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Determination of total phenolic content of raspberry and blackberry cultivars by immobilized horseradish peroxidase bioreactor
Journal of Food Composition and Analysis 24 (2011) 944–949
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Original Article
Determination of total phenolic content of raspberry and blackberry cultivars by immobilized horseradish peroxidase bioreactor Esra Is¸ık a, Saliha S¸ahin a, Cevdet Demir a,*, Cihat Tu¨rkben b a b
Department of Chemistry, Faculty of Science and Arts, University of Uludag, Go¨ru¨kle, Bursa 16059, Turkey Department of Horticulture, Faculty of Agriculture, University of Uludag, Bursa 16059, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 July 2010 Received in revised form 30 January 2011 Accepted 30 January 2011 Available online 12 February 2011
Raspberry and blackberry cultivars were assayed for total phenol content by enzymatic method using immobilized horseradish peroxidase (HRP). The HRP was immobilized on the styrene–divinylbenzene– polygluteraldehyde (STY–DVB–PGA) beads. Total phenol content determined by immobilized and free enzymatic methods was compared with those obtained by applying the Folin–Ciocalteu method. The total phenol content by immobilized HRP method ranged from 254 to 973 mg gallic acid equivalent (GAE) per 100 g fw as well as 105 to 435 mg ellagic acid equivalent (EAE) per 100 g fw in raspberry and blackberry cultivars. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical values were correlated with total phenol content in raspberry using immobilized enzymatic method as GAE (R2 = 0.7450, P = 0.05) and EAE (R2 = 0.7449, P = 0.05). All antioxidant activity values were highly correlated with total phenol content in blackberry using free and immobilized enzymatic methods as GAE (R2 = 0.9777 and 0.9223, P = 0.05 respectively) and EAE (R2 = 0.9979 and 0.9223, P = 0.05 respectively). A significant additivity was observed using individual phenolic compounds by immobilized enzymatic method as 95% (GAE) and 90% (EAE). These results show that the immobilized enzymatic assay can be applied to determine the total phenol content of berry fruits, which is more specific and not affected by interfering compounds. ß 2011 Elsevier Inc. All rights reserved.
Keywords: Raspberry (Rubus idaeus) Blackberry (Rubus fruticosus) DPPH Total phenol content Horseradish peroxidase Food analysis Food composition
1. Introduction Berry fruits are rich in phenolic compounds contents such as phenolic acids (Rommel and Wrolstad, 1993; Ha¨kkinen et al., 1998), flavonoids (Ha¨kkinen et al., 1998) and anthocyanins (Rommel and Wrolstad, 1993), which have been demonstrated that considerable antioxidant properties in vivo and in vitro in berries (Heinonen et al., 1998). The phenolic contents of berries are therefore an important parameter for the evaluation of their antioxidant properties and quality. Berries contain a diverse range of phenolic compounds with biological properties such as anticancer, anti-neurodegenerative and anti-inflammatory activities (Seeram et al., 2006; Seeram, 2008). The total phenolic content of fruits is therefore an important parameter of their antioxidant properties. The usual determination of total phenolic content has been achieved using the Folin–Ciocalteu procedure of Singleton (Singleton and Rossi, 1965; Singleton et al., 1999; Cliffe et al., 1994). This method is based on the oxidation of the reduced molecules by a mixture of the two strong inorganic oxidant phosphotungstic and phosphomolybdic acids. Separating techniques have been used to isolate, identify and determine individual
* Corresponding author. Tel.: +90 224 2941727; fax: +90 224 2941889. E-mail address: [email protected] (C. Demir). 0889-1575/$ – see front matter ß 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2011.01.016
polyphenolic compounds in berries, but these methods appear to be relatively more expensive and time consuming (Sellappan et al., 2002; Tu¨rkben et al., 2010). Recently, enzymatic methods and biosensors have been reported for the determination of total phenol content in tea, wine and juices (Stevanato et al., 2004; Girelli et al., 2009a). It has been found that a great number of phenols can act as aromatic donor molecules (Adam et al., 1999; Veitch and Smith, 2001). Also, flavonoids containing phenol B rings (Chan et al., 1999) and in general dietary polyphenols (Galati et al., 2002) can be oxidized by peroxidase/H2O2 to give reactive aryloxy radicals. Therefore, enzymatic method appears to be a suitable measure of the antioxidant properties of polyphenols. The enzymatic method offers the advantages of a shorter measuring period, a greater specificity toward the phenolic compounds, and the absence of the interference substances. Several enzymes have been immobilized on the pore glass support (Girelli et al., 2009a,b) and polymeric beads (Aybastıer and Demir, 2010) to obtain stable and reusable bioreactor that may be used in spectrophotometric and high performance liquid chromatographic (HPLC) systems. Horseradish peroxidase (HRP) has been immobilized by adsorption on cinnamic carbohydrate esters and used for phenolic resin synthesis and bioremediation (Rojas-Melgarejo et al., 2004). Several methods have been developed to determine the antioxidant activity of fruits, vegetables as well as herbs. Two
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major mechanisms, namely hydrogen atom transfer (HAT) and single electron transfer (SET), are well known in the evaluation of antioxidant activity against free radicals. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay is one of the methods utilizing both hydrogen atom transfer and single electron transfer mechanism. It is considered to be predominantly based on electron transfer reaction whereas hydrogen-atom transfer reaction is only a marginal pathway (Chen, 2006). In the present study, we propose a new immobilized enzymatic method for the determination of the total phenolic content in raspberry and blackberry fruits The HRP was immobilized on the styrene–divinylbenzene–polygluteraldehyde (STY–DVB–PGA) beads that were synthesized as in our previous paper (Aybastıer and Demir, 2010). The total phenol contents determined by free and immobilized enzymatic methods were compared with those obtained by applying the Folin method. Furthermore, the immobilized enzymatic method was validated by measuring the total phenol contents of individual phenolic compounds and their mixture, and summability of each method was evaluated. 2. Materials and methods 2.1. Chemicals Caffeic acid (98%) ferulic acid (98%), and p-coumaric acid (98%) were purchased from Merck (Darmstadt, Germany); phydroxybenzoic acid (99%), quercetin hydrate (95%), Folin– Ciocalteu phenol reagent, DPPH (2,2-diphenyl-1-picrylhydrazyl), gallic acid (98%), 4-aminoantipyrine (98%), and hydrogen peroxide (30%) were purchased from Sigma–Aldrich (St. Louis, USA); horseradish peroxidase donor: hydrogen peroxidase oxidoreductase, EC 1.11.1.7 type II (50 KU/mg of solid), was supplied from Sigma; ellagic acid (95%) was purchased from Fluka (Buchs, Switzerland). Methanol (HPLC grade) and hydrochloric acid (37%) were of analytical grade. All standard solutions were prepared in methanol (Merck, Darmstadt, Germany). 2.2. Plant materials Raspberry fruits (Rubus idaeus L.) of five cultivars (Aksu Kırmızısı, Rubin, Newburgh, Holland Boduru, Heritage) and blackberry fruits (Rubus fruticosus L.) of four cultivars (Bursa 1, Bursa 2, Jumbo, Chester) were collected from different commercial orchards in the region of Kestel (Bursa, Turkey), and handharvested at commercially mature stage during the growing season of July–August 2008. The fresh fruits were transported to the laboratory in the same day for sample preparation and analysis. The samples were stored at +4 8C for less than 2 days before extraction. It has been reported that berry fruits are stable about 10 days at +4 8C (Piljac-Zˇegarac and Dunja Sˇamec, 2010). 2.3. Extraction and hydrolysis Fresh raspberry and blackberry fruit samples in three lots of 5 g each of were separately blended with methanol. Ascorbic acid (80 mg) and 10 mL of 6 mol/L HCl (final concentration 1.2 mol/L HCl) were added to crushed berries. The mixture was stirred with magnetic stirrer and kept at room temperature for 16 h in dark. The hydrolyzed samples (total volume 50 mL) were separated from the solid matrix by filtration through sheets of qualitative filter paper. The hydrolyzed samples (total volume 50 mL) were separated from the solid matrix by filtration through sheets of qualitative filter paper. The extracts were used for the determination of total phenol contents by Folin, free and immobilized enzymatic methods and antioxidant activity. The extraction procedure was repeated three times (three extractions from one container of 500 g) for each
