Food Anal. Methods (2010) 3:144–153 DOI 10.1007/s12161-009-9102-3
Effect of Freezing and Frozen Storage on Phenolic Compounds of Raspberry and Blackberry Cultivars Cihat Türkben & Esra Sarıburun & Cevdet Demir & Vildan Uylaşer
Received: 25 May 2009 / Accepted: 11 August 2009 / Published online: 2 September 2009 # Springer Science + Business Media, LLC 2009
Abstract The phenolic compounds in raspberry and blackberry cultivars grown in Turkey were determined by liquid chromatography–mass spectrometry (MS)/MS in fresh, just-frozen, and stored fruits at −22 °C for 6 months period. The major phenolic compounds in water extracted samples were ellagic acid (1,350.36–727.9 mg/kg fresh fruit), ferulic acid (820.78–338.27 mg/kg fresh fruit), caffeic acid (754.85–202.78 mg/kg fresh fruit), p-coumaric acid (361.68–142.63 mg/kg fresh fruit), p-hydroxybenzoic acid (534.20–233.29 mg/kg fresh fruit), and quercetin (46.97–27.31 mg/kg fresh fruit) in raspberry and ellagic acid (1,828.07–1,555.13 mg/kg fresh fruit), ferulic acid (757.69–413.82 mg/kg fresh fruit), caffeic acid (736.85– 337.89 mg/kg fresh fruit), p-coumaric acid (877.45– 287.15 mg/kg fresh fruit), and quercetin (74.69–56.78 mg/kg fresh fruit) in blackberry. The varietal differences in the phenolic compound contents were larger among the blackberry cultivars (from 1,828.07 to 56.78 mg/kg fresh fruit) than among the raspberry cultivars (1,350.36 to 27.31 mg/kg fresh fruit). A significant decrease was observed in the content of p-hydroxybenzoic acid (from 534.20 to 114.30 mg/kg; Aksu
C. Türkben Department of Horticulture, Faculty of Agriculture, University of Uludag, Bursa 16059, Turkey E. Sarıburun : C. Demir (*) Department of Chemistry, Faculty of Science and Arts, University of Uludag, Bursa 16059, Turkey e-mail:
[email protected] V. Uylaşer Department of Food Engineering, Faculty of Agriculture, University of Uludag, Bursa 16059, Turkey
Kırmızısı) and the least decrease was in the content of caffeic acid (from 545.42 to 530.91 mg/kg; Heritage) in raspberry cultivars. On the other hand, ferulic acid (from 475.16 to 113.33 mg/kg) decreased significantly in blackberry (Bursa 2) after storage for 6 months. Keywords Raspberry . Blackberry . Freezing and Frozen Storage . Phenolic Compounds . LC–MS/MS
Introduction Raspberry (Rubus idaeus L.) and blackberry (Rubus fruticosus L.), member of the Rosaceae family, provide delicious fruits that can be consumed fresh or as ingredient in processed products such as ice cream, jam, jelly, marmalade, purées, fruit juices, liquors, etc. In Bursa (Turkey), raspberry and blackberry are two of the most important berries, grown extensively on a commercial basis primarily for export. Most of them are sold fresh, but after harvest, their fresh fruits are highly perishable with limited postharvest life, mainly due to decay (Botrytis cinerea) and firmness loss. Therefore, fresh fruits are preserved by storage in normal and controlled atmospheres (Haffner et al. 2002) or freezing from harvest to processing (De Ancos et al. 2000b). Berry fruits are rich in phenolic compounds contents such as ellagic acid (Rommel and Wrolstad 1993c; Häkkinen et al. 1999a, b), hydroxybenzoic acids (p-hydroxybenzoic acid, protocatechuic acid, vanillic acid, gallic acid, and syringic acid), hydroxycinnamic acids (p-coumaric acid, ferulic acid, caffeic acid; Rommel and Wrolstad 1993a; Häkkinen et al. 1999b), anthocyanins (Rommel and Wrolstad 1993a), and flavonols (quercetin, kaempferol, myricetin; Rommel and Wrolstad 1993b;
Food Anal. Methods (2010) 3:144–153
Häkkinen et al. 1999a, b). The phenolic compounds in berries have been reported to have antioxidant, anticancer, anti-inflammatory, and antineurodegenerative biological properties (Seeram et al. 2006b; Seeram 2008). Ellagic acid has been reported as the main phenolic compound in raspberry and blackberry in literature (Häkkinen et al. 1999b; Mertz et al. 2007; Sellappan et al. 2002). Bioavailability and pharmacokinestics of ellagic acid from pomegranate juice in human has been studied in literature (Seeram et al. 2004). Because of the biological properties associated with berry fruits, the identification of their phytochemicals is necessary for the evaluation of raspberry and blackberry consumption on human health. The effects of freezing and frozen storage on quality and quantity of ellagic acid, flavonols, and anthocyanin content in different cultivars of raspberries and blackberries have been studied in literature (De Ancos et al. 2000a, b; Häkkinen et al. 2000a, b; Mullen et al. 2002). However, there are no references about the affect of freezing and frozen storage on the hydroxycinnamic and hydroxybenzoic acids contents in berry fruits. The contents of phenolic compounds in berries are not only affected by genetic differences and preharvest environmental conditions but also by the degree of maturity at harvest, variety, growing area, and seasonal variations (Zadernowski et al. 2005). Several methods and chromatographic techniques have been reported for extraction and identification of phenolic compounds in berries (Määttä-Rııhınen et al. 2004; Häkkinen et al. 1998). Phenolic extracts of plant materials are always a mixture of different classes of phenolics that are