and some intact ones, all exhibiting marked irregularity in density, could be observed. Our observations obtained with TEM on the biological effect of ultrasound in S. cere¨ isiae at mild temperature confirmed these previous assumptions, showing a significant alteration in the inner structure of the whole cells. Microscopic observations were also performed when S. cere¨ isiae was treated for 2 min at 55⬚C, a lethal temperature for the yeast, with or without simultaneous application of sonication ŽWAs 90 ␮m. ŽFig. 6.. A Table 3 z a value estimation Ž␮m. for the relationship between the decimal reduction time and the wave amplitude of the ultrasonic treatment at 35 and 45⬚C pH

Temperature 35⬚C

5.6 3.0

45⬚C

za

R2 adj

za

R2 adj

238 189

0.88 0.96

128 322

0.89 0.90

S. Guerrero et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 31᎐39

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Fig. 5. TEM images of Saccharomyces cere¨ isiae. A, D, G, J ᎏ untreated cells; B, C, E, F, H, I, K, L ᎏ sonicated cells Ž20 kHz; 95.2 ␮m of amplitude; T : 45⬚C.. A: ultrathin section showing a natural bud scar and intact cell wall; B: puncturing of cell surface and less densely stained inner content; C: inner material radiating from the cell wall Žarrow.; D: very well differentiated organelles; E: discomposed inner structure but cell wall and cytoplasmic membrane not broken; F: swollen damaged cell wall, shrinkage of cytoplasmic material from cell wall, complete disruption of organelles; G: detail of electronically dense cell wall and a continuous plasma membrane; H: rests of cell wall and plasma membrane; I: disorganization of inner content, rupture of wall Žarrow. and plasma membrane with partial release of cellular content; J: daughter and mother yeast cells, clearly showing the nucleus, vacuoles, mitochondria and other organelles; K: complete disorganized inner content, swelling and rupture of cell walls. There are some subcellular fragments out of the cell; L: broken cell wall and cellular membrane Žarrow. and leakage of internal substances. Scale: E, H, J: 500 nm; G: 100 nm; A᎐D, F, I, K, L: 200 nm. cw, cell wall; pm, plasma membrane; n, nucleous; bs, bud scar; m, mitochondrion; nm, nuclear membrane; er, endoplasmic reticulum.

great structural difference between sonicated cells and cells with only heat treatment could not be observed. Heated sonicated cells and also heated cells showed some breakage of membranes and walls, but there were not wall debris and the contents did not leach out of the cells as in the cells treated at 45⬚C. In both samples the contents appeared coagulated and the lumen looked very vacuolized and coarse. These findings explain the

similar inactivation parameters for both thermosonication and only thermal treatment at 55⬚C.

4. Conclusions The combination of ultrasound and mild heat treatment was effective against S. cere¨ isiae survival at tem-

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emerged from the benefits of using intermediate ultrasonic amplitudes Ž; 83᎐95 ␮m. since the higher ones Ž107 ␮m. did not increase the lethal effect and, on the contrary, as it could be observed during the experiences, released many titanium particles eroded from the sonication probe to the heated medium. These studies are now being enhanced by the inclusion of another lethal factor to reach more inactivation of the microorganism under the hurdle technology concept and by exploring the microorganism response in real food system.

Acknowledgements We acknowledge the financial support from Universidad de Buenos Aires, CONICET and ANPCyT, Argentina; Universidad de las Americas᎐Puebla and CO´ NACYT, Mexico and CYTED XI.3 Project, Spain. We also wish to thank Dr Arquımedes Bolondi and Ms ´ Blanca Garcıa ´ for their technical assistance at the C.I.C.V. Electron Microscopy Center ŽINTA, Argentina.. References

Fig. 6. TEM images of Saccharomyces cere¨ isiae. A, C, E: thermosonicated cells Ž20 kHz; 95.2 ␮m of amplitude; T s 55⬚C; 2.5 min.. B, D, F: thermal-treated cells ŽT s 55⬚C; 2.5 min.. A: inner content vacuolized and disrupted; intracellular organelles difficult to recognize; B: vacuolization of content Žarrow. and rupture of cell walls and membranes Žopen arrow.. Intracellular organelles deformed or disrupted; C: rupture of plasma membrane Žarrow.; D: severely damaged cell wall and inner structure; E: damage in the area of bud scar Žarrow.; broken wall Žopen arrow.; F: disappeared nuclear membrane; breaking of cell wall. Scale: A: 500 nm; B᎐F: 200 nm.

peratures lower than approximately 55⬚C. At 35⬚C S. cere¨ isae inactivation by ultrasound could be observed and at 45⬚C, this inactivation was significantly increased. Otherwise, at moderate temperatures, thermal process time andror temperatures could be reduced to achieve the same lethality values by the simultaneous application of ultrasound. At 55⬚C, no advantages were observed by adding ultrasound. The rate of yeast inactivation increased exponentially with increases in amplitude, but no differences were generally found between D values at pH 5.6 and 3.0. An interesting conclusion

Agab, M. A., & Collins, M. Ž1992a.. Effect of treatments environment Žtemperature, pH, water activity. on the heat resistance of yeasts. Journal of Food Science and Technology 29, 5᎐9. Agab, M. A., & Collins, M. Ž1992b.. Effect of heat treatment on aerobically and anaerobically grown or starved yeast cell in different pH menstrua. Journal of Food Science and Technology 29, 10᎐13. Ahmed, F. I. K., & Russell, C. Ž1975.. Synergism between ultrasonic waves and hydrogen peroxide in the killing of microorganisms. Journal of Applied Bacteriology 39, 31᎐40. Alliger, H. Ž1975.. Ultrasonic disruption. American Laboratory 10, 75᎐85. Bolondi, A., Gaggino, O. P. & Monesiglio, J. C. Ž1995.. Microscopıa ´ Electronica. Tecnicas Generales, C.I.C.V., INTA, Secretarıa ´ ´ ´ de Agricultura, Ganaderıa Argentina. ´ y Pesca, Republica ´ Ciccolini, L., Taillandier, P., Wilhem, A.M., Delmas, H., & Strehaiano, P. Ž1997.. Low frequency thermo-ultrasonication of Saccharomyces cere¨ isiae suspensions: effect of temperature and of ultrasonic power. Chemical Engineering Journal 65, 145᎐149. Garcıa, ´ M. L., Burgos, J., Sanz, B., & Ordonez, ´˜ J. Ž1989.. Effect of heat and ultrasonic waves on the survival of two strains of Bacillus subtilis. Journal of Applied Bacteriology 67, 619᎐628. Gould, G. W. Ž1995.. New methods of food preservation. Blackie, Glasgow, Scotland. Hagenson, L. C., & Doraiswamy, L. Ž1998.. Comparison of the effects of ultrasound and mechanical agitation on a reacting solid᎐liquid system. Chemical Engineering Science 53, 131᎐148. Kinsloe, H., Ackerman, E., & Reid, J. J. Ž1954.. Exposure of microorganisms to measured sound fields. Journal of Bacteriology 68, 373᎐380. Lillard, H. S. Ž1993.. Bactericidal effect of chlorine on attached Salmonellae with and without sonification. Journal of Food Protection 8, 716᎐717. Lopez-Malo, A., Guerrero, S., & Alzamora, S. M. Ž1999.. Saccha´ romyces cerevisiae thermal inactivation kinetics combined with ultrasound. Journal of Food Protection 62, 1215᎐1217.

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