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Evaluation of bioactive compounds of black mulberry juice after thermal, microwave, ultrasonic processing, and storage at different temperatures Bo Jiang, Nitin Mantri, Ya Hu, Jiayin Lu, Wu Jiang and Hongfei Lu Food Science and Technology International published online 10 June 2014 DOI: 10.1177/1082013214539153 The online version of this article can be found at: http://fst.sagepub.com/content/early/2014/06/10/1082013214539153
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Article
Evaluation of bioactive compounds of black mulberry juice after thermal, microwave, ultrasonic processing, and storage at different temperatures Bo Jiang1,2, Nitin Mantri3, Ya Hu4, Jiayin Lu5, Wu Jiang4 and Hongfei Lu1,4
Abstract The effect of different sterilization methods (thermal, microwave, and ultrasonic processing) on the main bioactive compounds and antioxidant activity of black mulberry juice during selected storage time (8 days) and temperatures (5, 15, and 25 C) was investigated. The antioxidant activity of thermal-treated juice depleted with storage time, whilst both ultrasound- and microwave-treated juices showed transient increase in antioxidant activity during the first 2 days that later decreased with storage time. Lower temperature storage preserved more bioactive compounds and antioxidant activity, especially in ultrasound sterilized samples. The activation energy values were 15.99, 13.07, and 12.81 kJ/mol for ultrasonic, microwave, and thermal pasteurization processes, respectively. In general, ultrasound-sterilized samples showed higher total phenolics, anthocyanin, and antioxidant activity compared to the microwave- and thermal-processed juice during the storage time especially at lower temperatures.
Keywords Black mulberry juice, anthocyanin content, antioxidant activity, thermal, ultrasound, storage Date received: 23 November 2012; accepted: 5 May 2014
INTRODUCTION Recently, fruit juice obtained from mulberry (Morus species) is increasingly being promoted and consumed because of the reported nutritional and health benefits (Hojjatpanah et al., 2011). It is a rich source of bioactive compounds including phenolic substances like flavonols and anthocyanins with high antioxidant activity (Isabelle et al., 2008). Anthocyanins are the most important phenolic compounds present in berry fruits that are of special significance because of their contribution to total antioxidant activity of the fruit (Hassimotto and Genovese 2007). The major anthocyanins identified in mulberries are cyanidine-3-glucoside and cyanidine-3-rutinoside (Hojjatpanah et al., 2011; Suh et al., 2003). These anthocyanins are unstable Food Science and Technology International 0(0) 1–8 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1082013214539153 fst.sagepub.com
and easily susceptible to degradation through a number of factors such as light, pH, oxygen, enzymes, ascorbic acid, and temperature during storage and especially heat processing (Hojjatpanah et al., 2011; Kennedy et al., 2001). The stability of anthocyanins found in foods decreases during processing and storage as temperature rises (Cavalcanti et al., 2011). Anthocyanins show great susceptibility toward pH 1
College of Life Science, Zhejiang Sci-Tech University, Hangzhou, China 2 College of Biology and Food Engineering, Changshu Institute of Technology, Changshu, China 3 Health Innovations Research Institute, School of Applied Sciences, RMIT University, Melbourne, Australia 4 College of Chemistry and Life Science, Zhejiang Normal University, Jinhua, China 5 Division of General Studies, University of Illinois at UrbanaChampaign, Champaign, IL, USA Corresponding author: Hongfei Lu, College of Life Science, Zhejiang Sci-Tech University, Hangzhou, 310018, China. Email: [email protected]
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Food Science and Technology International 0(0) being more stable in acidic media at low pH values than in alkaline solutions with high pH values (Brouillard, 1982; Cavalcanti et al., 2011). Sterilization is an essential process to improve the shelf-life and safety of mulberry juice. Conventional thermal processing of fruit juice remains the most widely adopted technology for shelf-life extension and preservation of fruit juice. However, it often leads to detrimental changes in the sensory qualities of the juice (Odriozola-Serrano et al., 2008). Ultrasound processing is a promising non-thermal processing technology and a potential alternative or supplement to traditional thermal pasteurization (Tiwari et al., 2009c). The use of sonication to provide fresh high-quality, microbiologically safe, and high-nutritional-value fruit juices continue to be an area of research as shown in recent studies conducted on orange juice (Valero et al., 2007), strawberry juice (Tiwari et al., 2008), and blackberry juice (Tiwari et al., 2009a). Microwave pasteurization offers similar benefits to conventional methods, but with an improved product quality and reduced time of exposure to energy (Can˜umir et al., 2002). Several studies have successfully been conducted on the microwave pasteurization of fruit juices, as it preserves the natural organoleptic characteristics of the juice and reduces the time of exposure to energy, with the subsequently lower risk of losing essential thermolabile nutrients (Igual et al., 2010). Evaluating the influence of different sterilization methods on the bioactive compounds of mulberry juice during storage is essential for determining the best conditions for preserving the bioactive properties of the juice. In addition, no information is available on the degradation kinetics of anthocyanins during the storage of ultrasound- and microwave-processed mulberry juice to predict the quality changes that occur during storage. Hence, the main objective of this study was to investigate the effect of different sterilization methods including thermal, microwave, and ultrasound sterilization on the quality attributes of black mulberry juice as well as the anthocyanin degradation kinetics of these differently processed mulberry juices during storage.
MATERIALS AND METHODS Sample preparation Black mulberry fruits (Morus nigra L) were purchased from a local fruit market in Jinghua (Zhejiang, China). Black mulberries were selected on the basis of a similar degree of ripeness and apparent fruit quality. The fruits were squeezed using a domestic juice extractor and immediately filtered using four layers of cheese cloth to remove the pulp before different sterilization treatments.