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sample. The same extracts were used for the determination of total phenol contents and antioxidant activity. 2.4. UV/vis spectroscopy Spectrophotometric measurements were performed on a UV/ vis spectrometer (Varian Cary 50) equipped with 10 mm quartz cuvettes. 2.5. Analytical methods The solution used in the Folin assay of phenolics was prepared as follows: Lowry A: 2% aqueous Na2CO3 in 0.1 M NaOH; Lowry B: 0.5% CuSO4 aqueous solution in 1% NaKC4H4O6 solution; Lowry C: prepared freshly as mixture (50 mL Lowry A + 1 mL Lowry B); Folin–Ciocalteu reagent was diluted with H2O at a volume ratio 1:3 prior to use. The amount of total phenols in the berries was determined with Folin–Ciocalteu reagent according to the method of Apak et al. (2008). A 0.1 mL of berry extract, 1.9 mL of H2O and 2.5 mL of Lowry C solution were mixed and the mixture was allowed to stand for 10 min. At the end of this period, 0.25 mL of Folin reagent was added, and 30 min was allowed for stabilization of the blue color. The absorbance was measured at 750 nm. Total phenol contents were calculated from standard curves of gallic acid and ellagic acid, and results were expressed as mg of gallic acid equivalent (GAE) per 100 g of fresh weight (fw) and ellagic acid equivalent (EAE) per 100 g of fresh weight (fw) separately. Total phenolic content of berries was determined with free horseradish peroxidase (HRP) assay. A solution constituted of 100 mL of berry extract, 900 mL of 2.5 mM HRP, 2 mL of 9 mM 4aminoantipyrine in 0.1 M phosphate buffer, pH 8.0 and 1 mL of 6 mM H2O2, final volume of 4 mL was spectrophotometrically monitored at 510 nm. All measurements were performed in triplicate. Total phenol contents were calculated from standard curves of gallic acid and ellagic acid, and results were expressed as mg of GAE per 100 g of fresh weight (fw) and EAE per 100 g of fresh weight (fw) separately. HRP was immobilized on the STY–DVB–PGA beads (0.6–1.4 mm particle size) by covalent attachment. Synthesis of STY–DVB–PGA and immobilization procedure has been described in our previous paper (Aybastıer and Demir, 2010). The method was described briefly as follows: glutaraldehyde was polymerized in aqueous solution at pH 10.5. The polymerized glutaraldehyde solution was added to the organic mixture containing styrene, divinylbenzene and span. Polymerization was conducted at 80 8C for 3 h. The polymer (120 mg of the STY–DVB–PGA) was placed in a polyethylene column (size 6 mm 1 mm) and the enzyme (21 mg HRP) in 100 mL of 0.1 M phosphate buffer (pH 8) as a carrier solution was recycled through the column by a peristaltic pump at 2.5 mL/min flow rate for 26 h to eliminate unbound protein the polymer was washed with 0.1 M phosphate buffer. The amount of immobilized enzyme (as protein) on the support was calculated from the difference in the amount of protein contents in the solution before and after immobilization using the Bradford assay method (Bradford, 1976). The protein concentrations were determined by spectrophotometric measurements at 595 nm. Coomassie brilliant blue solution was used as dye reagent. The amount of protein was calculated from a standard curve of bovine serum albumine (BSA). After coupling of the enzyme to the STY– DVB–PGA, 82% of protein was removed from the solution at 26 h. Total phenolic content of berries was determined by immobilized HRP assay. A carrier solution containing of 2.25 mM 4aminoantipyrine in 0.1 M phosphate buffer (pH 8.0) was passed from the system continuously using peristaltic pump operated at 2.5 mL/min flow rate. While the system was operating, a mixture of 0.1 mL sample and 1 mL of 2.97 mM H2O2 solution was added to
Folin
Free HRP
R
2500
İmmobilized HRP
2000 1500 1000 500
te r
Ju m bo
H
ol la nd
N
a
C
he s
rs a
2
1 Bu
Bu
rs a
du ru Bo
er ita H
ew
ub i
ge
n
0 Ak su
mg gallic acid/100 g fruit
the carrier solution. After enzymatic reaction, the absorbance of final quinone-imine-colored product was measured at 510 nm. All measurements were performed in triplicate. Total phenol contents were calculated from standard curves of gallic acid and ellagic acid, and results were expressed as mg of GAE per 100 g of fresh weight (fw) and EAE per 100 g of fresh weight (fw) separately. Free radical scavenging activity of berries was determined with DPPH reagent according to our previous paper (Sarıburun et al., 2010). The fruit extracts (0.1 mL) and 0.18 mL, 1 103 mol/L of DPPH were mixed in methanol, then the final volume was brought up to 3 mL with methanol. After the reaction was allowed to take place in the dark for 30 min, the absorbance was recorded at 515 nm against blank to determine the concentration of remaining DPPH. Standard curve was prepared using different concentrations of Trolox. The results were expressed as mmol Trolox equivalent (TE) per g fresh weight (fw).
bu rg h
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Raspberry cultivars
Blackberry cultivars
Fig. 1. Total phenol content expressed as GAE of the raspberry and blackberry cultivars according to the Folin, free HRP and immobilized HRP enzymatic methods. The reported values are the mean of triplicate experiments.
Folin
2000
Free HRP
İmm obilized HRP
1750 1500 1250 1000 750 500 250
bo m Ju
he C
Bu
rs
a
st er
2
1 sa Bu r
a
H
Bo
er ita
du
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n ub i R
ew bu
rg h
0 Ak su
nd ol la H
Total phenol contents were determined in five raspberry and four blackberry cultivars using Folin, free and immobilized horseradish peroxidase (HRP) methods by UV–vis spectrophotometry. The comparison between Folin and enzymatic methods is shown in Figs. 1 and 2. The results represent the total phenol contents after subtraction of ascorbic acid contribution, which was used during the extraction of phenolic compounds in raspberry and blackberry. The average contribution of ascorbic acid to the total phenol content was 4.0 mg gallic acid equivalent (GAE) per g berry sample for extracts. Ascorbic acid content was determined by the standard spectrophotometric method (TS 6397). The method is based upon the quantitative discoloration of 2,6-dichlorophenol indophenol by ascorbic acid. The contents of ascorbic acid ranged from 18 to 26 mg per 100 g fw in raspberry and ranged from 19 to 26 mg per 100 g fw in blackberry cultivars. The total phenol content by immobilized HRP method ranged from 254 to 973 mg GAE per 100 g fw as well as 105 to 435 mg EAE per 100 g fw in raspberry and blackberry cultivars. Of the selected raspberry and blackberry cultivars, Bursa 2 showed the highest total phenol content (973 mg GAE per 100 g fw or 435 mg ellagic acid equivalent (EAE) per 100 g fw) while Heritage has the lowest total phenol content of samples (254 mg GAE per 100 g fw or 105 mg EAE per 100 g fw). The total phenol content in blackberry was higher than those of raspberry cultivars. On the other hand, immobilized enzymatic method was well correlated with Folin method (R2 = 0.7330 as GAE and R2 = 0.7455 as EAE in berries), which is in agreement with those reported for the determination of total phenol content of tea in the literature (Girelli et al., 2009a). Gallic acid has been mostly used as a standard for the determination of total phenol content in berries (De Ancos et al., 2000; Sellappan et al., 2002; Pantelidis et al., 2007). On the other hand, ellagic acid appears to be more sensitive in enzymatic method than gallic acid due to the fact that we used higher concentration of gallic acid in order to reach the same range of absorbance as ellagic acid (Table 1). This can be attributed that
N
3. Results and discussion
mg ellagic acid/100 g fruit
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Ras pberry cultivars
Blackberry cultivars
Fig. 2. Total phenol content expressed as EAE equivalent of the raspberry and blackberry cultivars according to the Folin, free HRP and immobilized HRP enzymatic methods. The reported values are the mean of triplicate experiments.
