INFLUENCE OF THERMO-SONICATION ON … Vol 4/01. FQS/05. Full paper - Zoran...Strawberries (Fragaria × ananassa Duch. cv. Alba) have high levels of micronutrients and phytochemical

Journal of Hygienic Engineering and Design

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Original scientific paperUDC 663.813:634.75]:579.67

INFLUENCE OF THERMO-SONICATION ON MICROBIOLOGICAL SAFETY, COLOR AND ANTHOCYANINS CONTENT OF STRAWBERRY JUICE

Zoran Herceg1*, Vesna Lelas1, Anet Režek Jambrak1, Tomislava Vukušić1, Branka Levaj1

1Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia

*e-mail: [email protected]

Abstract

Ultrasonication is an alternative method of food preser-vation that has the advantage of inactivating microor-ganisms in food without causing the common side-ef-fects associated with conventional heat treatments, such as nutrient, color and flavor loss. In this work high intensity ultrasound was used to investigate inactiva-tion microorganisms, preservation the anthocyanins content and consequently color stability of strawberry juices.

Juice samples, cloudy (sample A) and clear (B) juices, were treated with ultrasonic probe of 12 mm in di-ameter and with 20 kHz frequencies. For ultrasounds treatment, three parameters (amplitude, treatment time and temperature of sample) varied according to the statistical experimental design. Centre composite design was used to optimize and design experimental parameters: temperature (25, 40 and 55 0C), amplitude (60, 90 and 120 µm) and time (3, 6, and 9 minutes). Thermal pasteurization (85 0C/2 min) reduces total anthocyanin content for 7.1 to 7.4% but inactivate all microorganisms compared to untreated juices. After treatment with ultrasound (20 0C for 3, 6 or 9 min.) or thermo-sonication (40 0C for 3, 6 or 9 min. and 55 0C for 3 or 6 min.), the degradation of anthocyanins was gen-erally less intensive and was 0.6 to 6.4% and inactivate from 58 to 94% of microorganisms compared to the untreated juices. Only in the case of ultrasonic treat-ment at a temperature of 55 0C and treatment time of 9 min. the total content of anthocyanins, compared to untreated juice, was reduced by 8.5 to 9.0%, and their degradation was greater than that of pasteurized juices but inactivate all microorganisms.

From the results it can be concluded that ultrasound treatment can replace pasteurization in terms of pre-serving total anthocyanin content, color stability and inactivation of microorganisms.

Key words: High power ultrasound, Anthocyanins, Microor-ganisms, Strawberry juice, Response surface methodology.

1. Introduction

Strawberries (Fragaria × ananassa Duch. cv. Alba) have high levels of micronutrients and phytochemical com-pounds, particularly anthocyanins, and phenolic com-pounds. The levels of these phytochemicals strongly influence the sensorial-organoleptic attributes and nutritional value. Pelargonidin-3-glucoside (P3G) is the major anthocyanin found in strawberries and is respon-sible for the attractive, bright red color of fresh straw-berries [20 and 21]. Juices prepared from strawberries contain a relatively high content of anthocyanins. Strawberry juice color is a key factor influencing con-sumer’s perception of quality. The anthocyanin content of fruits is greatly influenced by various genetic (culti-var), environmental and agronomic factors. Anthocya-nins are highly unstable and very susceptible to degra-dation. Their stability is greatly affected by processing conditions including pH, temperature, light, oxygen, solvents, and the presence of enzymes, flavonoids, pro-teins and metal ions. Ultrasound processing of juices is reported to have a minimal effect on the degradation of key quality parameters such as color and total an-thocyanin content in strawberry and blackberry juices. Sonication is also reported to enhance cloud stability of juice. Applications of ultrasound in food processing have been reviewed by Knorr et al. [6] and the effects of sonication on fruit juices [2 and 20]. The effect of ul-trasound on anthocyanins was studied for strawberry juice by Tiwari et al. [22 and 23]. They reported a slight increase (1 - 2%) in the P3G content of the juice at low-er amplitude levels and treatment times, which may be due to the extraction of bound anthocyanins from the suspended pulp. However, in a study by Tiwari et al. [24], the total anthocyanin content of the juice was de-graded when higher amplitude levels were employed, but the maximum observed degradation was < 5%.

In order to preserve all micronutrients in strawberry juice it is important to find an alternative food pres-


