inactivation of escherichia coli with power ultrasound in...

E102 JOURNAL OF FOOD SCIENCE—Vol. 71, Nr. 2, 2006Published on Web 2/23/2006

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Inactivation of Escherichia coli withPower Ultrasound in Apple CiderEEEEEDGARDGARDGARDGARDGAR U U U U UGARTEGARTEGARTEGARTEGARTE-R-R-R-R-ROMEROOMEROOMEROOMEROOMERO, H, H, H, H, HAOAOAOAOAO F F F F FENGENGENGENGENG, S, S, S, S, SCOTTCOTTCOTTCOTTCOTT E. M E. M E. M E. M E. MARTINARTINARTINARTINARTIN, K, K, K, K, KEITHEITHEITHEITHEITH R. C R. C R. C R. C R. CADWALLADERADWALLADERADWALLADERADWALLADERADWALLADER, , , , , ANDANDANDANDAND S S S S SCOTTCOTTCOTTCOTTCOTT J. R J. R J. R J. R J. ROBINSONOBINSONOBINSONOBINSONOBINSON

ABSTRAABSTRAABSTRAABSTRAABSTRACTCTCTCTCT: : : : : The use of acoustic enerThe use of acoustic enerThe use of acoustic enerThe use of acoustic enerThe use of acoustic energy to securgy to securgy to securgy to securgy to secure apple cider safety was explore apple cider safety was explore apple cider safety was explore apple cider safety was explore apple cider safety was explored. Ied. Ied. Ied. Ied. Inactivnactivnactivnactivnactivation tests wation tests wation tests wation tests wation tests wererererere pere pere pere pere per-----formed with formed with formed with formed with formed with EscherichiaEscherichiaEscherichiaEscherichiaEscherichia colicolicolicolicoli K12 at 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C with and without ultrasound, followed K12 at 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C with and without ultrasound, followed K12 at 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C with and without ultrasound, followed K12 at 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C with and without ultrasound, followed K12 at 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C with and without ultrasound, followedby a validation test with by a validation test with by a validation test with by a validation test with by a validation test with E. coliE. coliE. coliE. coliE. coli O157:H7 at 60 °C. The cell morphology was observed with environmental O157:H7 at 60 °C. The cell morphology was observed with environmental O157:H7 at 60 °C. The cell morphology was observed with environmental O157:H7 at 60 °C. The cell morphology was observed with environmental O157:H7 at 60 °C. The cell morphology was observed with environmentalscanning electron microscopy for samples treated at 40 °C and 60 °C. Physical quality attributes of the applescanning electron microscopy for samples treated at 40 °C and 60 °C. Physical quality attributes of the applescanning electron microscopy for samples treated at 40 °C and 60 °C. Physical quality attributes of the applescanning electron microscopy for samples treated at 40 °C and 60 °C. Physical quality attributes of the applescanning electron microscopy for samples treated at 40 °C and 60 °C. Physical quality attributes of the applecider (pH, titrcider (pH, titrcider (pH, titrcider (pH, titrcider (pH, titratable acidityatable acidityatable acidityatable acidityatable acidity, °B, °B, °B, °B, °Brrrrrix, turbidityix, turbidityix, turbidityix, turbidityix, turbidity, and color) w, and color) w, and color) w, and color) w, and color) wererererere compare compare compare compare compared for tred for tred for tred for tred for treated sampleseated sampleseated sampleseated sampleseated samples. . . . . The inactivThe inactivThe inactivThe inactivThe inactivation testsation testsation testsation testsation testsshowed that sonication increased showed that sonication increased showed that sonication increased showed that sonication increased showed that sonication increased E. coliE. coliE. coliE. coliE. coli K12 cell destruction by 5.3-log, 5.0-log, and 0.1-log cycles at 40 °C, K12 cell destruction by 5.3-log, 5.0-log, and 0.1-log cycles at 40 °C, K12 cell destruction by 5.3-log, 5.0-log, and 0.1-log cycles at 40 °C, K12 cell destruction by 5.3-log, 5.0-log, and 0.1-log cycles at 40 °C, K12 cell destruction by 5.3-log, 5.0-log, and 0.1-log cycles at 40 °C,50 °C, and 60 °C, r50 °C, and 60 °C, r50 °C, and 60 °C, r50 °C, and 60 °C, r50 °C, and 60 °C, respectivespectivespectivespectivespectivelyelyelyelyely..... The additional destrThe additional destrThe additional destrThe additional destrThe additional destruction due to sonication was moruction due to sonication was moruction due to sonication was moruction due to sonication was moruction due to sonication was more pre pre pre pre pronounced at sublethalonounced at sublethalonounced at sublethalonounced at sublethalonounced at sublethaltemperatures. At the lethal temperature of 60 °C, the rate of death by ultrasound was not significantly differenttemperatures. At the lethal temperature of 60 °C, the rate of death by ultrasound was not significantly differenttemperatures. At the lethal temperature of 60 °C, the rate of death by ultrasound was not significantly differenttemperatures. At the lethal temperature of 60 °C, the rate of death by ultrasound was not significantly differenttemperatures. At the lethal temperature of 60 °C, the rate of death by ultrasound was not significantly