531 Journal o f Food Protection, Vol. 78, No. 3, 2015, Pages 531-539 doi: 10.4315/0362-028X.JFP-14-290 Copyright © , International Association for Food Protection

Microbial Inactivation and Physicochemical Properties of Ultrasound Processed Pomegranate Juice giGDEM UYSAL PALA,' NUKHET NiLUFER DEMIREL ZORBA,1*

and

GULgiN OZCAN 2

1Department o f Food Engineering, Faculty o f Engineering, and departm ent o f Biology, Faculty o f Science and Literature, Qanakkale Onsekiz Mart University, Terzioglu, Qanakkale 17020, Turkey MS 14-290: Received 18 June 2014/Accepted 23 October 2014

ABSTRACT The effects of ultrasound treatment at various amplitudes (50, 75, and 100%) and times (0, 6, 12, 18, 24, and 30 min) on Escherichia coli ATCC 25922 (a surrogate for E. coli 0157:H7) and Saccharomyces cerevisiae ATCC 2366 levels and physicochemical characteristics (monomeric anthocyanins, color values, total phenolics, pH, and soluble solids) were determined in pomegranate juice. More than a 5-log inactivation of E. coli ATCC 25922 and a 1.36-log inactivation of 5. cerevisiae ATCC 2366 were achieved after 30 min of ultrasound tr eatment at 100% amplitude. The log-linear and Weibull models were successfully used to estimate the microbial inactivation as a function of ultrasound treatment time (R2 > 0.97). No significant changes were observed in total phenolics, pH, and soluble solids of the treated juice (P > 0.05). The ultrasound treatment for up to 30 min resulted in more than 92 and 89% anthocyanin retention at 75 and 100% amplitude, respectively. The redness (a*) of the juice did not change significantly after the ultrasound treatment at amplitudes of 75 and 100% for up to 24 and 12 min, respectively. No significant changes in L* and b* values were observed after ultrasound treatment at all amplitudes and after up to 30 min of treatment for 50 and 75% amplitudes. Small differences in juice color were noted based on total color difference scores.

Thermal processing is the most commonly used preservation technique for inactivating microorganisms and enzymes in fruit juices (3, 33, 64). However, the process may have undesirable effects on sensory attributes (color, aroma, and flavor) and bioactive compounds (ascorbic acid, phenolics, and pigments) of juices (43, 46). Legal requirements for food processing before consumption and increased consumer demand for nutritious foods have resulted in increased use of nonthermal techniques for natural fruit juice processing (3, 4, 39). Investigations into nonthermal techniques have been conducted to determine ways to minimize the changes in nutritional and sensory properties of foods. Ultrasound (US) processing is one of the nonthermal techniques used for preserving heat-sensitive food products such as fruit juice (20). US is defined as sound waves with frequencies higher than those that can be heard by the human ear (>20 kHz) (8). US induces cavitation by forming microscopic gas bubbles in liquid. When these bubbles burst, they create violent shock waves and generate free radicals through the cellular membrane, resulting in microbial inactivation (35, 37, 53, 58). Pomegranate (Punica granatum L.) is a perennial plant in the Punicaceae family (50) that is widely grown in the aegean and southeastern regions of Turkey and other Mediterranean countries (41). Pomegranate fruit and pomegranate juice (PJ) have received wide public attention * Author for correspondence. Tel: + 9 0 (286) 218 0018, Ext 2260; Fax: + 9 0 (286) 218 0541; E-mail: [email protected].

because of their high concentrations of phenolics (ellagic catechins, tannins, and anthocyanins) and the particularly high activity of their antioxidants (7, 26, 60). The deep red color of PJ is correlated with its anthocyanin concentration, which is one of the most important parameters affecting the sensory aspects of consumer acceptance (60). The compo­ nents of the natural microbial flora on pomegranates differ with harvest region, harvest time, and the type of pomegranate fruit. Lopez-Rubira et al. (35) reported that main microbial components on pomegranate arils are mesophilic bacteria (3.28 to 3.86 log CFU/g), psychrotrophic bacteria (3.96 to 4.55 log CFU/g), Enterobacteriaceae (2.2 to 3.2 log CFU/g), lactic acid bacteria (3.2 to 4 log CFU/g), and yeasts and molds (2 to 4 log CFU/g). Other have reported the presence of hepatitis A virus on pomegranate arils (12). Uysal Pala and Kirca Toklucu (63) reported an aerobic plate count of 3.37 log CFU/ml and a yeast and mold count of 1.95 log CFU/ml in PJ after clarification. During processing of fruit juice, the low pH of the juices may increase the acid resistance and survival of pathogen contaminants. Researchers have found that acidadapted Escherichia coli 0157:H7 cells survived much better in apple juice than did nonadapted cells and were more heat tolerant (48, 60). Oyarzabal et al. (40) found that E. coli 0157:H7 could survive in various juice concentrates for up to 12 weeks. In fruit juices, yeasts also can cause product spoilage, with resulting economical loss (47). Saccharomyces cerevisiae is a spoilage microorganism of