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ervation process, because heat pasteurization deteri-orates all this nutrients and lower sensorial quality of strawberry juices. To avoid unwanted effects of heat, many attempts have been made to design alternative procedures for food preservation and sanitation. Ultra-sound has been identified as a potential technology to meet the FDA requirement of a 5-log reduction in per-tinent microorganisms found in fruit juices [19]. Son-ication technology can improve the process through reduced processing time, higher throughput and low-er energy consumption [10, 13, 15]. Ultrasonication is a non-thermal method of food preservation that has the advantage of inactivating microorganisms in food without causing the common side-effects associated with conventional heat treatments. Food processing using ultrasound involves the transmission of energy at frequencies higher than 20 kHz. The low-intensity ul-trasound, which uses very small power levels (typically less than 1 W cm-2, with the frequency range of 5 - 10 MHz), causes no physical or chemical alterations in the properties of the treated material and thus can be used to measure the texture, composition, viscosity or con-centration of food. In contrast, the high-intensity ultra-sound, which uses much higher power levels (typically in the range of 10 - 1000 W cm-2, with the frequency of 20 - 100 kHz), causes physical disruption of the material to which it is applied and promotes certain chemical re-actions [15]. If ultrasound were to be used in any prac-tical application, it would most likely have to be used in conjunction with pressure treatment (mano-soni-cation), heat treatment (thermo-sonication) or both (mano-thermo-sonication). The enhanced mechanical disruption of cells is the reason for the enhanced kill-ing when ultrasound is combined with heat or pressure [10]. The objective of this study is to investigate the ef-fect of high intensity ultrasound and pasteurization on the content of anthocyanins, microorganisms in straw-berry juice and color of strawberry juice samples using an ultrasound frequency of 20 kHz under various con-ditions (treatment time, temperature and amplitude). The purpose of this investigation was to examine the influence of high intensity ultrasound on the stability of total anthocyanin content, color degradation and in-activation of microorganisms in strawberry juice.

2. Materials and methods

2.1 Standards and chemicals

Cyanidin 3-glucoside chloride and pelargonidin 3-glu-coside chloride were obtained from Fluka (Neu-Ulm, Germany). HPLC-grade methanol, ethanol and formic acids were obtained from Merck (Darmstad, Germany). Media, agars and reagents for microbiological analysis were obtained from Biolife (Milano, Italy).

2.2 Samples

Two different strawberry (Fragaria× ananassa Duch. cv. Alba) juice, one cloudy (Sample A) and one clear (Sam-ple B) was subjected to ultrasound treatments or pas-teurization. The pasteurized samples were denoted as AP and BP, untreated A and B and ultrasound treated as A1-A16 and B1-B16.

2.3 Juice preparationEach juice was made from approximately 10 kg of strawberries on the mini scale pilot-plant. After screen-ing, removal of stems, shower washing, strawberry pulp were subjected to maceration by enzyme (Endo-zym Pectofruit Press, AEB s.p.a. Brescia, Italia) and also pressed on hydraulic pack press (Sample A). After press-ing sample B was subjected to clarification by enzymes and centrifugation. (Backman J-21 B, UK). All samples were pasteurized in a flow pasteurizer for 2 minutes at 85 0C (Samples AP and BP) or by ultrasound (Samples 1-16) (Table 1).

2.4 Ultrasound treatmentsStrawberry juice (100 mL) was placed in a round bottom glass (200 mL), which served as the treatment chamber. An ultrasonic processor (S-4000, Misonix Sonicators, Newtown, Connecticut, USA), set at 600 W, 20 kHz, 12-260 μm with a 12 mm diameter probe, was introduced into the vessel. Ultrasonication was carried out with 60, 90 and 120 µm amplitude. The strawberry juice sam-ples were treated by ultrasonic 3, 6 and 9 minutes at 25 0C. In the case of thermo-sonication before ultrasonic treatment the samples were heated at 40 and 55 0C. Overheating of the samples was prevented by water cooling of the treatment chamber. Each experiment was conducted in triplicate. For this study, 16 samples of each juices were ultrasonically treated (Table 1).

2.5 Determination of acoustic power

The most widely accepted method for determining the power from an acoustic horn into an aqueous solution is the calorimetric technique described by Margulis and Maltsev [9]. This method involves taking a known volume of water and applying ultrasound (for ca. 3 min.) while monitoring the change in temperature with time for various ultrasonic amplitudes. The ultrasonic power can be readily determined from the following equation:

tTCmP p ∂

∂××=

/1/

AI = P / A /2/


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Where:

P is the ultrasonic power (W); m is the mass of the sample (kg);

Cp is the specific heat capacity of strawberry juices (3.368 kJ kg-1 C-1);

AI is the ultrasonic intensity (W cm-2) and

A is the surface of probe (cm2).

2.6 Anthocyanins extraction

Anthocyanins were extracted from sonicated and pas-teurized samples, by using 10 mL of sample and 20 mL of 30% (by volume) aqueous ethanol. The mixture was extracted for 30 min. at 70 0C in ultrasonic bath, filtered through Whatman No. 40 filter paper (Whatman In-ternational Ltd., Kent UK) using a funnel in volumetric flasks. The filtrates were adjusted to 50 mL in volumet-ric flask with 30 % aqueous ethanol [24].

2.6.1 Anthocyanins determination

Chromatographic separation and determination of an-thocyanins was performed using HPLC analysis Varian ProStar system equipped with a ProStar Solvent Deliv-ery Module 230, Injector Rheodyne 7125, ProStar 330 UV-VIS Photodiode Array detector (230 - 510 nm). Sep-aration of anthocyanins was performed on a Pinnacle II C-18 (250×4.6 mm i.d.,5 μm) including Pinnacle C-18 guard column (10×4 mm i.d.,5 μm) (Restek, Bellefon-te, USA). Two milliliter of the supernatant was filtered through 0.45 μm filter syringe (Nylon Membranes, Su-pelco, Inc. Bellefonte, PA, USA) before injected (20 μL) into the HPLC [22 and 23].

