Chlorogenic Acid Oxidation by a Crude Peroxidase Preparation: Biocatalytic Characteristics and Oxidation Products

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Food Bioprocess Technol (2012) 5:243–251 DOI 10.1007/s11947-009-0241-8

ORIGINAL PAPER

Chlorogenic Acid Oxidation by a Crude Peroxidase Preparation: Biocatalytic Characteristics and Oxidation Products Ali Osman & Ayman El Agha & Dimitris P. Makris & Panagiotis Kefalas

Received: 1 June 2009 / Accepted: 28 July 2009 / Published online: 21 August 2009 # Springer Science + Business Media, LLC 2009

Abstract Plant food residues including trimmings and peels might contain a range of enzymes capable of transforming bio-organic molecules, and thus they may have potential uses in several biocatalytic processes, including green organic synthesis, modification of food physicochemical properties, bioremediation, etc. Although the use of bacterial and fungal enzymes has gained interest in studies pertaining to biocatalytic applications, plant enzymes have been given less attention or even disregarded. In this view, this study aimed at investigating the use of a crude peroxidase (POD) preparation from onion solid by-products for oxidizing chlorogenic acid (CGA), a widespread phenolic acid, various derivatives of which may occur in foods and food wastes. The highest enzyme activity was observed at a pH value of 4, but considerable activity was also observed at pH 2. Favorable temperatures for increased activity varied between 5 and 20°C. Liquid chromatography–mass spectrometry analysis of a PODtreated CGA solution showed the formation of two major oxidation products, which were tentatively identified as CGA dimers.

A. Osman : A. El Agha : P. Kefalas Laboratory of Chemistry of Natural Products, Mediterranean Agronomic Institute of Chania (M.A.I.Ch.), P. O. Box 85, 73100 Chania, Greece D. P. Makris (*) Institute of Technology and Management of Agricultural Ecosystems (I.T.E.M.A.), Centre for Research and Technology— Thessaly (CE.RE.TE.TH.), 1st Industrial Area, 38500 Volos, Greece e-mail: [email protected]

Keywords Biocatalysis . Chlorogenic acid . Onion . Peroxidase Abbreviations 4-AAP 4-aminoantipyrine CA caffeic acid CGA chlorogenic acid DMF dimethyl formamide ESI electrospray ionization FA ferulic acid p-CouA p-coumaric acid POD peroxidase SD standard deviation TCA trichloroacetic acid

Introduction The very effective catalytic properties of enzymes have promoted their introduction into several industrial products and processes. Over the past few years, there has been an increasing trend for the utilization of enzymes in various industrial applications, including fine chemicals synthesis (Davis and Boyer 2001; Liu et al. 2004), detergent manufacturing, fuel alcohol production, textile processing, etc. (Kirk et al. 2002). Regarding the food industry, enzymes have been widely employed in a spectrum of processes, such as aroma development, fruit juice elaboration, and the production of novel oils and fats with improved physical and/or nutritional attributes (Villeneuve 2003; Figueroa-Espinosa and Villeneuve 2005). Substantial effort has been focused on microbial sources (Synowiecki et al. 2006) and, in this context, the search for novel functional food ingredients

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Fig. 1 POD activity variation in response to CGA concentration. Reaction conditions: [H2O2]=2 mM; [Total protein]=34 µg mL−1; pH 4; T=23±2°C

Fig. 3 Dependence of POD activity on pH. Reaction conditions: [CGA]=1 mM; [H2O2]=1.6 mM; [Total protein]=34 µg mL−1; T= 23±2°C

using combinatorial enzymatic derivatization (Gayot et al. 2003; Mellou et al. 2005; Torres de Pinedo et al. 2005; Stevenson et al. 2005; Matsuo et al. 2008) and also the replacement of synthetic raisins by enzymatically polymerized polyphenols (Uyama and Kobayashi 2006) have been attempted. Plant peroxidases (POD) have significantly been investigated not only with regard to their potential in eliminating phenolic pollutants (Hamid and Rahman 2009) or other toxic compounds (Tripathi and Mishra 2009) but also for their utility in the generation of novel, bio-based polyphenols with important biological activities, including sinapic acid (SA) dimers and other oligomers (Liu et al. 2007) and ferulic acid (FA)/resveratrol heterodimers (Yu et al. 2007). The ability of POD to form ferulic and caffeic

acid (CA) dimers has been shown using an enzyme preparation from Bupleurum salicifolium callus cultures (Luis et al. 2005), but more recently, the generation of FA (El Agha et al. 2008a) and hydrocaffeic acid (El Agha et al. 2008b) dimers, as well as CA tetramers (El Agha et al. 2009), has been possible using a POD preparation from onion solid wastes. Hydroxycinnamate dimers and/or oligomers occur widely in certain plant foods and food products, such as sage (Bors et al. 2004) and grains including wheat, rye, and buckwheat (Gallardo et al. 2006). The interest in hydroxycinnamates as bioactive components of the diet, as structural and functional components of plant cell walls, and as precursors for flavors in the food industry has expanded rapidly in the last 5– 10 years Therefore, the potential of plant POD in the

