osmosonication of blackberry juice: impact on selected pathogens, spoilage microorganisms, and main...

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Osmosonication of Blackberry Juice:Impact on Selected Pathogens, SpoilageMicroorganisms, and Main Quality ParametersEric Wong, Fabrice Vaillant, and Ana Perez

Abstract: Osmosonication combines ultrasound with nonthermal concentration. It was applied on tropical highlandblackberry (Rubus adenotrichus) juice over different periods of time to assess reductions in microorganism and the impacton main quality parameters. This juice had been inoculated with Salmonella spp., Shigella sp., a lactic acid bacterium,yeasts, and molds. It was then sonicated for 5.9 to 34.1 min at 20 kHz and 0.83 W/mL. Nonthermal concentrationwas simulated by mixing the juice with a concentrate to obtain 650 g TSS/kg. It was then stored at −18 ◦C for up to82 h. The lactic acid bacterium, yeasts, and molds were reduced by 1.60 to as much as 5.01 log10 CFU/mL, whereas,for pathogens, reductions were total ≥7.1 log10 CFU/mL after 24 h of storage, even for juice not sonicated, becauseof low pH. Color, antioxidant capacity, anthocyanins, and ellagitannins did not change significantly during sonicationtreatment up to 32 min. However, an off-flavor was detected after 8 min of sonication. Nonetheless, osmosonication canbe considered as an alternative to thermal processes for producing safe and high-quality concentrates.

Keywords: food safety, fruit juice, microbial survival, nonthermal concentration, sonication

Practical Application: Osmosonication represents a potential processing alternative for producing safe and high-qualityconcentrated fruit juice without applying thermal treatments. Findings reported in this article can also be applied byindustries when concentrating juices by classical means at relatively low temperature. It provides industries with amathematical model specific for blackberry juice, from which different combinations of sonication time and storage timeof concentrate can be chosen to achieve safety and quality goals.

IntroductionConsumers are increasingly demanding minimal processing of

natural fruit juices. In response, numerous innovative technologies,mainly low-temperature processing, have been developed. How-ever, 1 consequence is that the microbiological safety of thesebeverages is often at risk. Indeed, processing at relatively low tem-perature has been responsible for an increased number of outbreaksof foodborne illnesses in last decades (Beuchat 1996; CDC 1999;CDC 2000). Pathogens such as Salmonella spp., Shigella sp., Listeriamonocytogenes, and Escherichia coli O157:H7 have been associatedwith the consumption of unpasteurized apple juice, apple cider,tomato juice, and orange juice (Parish 1997; Krause and others2001).

The main challenge for food technologists is, therefore, to de-velop juice stabilization processes that comply with higher foodsafety requirements while preserving sensorial, nutritional, andfunctional properties (Gould 2001). The U.S. Food and Drug

MS 20100232 Submitted 3/3/2010, Accepted 5/25/2010. Authors Wong andPerez are with Centro Nac. de Ciencia y Tecnologıa de Alimentos (CITA), Univ. deCosta Rica (UCR), Ciudad Universitaria Rodrigo Facio, Codigo Postal 11501-2060,San Jose, Costa Rica. Author Vaillant is with Centre de Cooperation Internationale enRecherche Agronomique pour le Developpement (CIRAD), UMR 95 QUALISUD,TA B-95/16, 73 rue Jean-Francois Breton, 34398 Montpellier Cedex 5, France.Direct inquiries to author Vaillant (E-mail: [email protected]).

Administration (FDA) requires fruit juices to be treated nonther-mally, with a minimal reduction of 5 log10 for pathogens (FDA2001; Van Opstal and others 2006). Consequently, most nonther-mal processes need to combine with other hurdle technologiesto ensure a significant reduction of not only pathogens, but alsospoilage microorganisms that may reduce food quality.

Osmotic evaporation (OE) is an emerging technology that oper-ates at ambient temperatures but allows fruit juices to concentrateto 650 g TSS/kg (total soluble solids per kilogram) while preserv-ing all nonvolatile molecules and most aroma compounds (Barbeand others 1998; Vaillant and others 2001; Cisse and others 2005).Although such high osmotic pressure reduces microbial survival(Poirier and others 1998; Mille and others 2002; Mille and oth-ers 2005), such reduction may not be sufficient in terms of foodsafety. Other nonthermal hurdle technologies must, therefore, beincorporated to ensure that the microorganism load that naturallycontaminates fruit juices is sufficiently reduced.

Treatment of liquid food products with ultrasonic radiation,also referred as sonication, has been combined with other process-ing steps to reduce microbial contamination (Piyasena and oth-ers 2003). Oscillatory high pressure on fluids by ultrasonic wavesinduces membrane damages and even cell disruption during se-vere treatment. However, sonication is often not effective alonebut when combined with heat (thermosonication), high dynamicpressure (manosonication), or both (thermo-manosonication), itsignificantly reduces time, temperature, and dynamic pressure forthe same F-value (Pagan and others 1999; Wu and others 2008;

C© 2010 Institute of Food Technologists R©M468 Journal of Food Science � Vol. 75, Nr. 7, 2010 doi: 10.1111/j.1750-3841.2010.01730.x

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Osmosonication: safety and quality . . .