The elution was carried out at room temperature using 2.5% formic acid aqueous solution and methanol solu-tion mixed in various ratios; A - water/methanol (5 : 95), B - water/methanol (12 : 88) and C - water/methanol (80 : 20) with following gradients: first 150 min. 100% A, from 15 to 35 min. 100% B, from 35 to 50 min. 75% B and 25% C, from 50 to 52 min. 50% B and 50% C and from 52 till 60 min. 100% C. Flow rate was 1 mL min-

1 with a UV-VIS detector set at 510 nm. Calculation of the concentrations were based on a calibration curve made of external standards of investigated anthocya-nins by comparison of their retention times and peak area of standards. Chromatograms were recorded and processed with Varian ProStar Chromatography soft-ware.

2.7 Color determination

Juice color was measured using a Konica Minolta CM-3500d colorimeter (CM-3500d, Konica Minolta Holdings, Inc., Japan). The instrument (650/00 geometry, D25 opti-cal sensor, 100 observer) was calibrated using white (L) 92.8; a) -0.8, b) 0.1) and black reference tiles. The color

values were expressed as L* (whiteness or brightness/darkness), a* (redness/greenness), and b* (yellowness/blueness). Color measurements were taken in triplicate from each sample with the mean values reported [17].

2.8 Determination of microorganisms

Serial dilutions were made in Buffered Peptone Water (BPW) of samples taken from untreated, sonicated and thermo-sonicated and pasteurized strawberry juices to evaluate the content of microorganisms. In untreated, sonicated, thermo-sonicated and pasteurized straw-berry juices samples presence of Salmonella spp., En-terobacteriae and Escherichia coli was not determinate.

Enumeration of aerobic mesophilic bacteria - 1 cm3 (AMBC- aerobic mesophilic bacteria count) of samples or dilutions with Buffered Peptone Water (BPW) was put in sterile Petri dish and added Tryptic Glucose Yeast Agar melted and cooled at 45 0C - 46 0C. This agar was shuffled in a circular motion and let it cool, than incu-bate at 32 ± 1 0C/48 hours. After, colonies were counted and calculated the result, CFU cm-3 of sample.

Enumerations of yeasts (Y) and molds (M) - from undi-luted sample or certain dilution of sample in BPW up to 0.5 cm3 inoculate in solid Oxytetracycline Glucose Yeast Extract Agar (OGYE Agar). Incubation was done at 32 ± 1 0C/48 - 72 hours. After, colonies were counted and calculated the result, CFU cm-3 of sample.

Microbiological analyses were performed after pas-teurization, ultrasound or thermo-ultrasound treat-ment. All microbiological analyses were conducted in triplicate for each experiment.

2.9 Experimental methodology

A general factorial design (STATGRAPHICS Centurion, StatPoint technologies, Inc, VA 20186, USA) consists of 16 experimental trials [11]. It has been designed and chosen to obtain general observation of ultrasound treatment on content of anthocyanins and total micro-biological count in strawberry juice. In order to deter-mine influence of each factor and their combination on content of anthocyanins and total microbiological count in strawberry juice, central composite design (CCD), and face centered model was chosen. The ultra-sound factors of amplitude (μm), temperature (0C), and treatment time (min) were studied. Analysis of variance (ANOVA) was carried out to determine any significant differences (p < 0.05) among the applied treatments. The operating variables were considered at three levels namely, low (-1), central (0) and high (1). Accordingly, 16 experiments were conducted with experiments or-ganized in a factorial design (including factorial points, axial points and centre point) and the remaining in-volving the replication of the central point to get good


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estimate of experimental error. Repetition experiments were carried out after other experiments followed by order of runs designed by program [12]. Response (output) values were total anthocyanins in strawberry juice in mg kg-1, and total microbiological count in CFU cm-3. The designs were based on two-level full factori-al design, which were augmented with centre and star points [7]. The total number of experiments of the de-signs (N) can be calculated as follows:

N= Ni+No+Nj /3/

Where:

Ni = 2n is the number of experiments of the two level full fac-torial design;

No is the number of centre points and

Nj = 2 x n, is the number of star points.

2.9.1 Response surface methodology

The experimental results were analyzed by response surface methodology (RSM) using the software STAT-GRAPHICS Centurion (StatPoint technologies, Inc, VA 20186, USA). Calculations were done at 95% of confi-dence level. The ultrasound factors of amplitude - X1 (μm), temperature - X2 (ºC) and treatment time - X3 (min) were studied using RSM. In order to optimize the ultrasound treatment and investigate effects of above independent variables on content of anthocyanins and total microbiological count in strawberry juice, cen-tral-composite rotary design with the variables at three levels was used in the experiments (Table 1). Design matrix for the experiment and the regression model proposed for response was given below [8]:

∑ ∑ ∑= = ⟨

+++=4

1

4

1

42

0i i ji

jiijiiiii XXXXY bbbb /4/

Where:

β0 is the value of the fixed response at the central point of the experiment which is the point (0, 0, 0);

βi, βii and βij are the linear, quadratic and cross-products coef-ficients, respectively.