differentcompared with the thermal-alone treatment. The inactivation of compared with the thermal-alone treatment. The inactivation of compared with the thermal-alone treatment. The inactivation of compared with the thermal-alone treatment. The inactivation of compared with the thermal-alone treatment. The inactivation of E. coliE. coliE. coliE. coliE. coli K12 with heat was described by 1st-order K12 with heat was described by 1st-order K12 with heat was described by 1st-order K12 with heat was described by 1st-order K12 with heat was described by 1st-orderkinetics, especially at 50 °C and 60 °C. For ultrasound treatments, concave upward survival curves were ob-kinetics, especially at 50 °C and 60 °C. For ultrasound treatments, concave upward survival curves were ob-kinetics, especially at 50 °C and 60 °C. For ultrasound treatments, concave upward survival curves were ob-kinetics, especially at 50 °C and 60 °C. For ultrasound treatments, concave upward survival curves were ob-kinetics, especially at 50 °C and 60 °C. For ultrasound treatments, concave upward survival curves were ob-serserserserservvvvved, which had a shape factor in the red, which had a shape factor in the red, which had a shape factor in the red, which had a shape factor in the red, which had a shape factor in the range of 0.547 to 0.720 for a ange of 0.547 to 0.720 for a ange of 0.547 to 0.720 for a ange of 0.547 to 0.720 for a ange of 0.547 to 0.720 for a WWWWWeibull distreibull distreibull distreibull distreibull distribution model. Eibution model. Eibution model. Eibution model. Eibution model. Extensivxtensivxtensivxtensivxtensiveeeeedamage for ultrasound treated damage for ultrasound treated damage for ultrasound treated damage for ultrasound treated damage for ultrasound treated E. coliE. coliE. coliE. coliE. coli K12 cells, including cell perforation, was observed. Perforation is a unique K12 cells, including cell perforation, was observed. Perforation is a unique K12 cells, including cell perforation, was observed. Perforation is a unique K12 cells, including cell perforation, was observed. Perforation is a unique K12 cells, including cell perforation, was observed. Perforation is a uniquephenomenon found on ultrasound-treated cells that could be caused by liquid jets generated by cavitation.phenomenon found on ultrasound-treated cells that could be caused by liquid jets generated by cavitation.phenomenon found on ultrasound-treated cells that could be caused by liquid jets generated by cavitation.phenomenon found on ultrasound-treated cells that could be caused by liquid jets generated by cavitation.phenomenon found on ultrasound-treated cells that could be caused by liquid jets generated by cavitation.TTTTTitritritritritratable acidityatable acidityatable acidityatable acidityatable acidity, pH, and °B, pH, and °B, pH, and °B, pH, and °B, pH, and °Brrrrrix of the cider wix of the cider wix of the cider wix of the cider wix of the cider wererererere not affected be not affected be not affected be not affected be not affected by ultry ultry ultry ultry ultrasound trasound trasound trasound trasound treatment. Meatment. Meatment. Meatment. Meatment. Minor changes in colorinor changes in colorinor changes in colorinor changes in colorinor changes in colorand turbidity for ultrasound treated samples, especially for sonication at 40 °C for 17.7 min, were observed.and turbidity for ultrasound treated samples, especially for sonication at 40 °C for 17.7 min, were observed.and turbidity for ultrasound treated samples, especially for sonication at 40 °C for 17.7 min, were observed.and turbidity for ultrasound treated samples, especially for sonication at 40 °C for 17.7 min, were observed.and turbidity for ultrasound treated samples, especially for sonication at 40 °C for 17.7 min, were observed.

KKKKKeyworeyworeyworeyworeywords: pods: pods: pods: pods: powwwwwer ultrer ultrer ultrer ultrer ultrasound, asound, asound, asound, asound, EEEEEscherichia colischerichia colischerichia colischerichia colischerichia coli, apple cider, apple cider, apple cider, apple cider, apple cider, cell morphology, cell morphology, cell morphology, cell morphology, cell morphology, quality, quality, quality, quality, quality

Introduction

Apple cider, traditionally consumed as fresh juice from late sum-mer to early winter in the United States, is a sweet, amber gold-

en, and turbid juice squeezed from ripe apples. Previously, apple ci-der was considered a safe product because of its low pH (3.3 to 4.1),which acts as a natural barrier against most microorganisms (Mattickand Moyer 1983). Since 1996, however, at least 6 outbreaks of food-borne illness caused by Escherichia coli O157:H7 have been traced tothe consumption of unpasteurized apple cider or apple juice (DeWaaland Barlow 2002), which have raised public concern about the micro-bial safety of the product. In response to this concern, the U.S. Food& Drug Administration (USFDA) issued regulations requiring allwholesale juice and juice product manufacturers to provide a mini-mum 5-log reduction in the most resistant pathogen of public healthsignificance in their final products (USFDA 2001).

