DSpace at VNU: Comparison of enzyme-assisted and ultrasound-assisted extraction of vitamin C and phenolic compounds from acerola (Malpighia emarginataDC.) fruit

International Journal of Food Science and Technology 2012 Original article Comparison of enzyme-assisted and ultrasound-assisted extraction of vitamin C and phenolic compounds from acerola (Malpighia emarginata DC.) fruit Hong Van Le & Van Viet Man Le* Department of Food Technology, Ho Chi Minh City University of Technology, 268 Ly Thuong Kiet Street, District 10, Ho Chi Minh City, Vietnam (Received September 2011; Accepted in revised form January 2012) Summary This article describes a comparative study of enzyme and ultrasound techniques for the simultaneous extraction of vitamin C and phenolic compounds from acerola fruit Ultrasound-assisted extraction (UAE) took only to achieve the highest level of vitamin C and phenolic compounds as well as antioxidant activity of acerola juice, while enzyme-assisted extraction (EAE) took up to 120 to obtain the maximal values On the basis of kinetic model of second-order extraction, the extraction rate constant of vitamin C and phenolics in UAE increased approximately 3.1 and 2.7 times, respectively, in comparison with that in EAE In addition, the maximal level of vitamin C, phenolics and the antioxidant activity evaluated by 1,1diphenyl-2-picrylhydrazyl (DPPH), and 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) methods in UAE was 4.6%, 3.5%, 4.6% and 3.3%, respectively, higher than those in EAE Obviously, UAE is a useful method for the extraction of antioxidants from plant materials Keywords Acerola fruit, antioxidant activity, enzyme-assisted extraction, kinetic, phenolic compounds, ultrasound-assisted extraction, vitamin C Introduction Acerola (Malpighia glabra L or Malpighia emarginata DC.) is a fruit found from Central America to northern South America It has recently been introduced in subtropical areas throughout the world, including Southeast Asia The pulp is very juicy, especially possesses fruity and sweet flavour (Boulanger & Crouzet, 2001) This fruit is well known to be one of the best natural sources of ascorbic acid (vitamin C), and has become extremely popular in daily life (Hanamura et al., 2006) This fruit also contains phenolic compounds Both vitamin C and phenolic compounds are powerful antioxidants, and their biological functions prevent common degenerative processes (Hanamura et al., 2008) Acerola juice with high vitamin C and phenolic contents has, therefore, attracted much more interest in human diet (Matta et al., 2004) The classical techniques for juice extraction are pressing (Chemat et al., 2008) and enzymatic maceration (Kashyap et al., 2001; Matta et al., 2004; Lieu & Le, 2010) Nevertheless, these methods are often *Correspondent: E-mail: lvvman@hcmut.edu.vn time- and energy-consuming as well as their extraction efficiency is usually low Nowadays, a number of novel methods for target component extraction from plant materials have been applied, for example, supercritical fluid extraction (Pinelo et al., 2007), accelerated solvent extraction (Hossain et al., 2011), microwave-assisted extraction (Bai et al., 2010) and ultrasound-assisted extraction (UAE) (Vilkhu et al., 2008; Khan et al., 2010) Among these methods, the technology of UAE has shown high extraction efficiency, low-energy and solvent consumptions (Pan et al., 2011) In recent years, there have been several researches on the application of UAE of various bioactive compounds, for instance, UAE of phenolic compounds from strawberries (Herrera & Luque de Castro, 2005), coconut shell powder (Rodrigues & Pinto, 2007), citrus peel (Ma et al., 2009; Khan et al., 2010) and olive fruit (Jerman et al., 2010) However, so far, there has not been report on UAE of antioxidant-rich acerola juice yet, which has both high vitamin C and phenolic contents The objectives of this study were to (i) investigate the effects of enzyme-assisted extraction (EAE) and UAE variables on extraction yield of vitamin C and phenolic compounds from acerola fruit, (ii) determine the kinetic doi:10.1111/j.1365-2621.2012.02960.x Ó 2012 The Authors International Journal of Food Science and Technology Ó 2012 Institute of Food Science and Technology Comparison of enzyme-assisted and ultrasound-assisted extraction