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Fig. 4 Dependence of POD activity on temperature. Reaction conditions: [CGA]=1 mM; [H2O2]=1.6 mM; [Total protein]=34 µg mL−1; pH 4

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All solvents used for chromatography were high-performance liquid chromatography (HPLC) grade. CGA and 4aminoantipyrine (4-AAP) were from Sigma Chemical (St. Louis, MO, USA). Hydrogen peroxide (30%) and trichloroacetic acid (TCA) were from Merck (Germany). For pH 2 and 8, a 100-mM potassium chloride/HCl and a 100-mM boric acid/NaOH buffer were used, respectively. For the pH range 3–7, a 50-mM phosphate/citrate buffer was used.

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Fig. 5 POD activity as a function of total protein content. Reaction conditions: [CGA]=1 mM; [H2O2]=1.6 mM; pH 4; T=23±2°C

enzymatic production of functional bioactive compounds to be used in the food sector should not be discarded. In this context, this study was undertaken to investigate some basic properties of POD obtained from an inexpensive source, such as onion solid wastes, with regard to generating derivatives of chlorogenic acid (CGA), a widespread hydroxycinnamate, in order to start paving the road for the industrial application of this enzymatic process.

CGA

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Fig. 6 Chromatographic profile of a CGA solution treated with crude POD for 10 min. Reaction conditions: [CGA]=1 mM; [H2O2]=1.6 mM; pH 4; T=23± 2°C. CGA chlorogenic acid, 1 and 2 chlorogenic acid oxidation products. The inset picture shows the UV–Vis spectrum that corresponds to both 1 and 2

The onion solid waste used in this study was obtained from a local catering facility (Chania, Crete) after processing of brown-skin onion bulbs. The waste consisted of the apical trimmings of the bulbs, as well as the outer dry and semidry layers (Khiari et al. 2008). The cell-free, POD-active extract was prepared as previously described (El Agha et al. 2008a). The material was transferred to the laboratory immediately after processing and ground in a domestic blender. An aliquot of 2 g of the ground tissue was suspended in 15 mL buffer (citrate/phosphate, pH 4) under stirring, and the suspension was centrifuged at 3,000×g for 20 min and filtered through filter paper to remove cell debris. The clear supernatant obtained was treated with activated charcoal for decolorization

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Fig. 7 Mass spectra of product 1 (a) and 2 (b). Upper and lower spectra in each case were obtained at −12 and −50 eV collision energy, respectively

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Fig. 8 MS fragmentation pathway of the tentative C–C dimer of CGA

and filtered through celite under vacuum. The clear filtrate was used as the crude enzyme source. Peroxidase Activity A previously established protocol was employed (El Agha et al. 2008a). The assay mixture contained 0.25 mL 4-AAP (10 mM in water), 0.1 mL substrate (CGA), 100 mM in DMF, 0.1 mL H2O2, 0.5 mL buffer, and 0.1 mL enzyme extract. Absorbance was monitored at 510 nm for over 2 min against suitable blank. One enzyme unit was defined as ΔA510 per second. Control reactions by omitting H2O2 or using heat-inactivated homogenate were also carried out. In assays performed at different temperatures, all constituents of the reaction mixture were preincubated either in a refrigerator (5°C) or in a thermostated water bath (30–60°C). For all determinations, a computer-controlled HP 8452A diode array spectrophotometer was used.

(Whatman), and the filtrate was used for chromatographic analyses. HPLC-DAD Analysis The equipment utilized was an HP 1090 series II liquid chromatograph coupled with an HP 1090 diode array detector (DAD) and controlled by Agilent ChemStation software. The column was a LiChrosphere RP18, 5 µm, 250×4 mm (Merck), protected by a guard volume packed with the same material. Both columns were maintained at 40°C. Eluent A and eluent B were 1% formic acid and acetonitrile, respectively. The flow rate was 1 mL min−1, and the elution program used was as follows: 0–5 min, 5% B, 5–45 min, 100% B, 45–55 min, 100% B. Monitoring of the eluate was performed at 320 nm. Liquid Chromatography–Mass Spectrometry A Finnigan MAT Spectra System P4000 pump was used coupled with a UV6000LP DAD and a Finnigan AQA mass spectrometer. Analyses were carried out on a Superspher RP18, 125×2 mm, 4 µm, column (Macherey-Nagel, Germany), protected by a guard column packed with the same material, and maintained at 40°C. Analyses were carried out employing electrospray ionization (ESI) in negative ion mode with acquisition set at −12 and −50 eV, capillary voltage at 4 kV, source voltage at 4.9 kV, detector voltage at 650 V, and probe temperature at 400°C. Eluent A and eluent B were 2.5% acetic acid and methanol, respectively. The flow rate was 0.33 mL min−1, and the elution program used was as follows: 0–5 min, 0% B; 5–30 min, 100% B; 30–35 min, 100% B. Statistical Analyses All determinations were carried out at least in triplicate and values were averaged and given along the standard deviation (±SD). For all statistics, Microsoft Excel™ 2000 and SigmaPlot™ 9.0 were used.