Walkling-Ribeiro and others 2009; Lee and others 2009a, 2009b).In these cases, sonication appears to weaken microbial membranes(Knorr and others 2004), making microorganisms more sensitiveto external stresses such as heat, dynamic pressure, pH (Pagan andothers 1999; Piyasena and others 2003; Ulusoy and others 2007),and even osmotic pressure. A previous study (Wong and others2008) had demonstrated that sonication of a contaminated orangejuice concentrate enhanced the reduction of Salmonella spp. af-ter a short storage period, compared with a nonsonicated sample.By analogy with other combined processes, including ultrasoundtreatments, “osmosonication” was defined as the coupling of son-ication with a subsequent increase of osmotic pressure (Wong andothers 2008).

Treatment of heat-sensitive juices (that is, blackberry juice)(Cisse and others 2009) by osmosonication may represent an alter-native for the production of innovative functional food ingredients.Nonetheless, the real impact of osmosonication on food safety andquality remains to be determined.

The objective of the present research was to determine theeffect of osmosonication on selected pathogens, spoilage microor-ganisms, and main quality parameters for blackberry juice.

Materials and Methods

Blackberry juice and physicochemical parametersBlackberry fruits were obtained from organic plantations, and

harvested and frozen by the company APROCAM (Cartago, CostaRica). Fruits were then squeezed, using a Hydraulic Shop PressOTC (Y125) Tool (OTC, Owatonna, Minn., U.S.A.), yielding ajuice at 110 g TSS/kg (0.6 MPa). The juice was then concentratedin a Votator evaporator (Chemetron Corp., Louisville, Ky., U.S.A.)at 70 ◦C to a final concentration of 650 g TSS/kg, correspondingto an osmotic pressure of 12.6 MPa. Blackberry juice was preparedfrom the concentrated juice by diluting with sterilized water underaseptic conditions. When required, pH levels were adjusted witheither citric acid or sodium bicarbonate. Experiments related tocolor, oxygen radical absorbance capacity (ORAC), anthocyanins,and ellagitannins were performed with microfiltered blackberryjuice (110 g TSS/kg) provided by Coopeagrimar R.L. (Alajuela,Costa Rica).

Microbial strains, inocula preparation, and determinationof microbial logarithmic reductions

The bacterial strains used in this study were: A mixed culture(1:3 each) of Salmonella strains (S. Typhi [ATCC 6539], S. En-teritidis [ATCC 13076], and S. Typhimurium [ATCC 14028];(American Type Culture Collection, Manassas, Va., USA); a strainof Shigella sp., isolated in our laboratory from tropical highlandblackberries collected in the production region (Cartago, CostaRica); and a strain of Lactobacillus casei subsp. rhamnosus (ATCC11981) (a lactic acid bacterium).

These bacterial strains were grown from frozen stocks (brain-heart infusion broth, containing 15% glycerol; Oxoid, Basingstoke,U.K.) after inoculation on xylose lysine deoxycholate agar (XLD,Oxoid) for Salmonella and Shigella, and MRS broth (Oxoid) for L.casei subsp. Rhamnosus. Each strain was then aseptically transferredinto 5 mL of trypticase soy broth (TSB; Oxoid) and incubated for18 h at 35 ◦C. The starting bacterial concentration was determinedby serial dilutions (0.1% peptone solution; Scharlau, Barcelona,Spain), plated on XLD or MRS agar, and incubated for 24 h at35 ◦C. Typical colonies were then counted and the results areexpressed as log10 CFU/mL.

Similarly, a mixture of strains of the yeasts Saccharomyces cerevisiaeand Zygosaccharomyces sp., also isolated from the same tropical high-land blackberries and maintained on potato dextrose agar (PDA;pH = 3.5) were used. Colonies were transferred to 5 mL ofTSB (Oxoid) and incubated for 18 h at 35 ◦C to obtain a liquidinoculum. The starting yeast concentration was determined by se-rial dilutions (0.1% peptone solution, Scharlau, Barcelona, Spain),plated on PDA and incubated for 48 h at 35 ◦C.

A mixture of spores from the molds Aspergillus sp. and Penicil-lium sp., isolated from blackberries, was also used. The molds weregrown as for yeasts, except that they were incubated at 23 ◦C for5 d. Spores were suspended in peptone water (0.1% peptone solu-tion, Scharlau, Barcelona, Spain), and initial spore concentrationdetermined by plate count.

Samples of 250 mL of blackberry juice (110 g TSS/kg) wereeach inoculated with a specific volume of the bacterial or yeastculture, or mould spore solution, prepared as described above,to obtain final concentrations of about 6 to 7 log10 CFU/mL.The inoculum was evenly distributed within each sample, usinga homogenizer (IKA Works, Malaysia). Initial counts were cal-culated, taking into account inoculum concentration, volume ofinoculum used, and final sample volume. Final counts of treatedsamples were determined as described for initial inocula counts.Reductions were calculated by subtracting the final count fromthe initial count, and expressed as log10 CFU/mL.

Sonication treatmentsA continuous recirculating system was used to sonicate juice

samples (Figure 1). The system consisted of a thermostated bath(A) to maintain the sample’s temperature at 25 ± 2 ◦C during allexperiments. The sample (250 mL of juice) (B) was placed in abeaker with a thermometer (C) from which it was recirculatedcontinuously at a constant flow rate of 35 mL/min, using a peri-staltic pump (D) (Master-flex L/S, Cole-Parmer Instrument Co.,Vernon Hills, Ill., U.S.A.), through a stainless-steel, double-wall,cylindrical, continuous-flow cell (E), featured with an ultrasonicprobe of 12.7-mm dia positioned at the cell’s centre (H). Ultrasonictreatment was applied at 20 kHz, using an ultrasonic converter (F)that operated continuously. The ultrasonic processor featured acontroller (G) (Ultrasonic Processor Model CP 750, Cole Parmer,Vernon Hills, Ill., U.S.A.) with automatic tuning and frequencycontrol for delivering constant amplitude of ultrasonic vibration atthe probe tip. Amplitude was set at 40% (48 μm), giving rise in ourspecific case of blackberry juice, to the delivery of a real-energy in-put of 50 ± 0.2 W, corresponding to a power density treatment of0.83 W/mL (50W/60 mL) throughout the 60-mL volume ofjuice within the flow cell (E). The entire circuit, including flowcell (E), was autoclaved prior to treatments and between trials withmicroorganisms.