ellagic acid has more hydroxyl groups than gallic acid. Moreover, individual phenolic compounds have been determined in raspberry and blackberry cultivars in our previously study and ellagic acid was the main phenolic compound in these berries (Tu¨rkben et al., 2010). The Folin method values of raspberry and blackberry are higher than those of enzymatic assays. This comparison indicated that the total phenol content of a certain type of cultivars of raspberry and blackberry can be significantly different due to different assays. A variation in the content of total phenol of berries could be observed due to their content of anthocyanin and ascorbic acid, which have been determined in raspberry and blackberry (De Ancos et al., 2000; Wu and Prior, 2005; Chen et al., 2007; Pantelidis et al., 2007). The higher results of total phenol content may probably be explained by poor specificity of Folin method, which can give an
Table 1 Validation parameters of standard phenolic compounds by Folin, free and immobilized enzymatic methods. Methods
LOD (mg/L)
LOQ (mg/L)
Precision (RSD%)
Recovery (%)
Linearity (R2) and linear range (mg/L)
Folin–Ciocalteu (mmol GAE) Folin–Ciocalteu (mmol EAE) Free enzyme (mmol GAE) Free enzyme (mmol EAE) Immobilized enzyme (mmol GAE) Immobilized enzyme (mmol EAE)
1.09 0.55 2.65 0.27 1.68 1.38
3.63 1.83 8.83 0.90 5.60 4.60
3.15 3.88 2.14 2.44 1.72 1.43
78 107 80 86 90 91
0.9984 0.9977 0.9983 0.9982 0.9971 0.9978
LOD, limit of detection; LOQ, limit of quantification; RSD, relative standard deviation; GAE, gallic acid equivalent; EAE, ellagic acid equivalent.
(1.6–20) (1.6–15) (3.8–20) (1.8–10) (3.8–25) (1.8–15)
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Table 2 Total phenol content of standard phenolic compounds by Folin and immobilized enzymatic methods.a Phenolic compounds
Ellagic acid Ferulic acid Caffeic acid p-Coumaric acid p-Hydroxybenzoic acid Quercetin Total Mixture Additivity (%)
Folin method (mmol GAE)
Immobilized enzymatic method (mmol GAE)
Folin method (mmol EAE)
Immobilized enzymatic method (mmol EAE)
0.127 0.009 0.114 0.008 0.135 0.010 0.130 0.006 0.036 0.003 0.092 0.005
0.136 0.008 0.030 0.003 0.055 0.004 0.083 0.007 0.135 0.009 0.065 0.006
0.021 0.002 0.019 0.001 0.021 0.002 0.021 0.001 0.012 0.001 0.017 0.001
0.070 0.007 0.065 0.006 0.030 0.002 0.027 0.003 0.039 0.003 0.034 0.002
0.633 0.037 0.584 0.024 92
0.503 0.012 0.476 0.031 95
0.111 0.001 0.087 0.004 78
0.264 0.017 0.237 0.018 90
GAE, gallic acid equivalent; EAE, ellagic acid equivalent. a Mean of three determinations SD (standard deviation).
overestimated value of total phenol content in berry fruits. It has been reported that, between the nonphenolic compounds such as citric acid, ascorbic acid and sulfite are potential interfering substances on the determination of total phenol content by the Folin method. They contribute to increase the absorbance value of the phenolic compounds. However, experimental data show that total phenol content by enzymatic method is not affected by the presence of interfering substances such as ascorbic acid and citric acid (Stevanato et al., 2004). Furthermore, peroxidase could catalyze the degradation of the anthocyanidin as a substrate in the presence of hydrogen peroxide (Zhang et al., 2005). Thus, the enzyme can be inhibited by anthocyanins and the residual enzyme activity may not be enough to catalyze the total conversion of the phenolic compounds. HRP can also be inactivated by reactions between hydrogen peroxide and enzymatic reaction intermediates, and irreversible reactions between the enzyme and phenoxyl radicals formed by oxidation of phenolic compounds (RojasMelgarejo et al., 2004). Individual anthocyanins and anthocyanidins have been identified in raspberry and blackberry cultivars in our previous paper (Sarıburun et al., 2010). Five anthocyanins and five anthocyanidins have been identified, using liquid chromatography–mass spectrometry (LC–MS/MS), as cyanidin-3-(2G-glucosylrutinoside), cyanidin-3-sophoroside, cyanidin-3-glucoside, cyanidin-3-rutinoside, cyanidin, peonidin, malvidin, pelargonidin in water, and pelargonidin-3-rutinoside, pelargonidin and delphinidin in methanol extract of raspberry cultivars. On the other hand, three anthocyanidins such as cyanidin, malvidin and pelargonidin were identified in water and delphinidin was identified in methanol extract of blackberry. By comparison of the results obtained by free and immobilized enzymatic methods it appeared that a significant difference of total phenol content between two methods was obtained in both raspberry and blackberry samples (Figs. 1 and 2). The differences might be due to the inactivation of enzyme by interaction with the support and the coupling conditions. However, immobilized enzymatic method has several advantageous properties such as stability, repeatedly use in reactors, easily separation from soluble reaction products, reuse stability, which can effectively reduce the cost in applications and unused substrate, thus simplifying workup and preventing protein contamination of the final product (Temoc¸in and Yig˘itog˘lu, 2009; Aybastıer and Demir, 2010). In order to figure out which method is more accurate, individual contribution of phenolic compounds to total phenol content was investigated by Folin and immobilized enzymatic methods. Levels of total phenol content of individual phenolic compounds at 4.5 mM were ranged between 0.136 mmol GAE in ellagic acid by immobilized enzymatic method and 0.012 mmol EAE in phydroxybenzoic acid by Folin method (Table 2). The differences between total phenol contents of individual phenolic compounds
could be explained by the number and position of substituted hydroxyl or methoxyl groups and glycosylation around the flavonoid skeleton. It is well-known that the antioxidant properties of the polyphenols are correlated with the delocalization, on the aromatic ring, of the phenoxyl unpaired electron, which stabilizes the free radical. Folin method is based on the chemical oxidation of the reduced molecules by a mixture of the two strong inorganic oxidants, phosphotungstic acid and phosphomolybdic acids (Stevanato et al., 2004). In enzymatic method, in the presence of hydrogen peroxide, horseradish peroxidase is oxidized. The oxidized form of enzyme will come into contact with phenols, which will result in the formation of phenoxyl radicals. These radicals will further react with the aromatic amine group of 4aminoantipyrine, giving rise to the final quinone-imine colored product. The total contribution percentage of phenolic compounds using Folin method was 92% as GAE and 78% as EAE whereas the contribution percentage was 95% as GAE and 90% as EAE in immobilized enzymatic method. Total antioxidant capacity in the phenolic mixture was almost equal to the sum of antioxidant capacity of its individual phenolics (Heo et al., 2007). On the other hand, enzymatic method is convenient for routine use in the laboratories. It only requires 5 min reaction time and relatively simple preparation whereas Folin needs more time and quite complicated procedure. The immobilized enzyme can be used repeatedly for at least 10 samples. The antioxidant activity of fruit extracts was determined as Trolox equivalents (mmol TE/g fw) using the DPPH assay in raspberry and blackberry cultivars. Figs. 3 and 4 demonstrate the correlations between Folin, free HRP and immobilized HRP
Fig. 3. Correlation between Folin, free HRP and immobilized HRP enzymatic methods and DPPH values for raspberry cultivars. FLNGA: Folin gallic acid equivalent; FLNEA: Folin ellagic acid equivalent; FRGA: free enzyme gallic acid equivalent; FREA: free enzyme ellagic acid equivalent; IMGA: immobilized enzyme gallic acid equivalent; IMEA: immobilized enzyme ellagic acid equivalent.