Experimental design All fresh juice filtrated was randomly divided into three groups (three individuals per group) among which one group was treated by thermal sterilization, and others were treated with microwave and ultrasonic processing, respectively. The total phenols, anthocyanin content, and 2,20 -diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of black mulberry juice before and after three different pasteurization processes were measured to evaluate the effect of different methods of sterilization on the quality of mulberry juice. Treatments A 650 -W high-intensity ultrasonic processor (Ningo Scientz biotechnology Co. Ltd., Ningbo, China) with a 6-mm titanium probe whose immersion depth was 25 mm in a 100 mL test tube was used for juice sonication. The juice samples were sonicated for 30 min and processed at a constant frequency of 20 kHz (Bhat et al., 2011). In the case of ultrasonic-treated juice, samples of 50 mL were placed in a 100 mL jacketed vessel through which water at 20 1 C was circulated at a flow rate of 0.5 L/min. The microwave process was performed in a microwave oven (Galanz WD900ASL23-5 S, 23 L, 900 W). Each time, 50 mL of the juice was treated by microwave for 30 s (Igual et al., 2010). For thermal pasteurization, 50 mL of the juice was heated in glass tubes in a thermostatic water bath operating at 90 C for 30 s. After the different sterilization processes these black mulberry juices were rapidly cooled in an ice bath and were poured separately into sterile test tubes; then these samples of every sterilization treatment were stored in incubators at 5, 15, and 25 C, respectively in the dark for 8 days. Samples were randomly taken and analyzed for each index every day during storage. All analyzed samplings were carried out in triplicate. Chemicals and reagents Trolox and DPPH free radical were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA). Gallic acid and Folin–Ciocalteu reagent (purity 99%), were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Total phenols content determination The total phenols content was determined by using Folin–Ciocalteu reagent method, as described by our previous study (Lu et al., 2012). Total phenols assay was conducted by mixing 0.5 mL of black mulberry juice sample, 8.25 mL of deionized water, 0.5 mL of Folin–-Ciocalteu reagent, and 0.75 mL of 20%
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Jiang et al. Na2CO3. After 40 min of reaction in a water bath at 40 C, the absorbance of the reaction mixture was measured at 755 nm using a spectrophotometer. Results were expressed as milligrams gallic acid equivalents (GAE) per 100 mL of juice.
where MW is the molecular weight of cyanidin-3-glucoside, DF is the dilution factor, L is the path length in cm, and " is the molar extinction coefficient for cyanidin-3-glucoside. Results were expressed as milligram cyanidin-3-glucoside equivalents (CGE) per 100 mL of juice.
DPPH radical scavenging activity determination The DPPH free radical scavenging activity was evaluated according to the method described by Zheng et al. (2010). Briefly, 0.1 mL mulberry juice sample was mixed with 10 mL of 0.03 g/L DPPH (2,2-diphenyl-1-picrylhydrazyl) ethanol solution at room temperature. The diluted solution (0.1 mL) with 10 mL distilled water was used as control. The absorbance was measured at 517 nm after 30 min of reaction in the dark. Scavenging activity was measured in triplicate samples. The percentage of DPPH radical scavenging activity of each plant extract was calculated using the following equation
Statistical analysis
The data of total phenolic compounds, anthocyanin, and antioxidant activity were expressed as mean standard deviation (SD) from three sterilization treatments (each treatment with three samples) and each sample with three replications. Pearson’s correlation coefficient was used to calculate correlations among the data obtained. Statistical analyses were carried out using the statistical analysis systems (SAS, version 9.0) software package and Excel statistical tools (Microsoft software). The data were graphically plotted by using OriginLab (OriginPro, version 8.0). A0 ðA1 As Þ Experimental data were fitted to a first-order kinetic 100 Scavenging activity ð%Þ ¼ A0 model (equations (2) and (3)) to describe the evolution ð1Þ of anthocyanins in various processed mulberry juices during storage. This kinetic type was expressed by the following equations where A0 is the absorbance of the control solution (containing only DPPH), A1 is the absorbance of the DPPH C ¼ C0 expðktÞ ð3Þ solution containing plant extract, and As is the absorbance of the sample extract solution without DPPH. The value was measured in triplicate samples.
