Ultrasonics Sonochemistry 20 (2013) 1316–1323

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Ultrasoni cs Sonoch emistry

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Effect of ultrasound frequency on antioxidant activity, total phenolic and anthocyanin content of red raspberry puree

Amir Golmohamadi a, Gregory Möller a, Joseph Powers b, Caleb Nindo a,⇑a School of Food Science, University of Idaho, Moscow, ID 83844, USA b School of Food Science, Washington State University, Pullman, WA 99164, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 28 April 2012 Received in revised form 31 December 2012 Accepted 31 January 2013 Available online 27 February 2013

Keywords:UltrasoundExtractionAntioxidant activity Anthocyanin content

1350-4177/$ - see front matter � 2013 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.ultsonch.2013.01.020

⇑ Corresponding author. Tel.: +1 208 885 9683.E-mail address: cnindo@uidaho.edu (C. Nindo).

Ultrasound in the 20–1000 kHz range show unique propagation characteristics in fluid media and possess energy that can break down fruit matrices to facilitate the extraction of valuable bioactive compounds.Red raspberries carry significant amounts of specific antioxidants, including ellagitannins and anthocya- nins that are important for human health. The objective of this study was to investigat e the effects of ultrasound frequencies associated with cavitation (20 kHz) and microstreaming (490 and 986 kHz) on total antiox idant activity (AOA), total phenolics content (TPC), and total monomeric anthocyanin content (ACY) of red raspberry puree prepared from crushed berries. The pureed fruit was subjected to high- intensity (20 kHz) and higher frequency-low intensity (490 and 986 kHz) ultrasound for 30 min. The tem- perature of treated purees increased to a maximum of 56 �C with 986 kHz. Sonication at 20 and 490 kHz significantly (p < 0.05) affected the AOA, ACY, and TPC of red raspberry puree, while 986 kHz had no significant effect on ACY and AOA (p < 0.05). In all cases, ultrasound treatment had significant and positive effect on at least one of the measure d parameters up to 30 min. Sonication beyond 10 min (and up to 30 min) using 20 kHz either produced no change or caused a drop in AOA and ACY. However,for 986 and 20 kHz, TPC, increased by 10% and 9.5%, respectively after 30 min (p < 0.05) compared to the control. At 20 kHz, AOA and ACY increased by 17.3% and 12.6% after 10 min. It was demonstrated that 20 kHz ultrasound treatment , when limit ed to 10 min, was the most effect ive for extractio n of bioactive comp ounds in red raspberry compared to 490 and 986 kHz although the effect could be similar at the higher frequencies if different amplitude s are used.

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1. Introduction

Ultrasound is widely used in the areas of science and engineer- ing because it is a nonthermal technique with multiple capabilities that makes it suitable for different industria l applications, includ- ing the food industry. Sound frequencies equal to or higher than 20 kHz are defined as ultrasound [1]. Power ultrasound frequencies range from 20 kHz to 1 MHz, and are considered so because of abil- ity to deliver high energy to fluid medium. Within this range the ultrasound waves can generate cavitation and microstream ing (microscopic fluid movement) effects that cause changes in a liquid medium [2]. Cavitation is the phenomeno n associated with pro- duction, growth and collapse of bubbles during the compression and rarefaction cycles in sound propagat ions. This phenomeno ncreates eddy currents in the fluid near the vibrating bubbles and the eddy currents consecutively apply a distortion and rotational motion on nearby cells or particles.

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The application of ultrasoun d in the 20–1000 kHz range is di- vided into two bands, namely, (1) low power ultrasound having low amplitud e and high frequenc y (100–1000 kHz), (2) high power ultrasoun d generated at high amplitude and low frequency (20–100 kHz). This second type is used in food processing, welding,and cleaning processes because cavitation bubbles are more diffi-cult to form at the higher ultrasonic frequenc ies [1,3]. The mecha- nism of ultrasound assisted extraction (UAE) of bioactive compound s in plants involves breaking the cell walls, diffusion of the solvent through the cell matrix and washing out the cell con- tents. Ultrasound is beneficial in extractio n because it damages the plant cell wall and increases the solvent uptake by the cell dur- ing sonication thereby increasing the extraction yield [4]. Usually during this process the medium temperature increases due to cav- itation, asymmetric collapse of bubbles close to the surfaces of cell walls, and the production of micro jets. These episodes produce en- ergy that is partly absorbed by the medium and dissipated as heat,resulting in temperature increase. However, the temperature re- mains low (less than 70 �C) when the reactor is equipped with acooling system [5].