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4. Conclusions
Fig. 4. Correlation between Folin, free and immobilized enzymatic methods and DPPH values for blackberry cultivars. FLNGA: Folin gallic acid equivalent; FLNEA: Folin ellagic acid equivalent; FRGA: free enzyme gallic acid equivalent; FREA: free enzyme ellagic acid equivalent; IMGA: immobilized enzyme gallic acid equivalent; IMEA: immobilized enzyme ellagic acid equivalent.
We have developed an immobilized enzymatic method for the rapid determination of total phenol content in raspberry and blackberry fruits. The presented method has several advantageous properties such as reuse stability of immobilized HRP, operational cost, easy separation from the reaction mixture and not affected by the presence of interference substances. The applicability and reliability of this analytical approach was confirmed by method validation and successful analysis of raspberry and blackberry samples. Significant correlation with antioxidant activity assay (DPPH) shows that the immobilized enzymatic method can be applied to determine the total phenol content even for a specific phenol class determination. Thus, antioxidant activity of berries analysed appears to be largely influenced by these phenolic compounds. In conclusion, the developed method is suitable for routine use in laboratories and should be utilized for rapid analysis of fruit extracts.
References enzymatic methods and DPPH values. Antioxidant activity was highly correlated with total phenol content by Folin method as gallic acid (FLNGA) (R2FLNGA ¼ 0:7163) and ellagic acid equivalents (FLNEA) (R2FLNEA ¼ 0:7164) in blackberry (Fig. 4). When enzymatic methods of data were compared, high correlations were observed between immobilized enzymatic method as gallic acid (IMGA), ellagic acid (IMEA) and DPPH assay (R2IMGA ¼ 0:7450 and R2IMEA ¼ 0:7449) in raspberry (Fig. 3). On the other hand, the highest correlations were observed between free enzymatic as gallic acid (FRGA), ellagic acid (FREA), immobilized enzymatic methods and DPPH assay (R2FRGA ¼ 0:9777, R2FREA ¼ 0:9979, R2IMGA ¼ 0:9223 and R2IMEA ¼ 0:9223) in blackberry (Fig. 4). In addition, the correlations among enzymatic methods are all positive which indicate that higher the total phenol content of the raspberry and blackberry, the better antioxidant activity. The results confirm that the immobilized enzymatic method could be used for the determination of antioxidant capacity in berry samples. The validation of the quantitative determination of total phenolic contents by Folin and enzymatic methods was performed by limits of detection (LODs; 3s/m), recovery (%), linearity and precision of gallic and ellagic acids (Table 1), where s is the standard deviation of the replicates and m is the slope of the calibration curve. LODs ranged from 1.09 to 2.65 mg/L and ranged from 0.27 to 1.38 mg/L for gallic and ellagic acids, respectively. Lower range of calculated LODs using ellagic acid as standard corresponds to the presence of more hydroxyl group in the molecule. The higher LOD value for free enzymatic method (2.65 mmol GAE) could be due to the higher linearity range of the response. The linearity of the methods was studied with gallic and ellagic acids levels ranging from 1.6 to 25 mg/L. The responses were linear up to 15 mg/L for ellagic acid and 25 mg/L for gallic acid with R2 values ranging from 0.9977 to 0.9982 and ranging from 0.9971 to 0.9984 for both phenolic compounds, respectively. The precision and recovery of the methods were evaluated by spiking raspberry and blackberry samples with standard gallic (15 mg/L) and ellagic (25 mg/L) acids. The precision of the methods, expressed as relative standard deviation (RSD%), was estimated by measuring three replicates of each compound. Better precisions were obtained by immobilized enzymatic method for both gallic and ellagic acids. The mean percentage recoveries ranged from 78 (Folin method) to 90% (immobilized enzymatic method) for gallic acid and ranged from 86 (immobilized enzymatic method) to 107% (Folin method) for ellagic acid. High recoveries were obtained by immobilized enzymatic method with gallic and ellagic acids.
Adam, W., Lazarus, M., Saha-Moller, C.R., Weichold, O., Hoch, U., Haring, D., Schreier, P., 1999. Biotransformation with peroxidases. Advances in Biochemical Engineering and Biotechnology 63, 73–108. ¨ zyu¨rek, M., C¸elik, S.E., 2008. Mechanism of antioxidant capacity Apak, R., Gu¨c¸lu¨, K., O assays and the CUPRAC (cupric ion reducing antioxidant capacity) assay. Microchimica Acta 160, 413–419. ¨ ., Demir, C., 2010. Optimization of immobilization conditions of TherAybastıer, O momyces lanuginosus lipase on styrene–divinylbenzene copolymer using response surface methodology. Journal of Molecular Catalysis B: Enzymatic 63, 170–178. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254. Chan, T., Galati, G., O’Brien, P.J., 1999. Oxygen activation during peroxidase catalysed metabolism of flavones or flavanones. Chemico-Biological Interactions 122, 15–25. Chen, H., 2006. Polyphenolic composition of tea (Camellıa sinensis) and its relationship with antioxidant activity and processing. D.Phil. Dissertation, Available from: Graduate School-New Brunswick Rutgers, The State University of New Jersey. Chen, F., Sun, Y., Zhao, G., Liao, X., Hu, X., Wu, J., Wang, Z., 2007. Optimization of ultrasound-assisted extraction of anthocyanins in red raspberries and identification of anthocyanins in extract using high-performance liquid chromatography–mass spectrometry. Ultrasonics Sonochemistry 14, 767–778. Cliffe, S., Fawer, M.S., Maier, G., Takata, K., Ritter, G., 1994. Enzyme assays for the phenolic content of natural juices. Journal of Agricultural and Food Chemistry 42, 1824–1828. De Ancos, B., Gonza´les, E.M., Cano, M.P., 2000. Ellagic acid, vitamin C, and total phenolic contents and radical scavenging capacity affected by freezing and frozen storage in raspberry fruit. Journal of Agricultural and Food Chemistry 48, 4565–4570. Galati, G., Sabzevari, O., Wilson, J.X., O’Brien, P.J., 2002. Prooxidant activity and cellular effects of the phenoxyl radicals of dietary flavonoids and other polyphenolics. Toxicology 177, 91–104. Girelli, A.M., Mattei, E., Papaleo, D., 2009a. Tyrosinase immobilized reactor as a fast tool for polyphenolic index of tea. Journal of Food Composition and Analysis 22, 709–713. Girelli, A.M., Giuliani, T., Mattei, E., Papaleo, D., 2009b. Determination of an antioxidant capacity index by immobilized tyrosinase bioreactor. Journal of Agricultural and Food Chemistry 57, 5178–5186. Ha¨kkinen, S.H., Ka¨renlampi, S.O., Heinonen, I.M., Mykka¨nen, H.M., To¨rro¨nen, A.R., 1998. HPLC method for screening of flavonoids and phenolic acids in berries. Journal of Agricultural and Food Chemistry 77, 543–551. Heinonen, M., Lehtonen, P.J., Hopia, A.I., 1998. Antioxidant activity of berry and fruit wines and liquors. Journal of Agricultural and Food Chemistry 46, 25–31. Heo, H.J., Kim, Y.J., Chung, D., Kim, D.O., 2007. Antioxidant capacities of individual and combined phenolics in a model system. Food Chemistry 104, 87–92. Pantelidis, G.E., Vasilakakis, M., Manganaris, G.A., Diamantidis, G.R., 2007. Antioxidant capacity, phenol, anthocyanin and ascorbic acid contents in raspberries, blackberries, red currants, gooseberries and Cornelian cherries. Food Chemistry 102, 777–783. Rojas-Melgarejo, F., Rodrı´guez-Lo´pez, J.N., Garcı´a-Ca´novas, F., Garcı´a-Ruiz, P.A., 2004. Immobilization of horseradish peroxidase on cinnamic carbohydrate esters. Process Biochemistry 39, 1455–1464. Rommel, A., Wrolstad, R.E., 1993. Influence of acid and base hydrolysis on the phenolic composition of red raspberry juice. Journal of Agricultural and Food Chemistry 41, 1237–1241.
