LWT - Food Science and Technology

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1 LWT - Food Science and Technology 42 (2009) Contents lists available at ScienceDirect LWT - Food Science and Technology journal homepage: Effect of L-ascorbic acid, sugar, pectin and freeze thaw treatment on polyphenol content of frozen strawberries Jan Oszmiański *, Aneta Wojdy1o, Joanna Kolniak Department of Fruit and Vegetable Processing, Wroclaw University of Environmental and Life Science, 25 Norwida Street, Wroclaw, Poland article info abstract Article history: Received 19 December 2007 Received in revised form 13 July 2008 Accepted 20 July 2008 Keywords: Fragaria x ananassa Duch Polyphenols Anthocyanins Proanthocyanidins Ellagic acid Freezing and thawing conditions Microwave thawing The stability of phenolic compounds of three strawberry cultivars was evaluated for changes during prefreezing treatments, storage and various freezing and thawing conditions. Polyphenol content was determined by HPLC DAD FL. The sum of assayed polyphenolic (proanthocyanidin and monomeric flavan-3-ols, anthocyanins, ellagic acid, p-coumaric acid) represented mg/kg in Kent, mg/ kg in Elkat, and mg/kg in Senga Sengana. After freezing, % of polyphenols were lost; protective effects of prefreezing treatments were seen on anthocyanidins and proanthocyanidins: ascorbic acid was the most effective pretreatment, allowing retention of % of anthocyanins, and almost total recovery ( %) when associated with liquid nitrogen freezing. Pectin and sugar only allowed retention of % and % of the antocyanins, respectively. Thawing of the strawberries in a microwave oven (instead of 20 h at 20 C) had a further positive effect on retention of anthocyanins, proanthocyanins, (þ)-catechin and ellagic acid. Ó 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. 1. Introduction Strawberries (Fragaria x ananassa Duch.) are an important crop in temperate regions such as Poland in Central Europe. They are widely consumed fresh and in processed forms especially as frozen fruits. The strawberry occupies an important position in the Polish industry of fruit freezing. The large scale of frozen strawberry production can be attributed to the short storage life of fresh fruit and to the very wide scale of its use as a frozen product. Frozen strawberries are the most common starting material used in the industrial manufacturing of strawberry jam, juices and concentrates (Oszmiański, Wojdy1o, & Matuszewski, 2007; Wicklund et al., 2005). These attractive fruits are favored for their excellent taste and can be considered a very potent source of bioactive phenolic compounds including hydroxycinnamic acids, ellagic acid, ellagitannins, flavan- 3-ols, flavonols, and anthocyanins (Määttä-Riihinen, Kamal-Eldin, & Törrönen, 2004). Compared with other fruits, strawberries possess high antioxidant activity (Sun, Chu, Wu, & Liu, 2002). Guo, Cao, Sofic, and Prior (1997) found that strawberry had 1.3 times the antioxidant activity of oranges, 2 times that of red grapes, 5 times that of apples and bananas, and 13 times that of honeydew melon (Guo et al.,1997). Antioxidant activity of strawberry phenolics is important in the * Corresponding author. Tel./fax: þ address: oszm@wnoz.up.wroc.pl (J. Oszmiański). prevention of cancer, cardiovascular and other chronic diseases (Hannum, 2004). Recently, antioxidant activity, independent of in vitro antiproliferative activity, of strawberry extracts has been shown using the HepG2 human liver cancer cells (Meyers, Watkins, Pritts, & Hai- Liu, 2003). Reduction in antioxidant activity during processing and storage (Lindley, 1998) may reduce the health beneficial effects of such food products. Injury of raw material tissue, exposure to oxygen, enzyme activity (PPO), light and heat may reduce the antioxidant compound content. Strawberry phenolics such as pelargonidin, ellagic acid, p-coumaric acid, quercetin and keampferol derivatives are very unstable and undergo destruction during fruits transformation in frozen products especially in the thawing process by native and microbiological enzymes (Garrote & Bertone, 