Thermal pasteurization at 71.1 °C for 6 to 11 s, depending on ap-ple cultivars, has been used to provide the mandated 5-log reduc-tion in the production of apple juice and apple cider. While provid-ing a reliable reduction in microbial population, the thermalpasteurization involves high costs in operation, especially to smallcider mills, and degrades sensory and nutritional qualities (Splitts-toesser and others 1996). Alternative juice safety intervention tech-nologies working under mild treatment conditions, which are oftentermed as nonthermal technologies, have hence gained interest.Existing nonthermal technologies for commercial juice processinginclude the CiderSureTM and the Light ProcessTM ultraviolet sys-tems, and the Fresher Under Pressure® high-pressure processing

(HPP) system. Technologies under development include pulsedelectric field (PEF) (Ortega-Rivas and others 1998; Evrendilek andothers 2000; Iu and others 2001; Yeom and others 2002), electronbeam irradiation (EBI) (Wang and others 2004), pulsed broad spec-trum light (Dunn and others 1995), freezing-thawing method (Uljasand Ingham 1999; Ingham and Schoeller 2002), and hurdle con-cepts based on the use of preservatives (Comes and Beelman2002). In recent years, studies have documented the utilization ofultraviolet (UV) light as a nonthermal method for apple cider pro-cessing (Wright and others 2000; Basaran and others 2004;Quinteros-Ramos and others 2004). Currently, UV treatment ismainly used by small apple cider producers to achieve the 5-logreduction required by the USFDA.

Another alternative to thermal pasteurization is the use of ultra-sonic energy to inactivate foodborne pathogens. Ultrasound desig-nates sound waves with a frequency beyond human hearing (16 Hzto 20 MHz). Power ultrasound refers to ultrasonic waves in the fre-quency range of 20 to 100 kHz having sound intensities of 10 to 1000W/cm2 (Feng and Yang 2005). Harvey and Loomis (1929) first report-ed the lethal effect of ultrasonic waves on microorganisms in 1930s.However, the potential of using ultrasound to inactivate microbeswas not fully realized until the development of powerful, economic,and durable transducers. A number of studies have been conductedto examine the efficacy of ultrasound on inactivation of several mi-croorganisms, which include Listeria monocytogenes (Pagán and oth-ers 1999a, 1999b; Mañas and Pagán 2000; Baumann and others 2005;Ugarte and others 2005), E. coli (Scherba and others 1991; Hua andThompson 2000; Furuta and others 2004), Bacillus subtilis (Garcia andothers 1989; Sala and others 1995; Raso and others 1998a), Salmonel-la spp (Lee and others 1989; Sams and Feria 1991), and Shigella(Ugarte and others 2006). The inactivation tests were mainly con-ducted in a saline solution or in water, with ultrasound treatmentalone or in combination with heat and/or low pressure.

MS 20050599 Submitted 10/5/05, Revised 11/9/05, Accepted 11/28/05. Au-thors Ugarte-Romero, Feng, Martin, and Cadwallader are with Dept. ofFood Science and Human Nutrition, Univ. of Illinois at Urbana-Cham-paign, Urbana, IL 61801. Author Robinson is with The Imaging TechnologyGroup, Beckman Inst. for Advanced Science and Technology, Urbana, Ill.Direct inquiries to author Feng (E-mail: [email protected]).

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Inactivation of E. coli with power ultrasound . . .

Only a few ultrasonic microbial inactivation tests were conductedin fruit juice. Zenker and others (2003) performed an ultrasound-assisted thermal treatment at 60 °C to inactivate E. coli K12 in orangejuice (pH 5.9) and documented a killing rate of 2.5 log colony-formingunits (CFU)/min (D60 = 0.4 min), 3.5 times faster than the thermal-alone treatment at the same temperature. Rodgers and Ryser (2004)used a multi-step intervention strategy to secure the safety of applecider and found a 2 log reduction in the population of E. coli O157:H7by sonication at 44 to 48 kHz for 3 min. Baumann and others (2005)applied ultrasound at an acoustic energy density of 0.46 W/mL toreduce L. monocytogenes 10403S population in apple cider and insaline solution at 6 temperatures. They observed a slight increase inthe inactivation rate in apple cider (pH 3.4) compared with that in theacidified saline solution (pH 3.4) at 40 °C, 50 °C, and 55 °C. There isno published work in the literature on ultrasonic inactivation of E.coli in apple cider at both sub-lethal and lethal temperatures. Infor-mation about the effect of power ultrasound on the quality of juiceproducts is limited. Zenker and others (2003) examined color chang-es and ascorbic acid retention in orange juice. During a 35-d storage,a slower ascorbic acid degradation in ultrasound treated juice com-pared with that of thermal pasteurized samples was observed. Al-though it is well known that microbial inactivation by power ultra-sound is attributed to a phenomenon called cavitation and severalhypotheses have been proposed to explain the inactivation behav-ior, studies into the inactivation mechanisms through scanning elec-tron microscopy (SEM) have not been published.