While demonstrating the significant effects 3-dimen-sional fitted surfaces were drawn [5 and 7]. The mod-el was fitted by multiple linear regressions (MLR). The validity of the quadratic empirical model was tested with analysis of variance (ANOVA). The confidence level used was 95%.

3. Results and Discussion

In this study, two strawberry juices (one cloudy - sam-ple A and one clear - sample B) were prepared using different technological processes without blanching. Immediately after preparation of juices, the compo-sition of anthocyanins and total anthocyanin content were determined, and then the juice was pasteurized (85 0C for 2 min.) or treated with ultrasound or ther-mo-ultrasound. Different process parameters of ultra-sound (amplitude, time, temperature) have been com-pared to the classical thermal treatments. Acoustic in-tensity applied in sonication of juices varied from 12.65 to 67.68 W cm-2 (Table 1).

Table 1. Treatment time, amplitude, temperature, pow-er intensity and added energy during ultrasound treat-ments of juices

XAmplitude

X1 (µm)

Time (minute)

X2

Tempe-rature

X3(0C)

Power (W)

Intensity(W cm-2)

A,B - - - - -

AP, BP - - - - -

A 1,B 1 60 9 25 64.18-68.42 56.79-60.54

A 2,B 2 90 3 40 17.01-22.58 15.05-19.98

A 3,B 3 120 3 25 23.71-25.38 20.98-22.46

A 4,B 4 120 3 55 21.60-22.90 19.11-20.26

A 5,B 5 90 6 55 42.47-44.26 37.58-39.16

A 6,B 6 60 3 55 15.34-16.38 13.37-14.49

A 7,B 7 120 9 55 72.33-76.60 64.00-67.68

A 8,B 8 90 6 40 41.20-43.98 36.45-38.89

A 9,B 9 60 6 40 31.72-33.84 28.07-29.94

A 10,B 10 90 9 40 70.05-72.98 61.98-64.57

A 11,B 11 120 9 25 72.58-75.85 64.22-67.11

A 12,B 12 90 6 40 41.20-43.47 36.45-38.46

A 13,B 13 60 3 25 14.30-16.13 12.65-14.27

A 14,B 14 90 6 25 41.51-44.99 36.73-39.81

A 15,B 15 120 6 40 48.59-50.84 42.99-44.98

A 16,B 16 60 9 55 67.89-71.72 60.07-63.46

3.1 Anthocyanins degradation

Content of anthocyanins in untreated, pasteurized and sonicated juice samples A and B are listed in Tables 2 and 3.


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Table 2. Concentrations of anthocyanins (mg kg-1) in untreated, pasteurized and sonicated strawberry juices A

StrawberryJuice B

Cyanidin-3-O-glucoside(mg kg-1)

Cyanidin-3-O-rutinoside(mg kg-1)

Pelargonidin-3-O-glucoside(mg kg-1)

Pelargonidin-3-O-rutinoside(mg kg-1)

Σ(mg kg-1)

B 1.003 8.881 131.680 8.650 150.214

BP 0.852 7.612 123.870 7.346 139.680

B1 0.911 8.126 128.090 8.411 145.538B2 0.973 8.768 131.330 8.713 149.784

B3 0.941 8.251 131.840 8.649 149.681

B4 0.909 8.042 127.340 7.983 144.274

B5 0.883 8.007 127.010 7.748 143.648

B6 0.905 8.113 127.970 7.929 144.917

B7 0.808 7.523 122.010 7.137 137.478

B8 0.963 8.703 130.090 8.322 148.078

B9 0.951 8.693 129.450 8.182 147.276

B10 0.861 8.131 124.040 7.572 140.604

B11 0.823 7.931 124.660 7.588 141.002

B12 0.963 8.703 130.090 8.322 148.078

B13 0.990 8.562 131.240 8.575 149.367

B14 0.982 8.761 130.750 8.361 148.854

B15 0.921 8.421 128.040 8.429 145.811B16 0.793 7.207 121.140 7.098 136.238

ple A 139.989 to 153.318 mg kg-1; sample B 136.238 to 149.784 mg kg-1) is between untreated and pasteurized samples. Data show that total anthocyanin content de-creases with longer ultrasound treatment which has statistically significant influence (p < 0.05) (Figure 1).