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fruit-based products, can grow under a variety of conditions (61), and is one of yeast species most frequently isolated from fruit juices and their concentrates (16, 47). US treatment is a promising food processing method for enhancing the microbial safety and physicochemical qualities of fruit juice. The inactivation efficacy of US treatment depends on the type of microorganisms, time of exposure, power level, amount and composition of food, and treatment temperature (45). Studies on the use of a sonication process to inactivate microorganisms and en­ zymes and determine changes in some physicochemical attributes have been conducted with orange juice (55), apple juice (2 ), red grape juice (38 , 59), blackberry juice (56), cantaloupe melon juice (20), pineapple juice (15), grapefruit juice (1), tomato juice (3 , 4 ), cactus pear juice (70), and PJ (5 , 6 , 17). The resistance of microorganisms to inactivation can increase after they have been exposed to certain stresses (66 , 67). The objectives of this study were to determine the efficacy of a US system for inactivation of acid-adapted E. coli ATCC 25922 (a surrogate for E. coli 0157:H7) and S. cerevisiae ATCC 2366 in PJ and to use a model to evaluate the changes in some important quality parameters of PJ, such as anthocyanin concentration, color, total phenolics, and other physicochemical qualities, as a function of treatment time and amplitude. MATERIALS AND METHODS PJ preparation. Pomegranates (P . granatum cultivar Hicaz) were obtained from Antalya, Turkey, and processed to clarified juice according to Uysal Pala and Krrca Toklucu (63). Initial total aerobic mesophilic bacteria and yeast and mold counts of PJ were determined by plating on tryptic soy agar (TSA; Merck, Darmstadt, Germany) with incubation at 37°C for 24 h and on dichloran rosebengal chloramphenicol agar (Merck) with incubation at 25 °C for 3 to 5 days, respectively. Microbial strains and culture conditions. E. coli ATCC 25922 (Microbiologies, Grenoble, France), a nonpathogenic test strain, was used as a surrogate for E. coli 0157:H7 for the inoculation study. A loopful of E. coli ATCC 25922 stock culture was transferred into 10 ml of tryptic soy broth (TSB; Merck) and incubated at 37°C for 18 h. S. cerevisiae ATCC 2366 (Dr. H. D i^ e r Baysal, izmir Institute of High Technology, Urla/izmir, Turkey) was cultivated in 10 ml of Sabouraud dextrose broth (SDB; Difco, BD, Sparks, MD) and incubated at 30°C for 48 h. Acid exposure of cultures. The E. coli and S. cerevisiae cultures were adapted gradually to pH 3.3 to 3.5 with malic acid and citric acid (Sigma Aldrich, Munich, Germany), respectively according to Koutchma et al. (32) with some modifications. Working cultures were grown overnight in TSB and SDB at 37 and 30°C, respectively. Cells were then harvested by centrifuga­ tion at 5,000 x g for 10 min at 4°C. The cell pellets were washed twice with sterile physiological saline, resuspended in 10 ml of TSB and SDB acidified to pH 5.0 with malic or citric acid depending on the organism, and incubated at 37 and 30°C, respectively. The pH of the medium was gradually decreased further to 3.3 to 3.5 with malic or citric acid. Acid-adapted cultures were inoculated into 50 ml of TSB or SDB that contained 20% PJ and incubated at 37 and 30°C for E. coli and S. cerevisiae, respectively, for 18 h (42).