1989). Previous studies have focused on the free phenolic content and antioxidant activity of strawberry products. However, antioxidants can exist in both free and bound forms. Free anthocyanins, hydroxycinnamic acids, (þ)-catechin and flavonol glycoside do not bind to strawberry cell walls; it is known that proanthocyanidin polymers are bound selectively as in apples (Renard, Baron, Guyot, & Drilleau, 2001). Proanthocyanidins are also found in strawberry fruits as procyanidin and propelargonidin derivatives (Gu et al., 2004). Hebert et al. (2002) suggested that proanthocyanidin content in strawberry can be used as an indicator of grey mold resistance in order to screen strawberry selections and cultivars for improved shelf-life and quality. The proanthocyanidins are /$34.00 Ó 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi: /j.lwt

2 582 J. Oszmiański et al. / LWT - Food Science and Technology 42 (2009) receiving increased attention because of their much more potent antioxidant properties than those of simple monomeric phenolics, which may correspond to a health-protective action (Hagerman et al., 1998). Quantitative data on proanthocyanidins found in the literature are often underestimated; their extraction from crude materials is not quantitative because of their ability to bind to cell wall polysaccharides (Guyot, Marnet, Laraba, Sanoner, & Drilleau, 1998). Therefore, methods including their cleavage in acidic conditions followed by stabilization of the carbocation by a nucleophilic reagent are to be preferred (Rigaud, Perez-Ilzarbe, Ricardo Da Silva, & Cheynier, 1991). Among these methods, phloroglucinolysis, which can be applied directly to the crude plant material and provides pertinent information on the structure and concentration of proanthocyanidins, was chosen (Kennedy & Jones, 2001). Freezing strawberries is known as a good method for increasing shelf-life, but this berry undergo quality changes throughout the whole frozen food chain, i.e. freezing, subsequent frozen storage and thawing. Freezing and thawing methods altogether with, some additives can improve strawberry polyphenols and colour stability. The strawberries are usually pretreated by mixing them with dry sugar in order to increase the sugar content in the berries. It also reduces floating berries which can arise during further processing. Treatments decreasing the textural degradation of strawberry frozen tissues by removing part of water with sugar and the addition of a high molecular weight solute like pectin to frozen fruits might have a crystabilizing effect (Kmiecik, Jaworska, & Lisiewska, 2000). Strawberries can be pretreated before freezing by the added sucrose. This additive sweetens the product, contributes to retain volatile compounds, reduces the water amount to freeze and decreases the oxidation by acting as a barrier to oxygen (Suutarinen, Heiska, Moss, & Autio, 2000). A reduction in free water causes less cell wall rupture and rearrangement of the cell contents during freezing. Decreasing drip losses ensure better fruit appearance, flavour, aroma and polyphenol degradation. Antioxidants are regularly added to food in order to increase its shelf-life. Ascorbic acid is one of the most common antioxidant additives in fruit preserves, but not in strawberry products, since it can react with the anthocyanins leading to loss of colour and overall quality in strawberry syrups (Skrede, Wrolstad, Lea, & Enersen, 1992). But in frozen strawberries ascorbic acid may be useful as polyphenol oxidation protector during freezing, storage and thawing process. Carbohydrate as pectins can be useful as for as some polyphenol protectors. They may act as cooperative hydrogen bonding between the oxygen atom of the carbohydrate and the phenolic hydroxyl group and by hydrophobic interactions (Vernhet, Pellerin, Prieur, Oszmianski, & Moutounet, 1996). Low-methylated pectin added before strawberry freezing can be useful for gel forming in jam process. It is important to adjust the freezing thawing process variables in order to better preserve and retain the quality and antioxidant content of