a) juice A

b) juice B

Four major anthocyanins in strawberry juice were significantly influenced by ultrasound, namely cyani-din-3-O-glucoside, cyanidin-3-O-rutinoside, pelargo-nidin-3-O-glucoside and pelargonidin-3-O-rutinoside. Total anthocyanin content during sonication was influ-enced by three investigated factors, i.e. ultrasound am-plitude level (µm), sonication time (min.) and tempera-ture (0C). Their effects were either individual or inter-active [11]. The contribution of total anthocyanin con-tent in untreated samples are 150.214 (sample B) and 153.818 mg kg-1 (sample A), depending on the techno-logical process applied for their production. Samples which were pasteurized (marked with P) at 85 0C for 2 min. show the lowest levels of anthocyanins (139.680 mg kg-1 (A) and 142.472 mg kg-1 (B)), as compared to sonicated or untreated juices, namely because of the thermal treatment, which causes decomposition of an-thocyanins. Patras et al. [14] found that anthocyanins (cyanindin-3-glucoside and pelargonidin-3-glucoside) in blackberry and strawberry puree were significantly degraded by heat treatment, without blanching inac-tivation of polyphenol oxidase enzyme in the samples. Tiwari et al. [22] reported a slight increase (1 - 2%) in the pelargonidin-3-glucoside content of the juice at low-er ultrasound amplitude levels and treatment times, which may be due to the extraction of bound antho-cyanins from the suspended pulp. Similarly, weak ultra-sonic irradiation is reported to promote an increase in the amount of phenolic compounds found in red wine [4]. This behavior is not in agreement with our results. Total anthocyanin content of sonicated samples (sam-


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Figure 1. Standardized Pareto charts for total anthocyan-in content in sonicated strawberry juices

There is also statistically significant influence (p < 0.05) of temperature of ultrasound treatment on total antho-cyanin content. Increase of temperature during sonica-tion is reducing total anthocyanin content. Also, it has been observed that decrease of anthocyanin level is higher when sonication temperature is increased than when sonication period is prolonged (Figure 2).

a) Juice A

b) Juice B

Figure 2. Response surface plots for total anthocyanin content in sonicated strawberry juices at temperature

40 ˚C

Maximal degradation of anthocyanins in juices in this study was observed after ultrasound treatment at the amplitude of 90 or 120 mm for 9 min. at 55 0C (samples A7 and B16; Tables 2 and 3). Tiwari et al. [24] report-ed that after sonication, the content of anthocyanins compared to untreated (control) samples decreased for about 3.2%. In our investigation decrease in total an-thocyanin content after pasteurization was reduced by 7.1 to 7.4% compared to untreated juices. After treat-ment with ultrasound or thermo-ultrasound, degrada-tion of anthocyanins was generally less intensive and was 0.6 to 6.4% compared to the pasteurized juices. Only in the case of ultrasonic treatment at 55 0C for 9 min., total content of anthocyanins, compared to un-treated juice, was reduced from 8.5 to 9.0%, and their degradation was greater than that in pasteurized juices.

Higher degradation of anthocyanins in these samples results from the formation, growth, and rapid collapse of microscopic bubbles. Cavities formed by sonication may be filled with water vapor and gases dissolved in the juice, such as O2 and N2, which may be responsi-ble for oxidative degradation of anthocyanins, as well as free radicals. When ultrasound amplitude is higher, larger amount of energy and intensity is input in the sonicated system (juice), causing a collapse of cavita-tions bubbles. Then, vapor and gases from the cavities are dissipated in the systems, causing several chemical reactions. Also, anthocyanin degradation during ultra-sonic processing could be related to oxidation reac-tions, promoted by the interaction of free radicals such as hydroxyl (·OH) formed during sonication following the reaction (H2O ·OH+H·), which leads to chemical decomposition by opening of rings and formation of chalcone [24]. The major reaction path for the degrada-tion of polar compounds is pyrolysis within cavitations bubbles in the liquid or gas pockets trapped in the crevices of the solid boundaries in the liquid medium [2]. In Table 8 optimal conditions for ultrasonic treat-ment using response surface models and total antho-cyanins content at optimal processing parameters of amplitude, treatment time and temperature are given. Also, polynomial fit models for total anthocyanin con-tent in sonicated strawberry juices are shown.

Table 8.

a) Optimal conditions for ultrasonic treatment using response surface models and total anthocyanins content at optimal processing parameters of amplitude, treatment time and tem-perature

Source Juice A Juice B

X1 - amplitude / µm 85.38 91.56

X2 - treatment time / min 3.00 3.00

X3 – temperature / ˚C 25.03 25.00

Total anthocyanins at optimal values / (mg / kg) 118.276 118.786

b) Polynomial fit models for total anthocyanin content in soni-cated strawberry juices

Juices Models

A

Total anthocyanins = 114.865 + 0.0991·X1 + 0.0714·X2 – 0.0010·X3 – 0.00058·X1

2 – 0.00044·X1·X2 + 0.000029·X1·X3 – 0.0338·X2

2 – 0.00092·X2·X3 – 0.00098·X3

2

B

Total anthocyanins = 114.505 + 0.1153·X1 – 0.3284·X2 + 0.05428·X3 – 0.00062·X1

2 + 0.0011·X1·X2 – 0.00028·X1·X3 – 0.0021·X2

2 – 0.0026·X2·X3 – 0.00146·X3

2

X1-amplitude (µm) X2 - treatment time (min) X3- temperature (˚C)


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3.2 Color degradation

Values of color parameters: lightness (L*), the red - green value (a*) and the blue - yellow value (b*) for samples A and B are shown in Tables 4 and 5.