A. Golmohamadi et al. / Ultrasonics Sonochemistry 20 (2013) 1316–1323 1317

Ultrasonic extraction of various food components has been re- ported mainly for 20–40 kHz; and the sample size, extraction time and solvent volume generally used by many researchers for solid–liquid extraction is between 1–30 g, 10–60 min, and 50–200 ml,respectively [6]. For example, Tiwari et al. [7] applied a 1500 Wultrasonic processor at a constant frequenc y of 20 kHz to investi- gate the effect of ultrasoun d on the degradat ion of anthocyanin and ascorbic acid in strawberry juice.

Current research suggests that a diet rich in bioactive com- pounds (such as those provided by antioxidant s found in fruits and vegetables) can play a role in protecting the body from degen- erative diseases [8,9]. Therefore, it is recommended to include adaily intake of fruits and vegetables, although according to USDA,most Americans do not consume the quantities and varieties that are recommend ed. Fruit juices are more convenie nt and less expensive than whole fruits [10]. In 2010, the juice retail market in the US was valued at $16.2 billion and Americans consumed an average of 30.3 L per person [11]. Efficient extraction of bioac- tive compounds for preparation of juices and other products, while not correlate d with the absorption and effectivenes s of the antiox- idants in vivo, can provide functionality , delivery, and other op- tions that may not be possible with the consumptio n of whole fruits and vegetables [12]. Enhanced recovery of anthocyanins from fruit or vegetable matrices is also of interest to food proces- sors because of their use as colorants, additives to promote nutri- tional value of other foods, or even as nutritional supplements .

Many berries contain polyphenol s, anthocyanins and ascorbic acid [13,14] in varying quantities ; and cranberry, raspberry, and black currant are considered important sources of anthocyanins [15,16]. Raspberries are unique among the berry fruits because of a nutrition al profile of low calories, high fiber, and phytochem ical composition that include flavonoids, phenolic acids, lignans, and tannins. Among the phenolic compounds, raspberries carry signif- icant amounts of ellagitan nins and anthocyanins which contribute to their appealing color and health-promot ing properties [17].Xylitol, a low-calo rie sugar substitute with immense applications in human health products, is found in red raspberries and straw- berries. The level of xylitol in raspberries (Rubus idaeus ) per gram of fresh weight is approximat ely 400 lg compared to 7.5–280 pg in other berries [18]. The Pacific Northwest region of the United States is a major producer of red raspberry, especially western Washington and Oregon State where there is commerc ial produc- tion and processing [19]. Processe d red raspberry in State of Wash- ington alone accounts for nearly 95% of the US production (about30 million kg/year) [20]. Because of this economic importance , it is important to not only increase fresh fruit production, but to investigate alternative processin g techniques to improve juice extraction and anthocya nin recovery. In conventional juice extrac- tion, red raspberry fruit is crushed to form a fruit mash which is then treated with enzymes to increase the free-run juice, shorten the pressing time, and minimize energy consumptio n [21]. The juice yield is improved by about 20% when the mash is treated with enzymes [22], so UAE can be an appropriate process step to enhance extraction yield and increase the antioxidant capacity of extracts [23].

Small scale UAE has been practiced since the 1950s [6], and most recently Chen and others [24] reported results of a laboratory scale extraction of red raspberry anthocyanin s using ultrasound.However, optimizati on of the process to determine suitable fre- quencies and strategies for scale up are still poorly understood.The extent of cell disruption, juice yield and antioxidant activity as affected by ultrasound frequency, power intensity, and material structure [5,25] need further investigatio n. The effect of frequency on sonochemical reactions has been investigated using an interest- ing ultrasound system with a single transducer that was capable of generating 0–5 W of ultrasound power at multiple ultrasoun d fre-

quencies (20, 40, 150, 200, 300 and 450 kHz) [26]. In a different study [27], the degradat ion of alachlor (a herbicide) residues in water was more rapid at 300 kHz than at 20 kHz under similar acoustic energy input.