strawberries. In general, it is accepted that faster freezing and thawing causes less migration of water and less breakage of cells walls, and consequently less texture deterioration. Ultra-high freezing rates and thus minimized physical damage due to formation of small ice crystals can be achieved by freezing using liquid nitrogen. In this kind of process the nitrogen protects the oxidation of vitamin C and polyphenols. Microwave thawing is known as requiring shorter thawing time and smaller space for processing, and reduces drip loss, microbial problems and chemical deterioration. The unique property of microwaves to penetrate and produce heat deep within food materials make them potential in accelerating thawing (Virtanen, Goedeken, & Tong, 1997). The improvement on the temperature uniformity during microwave thawing is necessary. Using this apparatus, thawing time was reduced by as much as a factor of seven compared to convective thawing at atmospheric temperature when appropriate conditions were used (Tong, Lentz, & Lund, 1993). In this investigation we study how the sucrose, L-ascorbic acid, pectin prefreezing treatments and various freezing and thawing conditions affect the content of strawberry polyphenols. Our objective was three strawberry cultivars (Elkat, Kent and Senga Sengana), commonly used in the Polish jam and juice industry. 2. Materials and methods 2.1. Chemicals (þ)-catechin, ( )-epicatechin, acetic acid, L-ascorbic acid, phloroglucinol and methanol were purchased from Sigma Aldrich (Steinheim, Germany). Ellagic acid, p-coumaric acid, quercetin, kaempferol, cyanidin-3-o-glucoside, pelargonidin-3-o-glucoside, pelargonidin-3-o-rutinoside, pelargonidin-3-o-malonyl-glucoside were purchased from Extrasynthese (Lyon, France) Plant materials Samples of three strawberry (F. x ananassa) cultivars (Elkat, Kent and Senga Sengana) at processing maturity were harvested (Moscicko near Wroc1aw, Poland) and fresh fruits were processed at the laboratory scale in Wroclaw University, in June The preparation for freezing included the sorting and calibration of fruits with respect to size (20 25 mm in diameter) and colour, washing and draining on sieves, drying and detaching the petiole. The fruits (allotted in 500 g lots) were packed in flat polyethylene containers Preparation of frozen and thawed strawberry The materials added to the strawberries were powdered sugar, low-methylated pectin (NE A3 Pektowin; Jas1o, Poland), and L- ascorbic acid. Four variants were prepared for each strawberry cultivars: 1 without additional substances; 2 in sugar (500 g of fruit with 50 g of powdered sugar); 3 in sugar with pectin (500 g of fruit with 50 g of powdered sugar with 0.5 g of pectin preparation); 4 insugar with L-ascorbic acid (500 g of fruit with 50 g of powdered sugar with 0.5 g L-ascorbic acid). The samples were frozen in liquid nitrogen and a domestic freezer. After 6 months storage at 20 C strawberries were thawed during 20 h at 20 C and 5 min in microwave oven and examined HPLC analysis of polyphenols Analysis of proanthocyanidins after phloroglucinolysis and other phenolic compounds was carried out in the raw materials and freeze samples immediately after thawing by HPLC DAD FL methods. Three replicates (three containers of 500 g) were analysed for each treatment. Mixed thawed pulp of berries (5 g) was extracted with 25 ml of methanol acidified with 1% acetic acid. The extraction was performed by incubation for 20 min under sonication and with occasional shaking. This method has proved to be adequate for complete extraction. Next, the slurry was centrifuged at 19000g for 10 min and the supernatant was used for HPLC analysis. The analysis of anthocyanins, flavan-3-ols, phenolic acids and flavonol glycosides was carried out using an L-7455 liquid chromatograph with a diode array detector and an L-7100 quaternary pump equipped with a D-7000 HSM multisolvent delivery system (Merck-Hitachi, Tokyo, Japan). Separation was performed in Q3