Table 4. Values of parameters (L*, a* and b*) in untreated, pasteurized and sonicated strawberry juices A

Strawberryjuice A L*(D65) a*(D65) b*(D65)

A 46.0850 64.8125 79.3925

AP N/A N/A N/A

A1 39.9681 60.9673 68.0722

A2 42.8157 62.8670 74.0351

A3 44.7175 64.5425 77.0500

A4 36.1100 57.3175 62.2375

A5 37.3581 58.3481 62.0039

A6 38.9574 58.9937 63.1756

A7 35.7975 56.8750 61.6950

A8 41.6482 60.0689 70.4689

A9 41.7357 61.0370 71.4650

A10 39.8765 60.0421 69.3548

A11 39.3250 59.6600 67.7725

A12 41.6482 60.0689 70.4689

A13 45.1021 63.2050 75.8425

A14 44.0546 63.2364 75.0759

A15 36.9675 56.9675 63.7050

A16 35.5972 56.1959 61.7037

Table 5. Values of parameters (L*, a* and b*) in untreat-ed, pasteurized and sonicated strawberry juices B

Strawberryjuice B L*(D65) a*(D65) b*(D65)

B 46.8000 67.0525 80.6075

BP N/A N/A N/A

B1 47.9413 67.5445 84.0917B2 48.9058 67.0973 84.8761

B3 49.9125 67.6700 85.9900

B4 49.2775 67.8600 84.8975

B5 47.5850 67.3400 82.1874

B6 48.0013 67.8426 84.7318

B7 46.9710 67.6900 81.9975

B8 48.6351 67.1303 84.0691

B9 48.0358 67.0982 83.9639

B10 47.8711 67.9947 85.0038

B11 48.9200 67.7475 84.2925

B12 48.6351 67.1303 84.0691

B13 49.9699 67.8976 85.8295

B14 49.0023 67.7361 85.0993

B15 48.8175 67.7675 84.1025B16 47.0050 67.7501 82.0175

Color parameters during sonication were influenced by three investigated factors, i.e. ultrasound amplitude level (µm), sonication time (min.) and temperature (0C). Data show that values for maximal treatment condition of sonication (120 µm, 55 °C and 9 min) for cloudy sam-ple A decrease values (L*), (a*) and (b*) compare to un-treated sample for 22.33%, 12.25% and 22.30%, but in-crease values for clear sample B (L*), (a*) and (b*) com-pare to untreated sample for 0.37%, 0.95% and 1.72%. Influence of pasteurization on color degradation was not investigated in this study. Samples of cloudy juice A which are treated with less intensive conditions shows decrease in values (L*), (a*) and (b*) compare to untreated sample for 2.97 - 13.47%, 0.42 - 7.36% and 2.95 - 12.64%, and for clear juice B increase in values (L*), (a*) and (b*) compare to untreated sample for 2.29 - 6.65%, 0.92 - 1.41% and 5.54 - 6.67%.

From Figure 3, results of statistical analysis show that there is significant influence (p < 0.05) of temperature and treatment time on color parameters L, a and b for strawberry juice A.

a) Juice A


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b) Juice B

Figure 3. Standardized Pareto charts for value of param-eters L*, a* and b* in sonicated strawberry juices

Also, the same influence is for strawberry juice B, but there is not significant influence of investigated pa-rameters for parameter a. From Figure 4 and response surface plots it is obvious that there is decrease in col-or parameters of L, a* and b* in juice A with increasing treatment time and amplitude.

a) Juice A

b) Juice B

Figure 4. Response surface plots for value of parameters L*, a* and b* in sonicated strawberry juices at tempera-

ture 40 0C


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For juice B, the trend and influence is the same but op-posite for color parameter a. The difference is because of natural behavior of cloudy and clear juice.

In Table 6 optimal conditions for ultrasonic treatment using response surface models and value of parame-ters L*,a* and b* at optimal processing parameters of amplitude, treatment time and temperature are given, and also polynomial fit models for value of parameters L*,a* and b* in sonicated strawberry juices.