The objective of this study was to evaluate the efficacy of powerultrasound on inactivation of E. coli in apple cider at sub-lethal andlethal temperatures. Quality analyses at treatment conditions un-der which a 5-log reduction was achieved were carried out to dis-cern the effect of sonication on the quality of apple cider. Scanningelectron microscopy was used to examine cell damage caused by ul-trasound treatments.

Materials and Methods

Microorganism and inoculum preparationMicroorganism and inoculum preparationMicroorganism and inoculum preparationMicroorganism and inoculum preparationMicroorganism and inoculum preparationA frozen stock culture (–80 °C) of Escherichia coli K12 obtained

from the food microbiology culture collection at the Univ. of Illinoisat Urbana-Champaign was thawed at room temperature for 30 min.One milliliter of this stock was inoculated in 100 mL of tryptic soybroth (TSB, Difco, Sparks, Md., U.S.A.) and grown at 37 °C for 24 h;0.1 mL was taken from this culture (101 mL), inoculated in 9.9 mL ofTSB, and grown for 8 additional h at 37 °C. The resulting culture wasstored at –18 °C. After thawing at room temperature for 30 min, 1.5mL was taken from the thawed culture and inoculated into 150 mLof TSB in Sorvall bottles. The inoculated Sorvall bottles were incu-bated at 37 °C for 15 h to reach a cell density of 108 to 109 CFU/mLin stationary phase. The culture was then centrifuged (10000 × g) at4 °C for 10 min in a Sorvall RC-5C Refrigerated Superspeed Centri-fuge (Kendro Ind., Newtown, Conn., U.S.A.). The pellet was collect-ed and suspended in 1.5 mL of irradiated apple cider that was laterused as the inoculum in inactivation tests.

Apple cider sample preparationApple cider sample preparationApple cider sample preparationApple cider sample preparationApple cider sample preparationApple cider that was freshly pressed from Red Delicious apples

was obtained from a local cider mill. Cider samples were placed in250 mL sterile Wheaton glass bottles (Wheaton Science Products,Millville, N.J., U.S.A.) with Teflon caps. Samples used in inactivationstudies were irradiated at 2.5 kGy in the Nuclear Radiation Labora-tory at the Univ. of Illinois at Urbana-Champaign, followed by stor-age at –18 °C until use. Samples used in quality analysis werestored at –18 °C without any treatment and kept frozen until used.

Ultrasound inactivation testsUltrasound inactivation testsUltrasound inactivation testsUltrasound inactivation testsUltrasound inactivation testsSonication was carried out using a VC-750 ultrasound generator

(Sonics & Materials, Inc., Newtown, Conn., U.S.A.) at frequency of 20 kHzand acoustic energy density (AED) of 0.46 W/mL. A 100-mL jacketedvessel was used to hold the samples. Before and after each experiment,all equipment was sterilized by ultraviolet light irradiation for 5 min andrinsed with 70% ethanol left to air dry in a laminar flow chamber (Lab-conco, Kansas City, Mo., U.S.A.). Before E. coli inoculation, a 3-min pre-heating with ultrasound was used for all the treatments to bring thetemperature to a target temperature. A Lauda/Brinkmann RefrigeratedCirculator (K-2/R, Brinkmann Instrument Co, Westbury, N.Y., U.S.A.) wasused to assist heating and to stabilize the temperature to ±1 °C of thetarget temperature. After the preheating, 1 mL of resuspended cells wasinoculated in 99 mL of the pretreated apple cider. The timing of a treat-ment started as soon as the inoculum was transferred to the treatmentvessel. Cider samples (100 mL each) were subjected to sonication at 5temperatures (40 °C, 45 °C, 50 °C, 55 °C, and 60 °C). Treatment time fortests at 40 °C, 45 °C, and 50 °C was 20 min, with sampling of a 0.5-mLaliquot every 5 min. At 55 °C, the treatment time was 16 min with a sam-ple taken every 4 min, while for 60 °C, the treatment time was 4 min witha sampling interval of 30 s. The 0.5-mL aliquot was immediately cooledin chilled water, serially diluted in sterile 0.1% peptone water (1:10), plat-ed on TSA, and then incubated at 37 °C for 24 h. Viable counts weredetermined by the standard plate count technique. A validation testwas conducted at 60 °C using E. coli O157:H7 to compare the resistanceof the E. coli O157:H7 strain with that of the E. coli K12 strain.