H V Le and V V M Le parameters of vitamin C and phenolic extraction process, and (iii) compare the acerola juice quality obtained from both methods Materials and methods Materials Enzyme source Commercial enzyme preparation ‘Celluclast 1.5L’ produced by Trichoderma reesei was obtained from SigmaAldrich (Singapore) The enzyme activity is 1500 Novo Cellulase Unit per gram (NCU g)1) One NCU is the amount of enzyme which, under standard conditions, degrades carboxymethylcellulose to reducing carbohydrates with a reduction power corresponding to lmol glucose per minute (Arapoglou et al., 2010) The optimal temperature and pH of this enzyme preparation are 50–60 °C and 4.5–6.0, respectively (Sørensen et al., 2003) Plant material Acerola (Malpighia emarginata DC.) used in this study was purchased from a farm in Go Cong, Vietnam The fruits were harvested during the period from July to December in 2010 The bright orange fruits without disease symptoms were selected Chemicals 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) were purchased from Sigma-Aldrich (Singapore) Folin–Ciocalteu reagent, l-ascorbic acid, methanol, ethanol, gallic acid, potassium persulphate (K2S2O8), 85% phosphoric acid (H3PO4) solution, potassium dihydrogen phosphate (KH2PO4) and anhydrous sodium carbonate (Na2CO3) were obtained from Merck (Darmstadt, Germany) All reagents were of analytical grade Double-distilled water was used throughout experiments Extraction methods Acerola was destemmed, washed and crushed in a blender (Panasonic, MJ 70M, Selangor, Malaysia) In this study, water was used as the extraction solvent for both methods Water was considered to be an efficient extraction solvent for antioxidant production from some plant materials (Mezadri et al., 2008; Pan et al., 2011) Moreover, water is an environmentally friendly extraction solvent, and using water, we could obtain antioxidant-rich acerola juice, but not only antioxidant compounds (vitamin C and phenolics) in acerola fruit After crushing, acerola mash was mixed with water at the weight ratio of water to acerola mash of 2:1 and subsequently used for juice extraction International Journal of Food Science and Technology 2012 Enzyme-assisted extraction For each assay, samples of 30 g diluted acerola mash were taken and placed into 250-mL beakers, which were covered with aluminium-foil papers to prevent the oxidative change from light First series: Different amounts of Celluclast 1.5L were added into beakers of samples Enzyme concentrations were 0.15%, 0.3%, 0.45%, 0.6%, 0.75%, and 0.9% (v ⁄ w) These values were equivalent to 0, 2.7, 5.4, 8.1, 10.8, 13.5, and 16.2 NCU g)1 of diluted acerola mash (NCU g)1), respectively The samples were then incubated in the period of 30 Second series: An amount of Celluclast 1.5L (8.1 NCU g)1) was added into beakers of samples The extraction times were varied from 30 to 150 In both series, the control samples were untreated with enzyme preparation Extraction temperature was adjusted to 50 °C using a thermostatic water bath (Memmert, Jakarta, Indonesia) After the period of incubation, the mash was filtered through a cheese cloth The obtained suspension was separated by a refrigerated centrifuge (Sartorius, Sigma 3K30, Geneva, Switzerland) at 1370 g for 10 at 10 °C, and the supernatant was collected for further analysis Ultrasound-assisted extraction The UAE was performed with a horn-type ultrasonic probe with frequency of 20 kHz (Sonics and Materials Inc, VC750, Newtown, MA, USA) For each assay, samples of 30 g of diluted acerola mash were taken and placed into 100-mL beakers which were covered with aluminium-foil papers to prevent oxidative change from light First series: Ultrasonic power levels were adjusted to 150, 300, 450 and 600 W, respectively Because each sample contained 30 g diluted acerola mash, the ultrasonic power per gram of the material would be 5, 10, 15 and 20 W g)1, respectively The ultrasonic time was Second series: The ultrasonic power of 15 W g)1 was applied The ultrasonic times were ranged from to 10 In both series, the control samples were untreated with ultrasound Because the temperature of the samples was gradually increased during the UAE, a water bath with cooled water (5–8 °C) was used to maintain the sample temperature not to exceed 50 °C At the end of the