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Inoculation of PJ samples and enumeration of microor­ ganisms. An aliquot (10 ml) of each grown broth culture was centrifuged at 5,000 x g for 10 min at 4°C to obtain a level of about 108 to 109 CFU/ml in peptone water, and 1 ml of this dilution was added directly to PJ to achieve a final level of about 106 to 107 CFU/ml in PJ. E. coli ATCC 25922 was enumerated on TSA using appropriate dilutions with 0.1% peptone water after incubation at 37°C for 18 h, and S. cerevisiae was enumerated on SDA after incubation at 30°C for 48 h. US treatment. A US generator (500 W, 20 kHz; Vibra-Cell 505, Sonics & Materials, Inc., Newtown, CT) equipped with a 19mm-diameter US probe attached to a transducer was used for US processing of PJ (9). The US probe was immersed in 100 ml of PJ sample to a depth of 25 mm. The amplitude of the US probe was set to 50, 75, or 100% to control the energy input during the experiment. Pulse intervals of 5 s on and 5 s off were applied for up to 30 min. Samples were collected every 6 min. PJ temperature during sonication was kept under 35°C by using a water bath at 15°C and fluctuated between 14.3 and 30.8°C depending on the US amplitude. Different trials were used for inoculation studies and for determination of physicochemical changes. The sonication experiments were done in duplicate, and parallel samples were analyzed from each experiment (n = 2 x 2). Color determination. Color values of the sonicated PJ samples were measured with a colorimeter (model CR-400, Konica Minolta Sensing, Osaka, Japan) based on three color coordinates; L* (lightness), a* (redness to greenness), and b* (yellowness to blueness). Total color difference (TCD) was calculated using equation 1 (58): TCD = (L —Lo)2 + (a —ao)2 + (b —bo)2

(1)

where L0, a0, and b0 are the color values of untreated PJ. Total monomeric anthocyanin concentration. The concen­ tration of monomeric anthocyanin in PJ samples was determined using the pH differential method (63). Results were expressed as milligrams of cyanidin-3-glucoside per liter of PJ. Anthocyanin retention (%) was calculated using equation 2: Anthocyanin retention (%) = (ACNfresh ACNsonicatedjuice/ACNfresh) X 100

( 2)

Total phenolics concentration. The Folin-Ciocalteu method was used for analysis of total phenolics in PJ samples (63). Results were expressed as milligrams of gallic acid per liter of PJ. Total soluble solids and pH. Total soluble solids (°Brix) and pH of PJ samples were measured at 20°C with a pocket refractometer (PAL-1, Atago, Tokyo, Japan) and a pH meter (MP 225, Mettler Toledo, GiePen, Germany), respectively. Kinetic modeling. Microbial inactivation data were evaluated with both a log linear (first order) inactivation kinetics model (equation 3) and a Weibull model (64) (equation 4) using the GInaFIT tool (24): log(iV) = log(/V0) - fr/ln ( 10)

(3)

log(/V) = log(/V0) - ( / / 8 ) p

(4)

where N and N0 are the count of the microorganisms (CFU per milliliter) at times t and t0, respectively; k is the first-order inactivation constant (1/min); 8 is a scale parameter that indicates time for the first decimal reduction; and p is a shape parameter that

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E. c o li ATCC 25922

FIGURE 2. Inactivation of S. cerevisiae in pomegranate juice samples sonicated at different amplitudes: 50% (A), 75% (■ ), 100% (♦).

FIGURE 1. Inactivation of E. coli ATCC 25922 in pomegranate juice samples sonicated at different amplitudes: 50% (A), 75% ( ■ ) , 100% ( ♦ ) .

describes the shape of the curve as concave downward (p > 1) or upward (p < 1). Weibull model parameters (8 and p) also were used to calculate the time required for a 5-log reduction (t5D) as a scientific criterion for pasteurization of juices by a nonthermal technology (39) according to equation 5 as suggested by Patil et al. (42): t5o = § x (5 )1/p

(5)

Degradation of the monomeric anthocyanins during the sonication process was also evaluated by the first-order kinetics model C = Coexp( —kt)

(6)

where C and C0 are the anthocyanin concentrations (milligrams per liter) at reaction times t and t0, respectively, and k is the reaction rate constant (1/min). Statistical analysis. SAS version 8.2 (49) was used for the statistical analyses. The MIXED procedure was utilized with the repeated statement. A variance-covariance model was used to account for the correlations of all the observations arising from PJ samples (19, 31). The Tukey method was used to adjust the differences (62). RESULTS AND DISCUSSION Microbial inactivation in sonicated PJ. The initial microbial load in PJ was about 2 log CFU/ml in all trials (means: 2 log CFU/ml total aerobic mesophilic bacteria, 1.36 log CFU/ml yeasts and molds). Sterilization for the inoculation study was not considered necessary. The microbial flora of pomegranates is well-adapted to low pH, and several researchers have found that virulence and resistance of microorganisms increased with exposure to various stresses (66, 67); therefore, acid adaption was invoked in E. coli and S. cerevisiae to mimic the situation in PJ. Inactivation curves of acid-adapted E. coli ATCC 25922 and S. cerevisiae ATCC 2366 in PJ samples by US amplitude (50, 75, and 100%) and treatment time (0, 6, 12, 18, 24, and 30 min) are shown in Figures 1 and 2. Both microorganisms in the sonicated PJ samples decreased with increasing amplitudes and treatment times. E. coli counts in