Thermal inactivation testsThermal inactivation testsThermal inactivation testsThermal inactivation testsThermal inactivation testsThermal inactivation of E. coli K12 was performed at the same

temperatures (40 °C, 45 °C, 50 °C, 55 °C, and 60 °C) as in the ultrason-ic inactivation tests. The 3-min preheating with ultrasound was alsoused to bring 99-mL cider samples to the target temperatures. Thesame treatment vessel was used with a magnetic stirrer to minimizetemperature variation in the vessel to ±0.5 °C of the target tempera-tures, which were maintained with the Lauda/Brinkmann Refriger-ated Circulator. The inoculum was added to the treatment vesselwhen the temperature of the 99-mL samples reached the target val-ues. The treatment times and sampling intervals for thermal inacti-vation tests were the same as that in the ultrasound inactivationtests. Enumeration procedure was the same as described previously.

Quality analysisQuality analysisQuality analysisQuality analysisQuality analysisColor, turbidity, °Brix, titratable acidity, and pH of cider samples

were measured to examine the effect of sonication on product quality.A separate set of experiments, which included sonication at 40 °Cand 60 °C, thermal-alone at 60 °C, and the control (no treatment),was conducted for this purpose using nonirradiated apple cider.

Turbidity of the treated samples was measured with a portableHellige 966 turbidimeter (Orbeco Analytical System, Inc., Farm-ingdale, N.Y., U.S.A.). Three 23-mL samples were transferred to aglass vial and agitated before taking turbidity readings. Turbidityreadings expressed in nephelometric turbidity units (NTU) wererecorded from the turbidimeter after allowing a sample to stabilizefor over 15 s. After measuring turbidity, samples were transferred toa 30-mL beaker for pH measurement with a pH meter (AccumetResearch AR15, Fisher Scientific Co., Pittsburgh, Pa., U.S.A.).

Titratable acidity was determined according to the AOAC 942.15B(AOAC 1990) method. Three 20-mL samples were placed in a 250-mL beaker agitated with a magnetic stir bar. Distilled water (80 mL)was added to ensure that the electrode was completely submergedinto the sample. Titration was performed with 0.1 N NaOH to a pHbeyond 8.1. Values used for interpolation were in the range of 7.9and 8.3. Data were reported as percent of malic acid.

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Soluble solids were measured using an Abbe 3-L refractometer(Bausch & Lomb, Rochester, N.Y., U.S.A.). Refractive index was re-corded and converted to °Brix. Measurements were performed at23.3 ± 0.5 °C.

For color measurement, 3 5-mL samples were taken from each treat-ed sample and transferred to a 35-mm plastic dish (Corning tissueculture dish, Corning, N.Y., U.S.A.). The CIE L* (lightness), a* (red togreen), and b* (yellow to blue) values were measured with a huntercolorimeter (LabScan XE, Hunter Associates Laboratories, Inc., Reston,Va., U.S.A.). An average of 3 readings were recorded for each sample.

Scanning electron microscopyScanning electron microscopyScanning electron microscopyScanning electron microscopyScanning electron microscopyEnvironmental scanning electron microscopy (ESEM) was used

in this study to examine the extent of cell damage caused by differ-ent treatments. E. coli K12 cells were placed in apple cider and sub-jected to non-treatment (control), thermal treatment at 72 °C for 30s, ultrasound and thermal treatments at 40 °C for 3 min, and ultra-sound and thermal treatments at 60 °C for 1 min. At selected treat-ment times, a 5-mL sample was taken from the treatment vessel,centrifuged, and suspended in a fixative solution (1.25% glutaral-dehyde solution in 0.1 mol L sodium cacodylate buffer) for 4 h. Al-iquots were filtered through a 25-mm-dia, 0.25-�m porosity Ano-disc (Whatman, Maidstone, U.K.). Filters were rinsed 5 times, for 10min each time, in 0.1 mol L sodium cacodylate buffer. Post fixationwas conducted in 1% Osmium in 0.1 mol L sodium cacodylate bufferfor 90 min. Samples were rinsed 3 times in distilled water and thendehydrated in a graded ethanol series (50%, 75%, 90%, 100%, 100%,and 100%). After a critical point drying (CPD) with a critical pointdryer (Samdri PVT-3D, Tousimis Research Corp., Rockville, Md.,U.S.A.), samples were mounted onto a stub and coated with gold/palladium with a sputter coater (Desk II TSC Denton Vacuum,Moorestown, N.J., U.S.A.). Samples were observed with an ESEM(Philips XL30 ESEM-FEG, FEI Co., Eindhoven, The Netherlands).

StatisticsStatisticsStatisticsStatisticsStatisticsFor all statistical analyses, SAS software version 8E was used (SAS

Institute, Cary, N.C., U.S.A.). Data were analyzed by 1-way analysis ofvariance (ANOVA), and Fisher LSD was used to determine statistical

differences among treatment means. Experiments were conducted intriplicate, and standard deviations are given in the figures as error bars.