For microbial tests, 250 mL of juice at 110 g TSS/kg wereinoculated and sonicated for various periods. Only the effectivesonication times, corresponding to average flow time within theflow cell (E), was reported. After sonication, an aliquot (1 mL) ofjuice was then mixed with 100 mL of concentrated juice, previ-ously sterilized, to reach a final concentration of 650 ± 2 g TSS/kg(12.6 MPa). The mixture took place at ambient temperature tosimulate OE. Samples were analyzed immediately (storage time =0) or after storage at −18.0 ± 0.2 ◦C.

Physicochemical and sensory analysesTotal soluble solids, expressed as g TSS/kg, were measured at

23.0 ± 0.5 ◦C with an Abbe refractometer (ATAGO, Tokyo,

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Japan), following the procedure described by the Association ofOfficial Analytical Chemists (AOAC 1990). Juice density was alsodetermined according to AOAC methods (AOAC 1990). Wateractivity (aw) was determined with a water activity meter (AquaLabCS-2, Decagon Devices, Inc., Pullman, Wash., U.S.A.). Osmoticpressure (�, in MPa) was roughly estimated as:

∏≈ −RT ln aw

Vw

(1)

where,

R is the universal gas constant (8.3144 × 10−6 MPa.m3/mol.k)T the absolute temperature (◦K)aw the water activity

Vw is the partial molar volume of water (m3/mol).

Antioxidant capacity of juice samples was determined ashydrophilic-ORAC (H-ORAC) as previously reported (Ou andothers 2001). It was expressed in micromoles of Trolox equiva-lents per milliliter (μmol TE/mL). Cellular antioxidant activitywas also performed by means of an in vitro model using humanerythrocytes following the Erythrocyte Cellular Antioxidant as-say (ERYCA) method reported previously (Gonzalez and others2010). In this case, results were given as micromoles of quercetinequivalents per milliliter (μmol QE/mL).

Color parameters (L∗, a∗, b∗) were determined using a Hunter-Lab ColorFlex 430 colorimeter (Reston, Va., U.S.A.) and colorwas expressed as a tristimulus parameter (L∗), hue angle (H∗ =tan−1(b∗/a∗)), and chroma (C∗ = (a∗2 + b∗2)1/2). Color differ-ence (�E∗) between sonicated samples and nonsonicated sam-ple was calculated using the formula �E∗ = (�L∗2 + �C∗2 +�H∗2)1/2(Gonnet 1999). Nonsonicated juice was recirculated inthe same condition as the sonicated sample but the sonicationequipment was disconnected. Both anthocyanins, cyanidin 3-glucoside and cyanidin 3-(6′malonyl)-glucoside, and both ellag-itanins (lambertianin C and sanguiin H-6) tentatively identifiedin R. adenotrichus by HPLC-DAD-MS (Mertz and others 2007)

were determined by HPLC-DAD following the method reportedpreviously (Mertz and others 2007; Acosta-Montoya and others2010) and results were, respectively, expressed in mg/100 mL ofCyanidin 3-glucoside or ellagic acid equivalents. Sensory trian-gular difference tests were performed to determine differences inflavor among treated juice samples (2 equal and 1 different) thatwere presented to 25 trained panelists. In each test, the treatedsample (at different sonication times) was compared with an un-treated sample (control). A weighted least-squares analysis wasperformed to analyze the amount of correct answers obtained foreach sonication period (4, 8, 12, 21, or 32 min).

Experimental designsThe experiments were designed to determine the following

effects: 4 sonication periods (0, 6.8, 13.6, and 20.4 min) for re-ducing Salmonella spp., Shigella sp., or spoilage microorganisms (alactic acid bacterium, yeasts, and molds). Also, 2 levels of soni-cation periods (0 or 20.4 min), storage times (0 or 24 h), TSS(110 or 650 g TSS/kg), or pH (1.8 or 3.2) for reducing Salmonellaspp., Shigella sp., or spoilage microorganisms; 4 sonication periods(0, 12, 21, or 32 min) for affecting color parameters (L∗, a∗, b∗,�E∗); And 6 sonication periods (0, 4, 8, 12, 21, and 32 min) foraffecting antioxidant capacity (H-ORAC), anthocyanin (cyanidin3-glucoside and cyanidin 3-[60 malonyl]) content and ellagitannin(lambertianin C and sanguiin H-6) content. Times of treatmentwere chosen on the basis of preliminary trials.

Experiments were designed to be randomized with factorialarrangements where applicable and at least 2 replications. The sig-nificance of factors and interactions were analyzed with ANOVA.