the sonicated PJ significantly decreased after 12 and 18 min of treatment at 50, 75, and 100% amplitude (P < 0.05). After 30 min of US treatment at amplitudes of 50, 75, and 100%, E. coli counts decreased by 3.44 log CFU/ml (49.05% of initial level), 4.54 log CFU/ml (64.80% of initial level), and 6.64 log CFU/ml (95.66% of initial level), respectively. However, decreases in S. cerevisiae after US treatment at 50 and 75% amplitude were nonsignificant (P > 0.05) at all treatment times. US treatment at 100% amplitude for 24 and 30 min had a significant effect on inactivation of S. cerevisiae (P = 0.005 and 0.0004, respectively) and resulted in decreases of 1.08 log CFU/ml (18.99% of initial level) and 1.36 log CFU/ml (24% of initial level), respectively. These decreases were consider­ ably lower than those of E. coli in sonicated PJ. These results indicate that microorganism type is an important factor affecting inactivation by US treatment. Few studies have been conducted on spoilage by yeasts and the use of sonication in food products. Most of these studies involve inactivation by sonication in model systems or in microbi­ ological media, and results have indicated that mild heat treatment is needed for adequate inactivation of S. cerevisiae (11, 13, 28, 29, 34, 68). The physical disruption of the microbial cell and its inactivation as the result of US processing is mainly due to cavitation caused by ultrasonic waves, localized heating, and formation of free radicals (“ OH, + H, and H20 2) (3, 21, 36,53). The yeast cells in the present study (S . cerevisiae ATCC 2366) were clearly more resistant to inactivation by US processing than were the bacterial cells (E . coli ATCC 25922). Patil et al. (42) also reported that bacterial cells differ in their responses to US processing based on strain type, growth medium, and pH adaptation time. Wordon et al. (68) concluded that cavitation causes immediate death of S. cerevisiae cells by membrane injury; however, not all injured cells died, and some injured cells recovered. In addition, US treatment caused unidentified internal injuries that made the cells heat sensitive. The first-order kinetics and Weibull (a and (3) parameters were successfully described to estimate micro­ bial inactivation as a function of sonication time (Tables 1 and 2). The inactivation of E coli ATCC 25922 and S. cerevisiae ATCC 2366 was adequately described by the

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TABLE 1. Parameters o f first-order kinetics model for sonicated pomegranate juice by amplitude and microorganism Microorganism

Amplitude (%)

E. coli ATCC 25922

50 75 100 50 75 100

S. cerevisiae

k (mean + SE) (per min)“ 0.24 0.34 0.48 0.03 0.05 0.11

+ ± + + + +

0.04 0.02 0.02 0.002 0.01 0.01

R2h

RSME‘

0.977 0.992 0.974 0.979 0.790 0.993

0.206 0.168 0.425 0.022 0.141 0.051

a First-order inactivation constant. h Coefficient of determination, which equals 1 — SSE/SSTO where SSE is the sum of squared errors obtained by summing the squared differences between the experimental data and the identified model and SSTO is the sum of the squared differences between the measured values and the mean of these measured values. c Root mean sum of squared errors (the square root of MSE), which can be derived by dividing SSE by the number of degrees of freedom n — k, i.e., the number of data points n minus the number of degrees of freedom k (parameters and initial values).