Protein Determination Results Protein content was determined according to Bradford (1976) using bovine serum albumin as standard. Chlorogenic Acid Oxidation A solution of CGA (1 mM) was oxidized with crude POD (total protein content 34 µg mL−1) and H2O2 (1.6 mM) for 10 min at room temperature (24±2°C) and pH 4. Following this, 0.1 mL of a 10% TCA solution in EtOH was added, and the mixture was centrifuged at 5,000×g for 10 min. The clear supernatant was filtered through 0.45 µm syringe filters

Biocatalytic Characteristics In view of determining some basic parameters that profoundly affect the oxidation of CGA, assays regarding the optimal pH and temperature, as well as substrate (CGA) and cosubstrate (H2O2) concentration, were undertaken. The effect of CGA concentration on the enzyme activity can be seen in Fig. 1. Increasing concentrations of CGA were shown to provoke a proportional effect on the enzyme activity, up to 1 mM. Thereafter, a decline in activity was

248 Fig. 9 MS fragmentation pathway of the tentative O–O dimer of CGA

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observed. Likewise, increases in H2O2 concentration were shown to promote enzyme activity up to 1.6 mM (Fig. 2). The examination of the pH effect over a range varying from 2 to 8 revealed that maximal enzyme activity was expressed at pH 4, whereas notable decline was seen when reactions were carried out at pH higher than 5 (Fig. 3). Significant activity, which was approximately 75% of the maximal one, was also seen at pH 2. In a similar fashion, temperatures ranging from 5 to 20°C were favorable, but thereafter a rapid decline was recorded (Fig. 4), and enzyme activity was virtually trivial at 60°C. By implementing optimal conditions with regard to substrate, cosubstrate, pH, and temperature, different dilutions of the homogenate were assayed (Fig. 5). Increasing amounts of total protein in the reaction mixture provoked proportionally

higher enzyme activities in a linear manner (R2 =0.99), suggesting that the rate of CGA oxidation is directly proportional to the total enzyme concentration. Tentative Identification of CGA Oxidation Products A CGA solution (1 mM) was incubated with POD cell-free extract under optimal conditions ([H2O2]=1.6 mM, pH 4, T=23±2°C) for 10 min. The mixture was then analyzed by HPLC and LC-ESI/MS in negative ionization mode. In the trace obtained at 320 nm (Fig. 6), two major peaks were detected (peaks 1 and 2), which were found to have identical UV spectrum with λmax =320, 328 nm (inset picture). Peak 1 gave a molecular ion [M–H]− at m/z 705 and a diagnostic fragment at m/z 727, which was identified as a

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Nicell 2000). In the case of CGA, the molar ratio found was 1.6, which is higher than those reported for structurally similar hydroxycinnamate derivatives, including hydrocaffeic acid (El Agha et al. 2008a) and CA (El Agha et al. 2009), where the H2O2/substrate ratios determined were 1.33 and 0.8, respectively. However, this value is lower than that reported for FA, which was 2.56 (El Agha et al. 2008b). Furthermore, the horseradish peroxidase (HPR)mediated oxidation of several phenolic pollutants, including 2-chlorophenol, 4-chlorophenol, o-cresol, p-cresol, and 2,4dichlorophenol, showed that the ratios may vary from 0.7 to 0.9 (Caza et al. 1999). The deviation between measured and theoretical stoichiometries has been postulated to be the result of several mechanisms in which products of the catalytic process are polymers larger than dimers. However, as no molecules larger than dimers were detected in the POD-catalyzed oxidation of CGA, it would be reasonably assumed that, as molecules polymerize and grow inside, they become insoluble and precipitate, and therefore, it is unlikely that they can be detected. Effect of pH and Temperature