E. Is¸ık et al. / Journal of Food Composition and Analysis 24 (2011) 944–949 Sarıburun, E., S¸ahin, S., Demir, C., Tu¨rkben, C., Uylas¸er, V., 2010. Phenolic content and antioxidant activity of raspberry and blackberry cultivars. Journal of Food Science 75, 328–335. Seeram, N.P., Adams, L.S., Zhang, Y., Lee, R., Sand, D., Scheuller, H.S., Heber, D., 2006. Blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth and stimulate apoptosis of human cancer cell in vitro. Journal of Agricultural and Food Chemistry 54, 9329–9339. Seeram, N.P., 2008. Berry fruits: compositional elements, biochemical activities, and the impact of their intake on human health, performance, and disease. Journal of Agricultural and Food Chemistry 56, 627–629. Sellappan, S., Akoh, C.C., Krewer, G., 2002. Phenolic compounds and antioxidant capacity of Georgia-grown blueberries and blackberries. Journal of Agricultural and Food Chemistry 50, 2432–2438. Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdic and phosphotungstic acid reagents. American Journal of Enology and Viticulture 16, 144–147. Singleton, V.L., Orthofer, R., Lamuela-Ravento´s, R.M., 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteu reagent. Methods in Enzymology 299, 152–178.
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Stevanato, R., Fabris, S., Momo, F., 2004. New enzymatic method for the determination of total phenolic content in tea and wine. Journal of Agricultural and Food Chemistry 52, 6287–6293. Temoc¸in, Z., Yig˘itog˘lu, M., 2009. Studies on the activity and stability of immobilized horseradish peroxidase on poly(ethylene terephthalate) grafted acrylamide fiber. Bioprocess and Biosystems Engineering 32, 467–474. TS 6397, 1989. Fruits, Vegetables and Derived Products – Determination of Ascorbic Acid Content – Part 2: Routine Methods. . Tu¨rkben, C., Sarıburun, E., Demir, C., Uylas¸er, V., 2010. Effect of freezing and frozen storage on phenolic compounds of raspberry and blackberry cultivars. Food Analytical Methods 3, 144–153. Veitch, N.C., Smith, A.T., 2001. Horseradish peroxidase. Advanced Inorganic Chemistry 51, 107–162. Wu, X., Prior, R.L., 2005. Systematic identification and characterization of anthocyanins by HPLC–ESI-MS/MS in common foods in the United States: fruits and berries. Journal of Agricultural and Food Chemistry 53, 2589–2599. Zhang, Z., Pang, X., Xuewu, D., Ji, Z., Jiang, Y., 2005. Role of peroxidase in anthocyanin degradation in litchi fruit pericarp. Food Chemistry 90, 47–52. Piljac-Zˇegarac, J., Dunja Sˇamec, D., 2010. Antioxidant stability of small fruits in postharvest storage at room and refrigerator temperatures. Food Research International, doi:10.1016/j.foodres.2010.09.039.
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Original Article
Determination of total phenolic content of raspberry and blackberry cultivars by immobilized horseradish peroxidase bioreactor Esra Is¸ık a, Saliha S¸ahin a, Cevdet Demir a,*, Cihat Tu¨rkben b a b
Department of Chemistry, Faculty of Science and Arts, University of Uludag, Go¨ru¨kle, Bursa 16059, Turkey Department of Horticulture, Faculty of Agriculture, University of Uludag, Bursa 16059, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 July 2010 Received in revised form 30 January 2011 Accepted 30 January 2011 Available online 12 February 2011
Raspberry and blackberry cultivars were assayed for total phenol content by enzymatic method using immobilized horseradish peroxidase (HRP). The HRP was immobilized on the styrene–divinylbenzene– polygluteraldehyde (STY–DVB–PGA) beads. Total phenol content determined by immobilized and free enzymatic methods was compared with those obtained by applying the Folin–Ciocalteu method. The total phenol content by immobilized HRP method ranged from 254 to 973 mg gallic acid equivalent (GAE) per 100 g fw as well as 105 to 435 mg ellagic acid equivalent (EAE) per 100 g fw in raspberry and blackberry cultivars. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical values were correlated with total phenol content in raspberry using immobilized enzymatic method as GAE (R2 = 0.7450, P = 0.05) and EAE (R2 = 0.7449, P = 0.05). All antioxidant activity values were highly correlated with total phenol content in blackberry using free and immobilized enzymatic methods as GAE (R2 = 0.9777 and 0.9223, P = 0.05 respectively) and EAE (R2 = 0.9979 and 0.9223, P = 0.05 respectively). A significant additivity was observed using individual phenolic compounds by immobilized enzymatic method as 95% (GAE) and 90% (EAE). These results show that the immobilized enzymatic assay can be applied to determine the total phenol content of berry fruits, which is more specific and not affected by interfering compounds. ß 2011 Elsevier Inc. All rights reserved.