first-order kinetics and Weibull models with regression coefficients (R2) of 0.97 and higher except for inoculated PJ processed at 75% amplitude, for which the R2 values were 0.790 and 0.824, respectively. Patil et al. (42) reported that zero-order kinetics models were a good fit for US-induced E. coli (ATCC 25922 and NCTC 12900) inactivation results. Adekunte et al. (J) also found that yeast inactivation in tomato juice followed the Weibull distribution with a high regression coefficient (R2 > 0.98). The first-order kinetics parameters indicated that inactivation rate constants of E. coli and S. cerevisiae linearly increased as a function of amplitude (R2 = 0.981 and 0.923, respectively). However, the inactivation rate constants for E. coli were higher than those for S. cerevisiae at all amplitudes. In Table 2, the a value (a Weibull parameter) refers to the first decimal reduction time during US processing, expressed in minutes. These values decreased with increasing amplitude for both microorganisms. The a values for E. coli and S. cerevisiae were 2.60 and 21.55 min, respectively, during US processing at 100% amplitude. According to the shape parameter (|3) of the Weibull model, survival curves of E. coli at all amplitudes indicated upward concavity of the curve (P < 1), which indicated adaptation of the remaining cells to the US process (64). The times needed to achieve a 5-log inactivation (t5D) of E. coli ATCC 25922 and S. cerevisiae ATCC 2366 in PJ at all amplitudes are shown in Table 2. The t5D values of both microorganisms consider­ ably decreased with increasing amplitudes. Similarly, Patil et al. (42) reported that 5-log inactivation times (t5D) of acid-

adapted E. coli ATCC 25922 and E. coli 0157:H7 NCTC 12900 decreased with increasing amplitude (0.4, 7.5, and 37.5 pm). Research has revealed that microorganisms that survive a given stress, such as high acid, often gain resistance to that stress and others through cross-protection (67). Patil et al. (42) found that acid-adapted E. coli had higher resistance to US processing. Several researchers concluded that acid adaptation changes the protein expres­ sion profiles and membrane lipid composition (14, 30, 6669). Ryu and Beuchat (48) reported increased heat resistance in acid-adapted E. coli 0157:H7 in heat-treated fruit juices. Shearer et al. (51) reported that the heat resistance of S. cerevisiae obtained from spoiled acidic products was higher in fruit juices than in citrate buffer. Increased resistance to organic acids, which are used as preservatives, after weak acid adaptation was reported in S. cerevisiae (44, 65). This resistance is due to several factors, including intracellular pH arrangement and reconfiguration of the cell membrane (44, 52, 65, 66). Chu-Ky et al. (14) reported higher viability in the response of acid-adapted S. cerevisiae to freeze drying. In a study of US processing in PJ, the Z)-values of nonadapted S. cerevisiae cells were between 8.33 + 0.21 and 16.94 + 0.18 min (6). Similar results were reported by Demirel Zorba and Ergen (17). Giannattasio et al. (25) reported that acid stress adaptation causes increased intracellular catalase and superoxide dismutase activity in S. cerevisiae in media with high concentrations of acetic acid; however, no catalase activity was found in nonadapted cells. Thus, acid-adapted S.

TABLE 2. Weibull model parameters and time required to obtain a 5-log reduction in microorganisms in sonicated pomegranate juice Microorganism

Amplitude (%)

E. coli ATCC 25922

50 75 100 50 75 100

S. cerevisiae

a (mean + SE) (min)“

8.44 4.91 2.60 92.23 55.28 21.55

± ± ± ± ± +

2.40 0.49 0.96 20.30 30.83 1.35

P (mean ± SE)*

0.92 0.83 0.76 0.93 0.61 1.06

+ + + + + +

0.18 0.04 0.11 0.18 0.37 0.12

R2

RMSE

t5D (min)c

0.978 0.999 0.988 0.980 0.824 0.993

0.233 0.083 0.332 0.025 0.15 0.056

48.54 34.14 21.61 520.54 773.43 98.37

a a, (8) [time unit] is a scale parameter and can be denoted as the time for the first decimal reduction if p = 1. h P> p H is a shape parameter. For P (p) > 1, convex curves are obtained, and for [1 (p) < 1, concave curves are obtained. c Time required for a 5-log reduction.