3 J. Oszmiański et al. / LWT - Food Science and Technology 42 (2009) a Synergi Fusion RP-80A column (250 mm 4.6 mm, 4 mm; Phenomenex, Torrance, CA USA). The oven temperature was set to 30 C. The mobile phase was composed of solvent A (4.5% formic acid in water) and solvent B (pure acetonitrile). The programme began with a linear gradient from 0 to 25% B in 36 min, followed by washing and reconditioning of the column. The flow rate was 1 ml/ min and the runs were monitored at wavelengths of 280 nm (flavan-3-ols), 320 nm (hydroxycinnamates), 360 nm (flavonol glycosides and ellagic acid), and 510 nm (anthocyanins). Photodiode array spectra were measured over the wavelength range nm in steps of 2 nm. Retention times and spectra were compared with those of pure standards within that range. Calibration curves were constructed with (þ)-catechin, p-coumaric acid, ellagic acid, isoquercitin and pelargonidin-3-o-glucoside as standards (Extrasynthese, France) Analysis of proanthocyanidins by phloroglucinolysis Direct phloroglucinolysis of freeze-dried strawberry fruit was performed as described by Kennedy and Jones (2001) method. Portions (0.5 g) of homogeneous samples were precisely weighed into 1.5 ml Eppendorf vials and freeze-dried, then 0.8 ml of the methanolic solution of phloroglucinol (75 g/l) and ascorbic acid (15 g/l) were added. After addition of 0.4 ml of methanolic HCl (0.3 N), the vials were closed and incubated for 30 min at 50 C and vortex every 10 min. The reaction was stopped by placing the vials in an ice bath and withdrawing of 0.5 ml of reaction medium, subsequently transferred to another vial and neutralized by 0.5 ml of sodium acetate buffer 0.2 M. Next the vials were cooled in ice water and centrifuged immediately at g for 10 min at 4 C. Samples were stored at 4 C until reverse phase HPLC (RP-HPLC) analysis. All incubations were done in triplicate. Phloroglucinolysis products were separated in a Atlantis T3, 5 mm, 100A column (250 mm 4.6 mm; Waters). The liquid chromatograph was a Waters (Milford, MA, USA) system equipped with diode array and scanning fluorescence detectors. Solvent A (aqueous acetic acid, 25 ml/l) and solvent B (acetonitrile) were used in the following gradient: initial, 5% B; 0 15 min, 10% B linear; min, 60% B linear; followed by washing and reconditioning of the column. A flow rate of 1 ml/min and an oven at 15 C temperature, injection of the filtrate (20 ml) on the HPLC system. Compounds, for which reference standards were available, were identified on chromatograms according to their retention times and UV vis spectra. The fluorescence detection was recorded at excitation wavelength 278 nm and emission wavelength 360 nm. The calibration curves based on peak area were established using (þ)-catechin, ( )-epicatechin, (þ)-catechin-phloroglucinol and ( )-epicatechin-phloroglucinol adducts standards. The average degree of polymerization was measured by calculating the molar ratio of all the flavan-3-ol units (phloroglucinol adducts þ terminal units) to ( )-epicatechin and (þ)-catechin corresponding to terminal units. Quantification of the (þ)-catechin, ( )-epicatechin, (þ)-catechin-phloroglucinol and ( )-epicatechin-phloroglucinol adducts was achieved by using the calibration curves of the corresponding standards Statistical analyses for individual phenolics in strawberry jam samples Statistical analysis was conducted using Statistica version 6.0 (StatSoft Poland). Significant differences (p 0.05) between average responses were evaluated by using one-way ANOVA with Duncan test. Principal component analysis (PCA) was performed using XLSTAT (Addinisoft, France) on mean values of 16 samples and 6 variables. The PCA is a multilinear descriptive method. 