Keywords: Raspberry (Rubus idaeus) Blackberry (Rubus fruticosus) DPPH Total phenol content Horseradish peroxidase Food analysis Food composition
1. Introduction Berry fruits are rich in phenolic compounds contents such as phenolic acids (Rommel and Wrolstad, 1993; Ha¨kkinen et al., 1998), flavonoids (Ha¨kkinen et al., 1998) and anthocyanins (Rommel and Wrolstad, 1993), which have been demonstrated that considerable antioxidant properties in vivo and in vitro in berries (Heinonen et al., 1998). The phenolic contents of berries are therefore an important parameter for the evaluation of their antioxidant properties and quality. Berries contain a diverse range of phenolic compounds with biological properties such as anticancer, anti-neurodegenerative and anti-inflammatory activities (Seeram et al., 2006; Seeram, 2008). The total phenolic content of fruits is therefore an important parameter of their antioxidant properties. The usual determination of total phenolic content has been achieved using the Folin–Ciocalteu procedure of Singleton (Singleton and Rossi, 1965; Singleton et al., 1999; Cliffe et al., 1994). This method is based on the oxidation of the reduced molecules by a mixture of the two strong inorganic oxidant phosphotungstic and phosphomolybdic acids. Separating techniques have been used to isolate, identify and determine individual
* Corresponding author. Tel.: +90 224 2941727; fax: +90 224 2941889. E-mail address: [email protected] (C. Demir). 0889-1575/$ – see front matter ß 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2011.01.016
polyphenolic compounds in berries, but these methods appear to be relatively more expensive and time consuming (Sellappan et al., 2002; Tu¨rkben et al., 2010). Recently, enzymatic methods and biosensors have been reported for the determination of total phenol content in tea, wine and juices (Stevanato et al., 2004; Girelli et al., 2009a). It has been found that a great number of phenols can act as aromatic donor molecules (Adam et al., 1999; Veitch and Smith, 2001). Also, flavonoids containing phenol B rings (Chan et al., 1999) and in general dietary polyphenols (Galati et al., 2002) can be oxidized by peroxidase/H2O2 to give reactive aryloxy radicals. Therefore, enzymatic method appears to be a suitable measure of the antioxidant properties of polyphenols. The enzymatic method offers the advantages of a shorter measuring period, a greater specificity toward the phenolic compounds, and the absence of the interference substances. Several enzymes have been immobilized on the pore glass support (Girelli et al., 2009a,b) and polymeric beads (Aybastıer and Demir, 2010) to obtain stable and reusable bioreactor that may be used in spectrophotometric and high performance liquid chromatographic (HPLC) systems. Horseradish peroxidase (HRP) has been immobilized by adsorption on cinnamic carbohydrate esters and used for phenolic resin synthesis and bioremediation (Rojas-Melgarejo et al., 2004). Several methods have been developed to determine the antioxidant activity of fruits, vegetables as well as herbs. Two
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major mechanisms, namely hydrogen atom transfer (HAT) and single electron transfer (SET), are well known in the evaluation of antioxidant activity against free radicals. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay is one of the methods utilizing both hydrogen atom transfer and single electron transfer mechanism. It is considered to be predominantly based on electron transfer reaction whereas hydrogen-atom transfer reaction is only a marginal pathway (Chen, 2006). In the present study, we propose a new immobilized enzymatic method for the determination of the total phenolic content in raspberry and blackberry fruits The HRP was immobilized on the styrene–divinylbenzene–polygluteraldehyde (STY–DVB–PGA) beads that were synthesized as in our previous paper (Aybastıer and Demir, 2010). The total phenol contents determined by free and immobilized enzymatic methods were compared with those obtained by applying the Folin method. Furthermore, the immobilized enzymatic method was validated by measuring the total phenol contents of individual phenolic compounds and their mixture, and summability of each method was evaluated. 2. Materials and methods 2.1. Chemicals Caffeic acid (98%) ferulic acid (98%), and p-coumaric acid (98%) were purchased from Merck (Darmstadt, Germany); phydroxybenzoic acid (99%), quercetin hydrate (95%), Folin– Ciocalteu phenol reagent, DPPH (2,2-diphenyl-1-picrylhydrazyl), gallic acid (98%), 4-aminoantipyrine (98%), and hydrogen peroxide (30%) were purchased from Sigma–Aldrich (St. Louis, USA); horseradish peroxidase donor: hydrogen peroxidase oxidoreductase, EC 1.11.1.7 type II (50 KU/mg of solid), was supplied from Sigma; ellagic acid (95%) was purchased from Fluka (Buchs, Switzerland). Methanol (HPLC grade) and hydrochloric acid (37%) were of analytical grade. All standard solutions were prepared in methanol (Merck, Darmstadt, Germany). 2.2. Plant materials Raspberry fruits (Rubus idaeus L.) of five cultivars (Aksu Kırmızısı, Rubin, Newburgh, Holland Boduru, Heritage) and blackberry fruits (Rubus fruticosus L.) of four cultivars (Bursa 1, Bursa 2, Jumbo, Chester) were collected from different commercial orchards in the region of Kestel (Bursa, Turkey), and handharvested at commercially mature stage during the growing season of July–August 2008. The fresh fruits were transported to the laboratory in the same day for sample preparation and analysis. The samples were stored at +4 8C for less than 2 days before extraction. It has been reported that berry fruits are stable about 10 days at +4 8C (Piljac-Zˇegarac and Dunja Sˇamec, 2010). 2.3. Extraction and hydrolysis Fresh raspberry and blackberry fruit samples in three lots of 5 g each of were separately blended with methanol. Ascorbic acid (80 mg) and 10 mL of 6 mol/L HCl (final concentration 1.2 mol/L HCl) were added to crushed berries. The mixture was stirred with magnetic stirrer and kept at room temperature for 16 h in dark. The hydrolyzed samples (total volume 50 mL) were separated from the solid matrix by filtration through sheets of qualitative filter paper. The hydrolyzed samples (total volume 50 mL) were separated from the solid matrix by filtration through sheets of qualitative filter paper. The extracts were used for the determination of total phenol contents by Folin, free and immobilized enzymatic methods and antioxidant activity. The extraction procedure was repeated three times (three extractions from one container of 500 g) for each
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sample. The same extracts were used for the determination of total phenol contents and antioxidant activity. 2.4. UV/vis spectroscopy Spectrophotometric measurements were performed on a UV/ vis spectrometer (Varian Cary 50) equipped with 10 mm quartz cuvettes. 2.5. Analytical methods The solution used in the Folin assay of phenolics was prepared as follows: Lowry A: 2% aqueous Na2CO3 in 0.1 M NaOH; Lowry B: 0.5% CuSO4 aqueous solution in 1% NaKC4H4O6 solution; Lowry C: prepared freshly as mixture (50 mL Lowry A + 1 mL Lowry B); Folin–Ciocalteu reagent was diluted with H2O at a volume ratio 1:3 prior to use. The amount of total phenols in the berries was determined with Folin–Ciocalteu reagent according to the method of Apak et al. (2008). A 0.1 mL of berry extract, 1.9 mL of H2O and 2.5 mL of Lowry C solution were mixed and the mixture was allowed to stand for 10 min. At the end of this period, 0.25 mL of Folin reagent was added, and 30 min was allowed for stabilization of the blue color. The absorbance was measured at 750 nm. Total phenol contents were calculated from standard curves of gallic acid and ellagic acid, and results were expressed as mg of gallic acid equivalent (GAE) per 100 g of fresh weight (fw) and ellagic acid equivalent (EAE) per 100 g of fresh weight (fw) separately. Total phenolic content of berries was determined with free horseradish peroxidase (HRP) assay. A solution constituted of 100 mL of berry extract, 900 mL of 2.5 mM HRP, 2 mL of 9 mM 4aminoantipyrine in 0.1 M phosphate buffer, pH 8.0 and 1 mL of 6 mM H2O2, final volume of 4 mL was spectrophotometrically monitored at 510 nm. All measurements were performed in triplicate. Total phenol contents were calculated from standard curves of gallic acid and ellagic acid, and results were expressed as mg of GAE per 100 g of fresh weight (fw) and EAE per 100 g of fresh weight (fw) separately. HRP was immobilized on the STY–DVB–PGA beads (0.6–1.4 mm particle size) by covalent attachment. Synthesis of STY–DVB–PGA and immobilization procedure has been described in our previous paper (Aybastıer and Demir, 2010). The method was described briefly as follows: glutaraldehyde was polymerized in aqueous solution at pH 10.5. The polymerized glutaraldehyde solution was added to the organic mixture containing styrene, divinylbenzene and span. Polymerization was conducted at 80 8C for 3 h. The polymer (120 mg of the STY–DVB–PGA) was placed in a polyethylene column (size 6 mm 1 mm) and the enzyme (21 mg HRP) in 100 mL of 0.1 M phosphate buffer (pH 8) as a carrier solution was recycled through the column by a peristaltic pump at 2.5 mL/min flow rate for 26 h to eliminate unbound protein the polymer was washed with 0.1 M phosphate buffer. The amount of immobilized enzyme (as protein) on the support was calculated from the difference in the amount of protein contents in the solution before and after immobilization using the Bradford assay method (Bradford, 1976). The protein concentrations were determined by spectrophotometric measurements at 595 nm. Coomassie brilliant blue solution was used as dye reagent. The amount of protein was calculated from a standard curve of bovine serum albumine (BSA). After coupling of the enzyme to the STY– DVB–PGA, 82% of protein was removed from the solution at 26 h. Total phenolic content of berries was determined by immobilized HRP assay. A carrier solution containing of 2.25 mM 4aminoantipyrine in 0.1 M phosphate buffer (pH 8.0) was passed from the system continuously using peristaltic pump operated at 2.5 mL/min flow rate. While the system was operating, a mixture of 0.1 mL sample and 1 mL of 2.97 mM H2O2 solution was added to
Folin
Free HRP
R
2500
İmmobilized HRP
2000 1500 1000 500
te r
Ju m bo
H
ol la nd
N
a
C
he s
rs a
2
1 Bu
Bu
rs a
du ru Bo
er ita H
ew
ub i
ge
n
0 Ak su
mg gallic acid/100 g fruit
the carrier solution. After enzymatic reaction, the absorbance of final quinone-imine-colored product was measured at 510 nm. All measurements were performed in triplicate. Total phenol contents were calculated from standard curves of gallic acid and ellagic acid, and results were expressed as mg of GAE per 100 g of fresh weight (fw) and EAE per 100 g of fresh weight (fw) separately. Free radical scavenging activity of berries was determined with DPPH reagent according to our previous paper (Sarıburun et al., 2010). The fruit extracts (0.1 mL) and 0.18 mL, 1 103 mol/L of DPPH were mixed in methanol, then the final volume was brought up to 3 mL with methanol. After the reaction was allowed to take place in the dark for 30 min, the absorbance was recorded at 515 nm against blank to determine the concentration of remaining DPPH. Standard curve was prepared using different concentrations of Trolox. The results were expressed as mmol Trolox equivalent (TE) per g fresh weight (fw).