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cerevisiae cells may recover from the effects of free radicals created by the ultrasonic waves and catalase activity, and higher D-values were obtained. The two test microorganisms also responded differently to the US processing. The t5D values and kinetic parameters clearly indicated that the acid-adapted E. coli ATCC 25922 cells were more susceptible to US processing than were the acid-adapted S. cerevisiae cells. Similar results were obtained by Char et al. (13), who reported that the inactivation curves of single strains or E. coli, S. cerevisiae, and multistrain yeast cocktails in orange juice were best described by a first-order kinetics model. The strain cocktails had survival curves similar to those of the individual strains for both sonicated orange and apple juices, and the authors concluded that the inactivation curves were dependent on the type of microorganism and treatment. E. coli and its cocktail were more sensitive to these treatments than were S. cerevisiae or the yeast cocktail. The higher resistance of S. cerevisiae to US treatment is mainly due to the difference in cell wall characteristics. In gram-negative bacteria, such as E. coli, the cell walls are thin and mainly composed of a 2- to 7-nm peptidoglycan layer and a 7- to 8-nm outer membrane (22, 23). The cell wall of S. cerevisiae mainly consists of mannoproteins and p-1,6 glucans with a thickness of 90 to 154 nm (18). Therefore, based on the thickness of the cell wall, S. cerevisiae is expected to be more resistant to US treatment than is E. coli. In addition to microorganism type, frequency settings and power of the equipment, type of medium, and growth conditions also affected the results of the US treatment. Although the equipment used in the present study was similar to that used by Char et al. (13), higher inactivation rates were obtained for E. coli at amplitudes different than those used by Char et al. We obtained higher D-values than those reported by Patil et al. (42), who used US at lower amplitudes. These differences may be associated with the differences in the power of the equipment used, the types of microorganisms, the acid adaption conditions, and the media. Previous reports of the effect of pH on the efficacy of US treatment are contradictory; some authors found that acidic pH has a positive effect on inactivation, and others found no influence of pH. In our study, the average pH of untreated PJ was 3.20, and acid-adapted S. cerevisiae easily grew at that pH. During the US treatment, the temperature of the PJ did not exceed 35°C. Guerrero et al. (28) reported that D35°c-values were 19 to 31 min, with different amplitudes (60 to 90%) and system pH values. However, in our study we obtained similar D-values (19.67 min) only at the highest amplitude (100%). Use of other amplitudes resulted in much longer D3o"crvalues (>100 min). The effect of US processing on yeasts was affected by inoculum preparation and the yeast strain assayed. In addition, acid adaptation could increase the D-values. Gomez-Diaz et al. (27) studied the response of Zygosaccharomyces bailii to single and combined effects of US and UV-C light in apple juice and used a Weibull model to describe the inactivation kinetics. The processing condition, maximum amplitude, and maximum temperature were 20 kHz, 120 pm, and 35°C,

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535

respectively. They obtained 2-log reductions after 40 min of treatment. Similar reductions were obtained in our study for S. cerevisiae ATCC 2366 with US treatment at 20 kHz and 30°C after 30 min. Bermudez-Aguirre and Barbosa-Canovas (11) used pulsed and continuous thermosonication treat­ ments to study the inactivation kinetics of S. cerevisiae in three fruit juices. These authors concluded that low temperatures did not affect yeast inactivation, but pulsed treatment delayed yeast inactivation. In our study, we applied pulsed treatment to minimize changes in the physicochemical properties of PJ, and a delaying effect was observed similar to that reported by Bermudez-Aguirre and Barbosa-Canovas. These researchers also obtained the highest inactivation rate in grape juice followed by pineapple and cranberry juices and concluded that the composition of each juice provided some protective effect on the cells. Because PJ has a high level of phenolic compounds similar to cranberry juice, the same protective effect may have occurred during our treatments. Adekunte et al. (4) also studied the effects of US treatment (20 kHz, 61 pm, 45°C, and pulsed mode) on inactivation of Pichia fermentans in tomato juice and found approximately 7-log reductions within 10 min. In another study in which a continuous mode with a higher amplitude (20 kHz, 95.2 pm, and 45°C) was used, lower inactivation rates (3-log reductions) for S. cerevisiae were obtained after 30 min of treatment in Sabouraud broth (pH 5.6) (29). In our study, 1.36-log reductions were obtained at 30°C after 30 min of treatment. These and previous findings indicate that lower inactivation rates are associated with the protective effect of the components of PJ, yeast strain, and treatment conditions (e.g., temperature and acid adaptation). Changes in anthocyanins and color parameters of sonicated PJ. The color of juice is one of the most significant factors affecting sensorial acceptance by con­ sumers (10). The bright deep red color of PJ is related to the anthocyanin concentration, as indicated by its high a* value. The effects of US amplitude and treatment time on the anthocyanin concentration of PJ and anthocyanin retention during sonication are shown in Table 3. Monomeric anthocyanin concentration in PJ was stable during sonica­ tion at 50% amplitude for up to 30 min and at 75 and 100% amplitudes for up to 24 and 18 min, respectively, resulting in nonsignificant decreases (P > 0.05). However, signifi­ cant decreases in monomeric anthocyanin concentrations (P < 0.05) were observed at higher amplitudes (75 and 100%) and treatment times (>18 min). The retention of monomeric anthocyanin after 12 min of US processing was 98.5% (275.54 to 271.36 mg/liter), 97.3% (270.11 to 262.80 mg/ liter), and 95.9% (283.46 to 271.77 mg/liter) at 50, 75, and 100% amplitudes, respectively. These results agree with those of Tiwari et al. (56), who reported a significant retention (95%) of anthocyanins in blackberry juice sonicated at 100% amplitude for 10 min. Alighourchi et al. (6) found about 91 and 95% retention of total monomeric anthocyanins in PJ from Alak Saveh arils and whole Alak Saveh pomegranate, respectively, sonicated at 100% amplitude for 9 min. In the present study, the retentions at