3. Results and discussion The average phenolic contents of the three strawberry cultivars, Elkat, Kent and Senga Sengana used as raw materials in the presented investigation are shown in Table 1. Ten different compounds were identified and quantified in strawberries by HPLC DAD method by comparison of their UV vis spectra of available standards and report from the literature (Kosar, Kafkas, Paydas, & Baser, 2004; Määttä-Riihinen et al., 2004). The anthocyanin, p-coumaric acid, ellagic acid and flavonol derivatives, (þ)-catechin and proanthocyanidins were found in strawberries. The total phenolic content (considering the sum of all of the individual phenolics) ranged from mg/kg fresh fruits (cv. Senga Sengana) to mg/kg fresh fruits (cv. Elkat). Häkkinen et al. (1999) indicated that ellagic acid is the main phenolic compound in strawberry, forming 51% of the phenolic compounds analysed. Ellagic acid is a hydrolytic product of ellagitannins. In the presented investigation the ellagitannins were not determined but only free ellagic acid and its glucoside derivative. The content of total ellagic acid (ellagic acid and ellagic acid glucoside) ranged from 13.4 mg/kg fresh fruits (cv. Senga Sengana and cv. Kent) to 3.4 mg/kg fresh fruits (cv. Elkat). The concentration of ellagic acid and ellagic acid glucoside were present at same level as previously reported in strawberry fruits (Amakura, Umino, Tsuji, & Tonogai, 2000; Määttä-Riihinen et al., 2004; Wang, Zheng, & Galletta, 2002). Regarding proanthocyanidins, the highest concentrations were found in cv. Kent ( mg/kg fresh fruits) and the smallest in cv. Senga Sengana ( mg/kg fresh fruits). Proanthocyanidins were present at much higher levels than previously reported for strawberries (Aaby, Wrolstad, Ekeberg, & Skrede, 2007). This difference is due to our use of phloroglucinolysis, which allows quantification of all polymeric and oligomeric proanthocyanidins. Previous studies had reported only oligomeric proanthocyanidin content analysed directly by HPLC method (Aaby, Skrede, & Wrolstad, 2005; Määttä- Riihinen et al., 2004). Table 1 Concentration (mg/kg of fresh weight) of the phenolic compound in raw material used for frozen strawberry production Senga Sengana Kent Elkat Cyanidin-3-O-glucoside f e f Pelargonidin-3-O-glucoside b b b Pelargonidin-3-O-rutinoside e e k Pelargonidin-3-O-malonyl-glucoside c d c p-coumaroyl-glucoside c f c Ellagic acid g f f Flavonols e c e (þ)-catechin d d d Proanthocyanidins a a a Total Values are means standard deviation. n ¼ 3; different letters within a line denote significantly different mean values at p < 0.05.

4 584 J. Oszmiański et al. / LWT - Food Science and Technology 42 (2009) Significant difference composition in the concentration of p- coumaric acid among different cultivars from 12.9 mg/kg fresh fruits for cv. Kent to 80.9 mg/kg fresh fruits for cv. Elkat was found. The concentration of p-coumaric acid found here in cv. Senga Sengana was twice that reported by Häkkinen and Törrönen (2000). Qualitative differences were found in anthocyanin composition of the Elkat (Polish cultivar) lacked pelargonidin-3-o-rutinoside. Four anthocyanins were identified in cv. Senga Sengana and cv. Kent: cyanidin-3-o-glucoside, pelarognidin-3-o-glucoside, pelargonidin-3-o-malonyl-glucoside and pelargonidin-3-o-rutionside. Elkat cultivar (Polish origin) lacked pelargonidin-3-o-rutinoside and thus contained only three anthocyanins. Pelargonidin-3-Oglucoside was predominant in all cultivars and ranged from mg/kg for cv. Kent strawberries to mg/kg for cv. Elkat. The concentration of flavonoids such as kaempferol and quercetin ranged from 5.0 mg/kg to 38.5 mg/kg. Lugasi and Hovari (2002) reported that quercetin was present at a concentration of mg/kg. Variation in content of different phenolic in strawberries can be due to factors not only such as cultivar but also maturity, size, present of achenes, extraction solvent and procedure. During the 6 months storage the content of polyphenols in frozen control strawberries underwent decreases, which varied between 17.8% for cv. Senga Sengana and 36.6% for cv. Kent. Table 2 Effects of cultivar and of L-ascorbic acid (AA), sugar (S), pectin (P) pretreatment, freezing in liquid nitrogen (N 2 ) and in microwave oven thawing (m) on polyphenol content (mg/kg fw) of 6 month stored frozen strawberries Samples Phenolic compounds Anthocyanins p-coumaric acid Ellagic acid Flavonols (þ)-catechin Proanthocyanidins Cultivar Control a a a b bc f Control þ N a a a ab a cd S þ AA a a a ab ade bc S þ AAN a a a a ad a S þ P a a a c bce de S þ PN a a a ab bde a S a a a ab c e SN a a a ab de b KENT m-contr a a a b bc