Results and Discussion

Thermal and ultrasound treatment at 5 temperaturesThermal and ultrasound treatment at 5 temperaturesThermal and ultrasound treatment at 5 temperaturesThermal and ultrasound treatment at 5 temperaturesThermal and ultrasound treatment at 5 temperaturesInactivation of E. coli K12 for thermal-alone treatments at 40 °C

and 45 °C was negligible (Figure 1). At 50 °C, a 0.67-log reduction inE. coli K12 population was obtained after 20 min of treatment, whichis higher than the 0.3-log reduction in population of E. coli O157:H7reported by Steenstrup and Floros (2002) for a treatment in a blendapple cider with a °Brix of 12.5. The environment in which E. coli cellsare treated has a marked effect on their heat resistance. Apple cidersdiffer in composition, pH, acidity, and °Brix and these parameters allcontribute to the differences in thermal death times observed instudies using different cider samples. Po and others (2002) reportedthat when the °Brix of simulated apple cider samples increased from10.17 to 14.01, a 45% increase in the D value for the inactivation of E.coli O157:H7 was observed. The apple cider used in this study had a°Brix of 11.7, lower than that used by Steenstrup and Floros (2002).The differences in food matrix, microorganism, and ultrasound treat-ment system may be the cause for the higher inactivation that oc-curred for heat inactivation at 50 °C. An increase in inactivation be-came evident for heat treatment at 55 °C, while at 60 °C, a 5-logreduction was achieved in 4 min (Figure 1).

Inactivation of E. coli K12 with ultrasound at the 5 temperaturesall resulted in a 5-log reduction in the number of survival countswithin the sonication time selected for each treatment (Figure 2).Compared with the thermal-alone treatment (Figure 1), sonicationat low temperatures caused a more pronounced increase in the inac-tivation rate. At 40 °C, for example, thermal treatment for 20 min didnot produce any noticeable cell destruction. However, sonication atthis temperature for 20 min reduced the survival count of E. coli K12by 5.3 log cycles. To highlight the added reduction in E. coli K12 pop-ulation due to the application of ultrasound to cider samples, the logreduction between sonication and thermal treatment at the sametemperature is plotted in Figure 3 for 3 selected temperatures. At theend of each treatment, and comparing to the thermal inactivation

Figure 1—Thermalinactivation of Escheri-chia coli K12 in applecider at 5 temperatures(a) and the correspond-ing temperature historycurves (b)

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counterparts, sonication increased cell destruction by 5.3-, 5.0-, and0.1-log cycles at 40 °C, 50 °C, and 60 °C, respectively. It is worth not-ing that, at 60 °C (Figure 3c), instead of following the trend in Figure3a and 3b, a sharp decrease in the net inactivation of sonication wasobserved. At this temperature, a higher vapor pressure lowered thecavitation threshold but produced vapor-filled bubbles. The collapseof the vapor-filled bubbles would be cushioned by vapor, renderingit less effective in microbial inactivation (Mason 1999). This phe-nomenon was also observed in the ultrasonic inactivation tests withGram-positive L. monocytogenes Scott A cells in apple cider (Ugarteand others 2006) when the treatment temperature was at 65 °C.Ugarte and others (2006) suggested that an upper temperature limitexisted for thermosonication beyond which the additional inactiva-tion due to ultrasound treatment became negligible.

The thermosonication test at 60 °C with E. coli O157:H7 resulted ina 5.6 log reduction in survival count after 20 min of treatment (resultnot shown), which was slightly higher than the 5.1-log reduction for E.coli K12 at the same temperature. However, no significant differencein cell destruction between these 2 microorganisms was found. Thetest results with non-pathogenic E. coli K12 can hence be used to findthe treatment conditions for the inactivation of E. coli O157:H7.

Inactivation kineticsInactivation kineticsInactivation kineticsInactivation kineticsInactivation kineticsFor many nonthermal technologies, the 1st-order reaction model

used for the evaluation of D- and z-values has been found to beinappropriate (Rodrigo and others 2003). Several nonlinear modelshave been proposed and used to describe the microbial inactiva-tion for nonthermal processing technologies. The Weibull model is

Figure 2—Inactivation ofEscherichia coli K12 withpower ultrasound inapple cider at 5 tempera-tures (a) and the corre-sponding temperaturehistory curves (b)

Figure 3—Log reduction differencebetween sonication and thermal-alonetreatment for Escherichia coli K12 at40 °C (a), 50 °C (b), and 60 °C (c)

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one of them that has been extensively studied by Peleg and Cole(1998) and Hassani and others (2005). In this study, the Weibullequation (Eq. 1) proposed by van Boekel (2002) was used:

(1)

where t is the treatment time (min), b is the scale parameter (min), andn is the shape factor. A value of n < 1 indicates a concave upward sur-vival curve while for n > 1, a concave downward survival curve can beobserved. The data fitting results are presented in Table 1. For thermaltreatment at 40 °C and 45 °C, the inactivation was negligible andhence no Weibull parameters were obtained. The shape factor forthermal treatment at 50 °C and 60 °C are close to 1, indicating a nearlog-linear relationship. The n at 55 °C, however, has a value of 1.95 andthe survival curve at this temperature exhibits a shoulder (Figure 1a).For all ultrasound inactivation tests, an n value in the rage of 0.547 to0.720 was observed. The relatively large deviation from 1 for n valuesof the sonication tests indicates that there might be a progressivedecrease in the cavitation activity. In addition, the use of traditional D-and z-values for inactivation analysis would introduce relatively largeerrors. Although concave upward survival curves have been observedin several ultrasound inactivation studies (Faruta and others 2004;Stanley and others 2004), there was no reported work on the use ofWeibull model to analyze ultrasound inactivation data.

Cell microtopography observationsCell microtopography observationsCell microtopography observationsCell microtopography observationsCell microtopography observationsThe environmental scanning electron microscopy (ESEM) images

of the control (no treatment) and treated cells are shown in Figure 4.For the control, the E. coli K12 cells exhibited normal granular texturewith external oligo-saccharide strands (Figure 4a). The cells treated at72 °C had a nongranular surface and some minor pitting, but the cellintegrity was well maintained (Figure 4b). Heat causes loss of nutrientsand ions, ribosome aggregation, DNA strand breaks, and inactivationof essential enzymes. However, the damage may not be manifestedon cell surfaces (Mañas and Pagán 2005). Raso and others (1998b) alsoobserved no cell wall disintegration for heat treatment of Yersinia en-terocolitica at 63 °C when a 99% reduction in survival count wasachieved. When exposing E. coli K12 cells to thermal treatment at 72 °Cfor 30 s, an over 5-log reduction in survival population can be achieved.Therefore, the 4 cells in Figure 4b should be regarded as dead.

Cells treated at 40 °C for 3 min maintained their granular texturebut were absent of the oligo-saccharide strands (Figure 4c and 4e).Because the thermal inactivation of E. coli K12 was negligible in 20min, after 3-min of heating under this condition, similarly treatedcells from which the ESEM picture was taken were most likely viable.Cells subject to sonication at 40 °C for 3 min, however, exhibited atotally different morphology. As seen in Figure 4d, a hole on the cellsurface with a proximate diameter of 200 to 300 nm was observed.Intracellular materials may have leaked from the treated cells. The

Table 1—Weibull model parameters and regression coefficients for Escherichia coli K12 cells treated with and with-out ultrasound at 5 temperatures

Thermal Sonication

Microorganism Temperature b n R2 b n R2

Escherichia coli K12 40 — — — 0.39 0.65 0.9945 — — 0.56 0.56 0.72 1.0050 1.23 0.95 0.98 0.40 0.66 0.9955 7.48 1.98 1.00 0.15 0.55 0.9460 0.44 1.18 0.97 0.12 0.72 0.94

Figure 4—Escherichia coli K12 cells observed with envi-ronmental scanning electron microscopy (ESEM): (a) con-trol, (b) heat treatment at 72 °C for 30 s, (c) heat treat-ment at 40 °C for 3 min (80000 magnification), (d) sonica-tion at 40 °C for 3 min (80000 magnification), (e) heat treat-ment at 40 °C for 3 min (40000 magnification), (f) sonica-tion at 40 °C for 3 min (40000 magnification), (g) heat treat-ment at 60 °C for 1 min (80000 magnification), (h) sonica-tion at 60 °C for 1 min (80000 magnification), (i) heat treat-ment at 60 °C for 1 min (40000 magnification), and (j) soni-cation at 60 °C for 1 min (40000 magnification)

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granular texture was still maintained, indicating that the treatmentwas mild. The cell destruction under this condition was about 1.23 logcycles. The cell wall perforation may be attributed to a phenomenoncalled liquid jet, which is generated when a cavitating bubble col-lapses near a solid-liquid interface. An in-flow of liquid with a max-imum speed of up to 156 m/s may occur when the bubble implodes(Leighton 1994). The hole on E. coli K12 surface thus may be causedby high-velocity liquid jet impinging onto the cell surface that perfo-rated cell wall and the cytoplasmic membrane. Other types of dam-age on E. coli K12 surface, such as cell wall deformation and shrink-age, can also be observed (Figure 4f) for sonication at 40 °C.

At 60 °C, heat-alone treatment for 1 min caused bulging on cellsurface but the surface granules could still be observed (Figure 4g and4i). Ultrasound treatment at this temperature resulted in extensivedamage and marked changes in cell morphology (Figure 4h and 4f).The arrow in Figure 5h points out the collapsed and wrinkled areawhile in Figure 5f, the arrow indicates cell rupture and disintegrationwhere the cells appear to have discharged intracellular material. TheESEM images revealed that sonication-induced cell damage at elevat-ed temperatures, especially at 60 °C, was mostly mechanical. Theseobservations confirm the report of Raso and others (1998b) that ultra-sound inactivated microbial cells through breakage of cell.