A central composite rotable design (Montgomery 2005) forstorage time (X1) (14 to 82 h) and sonication time (X2) (5.9 to34.1 min), with 5 replications for the centre point (48 h [X1];20 min [X2]), was used to assess a 2nd-order response pattern forthe reduction of the lactic acid bacterium (L. casei subsp. rhamno-sus), yeasts (S. cerevisiae and Zygosaccharomyces sp.), or molds (As-pergillus sp. and Penicillium sp.). Response surfaces were presentedas a contour plot graph using SigmaPlot, version 7.0 (2001).

Figure 1–Sketch of the continuous recirculatingsystem used to sonicate fruit juice.

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The quality parameters chosen from the fit of the polynomialmodel equation were the coefficient of determination (R2) be-tween the actual and predicted response, the probability (P) of theabsence of at least 1 significant regression factor in the model, andthe probability (P lof ) of the lack of fit.

An ANOVA with Tukey’s test was performed to determinesignificant differences in the overall reductions observed for eachgroup of microorganisms.

All statistical analyses were conducted, using JMP 5.0.1 (SASInstitute, Cary, N.C., U.S.A.) at a significance level of 0.05. Fornonsignificant effects, the power of the test (1 − β) was reported.

Results and Discussion

Logarithmic reduction of Salmonella spp., Shigella sp.,and spoilage microorganisms

Table 1 shows logarithmic reductions of 2 pathogens (Salmonellaspp. and Shigella sp.) and a pool of spoilage microorganisms (lacticacid bacterium, yeasts, and molds) that were previously inoculatedinto a blackberry juice that was either subjected or not subjectedto sonication over different periods, at different pH, and at eitherlow (110 g TSS/kg) or high concentration (650 g TSS/kg). Forconcentrated juice, a nonthermal process was simulated by mixingthe inoculated juice at 110 g TSS/kg with a previously concen-trated juice to reach a final concentration of 650 g TSS/kg. Forexample, this occurs when running the OE system at a constantfeed and bleed flow rate, in which a small flow of juice (at about110 g TSS/kg), is fed into a much larger recirculating stream ofconcentrated juice (at about 650 g TSS/kg) (Vaillant and others2001). Some analyses were performed immediately after mixing(storage time = 0) or after 24 h of storage at −18 ◦C. The juice,when concentrated to 650 g TSS/kg, remains a viscous fluid, evenat −18 ◦C, with flow properties that permit transfers of matter,and thus exerting osmotic pressure.

Table 1 indicates that single-strength juice (110 g TSS/kg),with no sonication but low concentration, and stored at −18 ◦Cfor 24 h, had no significant reduction (P = 0.3450; 1 − β =0.99) of its microbial load. The same remark can be made forsonicated samples that were not concentrated (110 g TSS/kg),but stored for 24 h at −18 ◦C (P = 0.4580; 1 − β = 0.99).Even when applied for as long as 20.4 min to a juice that wasnot previously concentrated before storage, sonication appeared tohave no effect on the microbial load. However, when the juice’s

pH was reduced, the microbial load decreased slightly after 24 h ofstorage, indicating that microorganisms in sonicated juice becomemore sensitive to lower pH. When natural pH of concentrateis maintained (pH 1.8), a total reduction of both pathogens wasachieved after the concentrate was stored for 24 h, even thoughsonication was not applied.

If the concentration of contaminated juice to 650 g TSS/kg issimulated, then an important logarithmic reduction of microor-ganisms after 24 h storage can be observed for all the other testedconditions. In this case, the impact of combining ultrasound treat-ments with storage at high osmotic pressure cannot be observed.Probably, high osmotic pressure combined with the additionalstress induced by low pH was sufficient to destroy all pathogens.Similar treatments applied to concentrated orange juice reducedSalmonella spp. by only 2.68 log10 CFU/mL (Wong and others2008). As pH of both concentrates differed greatly (3.2 for theconcentrated orange juice at 650 g TSS/kg and only 1.8 for theconcentrated blackberry juice at 650 g TSS/kg), the low pH ofblackberry juice may explain this difference in the reduction ofmicroorganisms.

In fact, when the pH of concentrated blackberry juice is artifi-cially increased to 3.2 by adding sodium hydroxide, a significantlylower (P < 0.0001) impact on pathogen reduction after storageat high osmotic pressure was observed for Salmonella and Shigella.Additionally, the impact of sonication of juice, followed by con-centration to 650 g TSS/kg and 24 h of storage, can be observedunder these conditions. That is, logarithmic reductions double onaverage over 0 to 20.4 min of sonication treatment proving that alow pH of concentrate represents another stress that is additionalto high osmotic pressure (Patil and others 2009; Lee and others2009a, 2009b). Thus, due to the high acidity of blackberry juiceconcentrate, sonication is not needed to achieve a logarithmic re-duction higher than 5 at least in the case of Salmonella or Shigella.

For spoilage microorganisms, the panorama is different, be-cause no significant differences (P = 0.3165; 1 − β = 0.99)were observed between the effects of pH at 1.8 and 3.2, regard-less of sonication or storage conditions. This can be explained bythe higher resistance of spoilage microorganisms to acidic envi-ronments, compared with pathogens like Salmonella and Shigella( Jay and others 2006).

Because spoilage microorganisms can resist a low pH medium,we could better observe the effects of sonication. For theconcentrate, spoilage microorganisms reduced a little more as

Table 1–Logarithmic reductions of Salmonella spp. or Shigella sp. or spoilage microorganisms (log10 CFU/mL) according to sonicationtime of 110 g TSS/kg blackberry juice and TSS, pH, and time conditions of storage at −18 ◦C.