In the case of UAE, no published literature was found where the effect of high frequency ultrasound (>50 kHz) on antioxidant capacity and recovery of anthocyanin s from fruits was investi- gated. Many studies are available on the applicati on of 20–40 kHz ultrasound for cell disruption or extraction [6], but just like in sonochemistry, most of those studies are based on the availabil- ity of commerc ial ultrasoun d generators which are designed to function at lower frequencies (<100 kHz). There is a need to evalu- ate other frequencies as a step toward optimization and scale up ultrasoun d assisted extraction. Therefore, the objective of this study was to investigate the effect of three ultrasound frequencies (20, 490 and 986 kHz) on antioxidant activity, total phenolic s con- tent and anthocyanin content of red raspberry puree as a berry product with important health-prom oting attributes. The effect of higher frequencies to increase or decrease the recovery of antho- cyanins in red raspberry puree was tested.

2. Materials and methods

2.1. Materials

Reagent grade L-ascorbic acid powder (99.5% purity) and 1,1-di- phenyl-2- picrylhydrazyl (DPPH) free radical were obtained from Sigma Co. (St. Louis, MO). A kit (ACW kit) for the analysis of water soluble antioxidants was purchased from Analytik-Jen a AG, Ger- many. Ascorbic acid solution (AAS), used as model to determine antioxidant activity assays and ultrasound intensity in the fluid,was prepared by dissolving 27 mg of L-ascorbic acid in 100 ml dis- tilled water. Red raspberries were purchased from a local grocery store, crushed, blended and mixed thoroughly to obtain purees (with 10.9% solid content) with fine and smooth texture.

2.2. Sonication

For the sonication treatments, 150 ml samples of either ascorbic acid solution or red raspberry puree were used. Three different ultrasoun d frequenc ies (20, 490 and 986 kHz) were applied for treatment of ascorbic acid solution and red raspberry puree in batch systems. During those treatments, the reactor vessel jacket was filled with ice water to keep the temperature as low as possi- ble. Samples were collected at 10-min intervals from start up to 30 min.

2.2.1. Ultrasound treatment at 20 kHz A 400 W capacity batch sonication system (Branson Sonifier, S-

450A, Danbury , CT) with a 7 cm vibrating titanium tip was used for treatments with the probe immersed half way in the liquid. The samples were sonicated through continuous pulsation at 50% out- put power setting. A 500 ml beaker was put inside another plastic beaker containing a mixture of ice and water. A magnetic stirrer was placed at the bottom of the plastic beaker for continuous mix- ing of the cold water for effective cooling (Fig. 1). Samples were subjected to sonication for 30 min, and at each time interval, a5 ml sample was taken. Each fresh sample was maintained in an ice chest during the experiment, centrifuged at 1880 g at room temperat ure for 20 min, and the supernatant was collected and kept frozen at �20 �C for analysis afterwards.

2.2.2. Ultrasound treatment at 490 and 986 kHz The higher ultrasoun d frequenc ies (490 and 986 kHz) were pro-

duced by a custom-made ultrasound generator (Fig. 2) equipped

Fig. 1. Schematic of a 20 kHz ultrasound reactor for treatment of red raspberry puree (RP) and ascorbic acid solution (AAS).

Fig. 2. Schematic of 490 and 986 kHz ultrasound reactor and a provision for sample purging with argon gas.

1318 A. Golmohamadi et al. / Ultrasonics Sonochemistry 20 (2013) 1316–1323

with a parabolic stainless steel horn collimator designed to focus sound waves from a 3.2 mm thick and 35 mm diameter piezoelec- tric transduc er (APC-841, American Piezo Ceramics, Mackeyville,PA). The collimator was equipped with a copper backing that pro- vided the electrical input and base for the transducer. The collima- tor itself was connected to a stainless steel reaction chamber (27.0 ± 0.1 cm in length and 7.5 ± 0.0 cm in diameter) which was mounted inside concentric cylindrical cooling chamber [28]. The head space was purged by argon gas (also bubbled through the li- quid) to minimize the potential effect of oxygen on the berry anti- oxidants. The sonication time, sampling procedure, and sample preparation was similar to that at 20 kHz.