bu rg h
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Raspberry cultivars
Blackberry cultivars
Fig. 1. Total phenol content expressed as GAE of the raspberry and blackberry cultivars according to the Folin, free HRP and immobilized HRP enzymatic methods. The reported values are the mean of triplicate experiments.
Folin
2000
Free HRP
İmm obilized HRP
1750 1500 1250 1000 750 500 250
bo m Ju
he C
Bu
rs
a
st er
2
1 sa Bu r
a
H
Bo
er ita
du
ru
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n ub i R
ew bu
rg h
0 Ak su
nd ol la H
Total phenol contents were determined in five raspberry and four blackberry cultivars using Folin, free and immobilized horseradish peroxidase (HRP) methods by UV–vis spectrophotometry. The comparison between Folin and enzymatic methods is shown in Figs. 1 and 2. The results represent the total phenol contents after subtraction of ascorbic acid contribution, which was used during the extraction of phenolic compounds in raspberry and blackberry. The average contribution of ascorbic acid to the total phenol content was 4.0 mg gallic acid equivalent (GAE) per g berry sample for extracts. Ascorbic acid content was determined by the standard spectrophotometric method (TS 6397). The method is based upon the quantitative discoloration of 2,6-dichlorophenol indophenol by ascorbic acid. The contents of ascorbic acid ranged from 18 to 26 mg per 100 g fw in raspberry and ranged from 19 to 26 mg per 100 g fw in blackberry cultivars. The total phenol content by immobilized HRP method ranged from 254 to 973 mg GAE per 100 g fw as well as 105 to 435 mg EAE per 100 g fw in raspberry and blackberry cultivars. Of the selected raspberry and blackberry cultivars, Bursa 2 showed the highest total phenol content (973 mg GAE per 100 g fw or 435 mg ellagic acid equivalent (EAE) per 100 g fw) while Heritage has the lowest total phenol content of samples (254 mg GAE per 100 g fw or 105 mg EAE per 100 g fw). The total phenol content in blackberry was higher than those of raspberry cultivars. On the other hand, immobilized enzymatic method was well correlated with Folin method (R2 = 0.7330 as GAE and R2 = 0.7455 as EAE in berries), which is in agreement with those reported for the determination of total phenol content of tea in the literature (Girelli et al., 2009a). Gallic acid has been mostly used as a standard for the determination of total phenol content in berries (De Ancos et al., 2000; Sellappan et al., 2002; Pantelidis et al., 2007). On the other hand, ellagic acid appears to be more sensitive in enzymatic method than gallic acid due to the fact that we used higher concentration of gallic acid in order to reach the same range of absorbance as ellagic acid (Table 1). This can be attributed that
N
3. Results and discussion
mg ellagic acid/100 g fruit
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Ras pberry cultivars
Blackberry cultivars
Fig. 2. Total phenol content expressed as EAE equivalent of the raspberry and blackberry cultivars according to the Folin, free HRP and immobilized HRP enzymatic methods. The reported values are the mean of triplicate experiments.
ellagic acid has more hydroxyl groups than gallic acid. Moreover, individual phenolic compounds have been determined in raspberry and blackberry cultivars in our previously study and ellagic acid was the main phenolic compound in these berries (Tu¨rkben et al., 2010). The Folin method values of raspberry and blackberry are higher than those of enzymatic assays. This comparison indicated that the total phenol content of a certain type of cultivars of raspberry and blackberry can be significantly different due to different assays. A variation in the content of total phenol of berries could be observed due to their content of anthocyanin and ascorbic acid, which have been determined in raspberry and blackberry (De Ancos et al., 2000; Wu and Prior, 2005; Chen et al., 2007; Pantelidis et al., 2007). The higher results of total phenol content may probably be explained by poor specificity of Folin method, which can give an
Table 1 Validation parameters of standard phenolic compounds by Folin, free and immobilized enzymatic methods. Methods
LOD (mg/L)
LOQ (mg/L)
Precision (RSD%)
Recovery (%)
Linearity (R2) and linear range (mg/L)
Folin–Ciocalteu (mmol GAE) Folin–Ciocalteu (mmol EAE) Free enzyme (mmol GAE) Free enzyme (mmol EAE) Immobilized enzyme (mmol GAE) Immobilized enzyme (mmol EAE)
1.09 0.55 2.65 0.27 1.68 1.38
3.63 1.83 8.83 0.90 5.60 4.60
3.15 3.88 2.14 2.44 1.72 1.43
78 107 80 86 90 91
0.9984 0.9977 0.9983 0.9982 0.9971 0.9978
LOD, limit of detection; LOQ, limit of quantification; RSD, relative standard deviation; GAE, gallic acid equivalent; EAE, ellagic acid equivalent.
(1.6–20) (1.6–15) (3.8–20) (1.8–10) (3.8–25) (1.8–15)
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Table 2 Total phenol content of standard phenolic compounds by Folin and immobilized enzymatic methods.a Phenolic compounds
Ellagic acid Ferulic acid Caffeic acid p-Coumaric acid p-Hydroxybenzoic acid Quercetin Total Mixture Additivity (%)
Folin method (mmol GAE)
Immobilized enzymatic method (mmol GAE)
Folin method (mmol EAE)
Immobilized enzymatic method (mmol EAE)
0.127 0.009 0.114 0.008 0.135 0.010 0.130 0.006 0.036 0.003 0.092 0.005
0.136 0.008 0.030 0.003 0.055 0.004 0.083 0.007 0.135 0.009 0.065 0.006
0.021 0.002 0.019 0.001 0.021 0.002 0.021 0.001 0.012 0.001 0.017 0.001
0.070 0.007 0.065 0.006 0.030 0.002 0.027 0.003 0.039 0.003 0.034 0.002
0.633 0.037 0.584 0.024 92
0.503 0.012 0.476 0.031 95
0.111 0.001 0.087 0.004 78
0.264 0.017 0.237 0.018 90
GAE, gallic acid equivalent; EAE, ellagic acid equivalent. a Mean of three determinations SD (standard deviation).