process, the mash was also filtered and separated in the same way as shown in the EAE Determination of kinetic parameters of vitamin C and phenolic extraction from the second-order kinetic model To determine the extraction rate constant of vitamin C, the second-order rate law was applied (Pan et al., 2011) The general second-order model can be written as: Ó 2012 The Authors International Journal of Food Science and Technology Ó 2012 Institute of Food Science and Technology Comparison of enzyme-assisted and ultrasound-assisted extraction H V Le and V V M Le dCt ẳ k Ce Ct ị2 dt 1ị where, k is the second-order extraction rate constant (L g)1 min)1), Ce is the extraction capacity (the equilibrium concentration of vitamin C in acerola juice) (g L)1), and Ct is the concentration of vitamin C in acerola juice at a given extraction time (g L)1) The integrated rate law for a second-order extraction, under the boundary conditions t = to t and Ct = to Ct, can be written as an equation (2) or a linearised equation (3): C2e kt ỵ Ce kt 2ị t t ẳ ỵ Ct kC2e Ce 3ị Ct ẳ The initial extraction rate, h (g L)1 min)1), as Ct ⁄ t when t approaches 0, can be defined as equation (4): h ¼ k C2e ð4Þ The initial extraction rate, h, the extraction capacity, Ce, and the second-order extraction rate constant, k, can be determined experimentally from the slope and the intercept by plotting t ⁄ Ct vs t The kinetic parameters of phenolic extraction were also calculated in the same way of those of vitamin C extraction Comparison of acerola juice quality obtained from enzymeassisted extraction (EAE) and ultrasound-assisted extraction (UAE) Enzyme-assisted extraction and UAE were carried out in the appropriate conditions obtained from the above experiments of each method The two methods were compared on the increased percentage of vitamin C, phenolic compounds and the antioxidant activity of acerola juice using control sample as the base The control sample was untreated with both enzyme preparation and ultrasound Analytical methods LC-10AS, Kyoto, Japan), a UV detector (Shimadzu, SPD-6AV, Kyoto, Japan) and a C18 column (250 · 4.6 mm I.D., lm) (Shimadzu, Gemini 5u C18 110A, Kyoto, Japan) The procedure was carried out according to the method of Abushita et al (1997) with slight modifications The mobile phase was a mixture of two solvents: A (potassium dihydrogen phosphate in water adjusted to pH 2.8) and B (methanol) with the ratio of 9:1 (v ⁄ v) The constant flow rate was 1.5 mL min)1 The column was maintained at room temperature, and the injection volume was 20 lL Ascorbic acid elution was monitored at 245 nm The content of ascorbic acid in acerola juice was expressed as grams per litre (g L)1) and was calculated using an external calibration curve prepared with the standard ascorbic acid According to Mezadri et al (2008), ascorbic acid is the predominant form of vitamin C in acerola; hence, the content of ascorbic acid was used to present the content of vitamin C in acerola juice in our study Antioxidant activity The procedure for antioxidant activity evaluation followed the method of Thaipong et al (2006) with slight modifications For DPPH assay, briefly, the stock 0.1 mm DPPH violet solution was diluted with methanol to obtain an absorbance of 1.1 ± 0.02 units at 515 nm The reaction medium contained mL of diluted DPPH• solution and 265 lL diluted acerola juice for the sample or methanol solution of Trolox (25–700 lm) for the standard, or water for the blank The reaction mixture was kept at room temperature in the dark for 20 min, and the absorbance was measured at 515 nm Antioxidant activity of acerola juice was expressed as millimolar Trolox equivalent antioxidant capacity per litre (mm TEAC) For ABTS assay, once the radical was formed, the ABTS·+ solution was diluted with ethanol to obtain an absorbance of 0.7 ± 0.02 units at 734 nm The reaction then started by adding 400 lL diluted acerola juice to mL of diluted ABTS•+ radical cation solution, and the absorbance was measured after at 734 nm Standard Trolox solutions (25–500 lm) were also evaluated against the radical Antioxidant activity of acerola juice was expressed as mmol Trolox equivalent antioxidant capacity per litre (mm TEAC) Phenolic compounds Total phenolic content in acerola juice was determined as previously described by Luque-Rodrı´ guez et al (2007), using Folin–Ciocalteu reagent The results were expressed as the equivalent to grams of gallic acid per litre of acerola juice (g GAE L)1) Vitamin C In this study, vitamin C was quantified by HPLC The HPLC system used included a pump (Shimadzu, Statistical analysis All experiments were performed in triplicate The experimental results obtained were expressed as means ± SD (standard deviation) Mean values were considered significantly different when P < 0.05 Analysis of variance was performed using the software Statgraphics plus, version 7.0 (Manugistics, Inc., Rockville, MD, USA) Ó 2012 The Authors International Journal of Food Science and Technology Ó 2012 Institute of Food Science and Technology International Journal of Food Science and Technology 2012 Comparison of enzyme-assisted and ultrasound-assisted extraction H V Le and V V M Le 21.2 18.2 20.2 19.2 Phenolics 17.7 Vitamin C 17.2 2.7 5.4 8.1 10.8 13.5 16.2 Phenolics (g GAE L–1) (a) 19.2 18.2 27.2 25.2 18.7 23.2 18.2 21.2 17.7 Phenolics 19.2 Vitamin C 17.2 17.2 18.9 30 60 90 120 150 180 17.2 Time of enzymatic treatment (min) Enzyme concentration (NCU g–1) (b) 140 Antioxidant activity (mM TEAC) (b) 130 120 110 100 DPPH ABTS 90 Vitamin C (g L–1) 22.2 18.7 Vitamin C (g L–1) Phenolics (g GAE L–1) (a) Antioxidant activity (mM TEAC) 2.7 5.4 8.1 10.8 13.5 16.2 18.9 130 120 110 90 Enzyme concentration (NCU g–1) DPPH 100 ABTS 30 60 90 120 150 180 Time of enzymatic treatment (min) Figure Effect of enzyme concentration on (a) the level of vitamin C, phenolics, and (b) antioxidant activity of acerola juice Results and discussion Enzyme-assisted extraction Figure shows the changes in the antioxidant content and antioxidant activity of acerola juice with respect to enzyme concentration The level of the target components significantly improved with the increase in enzyme concentration and approached the highest value at enzyme concentration of 8.1 NCU g)1 Higher enzyme concentration did not result in higher vitamin C and phenolic contents According to Mosier et al (1999), cellulase preparation from T reesei contained many enzymes such as endoglucanase, cellobiohydrolase and b-glucosidase These enzymes would be expected to degrade structural cellulose that makes up 27% in the primary cell wall of the acerola pulp (Lima et al., 1996) That enhanced the efficiency of the extraction process International Journal of Food Science and Technology 2012 Figure Effect of enzymatic extraction time on (a) the level of vitamin C, phenolics, and (b) antioxidant activity of acerola juice Owing to the increase in vitamin C and phenolic contents, the antioxidant activity of acerola juice also raised with the increase in enzyme concentration However, the antioxidant activities determined by DPPH method were always lower than those measured by ABTS method It could be explained that anthocyanin, which is one of the main polyphenol components in acerola fruit, absorbs maximally at 520 nm (Zanatta et al., 2005); therefore, its colour would interference with the DPPH chromogen, which has maximum absorbance at 515 nm This would lead to the results in the relatively lower activity measured by DPPH method (Awika et al., 2003) The influence of enzymatic extraction time on the antioxidant content and activity of acerola juice is listed in Fig A positive effect on vitamin C and phenolic level was observed when the time of EAE was not longer Ó 2012 The Authors International Journal of Food Science and Technology Ó 2012 Institute of Food Science and Technology Comparison of enzyme-assisted and ultrasound-assisted extraction H V Le and V V M Le Ultrasound-assisted extraction Figure illustrates the effect of ultrasonic power on the level of vitamin C, phenolic compounds and antioxidant activity of acerola juice The antioxidant concentrations increased when the ultrasonic power augmented from to 15 W g)1 (P < 0.05); however, these values did not change significantly at higher ultrasonic power (P > 0.05) The mechanism of UAE is ascribed to the acoustic cavitation, which includes the formation, growth and implosive collapse of bubbles in a liquid (Chowdhury & Viraraghavan, 2009) The implosion of cavitation bubbles generates severe turbulence, high-velocity interparticle collisions and perturbation in microporous particles of the