2.3. Ultrasound power measurem ent

The acoustic energy supplied by the transducers and delivered into the liquid is eventually dissipated as heat resulting in a mod- erate temperature increase (�50 �C). Therefore, knowing the phys- ical properties of liquid and by measuring the temperature change as a function of time, an estimation of the acoustic power (W) can be obtained. According to Jambrak et al. [29], this rate of acoustic energy dissipation (P), which may be calculated per unit volume of liquid, is given by:

P ¼ m:Cp:dT dt

� �ð1Þ

where m: mass of the sonicat ed liquid (kg); Cp: specific heat capacity at a consta nt pressure (J/kg �C). Since water forms 90%

of the raspbe rry puree, Cp was considered approximat ely equal to that of water (4180 J/kg �C); dT/dt: is the rate of temperat ure increase, obtained from the slope of the initial region of the heat- ing curve.In order to monitor temperature within the puree, ther- mocouples were inserted inside three narrow (2 mm internal diamete r) glass tubes with the sensing tips protrudin g slightly out of the tubes. The tubes, position ed vertically with 1.5 cm spacing, were then inserted half way in the ascorbic acid solution or red raspberry puree to measure temperat ure at the center,near the wall of the reactor and at a midpoint location betwee nthe wall and the center. One minute before the end of sonication ,the material being treated was mixed to obtain the final uniform temperat ure.

2.4. Determina tion of antioxidan t activity

2.4.1. Photochemilu minescence (PCL) method The photochemil uminescence (PCL) method combines a fast

photoche mical excitation of radical generation with sensitive luminomete ric detection. The advantage is that it is highly sensi- tive, involves few preparation steps, and the measurement time is reduced to a few minutes per measurement [30]. A reagent (ACW) kit (Photochem�, Analytik Jena AG, Jena, Germany) was used to measure the hydrophi lic antioxidant activity with ascorbic acid as reference. A standard curve was plotted from zero to 3 nmol/L and concentratio n of every sample for analysis was ad- justed to be within the linear region of the curve.

A. Golmohamadi et al. / Ultrasonics Sonochemistry 20 (2013) 1316–1323 1319

2.4.2. DPPH decolorat ion method The DPPH decolora tion method is an established procedure to

measure the radical scavenging capacity of a compound. Briefly,2.5 mg of DPPH powder was dissolved in 100 ml of methano l.The solution was mixed completely, capped and covered with aluminum foil to protect it from light. The DPPH solution was prepared fresh daily. A UV/Visible spectrophotom eter with ‘‘UV Winlab’’ software (Perkin Elmer PTP6., San Jose, CA) was used to measure the antioxidant activity of ascorbic acid solution by monitoring the absorbance at 517 nm using methanol as blank.The percentage of DPPH quenched was calculated each minute as follows:

%DPPH quenched ¼ 1� AbsðsampleÞAbsðcontrolÞ

� �� �� 100% ð2Þ

This DPPH method was used only for the ascorbic acid solution in order to validate the PCL method for determining the antioxi- dant activity of red raspberry puree.

2.5. Determinati on of total monomeric anthocya nins

The red raspberry total monomeric anthocyanin (ACY) content was determined by the pH differential method – a spectrophoto- metric method that involves measureme nt of absorbance at pH 1.0 and 4.5 [31]. The absorbance of the extract was measured at 520 and 700 nm using model PTP6 spectrophot ometer (PerkinElmer, San Jose, CA). Total anthocyanin s content (mg/L) was expressed as cyanidin- 3-glucoside according to equation :

Anthocyanin content mg L

� �¼ A�MW� DF� 1000

e� Lð3Þ

where A = (A520 nm, pH 1.0 � A700 nm, pH 1.0 ) � (A520 nm, pH 4.5 � A700 nm,

pH 4.5 ) with each reading corrected for haze at 700 nm; MW:molecular weight of cyanidin- 3-glucoside = 449.2, g/mol; DF:dilution factor; e: molar absorptivit y of cyanid in-3-gluco -side = 26,900; and L: cell path length (1 cm).