Table 6.a) Optimal conditions for ultrasonic treatment using response surface models and value of parameters L*, a* and b* at optimal processing parameters of amplitude, treatment time and tem-perature

SourceL* a* b*

Juice A Juice B Juice A Juice B Juice A Juice B

X1 - amplitude / µm 81.47 120.0 87.03 120.00 87.37 100.70

X2 - treatment time / min 3.00 3.00 3.00 9.00 3.00 3.00

X3 – temperature / ˚C 25.00 25.00 25.00 25.00 25.00 30.30

value of parameter at optimal values 46.7887 50.1569 65.6192 67.9199 79.5114 86.0317

b) Polynomial fit models for value of parameters L*, a* and b* in sonicated strawberry juices

Juices Models

A

Value of parameter L* = 42.676 + 0.31509·X1 – 2.0175·X2 – 0.1315·X3 – 0.001936·X1

2 + 0.003874·X1·X2 – 0.0004498·X1·X3 + 0.0280·X2

2 + 0.01904·X2·X3 – 0.001723·X3

2

B

Value of parameter L* = 51.022 – 0.00449·X1– 0.21569·X2 – 0.025254·X3 + 0.000072·X1

2 – 0.0003807·X1·X2 + 0.000089·X1·X3 + 0.002997·X2

2 – 0.00078·X2·X3 – 0.000301·X3

2

A

Value of parameter a* = 61.8186 + 0.3255·X1– 1.9142·X2 – 0.2506·X3 – 0.001825·X1

2 – 0.000402·X1·X2 – 0.000285·X1·X3 + 0.08998·X2

2 + 0.01077·X2·X3 + 0.000655·X3

2

B

Value of parameter a* = 70.3774 – 0.01937·X1– 0.3087·X2 – 0.07105·X3 + 0.000104·X1

2 + 0.0004904·X1·X2 – 0.000005·X1·X3 + 0.0229·X2

2 + 0.000036·X2·X3 + 0.000882·X3

2

A

Value of parameter b* = 67.1627 + 0.5504·X1– 4.1962·X2 + 0.08252·X3 – 0.003061·X1

2 – 0.000803·X1·X2 – 0.000515·X1·X3 + 0.15055·X2

2 + 0.04176·X2·X3 – 0.00800·X3

2

B

Value of parameter b* = 84.5511 + 0.03341·X1– 1.0618·X2 + 0.1663·X3 – 0.0001536·X1

2 – 0.000202·X1·X2 – 0.0000599·X1·X3 + 0.08539·X2

2 – 0.00605·X2·X3 – 0.002347·X3

2

X1-amplitude (µm) X2 - treatment time (min) X3- temperature (˚C)

3.3 Inactivation of microorganisms High intensity ultrasound was used to investigate inac-tivation of microorganisms in strawberry juice samples.

The inactivation of aerobic mesophilic bacteria, yeasts and molds in juice samples after ultrasonic treatment was influenced by three investigated factors, i.e. ultra-sound amplitude level (µm), sonication time (min.) and temperature (0C).

Pasteurized samples had 100% efficiency for inactiva-tion of microorganisms. Samples which are sonicated in conditions of 9 min. and 55 0C also have maximal (100%) efficiency for inactivation of microorganisms (Tables 7 and 8).

Table 7. Aerobic mesophilic bacteria count (AMBC), count of yeasts (Y) and molds (M) (CFU cm-3) in untreated, pas-teurized and sonicated strawberry juices A

Strawberryjuice A

AMBC(CFU/cm3)

Y(CFU/cm3)

M(CFU/cm3)

A 140 10 0AP 0 0 0A1 22 2 0A2 25 0 0A3 60 3 0A4 13 0 0A5 14 0 0A6 20 0 0A7 0 0 0A8 35 0 0A9 40 0 0

A10 7 0 0A11 6 4 0A12 35 0 0A13 60 5 0A14 49 0 0A15 5 0 0A16 0 0 0

Table 8. Aerobic mesophilic bacteria count (AMBC), count of yeasts (Y) and molds (M) (CFU cm-3) in untreated, pas-teurized and sonicated strawberry juices B

Strawberryjuice B

AMBC(CFU/cm3)

Y(CFU/cm3)

M(CFU/cm3)

B 145 10 0BP 0 0 0B1 21 2 0B2 24 0 0B3 63 5 0B4 16 0 0B5 15 0 0B6 12 0 0B7 0 0 0B8 34 0 0B9 32 0 0

B10 8 0 0

B11 6 3 0

B12 34 0 0

B13 59 4 0

B14 36 1 0

B15 5 0 0B16 0 0 0


35

Less intensive treatment conditions inactivate microor-ganisms from 58 to 94% compared with untreated juice samples. Maximal accepted levels according to nation-al sanitary standards for food [16] are < 10 cfu cm-3 for aerobic mesophilic bacteria and < 1 CFU cm-3 for yeasts and molds. In Figure 5 statistical analysis shows that there is statistically significant (p < 0.05) influence of temperature and treatment time and interactions of those two factors on count of aerobic mesophilic bac-teria for juice A and B.

a) Juice A

b) Juice B

Figure 5. Standardized Pareto charts for count of aerobic mesophilic bacteria in sonicated strawberry juices

In Figure 6 results shows that with increasing treatment time and amplitude there is sharp decrease in count of aerobic mesophilic bacteria.

a) Juice A

b) Juice B

Figure 6. Response surface plots for count of aerobic mesophilic bacteria in sonicated strawberry juices at

temperature 40 ˚C

In Table 9 optimal conditions for ultrasonic treatment using response surface models and count of Aerobic mesophilic bacteria at optimal processing parameters of amplitude, treatment time and temperature are giv-en. Also, polynomial fit models for count of Aerobic mesophilic bacteria in sonicated strawberry juices are shown.