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TABLE 3. Changes in monomeric anthocyanin concentration, color values (L*, a*, and b*J, and total color difference (TCD) o f pomegranate juice during sonication a Treatment time (min) 0 6 12

18 24 30 0 6 12 18 24 30 0 6 12

18 24 30 ±SE

Anthocyanin concn (mg/liter)

Amplitude (%) 50 50 50 50 50 50 75 75 75 75 75 75 100 100 100 100 100 100

275.54 273.45 271.36 267.39 264.88 260.92 270.11 265.93 262.80 257.79 253.20 248.39 283.46 276.37 271.77 265.52 259.46 252.78 3.50

Anthocyanin retention (C/Co) 1 0.992 0.985 0.970 0.961 0.947 1 0.985 0.973 0.954 0.937 0.920 1 0.975 0.959 0.937 0.915 0.892

a a a a a a a ac ac ac ac bc a a ac ac bc bc

L* 10.29

a* a

1 0 .2 1 a

10.31 a 10.15 a 10.16 a 10.09 a 9.84 a 9.87 a 9.80 a 9.79 a 9.96 a 9.73 a 10.08 a 10.11 a 9.92 a 9.72 a 9.66 a 9.61 a 0.14

29.21 28.99 28.71 28.55 28.39 28.25 28.24 27.99 27.39 27.13 26.82 26.25 28.87 28.20 27.04 26.11 25.20 24.63 0.33

-b* a a a a a a a ac ac ac ac bc a a ac bc bc b

4.41 4.68 4.62 4.78 4.85 4.84 5.03 5.23 5.29 5.32 5.31 5.58 4.88 5.18 5.53 5.86 6.09 6.16 0.24

TCD a a a a a a

0.36 0.55 0.77 0.94 1.07

a a a a b

a a a a a a

0.34 a 0.90 b 1 .1 6 b 1.43 b 2.07 c

a a a a a a

0.75 a 1.95 b 2.95 c 3.88 d 4.45 d 0.11

“ Within each column, values with different letters are significantly different (P < 0.05).

the highest processing time (30 min) were 94.7% (275.54 to 260.92 mg/liter), 92% (270.11 to 248.39 mg/liter), and 89.2% (283.46 to 252.78 mg/liter) at 50, 75, and 100% amplitudes, respectively. Anthocyanin degradation under more severe sonication treatment conditions is possibly because of cavitational collapse of microscopic bubbles and production of oxidation products such as free radicals (H20 2 or hydroxyl radicals) (53, 56-58). Anthocyanin degradation during sonication of PJ followed the first-order kinetics model with high determi­ nation coefficients (>0.98) (Table 4). Similarly, Tiwari et al. (58) found that sonodegradation of strawberry anthocy­ anins followed the first-order kinetics model. Rate constants (~ k x 10 3 per min) linearly increased as a function of amplitude during sonication. These results indicate that amplitude is one of the important factors effecting the degradation rate of anthocyanin during sonication. Changes in color values (L*, a*, and b*) and TCD are shown in Table 3. Insignificant decreases in lightness (L*) and increases in blueness (-/?*) values of PJ treated by US at all amplitudes for up to 30 min were observed. Trends for redness (a*) of sonicated PJ were similar to those for anthocyanin concentration. Significant reductions in a* values were observed after 30 and 18 min of sonication at 75 and 100% amplitudes, respectively. TCD scores indicate TABLE 4. First-order kinetics parameters fo r degradation o f anthocyanins in sonicated pomegranate juice Amplitude (%)

—k x 10 3 (per min)0

R2

50 75 100

1.84 2.76 3.69

0.987 0.996 0.998

a First-order inactivation constant.