Storage TSS ga Sonication Storage time SpoilageTSS/Kga time (min) (−18 ◦C) (h) Storage pH Salmonella spp.b Shigella sp.c microorganismsd

110 (0.6 MPa) 0 24 1.8 0.2 ± 0.2 0.09 ± 0.03 0.4 ± 0.3110 (0.6 MPa) 0 24 3.2 0.08 ± 0.01 0.02 ± 0.01 0.3 ± 0.2110 (0.6 MPa) 20.4 24 1.8 0.8 ± 0.1 1.0 ± 0.4 0.9 ± 0.1110 (0.6 MPa) 20.4 24 3.2 0.9 ± 0.2 0.6 ± 0.2 0.4 ± 0.2110 (0.6 MPa) 20.4 0 1.8 0 0.02 ± 0.01 0.05 ± 0.02110 (0.6 MPa) 20.4 0 3.2 0 0 0

650 (12.6 MPa) 0 24 1.8 ≥7.0 ≥7.1 1.6 ± 0.2650 (12.6 MPa) 0 24 3.2 2.0 ± 0.2 1.8 ± 0.3 2.1 ± 0.5650 (12.6 MPa) 6.8 24 1.8 ≥7.0 ≥7.1 2.2 ± 0.2650 (12.6 MPa) 13.6 24 1.8 ≥7.0 ≥7.1 2.7 ± 0.3650 (12.6 MPa) 20.4 24 1.8 ≥7.0 ≥7.1 3.0 ± 0.2650 (12.6 MPa) 20.4 24 3.2 4.0 ± 0.2 3.9 ± 0.3 3.1 ± 0.5aOsmotic pressure value shown in parenthesis.bInitial inoculation level of 7.0 log10 CFU/mL.cInitial inoculation level of 7.1 log10 CFU/mL.dInitial inoculation level of 6.7 log10 CFU/mL (mixed strain containing Lactobacillus casei subsp. Rhamnosus, Saccharomyces cerevisiae, Zygosaccharomyces sp., Aspergillus sp., and Penicilliumsp.).


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sonication time increased when samples were analyzed after 24 hof storage. All reductions found for sonicated samples (6.8, 13.6,and 20.4 min) were significantly higher (P < 0.05) than the re-ductions observed for nonsonicated samples. Hence, a synergisticeffect is observed between sonication time and 24-h storage ofconcentrated blackberry juice at high osmotic pressure.

Modeling logarithmic reductions for the lactic acidbacterium, yeasts, and molds

Figure 2 to 4 show the logarithmic reductions of the lactic acidbacterium (L. casei subsp. rhamnosus), yeasts (S. cerevisiae and Zy-

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Figure 2–Contour plot showing the effect of storage time and sonicationtime on the log reduction of a lactic acid bacterium.

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Figure 3–Contour plot showing the effect of storage time and sonicationtime on the log reduction of yeasts.

gosaccharomyces sp.), and molds (Aspergillus sp. and Penicillium sp.)according to a central composite design. Sonication times werevaried between 5.9 and 34.1 min, and storage times between 14and 82 h. Table 2 reports the fitting parameters of the 2nd-orderpolynomial equations, as well as the regression coefficients (R2),probability (P), and probability of lack of fit (Plof ). These wereconsidered acceptable. Figure 2 to 4 present a contour plot of theresponse surface for the logarithmic reductions. For all microor-ganisms tested, the influence of sonication time is predominantover storage time, as indicated by contour curves being almostparallel to the storage time axis.

On the basis of routine microbial counts implemented for trop-ical highland blackberry juice in our laboratory, yeasts, molds, andlactic acid bacteria usually range from 1.8 to 3.2 log10 CFU/mLbefore processing. Hence, a target reduction of 3 log10 CFU/mLwas set for these spoilage microorganisms. Under the conditionsstudied, the target reduction can be achieved for yeasts and moldsafter short sonication times of about 15 min and 30 to 40 h of stor-age. However, the lactic acid bacterium appeared more resistant to

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Figure 4–Contour plot showing the effect of storage time and sonicationtime on the log reduction of molds.

Table 2–Estimates for equation parameters and significance indi-cators for the model of reduction of lactic acid bacteria,a yeasts,b

and moldsc (log10 CFU/mL) according to the central compositedesign for sonication time (x) and storage time (y).

Equation parameter Estimateor significance (lactic acid Estimate Estimateindicator bacteria) (yeasts) (molds)

Y0 0.8179 1.2009 1.3134A (factor x) 0.0594 0.0876 0.0963B (factor y) 0.0174 0.0251 0.0275C (factor x2) −0.0005 −0.0008 −0.0009D (factor y2) −0.0001 −0.0001 −0.0002E (factor xy) 0.0000 0.0000 0.0000R2 0.79 0.78 0.78P 0.0269 0.0285 0.0277P lof 0.1237 0.1322 0.1302aLactobacillus casei subsp. Rhamnosus.bSaccharomyces spp.cAspergillus spp. and Penicillium spp.


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sonication and, under these conditions, only 2 log10 CFU/mL re-ductions could be achieved. Longer sonication times were neededto achieve a reduction of even 3 log10 CFU/mL after 80 h ofstorage.