Table 9.

a) Optimal conditions for ultrasonic treatment using response surface models and count of Aerobic mesophilic bacteria at op-timal processing parameters of amplitude, treatment time and temperature

Source Juice A Juice B

X1 - amplitude / µm 120.0 120.0

X2 - treatment time / min 9.00 8.99

X3 – temperature / 0C 51.795 51.28

Aerobic mesophilic bacteria count at optimal values / (CFU/cm3) - 2.811 - 2.145

b) Polynomial fit models for count of Aerobic mesophilic bacte-ria in sonicated strawberry juices

Juices Models

A

Count of Aerobic mesophilic bacteria = 92.968 + 0.79925·X1 – 7.6244·X2 – 2.8926·X3– 0.005153·X1

2 – 0.0125·X1·X2 + 0.0025·X1·X3 – 0.0153·X2

2 + 0.14167·X2·X3 + 0.01272·X32

B

Count of Aerobic mesophilic bacteria = 116.739 + 0.5935·X1 – 9.5204·X2 – 3.2616·X3 – 0.00400·X1

2 – 0.01528·X1·X2 + 0.00306·X1·X3 + 0.0441·X2

2 + 0.175·X2·X3 + 0.01287·X32

X1-amplitude (µm) X2 - treatment time (min) X3- temperature (0C)


36

According to standard, only juice samples A and B which are treated under maximum treatment condi-tions fulfill sanitary and health demands. It is import-ant to say that untreated samples A and B are produced from not blanched pulp and have a high amount of microorganism. Because of initial contamination, only ultrasound treatment under maximal conditions is effi-cient. Probably, if we would use samples from blanched pulp then it would not be required such intensive son-ication treatment conditions for inactivation of mi-croorganisms. Some authors have suggested that the efficacy of ultrasonic treatment for inactivation of bac-teria by effects of cavitation could be minimized with an increase in temperature. This fact could be proba-bly the result of an increased thermal effect that would masque the effect of sonication, and/or a decrease of the violence of implosion due to the increased vapor pressure at higher temperatures. More bubbles are formed but these are smaller and the violence of im-plosion decreases [18]. This behavior is not in agree-ment with our results. Although the cavitation effect could be minimized by the increase of temperature, in the case of milk, the concentration of solids in sus-pension could play an important role and improve the cavitation intensity [3 and 25]. Garcia et al. [3] observed that at high temperatures, there were no advantages of thermoultrasonication for inactivation of bacteria in milk, since the z-values of thermoultrasonication and thermal destruction were very similar. Although the mechanism is not clear, they attributed these results to the concentration of solids presented in milk. Ciccolini et al. [1] studied the survival of S. cerevisiae suspended in water at 45, 50 and 55 0C at different ultrasonic pow-ers, and found that application of ultrasonic waves at non-lethal temperature (45 0C) did not display a deac-tivation action while synergy between ultrasound and heat was confirmed at the higher temperatures.

4. Conclusions

- The purpose of this investigation is to find out how high intensity ultrasound effect on anthocyanins, color pigments and microorganisms in strawberry juice samples. For determination degradation/in-activation of ultrasound we investigated three fac-tors, i.e. ultrasound amplitude level (µm), sonication time (min.) and temperature (0C).

- Degradation of anthocyanins is generally less than compare to classical thermal pasteurization. Only in maximal treatment conditions degradation of anthocyanins is greater compare to the pasteuriza-tion.

- Color parameters was slightly increased for clear samples (B) and slightly decreased for cloudy sam-ples (A).

- Maximum deviation of color parameters was under maximal treatment conditions.

- Maximal efficiency for inactivation of microorgan-isms compare to untreated samples was in pasteur-ized samples and in samples which was under maxi-mal treatment conditions. In less invasive treatment conditions inactivation efficiency was from 58 to 94%.

- The modeling approaches by response surface methodology (RSM) developed in this study exploit data in order to identify the optimal processing pa-rameters for lowering the degradation of anthocy-anins, degradation of color and inactivation of mi-croorganisms in strawberry juice during ultrasound processing.

- This work demonstrates that thermo-sonication in-fluences the content of anthocyanins, color param-eters and inactivation of microorganisms in straw-berry juices and that RSM may be used to optimize critical process parameters to obtain juices with high retention levels of bioactive compounds and high level of inactivation of microorganisms.

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Strehaiano P. (1997). Low frequency thermo-ultrasoni-cation of Saccharomyces cerevisiae suspensions: effect of temperature and of ultrasonic power. Chemical Engineer-ing Journal, 65, pp. 145 - 149.

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INFLUENCE OF THERMO-SONICATION ON … Vol 4/01. FQS/05. Full paper - Zoran...Strawberries (Fragaria × ananassa Duch. cv. Alba) have high levels of micronutrients and phytochemical