T1=2 ¼ ln 0:5=k
Anthocyanins content determination Total anthocyanins content of each sample was measured using the modified pH differential method described by Zheng and Lu (2011), using two buffer systems: potassium chloride 0.025 M at pH 1.0 and sodium acetate 0.4 M at pH 4.5. Briefly, 1 mL of sample was transferred to a 10 mL volumetric flask and made up with each buffer. The absorbance of each equilibrated solution was then measured at 510 and 700 nm, using a UV–Vis spectrophotometer. Quartz cuvettes of 1-cm path length were used, and all measurements were carried out at room temperature (25 C). Absorbance readings were made against distilled water as a blank. The total anthocyanins content was calculated on the basis of cyanidin-3-glucoside (Alasalvar et al., 2005) with a molecular weight of 445.2 g/mol and an extinction coefficient of 29,600 L/mol cm (Giusti and Wrolstad, 2001), as
AC ¼ ½ðA510 A700 ÞPH 1:0 ðA510 A700 ÞPH 4:5 MWDF1000="L
ð2Þ
ð4Þ
where C and C0 are anthocyanins content at time t and zero (day), respectively and k is the first-order rate constant, while T1/2 is the half-life and t is the storage time. Temperature-dependence of anthocyanin degradation rate constant was determined by the Arrhenius equation
k ¼ A expðEa =RTÞ
ð5Þ
where A is the frequency factor (per day), Ea is the activation energy (kJ/mol), R is the universal gas constant (8.314 J/mol/K), and T is the absolute temperature (in Kelvin, K). In addition, temperature quotients (Q10) were calculated from the following equations
Q10 ¼ kðT þ 10Þ=kðTÞ
ð6Þ
where k(T þ 10) is the rate constant at (T þ 10) and k(T) is the rate constant at T. 3
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RESULTS AND DISCUSSION Changes in total polyphenol The content of total phenolics during the 8 days of storage ranged from 228.9 to 252.0, 231.0 to 253.3, and 237.5 to 258.1 mg per 100 mL for conventional thermal-, microwave-, and ultrasound-treated juice, respectively (Figure 1). Ultrasonic and microwave processes could avoid the decrease in the total phenolic content more availably when compared to the thermal pasteurization (Figure 1). This is probably because the polyphenoloxidase, peroxidase, and b-glucosidase in the juice samples have been inactivated more heavily
by ultrasonic and microwave treatments than by thermal process (Igual et al., 2010; Zheng and Lu, 2011). Furthermore, the possible reason for the observed highest values of phenolic compounds in the samples after ultrasonic processing might be attributed to the addition of sonochemically generated hydroxyl radicals (OH) to the aromatic ring of the phenolic compounds which has been reported to enhance the antioxidant activity of the phenolics (Ashokkumar et al., 2008; Bhat et al., 2011). Furthermore, in the first 5 days, the total phenolic content of all mulberry juices decreased. However,
Total phenols (mg/100 mL)
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Figure 1. Effect of thermal (), microwave (), and ultrasound (r) sterilizations on total phenols of black mulberry juice during storage at (a) 5 C, (b) 15 C, and (c) 25 C (p < 0.05).
Figure 2. Effect of thermal (), microwave (), and ultrasound (r) sterilizations on anthocyanins of black mulberry juice during storage at (a) 5 C, (b) 15 C, and (c) 25 C.
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Jiang et al. prolonged storage of the juices at selected temperatures resulted in increase in the total phenolic content toward the end of storage period for all juices (Figure 1). Similar behaviors were observed in grapefruit juice (Igual et al., 2010), blueberry juices (Barba et al., 2012), and six dark fruit juices (Piljac-Zˇegarac et al., 2009) during storage. This decrease of phenols during storage can be explained in such a way that some compounds are formedthat react with the unspecific Folin– Ciocalteu reagent and significantly decrease the phenolic content (Escarpa and Gonza´lez, 2001; Vinson et al., 2001). Some studies found the existence of a little peroxidase in the juices processed by pasteurization treatment, which promote to react with oxidation and significantly enhance phenolic content (Igual et al., 2010; Zheng and Lu, 2011). Other possible reason of increase in the total phenolic content with prolonged storage is that the formation of novel compounds such as Maillard reaction products had antioxidant activity (Manzocco et al., 2001). Changes in anthocyanins The initial anthocyanin content expressed as cyaniding3-glucoside was 89.61 mg per 100 mL (Figure 2). For all methods, the degradation rate of black mulberry anthocyanins increased with storage time and temperature. However, anthocyanin loss was greater in thermal sterilization compared to microwave and ultrasound sterilized juices during all temperature storage. Anthocyanin content decreased by 36%, 37%, and 45%, respectively when thermally processed juice was stored at 5, 15, and 25 C for 8 days. Comparatively, anthocyanin content of microwave and ultrasound sterilized juices reduced between 28–38% and 24– 34%, respectively during the same storage period
(Figure 2). Different studies have demonstrated the effectiveness of ultrasound in achieving high anthocyanin retention (Ashokkumar et al., 2008; Bhat et al., 2011; Tiwari et al., 2009a,b). Tiwari et al. (2009b) suggested that the level of anthocyanin degradation due to sonication was relatively low and compares favorably to thermal processing. They also demonstrated that sonication could be employed as a preservation technique for blackberry (Tiwari et al., 2009a) where anthocyanin retention is desired. The kinetic parameters of anthocyanin degradation during storage at selected temperatures are shown in Table 1. The degradation of anthocyanins in all processed black mulberry juice samples stored at selected temperatures were fitted to a first-order kinetic model (Figure 2), confirming the results of previous studies reporting the same model in other fruit juices during storage (Alighourchi and Barzegar, 2009; Kirca et al., 2007; Wang and Xu, 2007). Anthocyanin content was found to follow first-order degradation with R2 > 0.945. For all sterilization methods, the degradation rate increased at higher storage temperatures, which suggested that high temperature accelerated the anthocyanin degradation (Kirca et al., 2007). The lowest degradation rate value of anthocyanins was for ultrasound followed by microwave and least for thermal sterilization. Similar results were observed in grape and blackberry juices (Tiwari et al. 2009a,b). The possible reason was that the higher retention in anthocyanins may be caused by cavitation, which governs various physical, chemical, or biological reactions (Tiwari et al. 2009a). To determine the effect of storage temperature on the parameters, the constants obtained from equation (4) were fitted to an Arrhenius-type equation (Figure 3). The calculated activation energy values of the samples
Table 1. Kinetic parameters for anthocyanins of black mulberry juice treated by different sterilization methods during storage
Treatment Thermal
Microwave
Ultrasound
Q10
Temperature ( C)
K 102 (days)
T1/2 (days)
Ea (kJ/mol)
5–15 C
15–25 C
5–25 C
5 15 25 5 15 25 5 15 25
5.25 014 5.88 0.19 7.75 0.23 4.06 0.065 4.61 0.11 6.03 0.12 3.18 0.11 3.68 0.089 5.07 0.14
13.20 0.36 11.79 0.37 8.94 0.26 17.07 0.28 15.03 0.35 11.49 0.23 21.79 0.75 18.83 0.46 13.69 0.37
12.81 0.88
1.12 0.040
1.32 0.049
1.21 0.021
13.07 1.37
1.14 0.028
1.31 0.045
1.22 0.014
15.99 1.52
1.16 0.042
1.38 0.043
1.27 0.024
Data are the means SD of three samples, each with three replicates.