0.6

0.8

1.0

nge

in A

OA

(C

/C0)

2.6. Total phenolics content of red raspberry puree

The total phenolic s content (TPC) of sonicated red raspberry was determined according to the Folin–Ciocalteu’s method [32].Briefly, 20 ll of diluted sample was mixed with 1580 ll of DI water and 100 ll of 2 N Folin–Ciocalteu’s reagent. The mixture was left to stand for 3 min at room temperat ure before adding 300 ll sodium carbonate solution (20% w/v). The cuvettes were incubated at 40 �Cfor 30 min and absorbance read at 765 nm using deionized water as blank. The TPC was reported as mg Gallic acid equivalent per liter (mg/L). The standard curve was plotted within 50–500 mg/L of Gallic acid in DI water. The stock solution (0.5 g/100 ml) was prepared in 10% aqueous ethanol.

0.0

0.2

0.4

10 20 30

Cha

Sonication time (min)

20kHz 490 kHz 986 kHz

Fig. 3. Change in antioxidant activity (AOA) of ascorbic acid solution measured with PCL assay up to 30 min sonication at 986, 490, and 20 kHz. Each column represents mean of three individual experiments (p < 0.05) and bars are standard error.

2.7. Statistical analysis

All experiments were repeated and data reported represents three measure ments from three independent ultrasound treat- ments or chemical tests. The effect of ultrasound on each of the tested paramete rs was determined by analysis of variance (ANOVA) using SAS statistical analysis program version 9.0 (SAS, Cary, NC). The significance level (p-value of 0.05) was used to assess whether the individual treatments presented statistical ly different results. The difference among group means was tested with Duncan’s test.

3. Results and discussion

3.1. Effect of ultrasound on the antioxidan t activity of ascorbic acid

The DPPH and PCL analysis showed that the antioxidant activity of the ascorbic acid solution did not change significantly (p < 0.05)when low frequenc y high intensity ultrasound (20 kHz) or medium intensity ultrasound frequenc y (490 kHz) was used. However, the PCL analysis showed that there was significant drop in antioxidant activity of ascorbic acid solution after 30 min of sonication at the highest frequency (986 kHz) investigated (Fig. 3). This apparent drop was not observed with the DPPH assay (data not shown). At 986 kHz, the PCL assay showed a 7.5% decrease in antioxidant activity after 10 min and then another drop (47.6%) was recorded after 30 min. There was a good correlation between the results of antioxidant activity measured with DPPH and PCL methods except for the data obtained at 986 kHz. Because of this observed correla- tion, the PCL method was chosen to analyze the raspberry puree as it was possible to measure changes in antioxidant activity down to concentr ations as low as 0.5 nmol of ascorbic acid.

Ultrasound is reported to have a minimal effect on the quality of fruit juices that contain heat labile vitamins [33]. In one study, the ascorbic acid content of orange juice treated with ultrasound de- creased by only 5% when a maximum acoustic energy density of 0.81 W/cm 3 was used [34]. However , ascorbic acid degradation is greatly affected by temperature and the loss is more rapid at high- er temperatures. The temperature used in this study did not exceed 34 �C at the two lower frequenc ies (20 and 490 kHz), so the effect of temperature on extractio n was eliminated and any change in antioxidant activity could only be attributed to ultrasound. In the case of 986 kHz, the observed drop was probably due to the syner- gic effect of the ultrasound and temperature increase. Vikram et al.[35] found that with conventional heating of orange juice at 50 and 75 �C, it took 65.7 and 27.0 min, respectivel y, for ascorbic acid con- centration to decrease to one tenth of its initial value. Ascorbic acid degradat ion during ultrasound treatment has been attributed mainly to free radical production and extreme physical conditions experienced under power ultrasound treatments [36].

3.2. Effect of ultrasound on product temperature

Table 1 shows changes in temperature of ascorbic acid solution and red raspberry puree during sonication at the three tested

0.0

0.2

0.4

0.6

0.8

1.0

100 600 1200 1800

Pow

er in

put d

ensi

ty (

W/c

m3 )

Sonication time (sec)

20 kHz 490 kHz 986 kHz

Fig. 4. Relation between change in power (P), antioxidant activity (AOA) and anthocyanins content (ACY) at 20 kHz showing significant (p < 0.05) increase in the AOA and ACY at 10 min compared to the base. Each column is the mean of three individual experiments and bars show the standard error.