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Fig. 10 Putative, generalized oxidation pathway of CGA leading to the formation of dimers

sodium adduct [M–2H+Na]− (Fig. 7a). A dihydromonomer radical [M•]− was also seen at m/z 352. Upon increased collision energy (Fig. 7a, lower spectrum), fragmentation resulted in the generation of the daughter ion with m/z 513, which was also accompanied by a small proportion of its Na+ adduct (m/z 535). This fragmentation pattern was consistent with a C–C linked dimer, as shown in Fig. 8. Peak 2 yielded the same molecular ion and Na+ adduct at m/z 705 and 727, respectively (Fig. 7b), but formation of the dihydromonomer radical [M•]−, as well as of the monomer (m/z 353), was more pronounced. The radical with m/z 382 was thought to derive from the fragment m/z 513, following cleavage of the quinic acid moiety, as shown in Fig. 9. Removal of the keto-bearing side chain from m/z 513 would also afford the ion with m/z 459. This fragmentation pattern appeared to be consistent with an O–O linked dimer.

Discussion Biocatalytic Properties CGA/H2O2 Concentration Ratio The stoichiometry between H2O2 and simple phenolic substrates in POD-catalyzed reactions has been reported to be one-to-one (Hewson and Dunford 1976; Zhang and

The investigation of a crude onion POD showed that quercetin oxidation might be favored at pH 8 (Takahama and Hirota 2000), but this result was contrasted by more recent data indicating that quercetin oxidation rate peaks at pH 4 (Osman et al. 2008). The pH optimum found for CGA is thus in line with this outcome, but also with the one found for hydrocaffeic acid and FA (El Agha et al. 2008a, b). Contrary to that, favorable temperatures were found to lie between 5 and 20°C, which is significantly lower than that found for quercetin (Osman et al. 2008) and hydrocaffeic acid (40°C; El Agha et al. 2008a) and also lower than those observed for ferulic and CA (30°C; El Agha et al. 2008b, 2009). Oxidation Mechanism of CGA POD from various sources are known to effectively oxidize hydroxycinnamate derivatives. This has been well documented by experiments with pear (Pyrus communis) POD and CGA, CA, FA, and p-coumaric acid (p-CouA; RichardForget and Gauillard 1997); 3,4-dihydroxyphenylalanine and HPR (Takahama and Yoshitama 1998); CA, FA, pCouA, and SA and potato (Solanum tuberosum) POD (Arrieta-Baez and Stark 2006); and CA, FA, and p-CouA with POD originating from soybean (Glycine max), maize (Zea mays), and horseradish (Šukalović et al. 2008). The oxidation of o-diphenols, such as CGA, by polyphenol oxidases is known to proceed via the formation of o-quinones (Pierpoint 1966), but o-quinones can also be

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formed by POD catalysis, after a disproportionation step (Richard-Forget and Gauillard 1997; El Agha et al. 2008a). That is because the primary CGA oxidation products formed upon POD action are radicals (Yamasaki and Grace 1998; Ralph et al. 2004). Following their formation, these radicals may combine in a variety of dimers. In the case presented herein, it was made clear that CGA oxidation by onion POD may afford at least two different types of dimers. Based on the evidence provided by the liquid chromatography–mass spectrometry (LC-MS) studies, these dimers might form through a C–C or an O–O bond, although the possibility of a C–O bond cannot be ruled out. The finding that both derivatives were shown to occur at relatively equal amounts, judging by the peak areas at 320 nm, might indicate that there is no preferable pathway and that radical couplings could be, to some extent, random. This hypothesis is consistent with other investigations that demonstrated the formation of a plethora of diferulates upon the action of POD (Yamasaki and Grace 1998). Although the oxidation of a variety of mixtures of various hydroxycinnamates by HRP revealed the generation of dehydrodimers (Arrieta-Baez and Stark 2006), the fact that both CGA dimers detected in mixtures oxidized by onion POD maintained the original CGA UV–Vis spectrum rather suggests that no further rearrangements occurred within the dimer molecules. Thus, these dimers might derive from simple radical–radical coupling (Fig. 10). This assumption, however, remains to be elucidated once the dimers are isolated and fully characterized by hyphenated spectroscopic techniques.

Conclusions In the study presented herein, the use of a POD-active, crude preparation from onion solid wastes is proposed for the first time as an alternative source of an oxidative enzyme that could have a prospect in biocatalytic applications involving CGA. The preliminary investigations carried out showed that conditions for optimal enzyme activity with regard to an abundant phenolic, CGA, might lie within a range of 4–5 and 5–20°C, for pH and temperature, respectively. Efforts to elucidate the oxidative behavior employing LC-MS provided strong evidence that the oxidative pathway(s) implicated may have common features with those encountered in the oxidation of FA and that the products formed are CGA dimers.

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