Quality comparisonQuality comparisonQuality comparisonQuality comparisonQuality comparisonThe treatment times in quality analyses for thermal and sonica-

tion tests were chosen based on the time required to obtain a 5-logreduction in E. coli K12 population. The inactivation data at 40 °Cand 60 °C are plotted in Figure 5. The intersection of a horizontalline corresponding to a 5-log reduction and the upper bound of a

95% confidence interval for a linear regression of the inactivationdata gives the time needed for the quality analysis. The treatmenttimes for the quality tests obtained this way are listed in Table 2.

The turbidity values of cider treated with ultrasound at 40 °C and60 °C are significantly (P < 0.05) lower than the control and thermal treat-ment at 60 °C (Table 3). There was no difference between control andheat treatment at 60 °C. The turbidity of apple cider is a measure ofcloudiness and is related to suspended particles. Ultrasound may haveaffected those particles in 2 ways. For the single frequency (20 kHz) sys-tem used in this study, standing waves could be formed in the treatmentchamber and particles in the ultrasonic wave field would move to a pres-sure node and get enriched, which would cause a separation of theparticles (Groschl 1998). On the other hand, ultrasound treatment mayalso break down the particles to cause a reduction in particle size and tochange the transmittance. Both would contribute to a decrease in theturbidity of cider samples. The longer sonication time at 40 °C (17.7 min)thus resulted in a relatively high loss in turbidity. The pH of cider sam-ples treated with different methods was not significantly different. Sol-uble solid contents of the samples were in a narrow rage of 11.6 to 11.8°Brix and there was no significant difference observed. The changes intitratable acidity were in a very small range (0.16 to 0.17) among thesamples and no significant difference was found among the treat-ments. Color changes expressed by Hunter L values were all significant-ly different, although the relative changes with respect to the controlwere less than 8.6% for all treatments. The color change or darkening inapple cider is caused by enzymatic browning involving polyphenol ox-idase (PPO), which is related to particulate fractions (Zárate-Rodríguezand others 2000). Removal of suspended particles and inactivation ofPPO will mitigate the color changes. In this study, all treated samplesexhibited a slightly less dark color from L value readings compared withthe control, probably due to PPO inactivation and particle separation.

Conclusions

Power ultrasound enhanced the inactivation of E. coli K12 in ap-ple cider, especially at sublethal temperatures (40 °C, 45 °C, and

50 °C), compared with the thermal-only treatments at the sametemperatures. While the survival curves of E. coli K12 for thermalinactivation were mostly log-linear, the sonication tests exhibiteda non-log-linear inactivation behavior that could be described by

Table 2—Treatment times for quality analysis tests esti-mated from Figure 5

Treatment Time (min)

Control 0Sonication at 60 °C 3.6Sonication at 40 °C 17.7Heat at 60 °C 4.2

Figure 5—Treatment timeselection for quality analysistests: (a) sonication at 60 °C,(b) sonication at 40 °C, and (c)thermal treatment at 60 °C.

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the Weibull model. The ESEM images of ultrasound treated E. coliK12 cells revealed extensive surface damage. Besides large area ofcollapse and rupture, a unique perforation on cell wall was ob-served for sonication at 40 °C, which might have been caused by theliquid jet originated from the implosion of an asymmetric cavitat-ing bubble. E. coli O157:H7 showed a similar resistance to ultra-sound treatment at 60 °C, compared with E. coli K12. For ultra-sound-treated cider samples, the changes in pH, titratable acidity,and °Brix were not significantly different from those of the control.Significant differences in color and turbidity between the ultra-sound-treated samples and the control were found. Based on theresults of this study, power ultrasound treatment can provide the5-log reduction in apple cider required by the FDA and may proveuseful as an alternative to traditional pasteurization method.

AcknowledgmentsThis study was supported by the Illinois Council on Food and Agri-cultural Research (project nr 03E-125-4). The authors thank Cathe-rine Landers, Flavia A. Ramirez, Carlos R. Lemuz Gutierrez, andMeredith Agle for their technical and laboratory assistance duringexperimentation.

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Table 3—Turbidity, pH, °Brix, titratable acidity, and L values for apple cider samples treated with ultrasound and heatin comparison with the control

Control Sonication at 40 °C Sonication at 60 °C Heat treatment at 60 °Ca

Turbidity (NTU) 981.7a 780.2b 903.3c 943.6apH 4.06a 4.06a 4.05a 4.07a°Brix 11.7a 11.6a 11.6a 11.8aTitratable acidity (% of malic acid) 0.17a 0.16a 0.16a 0.17aL value 21.0a 21.6b 22.8c 22.1daDifferent letters on the same row indicate significant differences (P < 0.05).

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