Table 1Temperature (�C) of red raspberry puree and ascorbic acid solution during ultraso und treatment in a reactor with ice bath cooling.

Samples 20 kHz 490 kHz 986 kHz

A1 24.5 ± 0.1 20.6 ± 0.5 19.1 ± 0.5 A2 33.8 ± 0.5 21.0 ± 0.7 48.0 ± 0.1 J1 23.6 ± 0.7 17.0 ± 0.9 25.5 ± 0.3 J2 44.6 ± 0.6 26.2 ± 0.6 56.5 ± 0.2

A1: ascorbic acid solution before treatment; A2: ascorbic acid solution after treat- ment (30 min); J1: red raspberry juice before treatment; J2: red raspberry juice after treatment (30 min). Each value is the mean of three measurements.

0.0

0.2

0.4

0.6

0.8

1.0

0 10 20 30

Pow

er (

W/c

m3 )

, AO

A (

mm

ol/1

00m

l) o

r AC

Y

(g/l)

Sonication time (min)

Power(watt/ml) AOA(mmol/100ml) ACY(g/l)

bb

ba

c

e ed

ig. 5. Change in the power density during sonication of ascorbic acid solution at 0, 490 and 986 kHz. Each column is the mean of three individual experiments and ars show the standard error.

1320 A. Golmohamadi et al. / Ultrasonics Sonochemistry 20 (2013) 1316–1323

frequencies. The results showed that with ascorbic acid solution,only a 9.3 �C temperat ure increase was recorded after 30 min of sonication using the 20 kHz system (with cooling). However, under the same setting, a significant increase (48 �C) in temperature was observed without cooling. This depression of temperature increase showed that the designed cooling system was very efficient during the treatment of ascorbic acid solution. The power intensity com- parison of three tested frequenc ies in ascorbic acid solution showed that within the first 100 s, 986 kHz created almost two or- ders of magnitude higher acoustic energy than 20 kHz and 3.5 times higher than 490 kHz. After 10 min the increase in medium temperature was much lower and the acoustic power delivered al- most leveled off (Fig. 4). A similar pattern was observed with red raspberry puree (data not shown).

The temperat ure increased by about 44.6 �C (at an average rate of 0.7 �C/min) during a 30 min sonication of red raspberry puree at 20 kHz. Sonication of ascorbic acid solution at 986 kHz (with the ice bath cooling) resulted in a final temperature very close to 50 �C after 30 min. The same experiment, without cooling, resulted in a final temperature of 83.3 �C. The maximum temperat ure of the puree was recorded close to the sonotrode or transducer. Temper- ature increases were higher within the first 10 min (contributing to almost 80–85% of total increase) than in the next 20 min when the change was more gradual.

High temperature s coupled with exposure to molecula r oxygen may degrade certain groups of bioactive compound s including anthocyanin s which are the major group in red raspberry. How- ever, the degradation of anthocyanins increases with processing time and temperat ure and the effect of the latter was particularly true above 60 �C. The half-life of cynidin-3 -glucoside at 60 �C and

F2b

90 �C was reported as 16.7 and 2.9 h, respectively [37]. Now, the highest temperature of red raspberry puree after sonication for 30 min at 986 kHz was 55.4 �C, suggestin g that any effects on ACY content would be minimal.

It is known that elevated temperat ures enhance extractio n effi-ciency as the diffusion coefficient is increased and solubility of the target compound s into the solvent is boosted. Additionally, cell wall structure also gets weakened, thus facilitating the extraction of more from cellular compounds [38,39]. Therefore, an increase in temperat ure may not always lead to undesirabl e effects, but abalance must be struck between extraction yield and possible loss of antioxidant activity. Blueberry anthocya nins are shown to be al- most stable in the juice up to 60 �C; and after 120 min of heating,only 10% loss in total anthocya nin content was observed [40]. The anthocya nins of blackberry juice and blood orange juice showed anegligible drop (<1%) at temperature s below 70 �C, but the drop in- creased significantly at temperature s above 70 �C [41].