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varied with the different methods of sterilization (Table 1). The values were 12.81 kJ/mol for thermal, 13.07 kJ/mol for microwave, and 15.99 kJ/mol for ultrasound sterilization. Higher activation energy implied that the degradation of anthocyanins in ultrasonic sterilized samples is more stable to temperature elevations (Vikram et al., 2005). Temperature quotient (Q10) values were also calculated for the temperature ranges of 5–15, 15–25, and 5–25 C (Table 1). According to these values, the least effect of temperature rise on anthocyanin degradation was observed in thermal sterilized juice, followed by microwave and ultrasound sterilized samples, respectively indicating that degradation of anthocyanin in ultrasonic processing needed higher energy.
As shown in Figure 4, the antioxidant activity of thermal-treated juice depleted with storage time, whereas both ultrasound- and microwave-treated juices showed increase in antioxidant activity during the first 2 days of storage. Moreover, at the end of the storage, all the sterilized juices exhibited a significant decrease in antioxidants varying from 30% to 42% for thermally treated juice, 30% to 40% for microwave-treated juice, and 23% to 35% for ultrasound-sterilized juice. Increase in antioxidant activity during the first few days of storage has been previously reported for dark fruit juices (Piljac-Zˇegarac et al., 2009) and during the first few hours of incubation of apple juice (Van der Sluis et al., 2005) at elevated temperature. According to Pinelo et al. (2004), the increase in the activity may be explained by the strong tendency of polyphenols to undergo polymerization reactions, whereby the resulting oligomers possess larger areas available for charge
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Figure 3. The Arrhenius plots for degradation of anthocyanins in black mulberry juice processed by thermal (), microwave (), and ultrasound (r) sterilization methods during storage.
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Figure 4. Effect of thermal (), microwave (), and ultrasound (r) sterilizations on antioxidant activity of black mulberry juice during storage at (a) 5 C, (b) 15 C, and (c) 25 C (p < 0.05).
delocalization. When the degree of polymerization exceeds a critical value, the increased molecular complexity and steric hindrance reduce the availability of hydroxyl groups in the reaction with the DPPH radicals. This causes a resultant decrease in the antiradical capacity. This may explain the observed decrease in antioxidant activity of our mulberry juices, which decreased after the initial transient increase. The antioxidant capacity is related to the amount and composition of bioactive compounds present in food (Odriozola-Serrano et al., 2008). In this study, for all sterilization methods used, the DPPH values
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Jiang et al. correlated well with anthocyanin levels (R2 ¼ 0.794– 0.816). This demonstrates that anthocyanin is one of the main antioxidant compounds in black mulberry juice. However, there was no significant correlation between the total phenolic content and antioxidant activity of thermal processing (R2 ¼ 0.581), microwave (R2 ¼ 0.509), and ultrasound (R2 ¼ 0.279) sterilized juices. These Endings are in agreement with those of other researchers who observed no significant correlation between total phenolic content and radical scavenging activity in currant, pomegranate, cherry, and a few berry species. (Amakura et al., 2000; Piljac-Zˇegarac et al., 2009).
CONCLUSIONS This study investigated the effect of thermal, microwave, and ultrasound sterilization methods on the phytochemical content of black mulberry juice when stored at different temperatures. The higher stability of nutritional quality of black mulberry juice was achieved at the lowest temperature. Anthocyanin degradation of all sterilized juices followed the first-order reaction kinetics. Variation of degradation rate constants with temperature obeyed the Arrhenius relationship. In the black mulberry juice anthocyanins, total phenolics, and antioxidant activity degraded more quickly with increasing temperature during storage. For all storage temperatures, ultrasound sterilized juice was found to have higher retention of total phenolics, anthocyanins, and antioxidant activity compared to microwave and thermally processed juice. This work demonstrates that ultrasonic processing should be a preferred method for sterilizing mulberry juice products where retention of nutritional quality is desired upon storage. AUTHOR CONTRIBUTIONS Jiang B, Mantri N, and Hu Y contributed equally to this work.
FUNDING This work was supported by Zhejiang Provincial University Top Key Discipline of Biology, Science and Technology Key Project of Jiangsu Province (BE2012416), Six Key Talents Program of Jiangsu (2011NY032), and Science and Technology Project of Changshu City (CN201302).
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Tiwari BK, O’Donnell CP and Cullen PJ. (2009b). Effect of non thermal processing technologies on the anthocyanin content of fruit juices. Trends in Food Science and Technology 20(3–4): 137–145. Tiwari BK, O’Donnell CP, Muthukumarappan K and Cullen PJ. (2009c). Ascorbic acid degradation kinetics of sonicated orange juice during storage and comparison with thermally pasteurised juice. LWT-Food Science and Technology 42(3): 700–704. Valero M, Recrosio N, Saura D, Mun˜oz N, Martı´ N and Lizama V. (2007). Effects of ultrasonic treatments in orange juice processing. Journal of Food Engineering 80(2): 509–516. Van der Sluis AA, Dekker M and Van Boekel MAJS. (2005). Activity and concentration of polyphenolic antioxidants in apple juice. 3. Stability during storage. Journal of Agricultural and Food Chemistry 53(4): 1073–1080. Vikram VB, Ramesh MN and Prapulla SG. (2005). Thermal degradation kinetics of nutrients in orange juice heated by electromagnetic and conventional methods. Journal of Food Engineering 69(1): 31–40. Vinson JA, Su X, Zubik L and Bose P. (2001). Phenol antioxidant quantity and quality in foods: Fruits. Journal of Agricultural and Food Chemistry 49: 5315–5321. Wang WD and Xu SY. (2007). Degradation kinetics of anthocyanins in blackberry juice and concentrate. Journal of Food Engineering 82(3): 271–275. Zheng H and Lu H. (2011). Effect of microwave pretreatment on the kinetics of ascorbic acid degradation and peroxidase inactivation in different parts of green asparagus (Asparagus officinalis L.) during water blanching. Food Chemistry 128(4): 1087–1093. Zheng H, Lu H, Zheng Y, Lou H and Chen C. (2010). Automatic sorting of Chinese jujube (Zizyphus jujuba Mill. cv. ‘hongxing’) using chlorophyll fluorescence and support vector machine. Journal of Food Engineering 101(4): 402–408.