total differences in visual color of a juice and can be classified as slightly noticeable (< 1.5), noticeable (1.5 to 3), and visible (>3) (59). According to TCD scores, the 50 and 75% amplitude treatments caused small differences in PJ color. TCD scores of PJ sonicated at 50% amplitude for up to 30 min and at 75% amplitude for up to 24 min were slightly noticeable (3) range. Approximately a 5-log reduction in E. coli ATCC 25922 and 91.5% retention of monomeric anthocyanin also occurred at that processing time and amplitude level. Uysal Pala and Kirca Toklucu (63) reported 3.89% loss (96.11% retention) in monomeric anthocyanins and a 6.15-log reduction of E. coli ATCC 25922 in PJ after UV-C treatment at 37.41 J/ml. Changes in phenolics of sonicated PJ. Phenolics as secondary metabolites of plants are essential bioactive compounds and are responsible for color, flavor, and antioxidant effects of the fruits and their juices (13, 19, 30). PJ extracted from the Hicaz variety is rich in phenolic compounds. In the present study, the mean ( + SE) value of phenolics determined before treatments was 1,482.4 + 40.8 mg/liter, comparable to the 1,742 + 22.2 mg/liter reported by Uysal Pala and Toklucu (63). Amplitudes (50, 75, and 100%), processing times (6 to 30 min), and theninteractions did not have significant effects (P > 0.05) on the concentrations of phenolics (1,471.4 ± 40.8 to 1468.4 ± 40.8 mg gallic acid per liter of PJ at 50% amplitude, 1,484.4 + 40.8 to 1,482.4 + 40.8 mg gallic acid per liter at 75% amplitude, and 1,491.4 ± 40.8 to 1,490.4 + 40.8 mg gallic acid per liter at 100% amplitude for 30 min).

J. Food Prot., Vol. 78, No. 3

Similarly, Fonteles et al. (20) and Costa et al. (15) reported no significant effects of sonication times (2 to 10 min) and amplitude (20, 60, and 100%) on phenolics of cantaloupe melon juice and pineapple juice, respectively. However, significant increases in phenolics of apple juice after sonication at 25 kHz and 20°C for 30, 60, and 90 min were reported by Abid et al. (2). Zafra-Rojas et al. (70) also found significant increases in phenolic compounds of cactus pear juice treated with US at 20 kHz and 80% amplitude for 3, 5, and 8 min, but the phenolic concentrations of juice sonicated for 10 and 15 min were not significantly different from those of the control. These authors suggested that these increases were the result of the release of phenolics from plant cells present in the juices due to bubble cavitation during the sonication process. Release of more phenolics during sonication might be occurring in pulpy juices compared with clear- juices. However, Demirel Zorba and Ergen (17) and Alighourchi et al. (6) also reported increases in both total anthocyanins and total phenolics in sonicated PJ, depending on treatment time. Soluble solids and pH of sonicated PJ. The pH of the PJ samples (3.19 for 50% amplitude and 3.21 for 75 and 100% amplitudes) was unchanged after sonication. No significant changes were observed in the soluble solids of PJ at 50% (17.65 to 17.57 °Brix) and 100% (17.40 to 17.49 °Brix) amplitudes (P > 0.05). Similar results were reported for red grape juice by Tiwari et al. (59), blackberry juice by Tiwari et al. (56), orange juice by Tiwari et al. (54), apple juice by Abid et al. (2), and grapefruit juice by Aadil et al.

PROPERTIES OF SONICATED POMEGRANATE JUICE

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( 1).

In this study, US processing achieved more than a 5-log reduction in E. coli ATCC 25922 as a surrogate for E. coli 0157:H7 inoculated into PJ, which meets the U.S. Food and Drug Administration guidelines regarding pathogen reduc­ tion in fruit juices. The effect of US treatment on S. cerevisiae inoculated into PJ was more limited, resulting in only a 1.36-log reduction under the maximum processing conditions, which is why additional treatments such as mild heat, pressure, or a natural antimicrobial aie required to control yeast in sonicated PJ. However, the color of the PJ was significantly affected at higher US amplitudes and treatment times, and visible color differences occurred under the maximum processing conditions. However, total phenolics did not change significantly. Overall, the results of this study indicate that US technology has the potential to improve the safety and quality of PJ depending on the power levels and treatment times used.

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ACKNOWLEDGMENTS The authors thank Associate Doctor Onder Ayyildiz and Research Assistant Burcu Deri for their technical assistance with the US system. Prof. Dr. Cengiz Caner and Prof. Dr. Akm Pala are also acknowledged for language editing.

1.

Aadil, R. M„ X.-A. Zeng, Z. Han, and D.-W. Sun. 2013. Effects of ultrasound treatments on quality of grapefruit juice. Food Chem. 141: 3201-3206.

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