3.3. Change in total monomeric anthocyanin content

Total monomer ic anthocyanin (ACY) content of red raspberry puree increased by 12.6% at 20 kHz (Fig. 5) and by 6.7% at 490 kHz after 10 min sonicatio n (p < 0.05) (Table 2). However, at 20 kHz, no further significant increase was recorded after 20 min.At 986 kHz no significant change in ACY content was observed .Holtung et al. [42] also found no significant change in TPC and ACY content after black currant press residue was sonicated at 20, 40, 55 and 80 �C for 15 and 30 min. Tiwari et al. [7] similarlyobserved only a slight increase (<1%) in the anthocyanin content of strawber ry juice sonicated for less than five minutes at 20 kHz and lower amplitudes (<60% of full scale). Chen et al. [24] sug-gested keeping the temperature below 40 �C to optimize the extractio n of anthocyanin s from red raspberry. In our case, the temperat ure of red raspberry puree reached 35 �C within the first10 min of sonication, and after 20 min it was close to 40 �C (whichwas within the optimal temperature range). This increase in tem- perature improved the mass transfer, resulting in more extractio n.The acoustic power intensity increased by 80–85% within the first10 min to positively affect the extraction rate. The extractable and/ or measurable anthocyanin content can be increased via sonication as was observed within the first 10 min when cavitation effects caused significant cell disruption. Thereafter, other factors such as solvent type may play a greater role.

Tabl

e 2

Effe

ct o

f ul

tras

ound

fre

quen

cy a

nd t

ime

on t

otal

ant

ioxi

dant

act

ivit

y (A

OA

),to

tal

phen

olic

con

tent

(TP

C)an

d m

onom

eric

ant

hocy

anin

con

tent

(A

CY)

of r

ed r

aspb

erry

pur

ee.

Freq

uen

cy (

kHz)

Tota

l an

tiox

idan

t ac

tivi

ty (l

mol

/l)

Tota

l ph

enol

ic c

onte

nt

(mg/

l)M

onom

eric

an

thoc

yan

in c

onte

nt

(mg/

l)

010

20

30

0

10

20

30

010

20

30

20

6189

±94

c72

60 ±

195 a

7464

±89

a68

08 ±

12 b

1454

±29

b15

29 ±

28 ab

1594

±57

a16

28 ±

30 a

282

±3b

317

±8a

291

±3b

248

±8b

490

3388

±58

b36

56 ±

84 a

3884

±64

a37

58 ±

131 a

1161

±35

a11

30 ±

28 a

1151

±24

a10

97 ±

6a31

9 ±

3b34

1 ±

4a34

1 ±

0a32

8 ±

4b

986

3412

±61

a35

47 ±

71 a

3522

±10

2 a34

06 ±

63 a

1107

±47

b11

03 ±

14 b

1086

±17

b12

28 ±

38 a

305

±10

a31

5 ±

2ab29

3 ±

10 ab

289

±10

a

Mea

n ±

stan

dard

err

or o

f th

r ee

mea

sure

men

ts.M

ean

s in

eac

h r

ow w

ith

dif

fere

nt

lett

ers

are

sign

ifica

ntl

ydi

ffer

ent

(p<

0.05

).

A. Golmohamadi et al. / Ultrasonics Sonochemistry 20 (2013) 1316–1323 1321

Chen et al. [24] showed that at temperat ures below 40 �C, the optimum extraction time was 3.3 min with 400 W power input.This processin g condition extracted 34.5 mg of anthocyanin s(cyanidin-3-glucoside equivalent) per 100 g of fresh fruit which was similar to the yield after 53 min of conventional extraction at 71 �C. The improvem ent in extractio n time was attributed to cavitation which increases the number of damaged cells and/or mass transfer from cell matrix to solvent. The power input for 20 and 986 kHz was similar, but the 986 kHz treatment had minimal effect on the puree because, as the sonication frequency ap- proached 1 MHz, the cavitation was not sufficient to disrupt the cell walls. Therefore, no noticeable extractio n occurred and hence the level of anthocya nins remained constant.