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Evaluation of bioactive compounds of black mulberry juice after thermal, microwave, ultrasonic processing, and storage at different temperatures Bo Jiang, Nitin Mantri, Ya Hu, Jiayin Lu, Wu Jiang and Hongfei Lu Food Science and Technology International published online 10 June 2014 DOI: 10.1177/1082013214539153 The online version of this article can be found at: http://fst.sagepub.com/content/early/2014/06/10/1082013214539153
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Article
Evaluation of bioactive compounds of black mulberry juice after thermal, microwave, ultrasonic processing, and storage at different temperatures Bo Jiang1,2, Nitin Mantri3, Ya Hu4, Jiayin Lu5, Wu Jiang4 and Hongfei Lu1,4
Abstract The effect of different sterilization methods (thermal, microwave, and ultrasonic processing) on the main bioactive compounds and antioxidant activity of black mulberry juice during selected storage time (8 days) and temperatures (5, 15, and 25 C) was investigated. The antioxidant activity of thermal-treated juice depleted with storage time, whilst both ultrasound- and microwave-treated juices showed transient increase in antioxidant activity during the first 2 days that later decreased with storage time. Lower temperature storage preserved more bioactive compounds and antioxidant activity, especially in ultrasound sterilized samples. The activation energy values were 15.99, 13.07, and 12.81 kJ/mol for ultrasonic, microwave, and thermal pasteurization processes, respectively. In general, ultrasound-sterilized samples showed higher total phenolics, anthocyanin, and antioxidant activity compared to the microwave- and thermal-processed juice during the storage time especially at lower temperatures.
Keywords Black mulberry juice, anthocyanin content, antioxidant activity, thermal, ultrasound, storage Date received: 23 November 2012; accepted: 5 May 2014
INTRODUCTION Recently, fruit juice obtained from mulberry (Morus species) is increasingly being promoted and consumed because of the reported nutritional and health benefits (Hojjatpanah et al., 2011). It is a rich source of bioactive compounds including phenolic substances like flavonols and anthocyanins with high antioxidant activity (Isabelle et al., 2008). Anthocyanins are the most important phenolic compounds present in berry fruits that are of special significance because of their contribution to total antioxidant activity of the fruit (Hassimotto and Genovese 2007). The major anthocyanins identified in mulberries are cyanidine-3-glucoside and cyanidine-3-rutinoside (Hojjatpanah et al., 2011; Suh et al., 2003). These anthocyanins are unstable Food Science and Technology International 0(0) 1–8 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1082013214539153 fst.sagepub.com
and easily susceptible to degradation through a number of factors such as light, pH, oxygen, enzymes, ascorbic acid, and temperature during storage and especially heat processing (Hojjatpanah et al., 2011; Kennedy et al., 2001). The stability of anthocyanins found in foods decreases during processing and storage as temperature rises (Cavalcanti et al., 2011). Anthocyanins show great susceptibility toward pH 1
College of Life Science, Zhejiang Sci-Tech University, Hangzhou, China 2 College of Biology and Food Engineering, Changshu Institute of Technology, Changshu, China 3 Health Innovations Research Institute, School of Applied Sciences, RMIT University, Melbourne, Australia 4 College of Chemistry and Life Science, Zhejiang Normal University, Jinhua, China 5 Division of General Studies, University of Illinois at UrbanaChampaign, Champaign, IL, USA Corresponding author: Hongfei Lu, College of Life Science, Zhejiang Sci-Tech University, Hangzhou, 310018, China. Email: [email protected]
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Food Science and Technology International 0(0) being more stable in acidic media at low pH values than in alkaline solutions with high pH values (Brouillard, 1982; Cavalcanti et al., 2011). Sterilization is an essential process to improve the shelf-life and safety of mulberry juice. Conventional thermal processing of fruit juice remains the most widely adopted technology for shelf-life extension and preservation of fruit juice. However, it often leads to detrimental changes in the sensory qualities of the juice (Odriozola-Serrano et al., 2008). Ultrasound processing is a promising non-thermal processing technology and a potential alternative or supplement to traditional thermal pasteurization (Tiwari et al., 2009c). The use of sonication to provide fresh high-quality, microbiologically safe, and high-nutritional-value fruit juices continue to be an area of research as shown in recent studies conducted on orange juice (Valero et al., 2007), strawberry juice (Tiwari et al., 2008), and blackberry juice (Tiwari et al., 2009a). Microwave pasteurization offers similar benefits to conventional methods, but with an improved product quality and reduced time of exposure to energy (Can˜umir et al., 2002). Several studies have successfully been conducted on the microwave pasteurization of fruit juices, as it preserves the natural organoleptic characteristics of the juice and reduces the time of exposure to energy, with the subsequently lower risk of losing essential thermolabile nutrients (Igual et al., 2010). Evaluating the influence of different sterilization methods on the bioactive compounds of mulberry juice during storage is essential for determining the best conditions for preserving the bioactive properties of the juice. In addition, no information is available on the degradation kinetics of anthocyanins during the storage of ultrasound- and microwave-processed mulberry juice to predict the quality changes that occur during storage. Hence, the main objective of this study was to investigate the effect of different sterilization methods including thermal, microwave, and ultrasound sterilization on the quality attributes of black mulberry juice as well as the anthocyanin degradation kinetics of these differently processed mulberry juices during storage.