Analysis of the data indicates that reductions (1.69 to 3.12 log10

CFU/mL) for the lactic acid bacterium L. casei subsp. rhamnosuswere significantly smaller (P < 0.05) than the reductions deter-mined for yeasts (2.47 to 4.57 log10 CFU/mL) (S. cerevisiae andZygosaccharomyces sp.) and molds (2.71 to 5.01 log10 CFU/mL)(Aspergillus sp. and Penicillium sp.). No difference (P > 0.05) wasdetermined in observed logarithmic reductions between yeasts andmolds. Consequently, while the target reduction can be achievedfor yeasts and molds, reduction of lactic acid bacteria may be thelimiting factor when considering osmosonication to treat acidicfruit-juice concentrates.

Nonetheless, developed models would be useful for industry, asdifferent combinations of sonication times and storage times can bechosen to achieve similar microbial reductions. Food technologistsmay choose the combination that best fits their processes. Longersonication steps imply shorter storage periods and vice versa.

Effects of sonication on color, antioxidant capacity,anthocyanins, and ellagitannins

Juice of tropical highland blackberry (R. adenotrichus) was cho-sen as a model, because the juice is a rich source of thermosensitivephenolic compounds with very high antioxidant capacity (Mertzand others 2007; Hassimotto and others 2008; Mertz and oth-ers 2009; Acosta-Montoya and others 2010). Impact of sonicationtreatment on color (L∗, H∗, C∗, �E∗), antioxidant capacity, antho-cyanins, and ellagitannins after applying sonication, for 6 differenttimes of exposure (0, 4, 8, 12, 21, and 32 min), is presented inTable 3.

It can be observed that no significant differences were foundfor different sonication times for all quality parameters. Colorparameters (L∗, C∗, H∗) remained constant (P > 0.0675; 1 − β =0.98) for all times, including untreated samples. �E∗ also remainedconstant (P > 0.4072; 1 − β = 0.99) indicating the absence ofoverall color degradation between untreated and treated samples.

These results are confirmed by the analysis of the content ofanthocyanins in juices sonicated up to 32 min as no statisticaldifferences were observed for all treatment times, for both antho-cyanins, cyanidin 3-glucoside (P > 0.8634; 1 − β = 0.99) andcyanidin 3-(6′malonyl) glucoside (P > 0.8638; 1 − β = 0.99).

The same was observed for other phenolic compounds such asellagitanins like lambertianin C (P > 0.8626; 1 − β = 0.99) andsanguiin H-6 (P > 0.9257; 1 − β = 0.99). In good agreement withthese results, the antioxidant capacity (H-ORAC) (P = 0.3338;1 − β = 0.84) and the antioxidant activity (ERYCA) (P = 0.1479;1 − β = 0.99) were also found to remain almost constant for alltreatment times analyzed.

Contrary to the previous study on the impact of sonicationon microorganisms in pressed blackberry juice containing 2% to3 % suspended pulp, these treatments were performed on micro-filtered juice (0% suspended pulp) to avoid interference due tothe possible extraction of bound phenolics from the suspendedpulp that could have offset possible loses during sonication (Ti-wari and others 2009a). In this case, our experiments prove thatultrasonic irradiation has no significant effect on the degradationof color, on the content of thermosensitive phenolic compoundsincluding anthocyanins and even on antioxidant activity assessedeither by chemical method (H-ORAC) or using an in-vitro cellu-lar model (ERYCA). This is in accordance with other studies thathave shown good retention percentages (> 95%) of anthocyaninsafter similar sonication treatments (Tiwari and others 2008, 2009a,2009b, 2010). It confirms that although the propagation of ultra-sonic waves in liquids induces cavitation giving rise at the level ofmicro-bubble to very high temperatures, pressures, and shearingstresses, this process behaves like a nonthermal technology as nosignificant rise in macro-temperature is registered and most qualityparameter including functional quality are preserved.

Effect of sonication period on sensorial qualityTable 4 shows the results of triangular difference tests performed

for sonicated samples, compared with untreated samples. This anal-ysis was performed only on sonicated single-strength juices. A

Table 4–Proportion of correct answers in the triangular differ-ence test and associated individual probability according to son-ication time of 110 g TSS/kg blackberry juice.

Sonication Proportion of Associated individualtime (min) correct answers probability (p)

4 0.48 >0.058 0.60 ≤0.0112 0.90 ≤0.0121 0.85 ≤0.0132 0.95 ≤0.01

Table 3– Color parameters (L∗, C∗, H∗, �E∗), antioxidant capacity (H-ORAC), content of cyaniding 3-glucoside, cyaniding 3-(6′malonyl)-glucoside, lambertianin C, and sanguiin H-6 according to sonication time of 110 g TSS/kg blackberry juice.


Quality parameter 0 4 8 12 21 32

L∗ 1.29 ± 0.08 ND3 ND3 1.0 ± 0.1 1.03 ± 0.07 0.9 ± 0.2C∗ 4.3 ± 0.1 ND3 ND3 3.6 ± 0.2 3.9 ± 0.1 3.39 ± 0.07H∗ 0.22 ± 0.03 ND3 ND3 0.34 ± 0.04 0.17 ± 0.03 0.22 ± 0.01�E∗ 0 ND3 ND3 1.0 ± 0.1 0.51 ± 0.07 0.8 ± 0.2Cyanidin 3-glucoside (mg Cy-glu1.100/mL) 110 ± 3 116 ± 1 116 ± 4 114 ± 4 114.3 ± 0.9 112 ± 3Cyanidin 3-(6’malonyl) glucoside (mg