f m-contrn a a a ab a cd m-s þ AA a a a ab ade bc m-s þ AAN a a a a ad a m-s þ P a a a c bce de m-s þ PN a a a ab bde a m-s a a a ab c e m-sn a a a ab de b ELKAT Control a bc a ab a c Control þ N a abc b ab a c S þ AA a abc b ab a a S þ AAN a abc ab ab a a S þ P a c b b a bc S þ PN a ab ab ab a ab S a abc ab a a bc SN a a ab a a bc m-contr a bc a ab a c m-contrn a abc b ab a c ms þ AA a abc b ab a a m-s þ AAN a abc ab ab a a m-s þ P a c b b a bc m-s þ PN a ab ab ab a ab m-s a abc ab a a bc m-sn a a ab a a bc Senga sengana Control a a a a a b Control þ N a a a a a ab S þ AA a a a a a a S þ AAN a a a a a a S þ P a a a a a ab S þ PN a a a a a a S a a a a a ab SN a a a a a a m-contr a a a a a b m-contrn a a a a a ab ms þ AA a a a a a a m-s þ AAN a a a a a a m-s þ P a a a a a ab m-s þ PN a a a a a a m-s a a a a a ab m-sn a a a a a a Values are means standard deviation. n ¼ 3; different values within a column at p < 0.05 for each cultivars.

5 J. Oszmiański et al. / LWT - Food Science and Technology 42 (2009) In relation to the control samples (frozen and thawed by traditional methods) freezing in liquid nitrogen and thawing in microwave oven allowed an increase of the retention of polyphenols in all strawberry varieties ( %). The prefreezing treatments with sucrose, low-methoxy pectin, and L-ascorbic acid had some protective effect on the polyphenol content in the final strawberries (Table 2). These substances were added to the fruits as powders. Their different combinations were based on the data from the literature, which postulated their greater effectiveness in the combined form (Garrote & Bertone, 1989; Kmiecik et al., 2000). Detailed information on the effects of prefreezing treatments with sucrose, low-methoxy pectin, and L-ascorbic acid, modes of freezing and defreezing, on the different classes of phenolic compounds are shown in Table 2 and Figs. 1 and 2. The highest polyphenol contents were found in cv. Elkat and cv. Senga Sengana samples in the combinations with pectin and sucrose ( mg/ kg and mg/kg, respectively). The smaller losses in polyphenols than in samples with sugar alone can be attributed to the formation of a natural coating of pectin on the fruits, limiting the access of oxygen to their tissue. The ascorbic acid and sugar combination was the most effective in cv. Kent which contained more than mg/kg of these compounds. This corroborates the opinion of Robards, Prenzler, Tucker, Swatsitang, and Glover (1999) that this acid inhibits the reaction of browning and effects the regeneration of polyphenols, though it can enhance the degradation of anthocyanins. The anthocyanin amounts were about half smaller in cv. Kent than in cv. Senga Sengana and cv. Elkat (Table 1). It was observed that all additives and treatment (frozen and defrost methods) for all strawberry cultivars had significantly (p < 0.05) influenced on content proanthocyanidins after 6 month stored frozen fruits. (þ)-catechin was the least stable among the phenolic compounds. After 6 months at 20 C and defrost during 20 h at 20 C averaging 54, 63 and 45% of the start levels, remained in the control samples of cv. Kent, cv. Elkat and cv. Senga Sengana, respectively. The degradation of (þ)-catechin may be explained by enzymatic oxidation, as this ortho-diphenolic compound is known to be a major substrate for oxidases (Lopez-Serrano & Ros Barcelo, 2002; Pourcel, Routaboul, Cheynier, Lepiniec, & Debeaujon, 2007). The sugar, pectin and ascorbic acid pretreatment, in the current experiment, had some protective effect on (þ)-catechin content, but only significantly (p < 0.05) on (þ)-catechin in Kent cv. Fig. 1. Principal component analysis of frozen in liquid nitrogen (N) and in refrigerator cv. Senga Sengana strawberries without (c-control) and with sugar (s), pectin (p) and L-ascorbic acid (a) prefreezing treatments stored 6 month stored at 20 C thawed in microwave oven (m) and during 20 h at 20 C. The measured variables (phenolics: A anthocyanins; P-proanthocyanidins; EA-ellagic acid; (þ)c (þ)catechins; Q quercetin derivative; K keampferol derivative) are shown on the same plot. Fig. 2. Principal