MATERIALS AND METHODS Sample preparation Black mulberry fruits (Morus nigra L) were purchased from a local fruit market in Jinghua (Zhejiang, China). Black mulberries were selected on the basis of a similar degree of ripeness and apparent fruit quality. The fruits were squeezed using a domestic juice extractor and immediately filtered using four layers of cheese cloth to remove the pulp before different sterilization treatments.
Experimental design All fresh juice filtrated was randomly divided into three groups (three individuals per group) among which one group was treated by thermal sterilization, and others were treated with microwave and ultrasonic processing, respectively. The total phenols, anthocyanin content, and 2,20 -diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of black mulberry juice before and after three different pasteurization processes were measured to evaluate the effect of different methods of sterilization on the quality of mulberry juice. Treatments A 650 -W high-intensity ultrasonic processor (Ningo Scientz biotechnology Co. Ltd., Ningbo, China) with a 6-mm titanium probe whose immersion depth was 25 mm in a 100 mL test tube was used for juice sonication. The juice samples were sonicated for 30 min and processed at a constant frequency of 20 kHz (Bhat et al., 2011). In the case of ultrasonic-treated juice, samples of 50 mL were placed in a 100 mL jacketed vessel through which water at 20 1 C was circulated at a flow rate of 0.5 L/min. The microwave process was performed in a microwave oven (Galanz WD900ASL23-5 S, 23 L, 900 W). Each time, 50 mL of the juice was treated by microwave for 30 s (Igual et al., 2010). For thermal pasteurization, 50 mL of the juice was heated in glass tubes in a thermostatic water bath operating at 90 C for 30 s. After the different sterilization processes these black mulberry juices were rapidly cooled in an ice bath and were poured separately into sterile test tubes; then these samples of every sterilization treatment were stored in incubators at 5, 15, and 25 C, respectively in the dark for 8 days. Samples were randomly taken and analyzed for each index every day during storage. All analyzed samplings were carried out in triplicate. Chemicals and reagents Trolox and DPPH free radical were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA). Gallic acid and Folin–Ciocalteu reagent (purity 99%), were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Total phenols content determination The total phenols content was determined by using Folin–Ciocalteu reagent method, as described by our previous study (Lu et al., 2012). Total phenols assay was conducted by mixing 0.5 mL of black mulberry juice sample, 8.25 mL of deionized water, 0.5 mL of Folin–-Ciocalteu reagent, and 0.75 mL of 20%
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Jiang et al. Na2CO3. After 40 min of reaction in a water bath at 40 C, the absorbance of the reaction mixture was measured at 755 nm using a spectrophotometer. Results were expressed as milligrams gallic acid equivalents (GAE) per 100 mL of juice.
where MW is the molecular weight of cyanidin-3-glucoside, DF is the dilution factor, L is the path length in cm, and " is the molar extinction coefficient for cyanidin-3-glucoside. Results were expressed as milligram cyanidin-3-glucoside equivalents (CGE) per 100 mL of juice.
DPPH radical scavenging activity determination The DPPH free radical scavenging activity was evaluated according to the method described by Zheng et al. (2010). Briefly, 0.1 mL mulberry juice sample was mixed with 10 mL of 0.03 g/L DPPH (2,2-diphenyl-1-picrylhydrazyl) ethanol solution at room temperature. The diluted solution (0.1 mL) with 10 mL distilled water was used as control. The absorbance was measured at 517 nm after 30 min of reaction in the dark. Scavenging activity was measured in triplicate samples. The percentage of DPPH radical scavenging activity of each plant extract was calculated using the following equation
Statistical analysis
The data of total phenolic compounds, anthocyanin, and antioxidant activity were expressed as mean standard deviation (SD) from three sterilization treatments (each treatment with three samples) and each sample with three replications. Pearson’s correlation coefficient was used to calculate correlations among the data obtained. Statistical analyses were carried out using the statistical analysis systems (SAS, version 9.0) software package and Excel statistical tools (Microsoft software). The data were graphically plotted by using OriginLab (OriginPro, version 8.0). A0 ðA1 As Þ Experimental data were fitted to a first-order kinetic 100 Scavenging activity ð%Þ ¼ A0 model (equations (2) and (3)) to describe the evolution ð1Þ of anthocyanins in various processed mulberry juices during storage. This kinetic type was expressed by the following equations where A0 is the absorbance of the control solution (containing only DPPH), A1 is the absorbance of the DPPH C ¼ C0 expðktÞ ð3Þ solution containing plant extract, and As is the absorbance of the sample extract solution without DPPH. The value was measured in triplicate samples.