Cy-glu1.100/mL)7.4 ± 0.3 8.0 ± 0.3 7.9 ± 0.3 7.8 ± 0.4 7.70 ± 0.03 7.6 ± 0.1

Lambertianin C (mg EA2.100/mL) 40.7 ± 0.4 44.9 ± 0.1 44 ± 1 42 ± 3 43.5 ± 0.1 42.2 ± 0.9Sanguiin H-6 (mg EA2.100/mL) 35.1 ± 0.5 39 ± 1 37.9 ± 0.9 37 ± 2 37.3 ± 0.3 37 ± 7H-ORAC (μmol TE4/mL) 48 ± 5 53 ± 2 53 ± 3 53 ± 3 47 ± 3 49 ± 3ERYCA (μmol QE5/mL) 10.2 ± 0.9 10.7 ± 0.7 11.1 ± 0.8 14.2 ± 0.4 14 ± 1 11 ± 11Expressed as cyaniding 3-glucoside equivalents.2Expressed as ellagic acid (EA) equivalents.3Not determined.4Expressed as trolox equivalents.5Expressed as quercetin equivalents.


M:Food

Microbiology

&Safety


panel of trained analysts was given instructions to consider mostlyflavor when performing the sensorial analysis. For sonicationtreatments of up to 4 min, no significant differences could be de-tected. However, significant differences (P ≤ 0.01) were detectedfor sonication treatments of 8 min or more. An off-flavor, oftendescribed as a “cooked flavor” by some panelists, was reportedfor these samples. Degradation of existing aromatic compoundsor/and production of neoformed aromatic compounds may haveoccurred during sonication, presenting low-sensory perceptionthreshold.

If limited by sensorial quality, sonication of less than 8 minoffers modest possibilities for reducing the presence of spoilagemicroorganisms (Figure 2 to 4). For blackberry juice, other waveamplitudes for sonication treatment as well as discontinuous ul-trasonic irradiation may be tested to enhance microbial reductionand, at the same time, address better sensorial quality.

ConclusionResults obtained show that nonthermal concentration of high-

acid blackberry juice allows a considerable reduction of microbialloads and, when combined with sonication, microbial reductionscan also be enhanced without significantly affecting main physico-chemical and functional quality parameters. Therefore, osmoson-ication can be considered as an alternative to thermal stabilizationprocesses for producing high-quality juice concentrates.

Additionally, even classical processes to obtain juice concentratecan benefit from these findings. Actually, concentration using vac-uum evaporators is generally performed at relatively low temper-ature (<70 ◦C) and for safety reasons, an additional pasteurizationstep is required before storage of concentrate at −20 ± 2 ◦C. Onthe basis of results presented in this article, the implementationwithin a classical concentration line of a sonication step to replacethe additional thermal pasteurization should also be considered byindustries to better preserve overall quality of concentrates.

AcknowledgmentsThe authors wish to thank UNESCO (grant nr. 833.701-3)

for their financial support. This research project was also partiallyfunded by PAVUC-FP6-INCO project DEV-2, contract 015279.Authors are also grateful to Esteban Gonzalez and Paula Valverdefor ORAC and ERYCA analyses.

ReferencesAcosta-Montoya O, Vaillant F, Cozzano S, Mertz C, Perez AM, Castro MV. 2010. Phenolic

content and antioxidant capacity of tropical highland blackberry (Rubus adenotrichus Schltdl.)during three edible maturity stages. Food Chem 119(4):1497–501.

AOAC. 1990. Fruits and fruit products. 2nd ed. Arlington, Va.: K. Helrich. 928 p.Barbe AM, Bartley JP, Jacobs AL, Johnson RA. 1998. Retention of volatile organic

flavour/fragrance components in the concentration of liquid foods by osmotic distillation.J Memb Sci 145:67–75.

Beuchat LR. 1996. Pathogenic microorganisms associated with fresh produce. J Food Prot59:204–16.

CDC. 1999. Centers for Disease Control and Prevention-Outbreak of Salmonella serotypeMuenchen infections associated with unpasteurized orange juice: United States and Canada.Morb Mortal Wkly Rep 48:582–5.

CDC. 2000. Centers for Disease Control and Prevention (CDC)- surveillance summaries. MorbMortal Wkly Rep 49:1–51.

Cisse M, Vaillant F, Perez AM, Dornier M, Reynes M. 2005. The quality of orange juiceprocessed by coupling crossflow microfiltration and osmotic evaporation. Int J Food Sci andTechnol 40:105–16.

Cisse M, Vaillant F, Acosta O, Dhuique-Mayer C, Dornier M. 2009. Thermal degradationkinetics of anthocyanins from blood orange, blackberry and roselle using Arrhenius, Eyringand Ball Models. Food Chem 57:6285–91.

FDA. 2001. Food and Drug Administration (FDA) Final Rule: HACCP procedures for the safeand sanitary processing of juice. Federal register 66(13):6138–202.

Gonnet JF. 1999. Colour effects of co-pigmentation of anthocyanins revisited-2. A colorimetriclook at the solutions of cyanin co-pigmented by rutin using the CIELAB scale. Food Chem66:387–94.

Gonzalez E, Vaillant F, Rojas G, Perez A. 2010. Novel semiautomated method for assessing invitro cellular antioxidant activity using the light-scattering properties of human erythrocytes.J Agr Food Chem 58(3):1455–61.

Gould GW. 2001. New processing technologies: an overview. Proc Nutr Soc 60:463–74.Hassimotto NMA, Pinto MD, Lajolo FM. 2008. Antioxidant status in humans after consumption

of blackberry (Rubus fruticosus L.) juices with and without defatted milk. J Agric Food Chem56(24):11727–33.