materials, which accelerates the eddy diffusion and internal diffusion Moreover, cavitation within the proximity of solid surface causes surface erosion and particle breakdown This effect provides 23.2 (a) 19.2 18.7 21.2 20.2 18.2 17.2 19.2 Phenolics 17.7 Vitamin C 10 15 20 Vitamin C (g L–1) Phenolics (g GAE L–1) 22.2 18.2 25 17.2 Ultrasonic power (W g–1) (b) 130 Antioxidant activity (mM TEAC) than 120 A further increase in the extraction time resulted in significantly (P < 0.05) lower concentrations of vitamin C and phenolic compounds Hence, the antioxidant activity of acerola juice also declined when the biocatalytic time was prolonged To clearly understand the effect of the extraction time on the performance of vitamin C, an additional experiment was conducted with the standard solution of ascorbic acid under the same temperature and enzyme concentration The results (unpublished data) showed that the ascorbic acid level was stable during the first 30 and then slightly decreased about 3.2% after 120-min incubation as compared to the initial level Finally, a remarkable reduction of 10.9% in the ascorbic acid level was observed in the 150-min treated sample compared to that in the 120-min treated sample This was attributed to the thermo-oxidative degradation caused by high temperature and overlong extraction time It is interesting to note that the percentage reduction in vitamin C in the 150-min treated acerola juice compared to the 120 treated acerola juice was 10.8% (Fig 2) This similarity suggested that the extraction of vitamin C could not continue when the enzymatic extraction time was longer than 120 In summary, the results indicated that the efficient extraction period with enzyme concentration of 8.1 NCU g)1 was 120 Under these appropriate conditions, the level of vitamin C, phenolics and antioxidant activity of acerola juice based on DPPH and ABTS methods rose approximately 35.7%, 9.0%, 23.9% and 22.6%, respectively, in comparison with those of the control sample Although pectinase has been widely used in juice extraction (Kashyap et al., 2001; Lieu & Le, 2010), our results revealed that cellulolytic enzyme is also a potential biocatalyst to obtain antioxidant-rich fruit juice 120 110 100 DPPH ABTS 90 10 15 20 25 Ultrasonic power (W g–1) Figure Effect of ultrasonic power on (a) the level of vitamin C, phenolics, and (b) antioxidant activity of acerola juice exposure of new surfaces further increasing mass transfer (Vilkhu et al., 2008) Our results are consistent with previous findings (Ma et al., 2009; Pan et al., 2011) Ma et al (2009) and Pan et al (2011) reported that the yields of phenolic compounds from citrus and pomegranate peel depended significantly on the ultrasonic power level In our study, nevertheless, the content of vitamin C and phenolics kept unchanged when the ultrasonic power was higher than 15 W g)1 This may be attributed to the fact that the cavitation bubbles may grow too big to collapse or collapse weakly which could cause the reduction in the cavitation effect Also, many bubbles may hamper the propagation of the ultrasound wave (Sun et al., 2011) A positive correlation between the content of antioxidants and the antioxidant activity of acerola juice was observed (Fig 3a, b) The increase in biologically active compounds may lead to enhance the antioxidant activity Ó 2012 The Authors International Journal of Food Science and Technology Ó 2012 Institute of Food Science and Technology International Journal of Food Science and Technology 2012 Comparison of enzyme-assisted and ultrasound-assisted extraction H V Le and V V M Le Phenolics (g GAE L–1) 19.7 27.2 20.0 25.2 18.0 19.2 23.2 18.7 21.2 18.2 Phenolics 17.2 19.2 Vitamin C 17.7 10 12 Concentration of ascorbic acid standard solution (g L–1) 20.2 Vitamin C (g L–1) (a) 16.0 14.0 Uncontrolled temperature 12.0 Controlled temperature 17.2 10.0 Ultrasonic time (min) 10 12 Ultrasonic time (min) (b) 140 Antioxidant activity (mM TEAC) Figure Effect of ultrasonic time on the stability of vitamin C The ascorbic acid standard solution was sonicated at the ultrasonic power of 15 W g)1 under both uncontrolled and controlled temperature conditions 130 120 110 DPPH 100 90 ABTS 10 12 Ultrasonic time (min) Figure Effect of ultrasonic time on (a) the level of vitamin C, phenolics, and (b) antioxidant activity