T1=2 ¼ ln 0:5=k
Anthocyanins content determination Total anthocyanins content of each sample was measured using the modified pH differential method described by Zheng and Lu (2011), using two buffer systems: potassium chloride 0.025 M at pH 1.0 and sodium acetate 0.4 M at pH 4.5. Briefly, 1 mL of sample was transferred to a 10 mL volumetric flask and made up with each buffer. The absorbance of each equilibrated solution was then measured at 510 and 700 nm, using a UV–Vis spectrophotometer. Quartz cuvettes of 1-cm path length were used, and all measurements were carried out at room temperature (25 C). Absorbance readings were made against distilled water as a blank. The total anthocyanins content was calculated on the basis of cyanidin-3-glucoside (Alasalvar et al., 2005) with a molecular weight of 445.2 g/mol and an extinction coefficient of 29,600 L/mol cm (Giusti and Wrolstad, 2001), as
AC ¼ ½ðA510 A700 ÞPH 1:0 ðA510 A700 ÞPH 4:5 MWDF1000="L
ð2Þ
ð4Þ
where C and C0 are anthocyanins content at time t and zero (day), respectively and k is the first-order rate constant, while T1/2 is the half-life and t is the storage time. Temperature-dependence of anthocyanin degradation rate constant was determined by the Arrhenius equation
k ¼ A expðEa =RTÞ
ð5Þ
where A is the frequency factor (per day), Ea is the activation energy (kJ/mol), R is the universal gas constant (8.314 J/mol/K), and T is the absolute temperature (in Kelvin, K). In addition, temperature quotients (Q10) were calculated from the following equations
Q10 ¼ kðT þ 10Þ=kðTÞ
ð6Þ
where k(T þ 10) is the rate constant at (T þ 10) and k(T) is the rate constant at T. 3
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RESULTS AND DISCUSSION Changes in total polyphenol The content of total phenolics during the 8 days of storage ranged from 228.9 to 252.0, 231.0 to 253.3, and 237.5 to 258.1 mg per 100 mL for conventional thermal-, microwave-, and ultrasound-treated juice, respectively (Figure 1). Ultrasonic and microwave processes could avoid the decrease in the total phenolic content more availably when compared to the thermal pasteurization (Figure 1). This is probably because the polyphenoloxidase, peroxidase, and b-glucosidase in the juice samples have been inactivated more heavily
by ultrasonic and microwave treatments than by thermal process (Igual et al., 2010; Zheng and Lu, 2011). Furthermore, the possible reason for the observed highest values of phenolic compounds in the samples after ultrasonic processing might be attributed to the addition of sonochemically generated hydroxyl radicals (OH) to the aromatic ring of the phenolic compounds which has been reported to enhance the antioxidant activity of the phenolics (Ashokkumar et al., 2008; Bhat et al., 2011). Furthermore, in the first 5 days, the total phenolic content of all mulberry juices decreased. However,
Total phenols (mg/100 mL)
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Figure 1. Effect of thermal (), microwave (), and ultrasound (r) sterilizations on total phenols of black mulberry juice during storage at (a) 5 C, (b) 15 C, and (c) 25 C (p < 0.05).
Figure 2. Effect of thermal (), microwave (), and ultrasound (r) sterilizations on anthocyanins of black mulberry juice during storage at (a) 5 C, (b) 15 C, and (c) 25 C.
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Jiang et al. prolonged storage of the juices at selected temperatures resulted in increase in the total phenolic content toward the end of storage period for all juices (Figure 1). Similar behaviors were observed in grapefruit juice (Igual et al., 2010), blueberry juices (Barba et al., 2012), and six dark fruit juices (Piljac-Zˇegarac et al., 2009) during storage. This decrease of phenols during storage can be explained in such a way that some compounds are formedthat react with the unspecific Folin– Ciocalteu reagent and significantly decrease the phenolic content (Escarpa and Gonza´lez, 2001; Vinson et al., 2001). Some studies found the existence of a little peroxidase in the juices processed by pasteurization treatment, which promote to react with oxidation and significantly enhance phenolic content (Igual et al., 2010; Zheng and Lu, 2011). Other possible reason of increase in the total phenolic content with prolonged storage is that the formation of novel compounds such as Maillard reaction products had antioxidant activity (Manzocco et al., 2001). Changes in anthocyanins The initial anthocyanin content expressed as cyaniding3-glucoside was 89.61 mg per 100 mL (Figure 2). For all methods, the degradation rate of black mulberry anthocyanins increased with storage time and temperature. However, anthocyanin loss was greater in thermal sterilization compared to microwave and ultrasound sterilized juices during all temperature storage. Anthocyanin content decreased by 36%, 37%, and 45%, respectively when thermally processed juice was stored at 5, 15, and 25 C for 8 days. Comparatively, anthocyanin content of microwave and ultrasound sterilized juices reduced between 28–38% and 24– 34%, respectively during the same storage period
(Figure 2). Different studies have demonstrated the effectiveness of ultrasound in achieving high anthocyanin retention (Ashokkumar et al., 2008; Bhat et al., 2011; Tiwari et al., 2009a,b). Tiwari et al. (2009b) suggested that the level of anthocyanin degradation due to sonication was relatively low and compares favorably to thermal processing. They also demonstrated that sonication could be employed as a preservation technique for blackberry (Tiwari et al., 2009a) where anthocyanin retention is desired. The kinetic parameters of anthocyanin degradation during storage at selected temperatures are shown in Table 1. The degradation of anthocyanins in all processed black mulberry juice samples stored at selected temperatures were fitted to a first-order kinetic model (Figure 2), confirming the results of previous studies reporting the same model in other fruit juices during storage (Alighourchi and Barzegar, 2009; Kirca et al., 2007; Wang and Xu, 2007). Anthocyanin content was found to follow first-order degradation with R2 > 0.945. For all sterilization methods, the degradation rate increased at higher storage temperatures, which suggested that high temperature accelerated the anthocyanin degradation (Kirca et al., 2007). The lowest degradation rate value of anthocyanins was for ultrasound followed by microwave and least for thermal sterilization. Similar results were observed in grape and blackberry juices (Tiwari et al. 2009a,b). The possible reason was that the higher retention in anthocyanins may be caused by cavitation, which governs various physical, chemical, or biological reactions (Tiwari et al. 2009a). To determine the effect of storage temperature on the parameters, the constants obtained from equation (4) were fitted to an Arrhenius-type equation (Figure 3). The calculated activation energy values of the samples
Table 1. Kinetic parameters for anthocyanins of black mulberry juice treated by different sterilization methods during storage
Treatment Thermal
Microwave
Ultrasound
Q10
Temperature ( C)
K 102 (days)
T1/2 (days)
Ea (kJ/mol)
5–15 C
15–25 C
5–25 C
5 15 25 5 15 25 5 15 25
5.25 014 5.88 0.19 7.75 0.23 4.06 0.065 4.61 0.11 6.03 0.12 3.18 0.11 3.68 0.089 5.07 0.14
13.20 0.36 11.79 0.37 8.94 0.26 17.07 0.28 15.03 0.35 11.49 0.23 21.79 0.75 18.83 0.46 13.69 0.37
12.81 0.88
1.12 0.040
1.32 0.049
1.21 0.021
13.07 1.37
1.14 0.028
1.31 0.045
1.22 0.014
15.99 1.52
1.16 0.042
1.38 0.043
1.27 0.024
Data are the means SD of three samples, each with three replicates.