Jay JM, Loessner MJ, Golden DA. 2006. Modern food microbiology. New York: Springer.790 p.

Knorr D, Zenker M, Heinz V, Lee DU. 2004. Applications and ultrasonics in food potential ofprocessing. Trends Food Sci & Technol 15(5):261–6.

Krause G, Terzagian R, Hammond R. 2001. Outbreak of Salmonella serotype Anatum infectionassociated with unpasteurized orange juice. South Med J 94:1168–72.

Lee H, Zhou B, Feng H, Martin SE. 2009a. Effect of pH on inactivation of escherichia colik12 by sonication, manosonication, thermosonication, and manothermosonication. J FoodSci 74(4):191–8.

Lee H, Zhou B, Liang W, Feng H, Martin SE. 2009b. Inactivation of Escherichia coli cellswith sonication, manosonication, thermosonication, and manothermosonication: microbialresponses and kinetics modeling. J Food Eng 93(3):354–64.

Mertz C, Cheynier V, Gunata Z, Brat P. 2007. Analysis of phenolic compounds in two blackberryspecies (Rubus glaucus and Rubus adenotrichus) by high-performance liquid chromatographywith diode array detection and electrospray ion trap mass spectrometry. J Agric Food Chem55(21):8616–24.

Mertz C, Gancel AL, Gunata Z, Alter P, Mayer CD, Vaillant F, Perez AM, Ruales J, BratP. 2009. Phenolic compounds, carotenoids and antioxidant activity of three tropical frutis.J Food Compost Anal 22:381–7.

Mille Y, Beney L, Gervais P. 2002. Viability of Escherichia coli after combined osmotic andthermal treatment: a plasma membrane implication. Biochem Biophys Acta 1567:41–8.

Mille Y, Beney L, Gervais P. 2005. Compared tolerance to osmotic stress in various microor-ganisms: towards a survival prediction test. Biotechnol Bioeng 92:479–84.

Montgomery D. 2005. Design and analysis of experiments. 2nd ed. Boston: John Wiley & Sons.556 p.

Ou B, Hampsch-Woodill M, Prior R. 2001. Development and validation of an improved oxygenradical absorbance capacity assay using fluorescein as the fluorescent probe. J Agric Food Chem49:4619–26.

Pagan R, Manas P, Raso J, Condon S. 1999. Bacterial resistance to ultrasonic waves underpressure at nonlethal (manosonication) and lethal (manothermosonication) temperatures. ApplEnviron Microbiol 65(1):297–300.

Parish ME. 1997. Public health and nonpasteurized fruit juices. Crit Rev Microbiol 23:109–19.

Patil S, Bourke P, Kelly B, Frıas JM, Cullen PJ. 2009. The effects of acid adaptation on Escherichiacoli inactivation using power ultrasound. Innov Food Sci Emerg Technol 10(4):486–90.

Piyasena P, Mohareb E, McKellar RC. 2003. Inactivation of microbes using ultrasound: a review.Int J Food Microbiol 87:207–16.

Poirier I, Marechal PA, Evrard C, Gervais P. 1998. Escherichia coli and Lactobacillus plantarumresponses to osmotic stress. Appl Microbiol Biotechnol 50:704–9.

Tiwari BK, Muthukumarappan K, O’Donnel CP, Cullen PJ. 2008. Effects of sonication on thekinetics of orange juice quality parameters. J Agric food Chem 56:2423–8.

Tiwari BK, O’Donnell CP, Cullen PJ. 2009a. Effect of non thermal processing technologies onthe anthocyanin content of fruit juices. Trends in Food Science & Technology 20(3–4):137–45.

Tiwari BK, O’Donnell CP, Cullen PJ. 2009b. Effect of sonication on retention of anthocyaninsin blackberry juice. J Food Eng 93(2):166–71.

Tiwari BK, Patras A, Brunton N, Cullen PJ, O’Donnell CP. 2010. Effect of ultrasound processingon anthocyanins and color of red grape juice. Ultrason Sonochem 17(3):598–604.

Ulusoy BH, Colak H, Hampikyan H. 2007. The use of ultrasonic waves in food technology.Res J Agric Biol Sci 2(4):491–7.

Vaillant F, Jeanton E, Dornier M, Reynes GM, O’Brien M, Decloux M. 2001. Concentrationof passion fruit juice on an industrial pilot scale using osmotic evaporation. J Food Eng47:195–202.

Van Opstal I, Bagamboula CF, Theys T, Vanmuysen SCM, Michiels CW. 2006. Inactivation ofEscherichia coli and Shigella in acidic fruit and vegetables juices by peroxidase systems. J ApplMicrob 101:242–50.

Walkling-Ribeiro M, Noci F, Riener J, Cronin DA, Lyng JG, Morgan DJ. 2009. The Impact ofThermosonication and Pulsed Electric Fields on Staphylococcus aureus Inactivation and SelectedQuality Parameters in Orange Juice. Food Bioprocess Technol 2(4):422–30.

Wong E, Perez AM, Vaillant F. 2008. Combined effect of osmotic pressure and sonication onthe reduction of Salmonella spp. in concentrated orange juice. J Food Saf 28:499–513.

Wu J, Gamage TV, Vilkhu KS, Simons LK, Mawson R. 2008. Effect of thermosonication onquality improvement of tomato juice. Innov Food Sci Emerg Technol 9(2):186–95.


osmosonication of blackberry juice: impact on selected pathogens, spoilage microorganisms, and main...

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