of acerola juice of the extract Besides, ultrasound could separate sugar moiety from acerola glycosides to create aglycones It was reported that aglycones are more potent antioxidants than their corresponding glycosides (London˜oLondon˜o et al., 2010) Furthermore, the moderate sonochemical hydroxylation of phenolic compounds, which was caused by hydroxyl radicals produced during the sonolysis of water, could improve their antioxidant properties (Ashokkumar et al., 2008) The results in Fig have shown that prolongation of the ultrasonic time augmented the level of vitamin C, phenolics and antioxidant activity of acerola juice; maximum of these values achieved after 6-min sonication The vitamin C content rose approximately 40.3% compared to that in the control sample, while the increase in phenolic content was much lower with just roughly 12.5% (Fig 4a) The antioxidant activities of International Journal of Food Science and Technology 2012 acerola juice measured by DPPH and ABTS methods under this condition also increased 28.5% and 25.9%, respectively, as compared to those in the control sample (Fig 4b) To evaluate the stability of the antioxidants under sonication, the standard solution of ascorbic acid was exposed to ultrasound with the same conditions of UAE Because of polyphenol-protective ability of ascorbic acid (Altunkaya & Gokmen, 2009), we chose this component to investigate the stability of the antioxidants in acerola juice As seen from Fig 5, when the temperature was controlled to be inferior to 50 °C (the ultrasonic conditions in our study), the concentration of ascorbic acid in the standard solution did not change, whilst this value dramatically reduced up to 29.2% in the period of 10-min ultrasonic treatment without temperature control The reduction in ascorbic acid concentration under the uncontrolled temperature condition may be due to (i) the interaction of this compound with hydroxyl radicals, and ⁄ or (ii) the adverse effect of temperature on the structure of ascorbic acid Nonetheless, according to Entezari et al (2003) and Khan et al (2010), sonication at frequency of 20 kHz during short duration produced low concentration of hydroxyl radicals It was therefore concluded that the loss of ascorbic acid may be related to the decomposition of analyte caused by thermal degradation Thus, temperature was an important variable for ultrasonic extraction of vitamin C We suggested that the temperature of UAE for antioxidants in acerola fruit should not exceed 50 °C Ó 2012 The Authors International Journal of Food Science and Technology Ó 2012 Institute of Food Science and Technology Comparison of enzyme-assisted and ultrasound-assisted extraction H V Le and V V M Le (a) 9.0 Extraction rate reciprocal (min L g–1) 8.0 7.0 Phenolics 6.0 Vitamin C 5.0 4.0 3.0 2.0 1.0 0.0 30 60 90 120 150 180 Time (min) (b) 0.6 Extraction rate reciprocal (min L g–1) 0.5 Phenolics 0.4 Vitamin C UAE were also observed to augment 9.5% and 4.5% for vitamin C and phenolic compounds, respectively, in comparison with those in EAE These results concluded that UAE efficiently enhances the extraction rate of antioxidants from acerola fruit Similar kinetic effects were evidenced by Chemat et al (2004) and Khan et al (2010) for UAE of polyphenols from orange peel and essential oils from caraway seeds, respectively Likewise, ultrasound could improve the second-order kinetic parameters of polyphenol extraction from pomegranate peel (Pan et al., 2011) However, the values and the increase in the kinetic parameters estimated by these authors were much lower than those in our study It is possible that (i) the level of phenolic content in acerola fruit was higher than that in pomegranate peel, and (ii) phenolic compounds could be extracted from fruit easier than from peel As seen from Table 1, all kinetic parameters for vitamin C were always remarkably higher than those for phenolic compounds Thus, vitamin C could be extracted more efficiently than phenolics regardless of extraction methods The second-order model fitted well the experimental results owing to the obtained high coefficient of determination (Table 1) This confirmed that there are two main stages during the antioxidant extraction from acerola fruit 0.3 Comparison of acerola juice quality obtained from enzymeassisted extraction (EAE) and ultrasound-assisted extraction (UAE) 0.2 0.1 0.0 10 12 Time (min) Figure Extraction rate reciprocal (t ⁄ Ct) of antioxidants at different extraction times (t) in (a) enzyme-assisted extraction (EAE) and (b) ultrasound-assisted extraction (UAE) Extraction kinetics of enzyme-assisted extraction (EAE) and ultrasound-assisted extraction (UAE) Figure illustrates the linearised forms of the secondorder model for the two extraction methods The extraction capacity, Ce, the initial extraction rate, h, the extraction rate constant, k, and the coefficient of determination, R2, given for the two methods in Table 1, are in accordance with the graphs in Fig The results indicated that Ce, h and k were higher in UAE than those in EAE The values of the rate constant, k, were found approximately 3.1 times for vitamin C and 2.7 times for phenolics as fast in UAE as in EAE; in the same ways, the values of the extraction capacity, Ce, in In comparison with EAE, UAE improved the level of phenolic compounds and vitamin C as well as antioxidant activity of acerola juice (Table 2) Regarding vitamin C, UAE increased its value 4.6% more than EAE Besides, phenolic level in UAE was also 3.5% higher than that in EAE Our results are in agreement with many findings that UAE enhanced the yield of bioactive constituents in comparison with the conventional techniques (Vilkhu et al., 2008; Khan et al., 2010) As seen in Table 2, both EAE and UAE enhanced greater vitamin C content than phenolic content The possible reasons are that (i) vitamin C is more abundant than phenolic compounds in acerola fruit (Mezadri et al., 2008), and (ii) phenolic compounds can link with various compounds of cell walls such as polysaccharides or proteins (Lieu & Le, 2010), which may make phenolic extraction more difficult than vitamin C extraction Owing to the increase in vitamin C and phenolic contents, the increase in antioxidant activity of acerola juice in UAE was also higher in comparison with that in EAE (Table 2) It should be noted that ultrasound improved not only the content of antioxidants but also the total antioxidant activity of these compounds in our study Further experimental work on identifying the Ó 2012 The Authors International Journal of Food Science and Technology Ó 2012 Institute of Food Science and Technology International Journal of Food Science and Technology 2012 Comparison of enzyme-assisted and ultrasound-assisted extraction H V Le and V V M Le Table Comparison of the second-order kinetic parameters of antioxidant extraction in enzyme-assisted extraction (EAE) and ultrasound-assisted extraction (UAE) Component Vitamin C Phenolic compounds Extraction method Extraction capacity, Ce (g L)1) Initial extraction rate, h (g L)1 min)1) Extraction rate constant, k (L g)1 min)1) R2 EAE UAE EAE UAE 23.42 25.64 18.87 19.72 333.3 1250.0 114.9 333.3 0.61 1.90 0.32 0.86 0.992 0.998 1.000 1.000 Table Comparison of acerola juice quality obtained from enzymeassisted extraction (EAE) and ultrasound-assisted extraction (UAE) Increased level of components* (%) Antioxidant activity Extraction method Vitamin C Phenolics DPPH ABTS EAE UAE 35.7a 40.3b 9.0a 12.5b 23.9a 28.5b 22.6a 25.9b EAE: 1:2 (w ⁄ w) acerola mash ⁄ water ratio, enzyme concentration of 8.1 NCU g)1, and extraction time of 120 at 50 °C; UAE: 1:2 (w ⁄ w) acerola mash ⁄ water ratio, ultrasonic power of 15 W g)1 and ultrasonic time of at 50 °C Different small letters in the same column mean significant difference at P < 0.05 *Compared to the control sample types of phenolic compounds has to be carried out for profound understanding Conclusions Ultrasound-assisted extraction has been shown to be an efficient method for the extraction of vitamin C and phenolic compounds from acerola fruit compared to EAE The results indicated that not only higher levels of vitamin C and phenolics were achieved but also higher antioxidant activity 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by plotting t ⁄ Ct vs t The kinetic parameters of phenolic extraction were also calculated in the same way of those of vitamin C extraction Comparison of acerola. .. concentration of vitamin C in acerola juice) (g L)1), and Ct is the concentration of vitamin C in acerola juice at a given extraction time (g L)1) The integrated rate law for a second-order extraction,