overestimated value of total phenol content in berry fruits. It has been reported that, between the nonphenolic compounds such as citric acid, ascorbic acid and sulfite are potential interfering substances on the determination of total phenol content by the Folin method. They contribute to increase the absorbance value of the phenolic compounds. However, experimental data show that total phenol content by enzymatic method is not affected by the presence of interfering substances such as ascorbic acid and citric acid (Stevanato et al., 2004). Furthermore, peroxidase could catalyze the degradation of the anthocyanidin as a substrate in the presence of hydrogen peroxide (Zhang et al., 2005). Thus, the enzyme can be inhibited by anthocyanins and the residual enzyme activity may not be enough to catalyze the total conversion of the phenolic compounds. HRP can also be inactivated by reactions between hydrogen peroxide and enzymatic reaction intermediates, and irreversible reactions between the enzyme and phenoxyl radicals formed by oxidation of phenolic compounds (RojasMelgarejo et al., 2004). Individual anthocyanins and anthocyanidins have been identified in raspberry and blackberry cultivars in our previous paper (Sarıburun et al., 2010). Five anthocyanins and five anthocyanidins have been identified, using liquid chromatography–mass spectrometry (LC–MS/MS), as cyanidin-3-(2G-glucosylrutinoside), cyanidin-3-sophoroside, cyanidin-3-glucoside, cyanidin-3-rutinoside, cyanidin, peonidin, malvidin, pelargonidin in water, and pelargonidin-3-rutinoside, pelargonidin and delphinidin in methanol extract of raspberry cultivars. On the other hand, three anthocyanidins such as cyanidin, malvidin and pelargonidin were identified in water and delphinidin was identified in methanol extract of blackberry. By comparison of the results obtained by free and immobilized enzymatic methods it appeared that a significant difference of total phenol content between two methods was obtained in both raspberry and blackberry samples (Figs. 1 and 2). The differences might be due to the inactivation of enzyme by interaction with the support and the coupling conditions. However, immobilized enzymatic method has several advantageous properties such as stability, repeatedly use in reactors, easily separation from soluble reaction products, reuse stability, which can effectively reduce the cost in applications and unused substrate, thus simplifying workup and preventing protein contamination of the final product (Temoc¸in and Yig˘itog˘lu, 2009; Aybastıer and Demir, 2010). In order to figure out which method is more accurate, individual contribution of phenolic compounds to total phenol content was investigated by Folin and immobilized enzymatic methods. Levels of total phenol content of individual phenolic compounds at 4.5 mM were ranged between 0.136 mmol GAE in ellagic acid by immobilized enzymatic method and 0.012 mmol EAE in phydroxybenzoic acid by Folin method (Table 2). The differences between total phenol contents of individual phenolic compounds
could be explained by the number and position of substituted hydroxyl or methoxyl groups and glycosylation around the flavonoid skeleton. It is well-known that the antioxidant properties of the polyphenols are correlated with the delocalization, on the aromatic ring, of the phenoxyl unpaired electron, which stabilizes the free radical. Folin method is based on the chemical oxidation of the reduced molecules by a mixture of the two strong inorganic oxidants, phosphotungstic acid and phosphomolybdic acids (Stevanato et al., 2004). In enzymatic method, in the presence of hydrogen peroxide, horseradish peroxidase is oxidized. The oxidized form of enzyme will come into contact with phenols, which will result in the formation of phenoxyl radicals. These radicals will further react with the aromatic amine group of 4aminoantipyrine, giving rise to the final quinone-imine colored product. The total contribution percentage of phenolic compounds using Folin method was 92% as GAE and 78% as EAE whereas the contribution percentage was 95% as GAE and 90% as EAE in immobilized enzymatic method. Total antioxidant capacity in the phenolic mixture was almost equal to the sum of antioxidant capacity of its individual phenolics (Heo et al., 2007). On the other hand, enzymatic method is convenient for routine use in the laboratories. It only requires 5 min reaction time and relatively simple preparation whereas Folin needs more time and quite complicated procedure. The immobilized enzyme can be used repeatedly for at least 10 samples. The antioxidant activity of fruit extracts was determined as Trolox equivalents (mmol TE/g fw) using the DPPH assay in raspberry and blackberry cultivars. Figs. 3 and 4 demonstrate the correlations between Folin, free HRP and immobilized HRP
Fig. 3. Correlation between Folin, free HRP and immobilized HRP enzymatic methods and DPPH values for raspberry cultivars. FLNGA: Folin gallic acid equivalent; FLNEA: Folin ellagic acid equivalent; FRGA: free enzyme gallic acid equivalent; FREA: free enzyme ellagic acid equivalent; IMGA: immobilized enzyme gallic acid equivalent; IMEA: immobilized enzyme ellagic acid equivalent.
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4. Conclusions
Fig. 4. Correlation between Folin, free and immobilized enzymatic methods and DPPH values for blackberry cultivars. FLNGA: Folin gallic acid equivalent; FLNEA: Folin ellagic acid equivalent; FRGA: free enzyme gallic acid equivalent; FREA: free enzyme ellagic acid equivalent; IMGA: immobilized enzyme gallic acid equivalent; IMEA: immobilized enzyme ellagic acid equivalent.
We have developed an immobilized enzymatic method for the rapid determination of total phenol content in raspberry and blackberry fruits. The presented method has several advantageous properties such as reuse stability of immobilized HRP, operational cost, easy separation from the reaction mixture and not affected by the presence of interference substances. The applicability and reliability of this analytical approach was confirmed by method validation and successful analysis of raspberry and blackberry samples. Significant correlation with antioxidant activity assay (DPPH) shows that the immobilized enzymatic method can be applied to determine the total phenol content even for a specific phenol class determination. Thus, antioxidant activity of berries analysed appears to be largely influenced by these phenolic compounds. In conclusion, the developed method is suitable for routine use in laboratories and should be utilized for rapid analysis of fruit extracts.
References enzymatic methods and DPPH values. Antioxidant activity was highly correlated with total phenol content by Folin method as gallic acid (FLNGA) (R2FLNGA ¼ 0:7163) and ellagic acid equivalents (FLNEA) (R2FLNEA ¼ 0:7164) in blackberry (Fig. 4). When enzymatic methods of data were compared, high correlations were observed between immobilized enzymatic method as gallic acid (IMGA), ellagic acid (IMEA) and DPPH assay (R2IMGA ¼ 0:7450 and R2IMEA ¼ 0:7449) in raspberry (Fig. 3). On the other hand, the highest correlations were observed between free enzymatic as gallic acid (FRGA), ellagic acid (FREA), immobilized enzymatic methods and DPPH assay (R2FRGA ¼ 0:9777, R2FREA ¼ 0:9979, R2IMGA ¼ 0:9223 and R2IMEA ¼ 0:9223) in blackberry (Fig. 4). In addition, the correlations among enzymatic methods are all positive which indicate that higher the total phenol content of the raspberry and blackberry, the better antioxidant activity. The results confirm that the immobilized enzymatic method could be used for the determination of antioxidant capacity in berry samples. The validation of the quantitative determination of total phenolic contents by Folin and enzymatic methods was performed by limits of detection (LODs; 3s/m), recovery (%), linearity and precision of gallic and ellagic acids (Table 1), where s is the standard deviation of the replicates and m is the slope of the calibration curve. LODs ranged from 1.09 to 2.65 mg/L and ranged from 0.27 to 1.38 mg/L for gallic and ellagic acids, respectively. Lower range of calculated LODs using ellagic acid as standard corresponds to the presence of more hydroxyl group in the molecule. The higher LOD value for free enzymatic method (2.65 mmol GAE) could be due to the higher linearity range of the response. The linearity of the methods was studied with gallic and ellagic acids levels ranging from 1.6 to 25 mg/L. The responses were linear up to 15 mg/L for ellagic acid and 25 mg/L for gallic acid with R2 values ranging from 0.9977 to 0.9982 and ranging from 0.9971 to 0.9984 for both phenolic compounds, respectively. The precision and recovery of the methods were evaluated by spiking raspberry and blackberry samples with standard gallic (15 mg/L) and ellagic (25 mg/L) acids. The precision of the methods, expressed as relative standard deviation (RSD%), was estimated by measuring three replicates of each compound. Better precisions were obtained by immobilized enzymatic method for both gallic and ellagic acids. The mean percentage recoveries ranged from 78 (Folin method) to 90% (immobilized enzymatic method) for gallic acid and ranged from 86 (immobilized enzymatic method) to 107% (Folin method) for ellagic acid. High recoveries were obtained by immobilized enzymatic method with gallic and ellagic acids.
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