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(a) 32
3.4
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3.40
1/T
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3.50
3.55
3.60
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26
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24
varied with the different methods of sterilization (Table 1). The values were 12.81 kJ/mol for thermal, 13.07 kJ/mol for microwave, and 15.99 kJ/mol for ultrasound sterilization. Higher activation energy implied that the degradation of anthocyanins in ultrasonic sterilized samples is more stable to temperature elevations (Vikram et al., 2005). Temperature quotient (Q10) values were also calculated for the temperature ranges of 5–15, 15–25, and 5–25 C (Table 1). According to these values, the least effect of temperature rise on anthocyanin degradation was observed in thermal sterilized juice, followed by microwave and ultrasound sterilized samples, respectively indicating that degradation of anthocyanin in ultrasonic processing needed higher energy.
As shown in Figure 4, the antioxidant activity of thermal-treated juice depleted with storage time, whereas both ultrasound- and microwave-treated juices showed increase in antioxidant activity during the first 2 days of storage. Moreover, at the end of the storage, all the sterilized juices exhibited a significant decrease in antioxidants varying from 30% to 42% for thermally treated juice, 30% to 40% for microwave-treated juice, and 23% to 35% for ultrasound-sterilized juice. Increase in antioxidant activity during the first few days of storage has been previously reported for dark fruit juices (Piljac-Zˇegarac et al., 2009) and during the first few hours of incubation of apple juice (Van der Sluis et al., 2005) at elevated temperature. According to Pinelo et al. (2004), the increase in the activity may be explained by the strong tendency of polyphenols to undergo polymerization reactions, whereby the resulting oligomers possess larger areas available for charge
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Figure 3. The Arrhenius plots for degradation of anthocyanins in black mulberry juice processed by thermal (), microwave (), and ultrasound (r) sterilization methods during storage.
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Figure 4. Effect of thermal (), microwave (), and ultrasound (r) sterilizations on antioxidant activity of black mulberry juice during storage at (a) 5 C, (b) 15 C, and (c) 25 C (p < 0.05).
delocalization. When the degree of polymerization exceeds a critical value, the increased molecular complexity and steric hindrance reduce the availability of hydroxyl groups in the reaction with the DPPH radicals. This causes a resultant decrease in the antiradical capacity. This may explain the observed decrease in antioxidant activity of our mulberry juices, which decreased after the initial transient increase. The antioxidant capacity is related to the amount and composition of bioactive compounds present in food (Odriozola-Serrano et al., 2008). In this study, for all sterilization methods used, the DPPH values
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Jiang et al. correlated well with anthocyanin levels (R2 ¼ 0.794– 0.816). This demonstrates that anthocyanin is one of the main antioxidant compounds in black mulberry juice. However, there was no significant correlation between the total phenolic content and antioxidant activity of thermal processing (R2 ¼ 0.581), microwave (R2 ¼ 0.509), and ultrasound (R2 ¼ 0.279) sterilized juices. These Endings are in agreement with those of other researchers who observed no significant correlation between total phenolic content and radical scavenging activity in currant, pomegranate, cherry, and a few berry species. (Amakura et al., 2000; Piljac-Zˇegarac et al., 2009).
CONCLUSIONS This study investigated the effect of thermal, microwave, and ultrasound sterilization methods on the phytochemical content of black mulberry juice when stored at different temperatures. The higher stability of nutritional quality of black mulberry juice was achieved at the lowest temperature. Anthocyanin degradation of all sterilized juices followed the first-order reaction kinetics. Variation of degradation rate constants with temperature obeyed the Arrhenius relationship. In the black mulberry juice anthocyanins, total phenolics, and antioxidant activity degraded more quickly with increasing temperature during storage. For all storage temperatures, ultrasound sterilized juice was found to have higher retention of total phenolics, anthocyanins, and antioxidant activity compared to microwave and thermally processed juice. This work demonstrates that ultrasonic processing should be a preferred method for sterilizing mulberry juice products where retention of nutritional quality is desired upon storage. AUTHOR CONTRIBUTIONS Jiang B, Mantri N, and Hu Y contributed equally to this work.
FUNDING This work was supported by Zhejiang Provincial University Top Key Discipline of Biology, Science and Technology Key Project of Jiangsu Province (BE2012416), Six Key Talents Program of Jiangsu (2011NY032), and Science and Technology Project of Changshu City (CN201302).
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