soluble in the solvent system used. Most common solvents are aqueous mixtures with methanol, ethyl acetate, and acetone. Furthermore, hydrolysis of glycoside bonds is often used in the extraction procedure, and thereby, essential information of total phenolic compounds as aglycones can be obtained (Hertog et al. 1992; Häkkinen et al. 2000a; Zafrilla et al. 2001). Various chromatographic techniques have been employed for separation and quantification of phenolic acids. High-performance liquid chromatography (HPLC) has been most widely used for both separation and quantification of phenolic compounds (Hertog et al. 1992; Häkkinen et al. 1999b, 2000a; De Ancos et al. 2000a). The use of liquid chromatography with mass spectrometry (LC–MS) detection provides useful structural information and allows for tentative compound identification when peaks have similar retention times and similar UV absorption spectra. In addition, LC–MS/MS is also useful for distinguishing compounds with identical molecular weights such as quercetin and ellagic acid (Seeram et al. 2006a; Mertz et al. 2007). The objective of this study was to evaluate the effect of freezing and frozen storage on the concentration of individual phenolic acids (ellagic, ferulic, caffeic,
145
p-coumaric, p-hydroxybenzoic, and protocatechuic acids) and flavonoids (quercetin, kaempferol, myricetin, and (+)catechin) in raspberry and blackberry which they have high content of phenolic compounds. The phenolic acids and flavonoids in five raspberry and four blackberry cultivars were identified and quantified in water and methanol extracts after acid hydrolysis.
Materials and Methods Reagents and Standards Caffeic acid, ferulic acid, p-coumaric acid, p-hydroxybenzoic acid, and protocatechuic acid were purchased from Merck (Darmstadt, Germany); (+)-catechin hydrate, kaempferol, and quercetin hydrate were purchased from Sigma-Aldrich (St. Louis, MO, USA); and ellagic acid and myricetin were purchased from Fluka (Buchs, Switzerland). Methanol and hydrochloric acid were of analytical grade. All standard solutions were prepared in methanol (Merck, Darmstadt, Germany). HPLC grade formic acid and acetonitrile were purchased from Merck (Darmstadt, Germany). Sampling Raspberry fruits (R. idaeus L.) of five cultivars (Aksu Kırmızısı, Rubin, Newburgh, Holland Boduru, Heritage) and blackberry fruits (R. 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 hand-harvested at commercially mature stage during the growing season of July–August 2007. The fruits (allotted in 0.5 kg lots) were packed in flat polyethylene containers. The fresh and just-frozen (frozen at a commercial frozen storage, Mevsim Foods Industry, at −35 °C in air blast freezing for 5 h) fruits were transported to the laboratory for sample preparation. Another part of polyethylene containers (0.5 kg lots) containing just-frozen fruits was stored at −22 °C for 6 months for further use. Extraction and Hydrolysis of the Phenolic Compounds Frozen berries were thawed at room temperature (23 °C) in dark for 30 min. Fresh, just-frozen, and frozen berries were blanched separately using water or methanol for 5 min. Blanched berries were milled in a household blender in three lots of 5 g each. 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
146
Food Anal. Methods (2010) 3:144–153
qualitative filter paper. A 15-mL portion of methanol extract was evaporated at 30 °C until 3 mL left using rotary evaporator. The filtrates were further passed through 0.45-μm membrane filters before LC–MS/MS analysis. Three replicates (three extractions from one container of 500 g) were analyzed for each treatment.
and mean separations with least significant difference were applied using the MSTAT-C software package, version 2.1 (Michigan State University, Kalamazoo, MI, USA).
Results and Discussion LC–MS/MS Conditions LC–MS/MS analysis was performed with an Agilent 1100 LC-MSD Trap SL Model LC–MS/MS system equipped with an autosampler. Chromatographic separations were carried out using a Zorbax SB C18 (50×4.6 mm, i.d. 1.8 μm) column. Mobile phase consists of 1% formic acid in water (solvent A) and acetonitrile (solvent B). Gradient conditions are 0–7 min 30% B, 7–27 min 50% B, and 27– 30 min 33% B; total run time is 30 min. The column was equilibrated for 10 min prior to each analysis. Flow rate was 0.2 mL/min and injection volume was 10 μL. Mass spectrometric data were acquired in positive mode with electrospray ionization source. The mass spectra were recorded in the range of m/z 100–500. Nitrogen was used both as drying gas and as nebulizing gas at flow rates of 5.00 L/min and 15.00 psi, respectively. The high-voltage capillary was held at 4,000 V. The mass spectra of each compound were obtained at extracted ion mode [M+H]+ as illustrated in Table 1. Peaks were identified on the basis of comparison of retention times and MS spectra with standards of ferulic acid, caffeic acid, p-coumaric acid, p-hydroxybenzoic acid, protocatechuic acid, ellagic acid, (+)-catechin, kaempferol, quercetin, and myricetin. Statistical Analysis The data were subjected to one-way analysis of variance (ANOVA) using the Minitab software package version 14 (University of Texas, Austin, TX, USA). Statistical differences at p