component analysis of frozen in liquid nitrogen (N) and in refrigerator of three strawberry cultivars cv. Senga Sengana, cv. Kent and cv. Elkat without and with sugar, pectin and L-ascorbic acid prefreezing treatments stored 6 month stored at 20 C thawed in microwave oven and during 20 h at 20 C. The measured variables (phenolics: A anthocyanins; P-proanthocyanidins; EA-ellagic acid; (þ)c (þ)catechins; Q quercetin derivative; K keampferol derivative; p-ca p-coumaric acid derivative) are shown on the same plot. Proanthocyanidins and anthocyanin were also degraded during freezing, storing and thawing of control samples prepared from cvs Kent ( 35 and 25%, respectively), Elkat ( 16 and 27%) and Senga sengana ( 19 and 8%). None of the additives or treatments (freezing and defrost method) had a significant impact (p < 0.05) on content of anthocyanins in stored frozen fruits. There were minor changes in the levels of p-coumaroyl glycosides, and flavonols in the defrosted strawberries. The concentrations of ellagic acid were much higher in most of the stored and thawed strawberries than in the fresh fruits. The increase of ellagic acid may be explained by release of ellagic acid after hydrolysis of the ellagitannins present. The same effect has been observed during strawberry puree storage (Aaby, Wrolstad et al., 2007). Principal component analysis (PCA) was performed on mean values of 6 phenolic groups and 16 variables of method preparations of cv. Senga Sengana samples and visualized by means of the PCA-biplot (Fig. 1). The closer the samples lay on the biplot, the more similarly the methods describe them. Respectively, the further the samples lay from each other, the more different they are with respect to the measured parameters. The difference between effects of the frozen strawberry preparation and thawing methods explained 68.9% of the total variation in principal component. The samples which were thawed in microwave oven had a much higher phenolic content (anthocyanins, proanthocyanins, (þ)-catechin and ellagic acid) than samples which was thawed during 20 h at 20 C. Probably enzymatic reaction could take place in destroyed strawberry tissues during long thawing process. It has been reported that catechin and PPO (polyphenol oxidase) readily react, resulting in reactive quinones which degrade anthocyanins, proanthocyanidins and other phenolics (Wesche-Ebeling & Montgomery, 1990). Both PPO and (þ)-catechin have been reported to be present a high concentration in strawberries (Lopez-Serrano & Ros Barcelo, 1995). To achieve a higher content of phenolic compounds in strawberries, they should be thawed by faster method in microwave oven. Also cv. Senga Sengana strawberry samples which were frozen in liquid nitrogen and thawed in microwave oven and treated with sugar and pectin or L-ascorbic acid were closest to the projections of the anthocyanins, (þ)-catechin and proanthocyanidins (Fig. 1). Taking into account the strawberry varieties and phenolic compounds in frozen and thawed and pretreated samples after the PCA analysis, the scatterplot shows a very good separation among the samples as a function of the cultivar (Fig. 2). Thus, it can be

6 586 J. Oszmiański et al. / LWT - Food Science and Technology 42 (2009) observed that the samples should be classified into two groups, one of them including the samples from the cv. Elkat and cv. Senga Sengana (with larger content of anthocyanins, p-coumaric acid and lower amount of proanthocyanidins), and the second including the samples from the cv. Kent (with lager content of proanthocyanidins and lower amount of anthocyanins and p-coumaric acid). In conclusion, the study revealed that the sugar, pectin and ascorbic acid pretreatment, as well as microwave thawing frozen strawberries, had some protective effect on many polyphenolic compounds. More efficient protection in present investigation was obtained for the anthocyanins, proanthocyanins and (þ)-catechin than for flavonols and phenolic acids. The cultivars had significant effect on polyphenol content in frozen strawberries. 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