3.4. Effect of sonication on total phenolic content of red raspberry puree

There was no significant change in TPC of red raspberry puree at all the ultrasound frequencies (p < 0.05) investiga ted except for sonicatio n at 986 kHz for 30 min where there was 10.9% increase (Table 2). This increase can be attributed to the greater overall temperat ure increase at this frequency. Viljanen et al. [43] re-ported that two major phenolics compounds could be found in red raspberry, with elagitanins and anthocyanin s accountin g for 51% and 31% of total phenolic s, respectivel y. Our results showed that none of the three ultrasound frequencies had any negative ef- fect on the total phenolics content of the red raspberry puree. No significant increase in total phenolics content was observed after 10 min of sonication using 20 and 490 kHz; however, the recovery of anthocyanins increased. This suggests that anthocyanins may not have contributed much to the observed total phenolics content of the red raspberry puree.

The increase in total phenolics at 986 kHz could be explained by temperat ure increase which can increase the transfer of the red raspberry phenolic compounds from the puree to the extraction solvent. This suggests that this approach could be used to extract the same compounds from pulp, seeds or both. It is probable that the synergic effect of temperature and sonication caused the in- crease in total phenolics content. In a study with black currant press-res idue [42], the effect of extraction time on total phenolics content and total monomeric anthocyanin s content was not depende nt on extraction temperature when the extraction med- ium was kept at lower temperat ures (�20 �C). However, at higher temperat ures (40, 55 and 80 �C), longer extractio n time increased the extraction of total phenolics and total monomer ic anthocya nins.

3.5. Total antioxidan t activity of red raspberry puree

The total antioxidant activity (AOA) of red raspberry puree sig- nificantly increased after 10 min (p < 0.05) of treatment at 20 (Fig. 5) and 490 kHz (Table 2). For both frequencies, no significant(p < 0.05) change was observed after 10 min except for the 20 kHz treatment where the antioxidant activity decreased by 6.6% but re- mained higher than that of the control after 30 min. Treatments at 986 kHz showed no significant change in antioxidant activity.These results revealed that power ultrasound had no deteriorative effect on antioxidants in red raspberry puree. However , results from frequenc ies tested did not support the hypothes is that in- crease in frequency can lead to an increase in the antioxidant activ- ity of the extracts. In addition, it can be inferred that the major contributor to antioxidant activity are anthocya nins, as the trend in antioxidant activity paralleled that of anthocya nin content. This agrees with the findings of Liu et al. [44] which showed that anthocya nins have major contributi on to berry color, and that

1322 A. Golmohamadi et al. / Ultrasonics Sonochemistry 20 (2013) 1316–1323

darker colored raspberry cultivars (e.g. Heritage) have higher anti- oxidant activity than the lighter colored Anne cultivar.

4. Conclusion

Results of treatments using three ultrasound frequencies (20,490 and 986 kHz) showed that none of the frequencies had nega- tive impact on major bioactive compound s in red raspberry puree.In addition, high intensity (20 kHz) and the higher frequenc y(490 kHz) ultrasound can increase the antioxidant activity and to- tal monomeric anthocyanin content of red raspberry puree due to better extraction. Ultrasound treatment at 490 kHz had less effect compared to 20 kHz while treatment at 986 kHz had no significanteffect on antioxidant activity and total monomeric anthocya nin content. The greatest positive impact was the increase in antioxi- dant activity and total monomeric anthocyanin content by 17.3%and 12.6%, respectively when 20 kHz was applied for 10 min. No decrease in the antioxidant activity of ascorbic acid was observed at 20 kHz. The positive effects of ultrasoun d assisted extraction de- creased at higher frequencies regardless of power input. The re- sults also showed that ultrasound extraction at the three tested frequencies was most efficient up to10 min. Treatment tempera- ture and 986 kHz had a synergic effect on the loss of vitamin C after 30 min of sonication although the antioxidant activity and total phenolic content of red raspberry puree did not change at this higher frequenc y. As frequenc y increased, there was less cavitation and the investiga ted bioactive compounds in red raspberry puree were less affected. Sonication at different amplitudes and power intensities may cause extraction effects similar to use of different frequencies. Therefore, other condition s for achieving maximum extraction should be investigated to define parameters that would be suitable for treatment of similar fruit purees.

Acknowled gement

This work has been funded by the USDA NIFA Grant #2009- 35503-0520 7. The authors would like to thank Dr. Farhad Foroudi for his valuable help with statistical analysis of the data.

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