THE EFFECT OF AIR-DRYING, FREEZE-DRYING AND STORAGE ON THE QUALITY AND ANTIOXIDANT ACTIVITY OF SOME SELECTED BERRIES

MAGDALENA MICHALCZYK1, RYSZARD MACURA and IWONA MATUSZAK Department of Refrigeration and Food Concentrates Faculty of Food Technology Agricultural University of Krakow ul. Balicka 122 30-149 Krakow, Poland
Accepted for Publication March 30, 2008

ABSTRACT The aim of this study was to analyze the effects of air-drying and freezedrying and subsequent storage of dried products on the content of polyphenols, anthocyanins and the antioxidant properties of selected berry fruits. The material was raspberry (Rubus ideaus L.), strawberry (Fragaria ananassa Duch) and bilberry (Vaccinum myrtillus). Despite exposure to atmospheric oxygen, the stored freeze-dried fruit retained the properties of the raw material better than the air-dried product. In the case of the latter, there were considerable differences in the retention of total polyphenolic and anthocyanin content as well as antioxidant properties in the three fruit species examined. In particular, bilberry maintained a high polyphenol and anthocyanin content and high antioxidant potential despite the greatest losses of these compounds.

PRACTICAL APPLICATIONS Consumer demand for food with health-promoting qualities is increasing. The food industry has intensied its efforts to provide high quality, semiprocessed products that full this requirement. The results presented in this work indicate that even after long-term storage and despite exposure to atmospheric oxygen, freeze-dried berries retain the antioxidant properties of the raw material to a very high degree. Therefore, lyophilisates can satisfy this particular need. Air-dried berries are much less stable during long-term

Corresponding author. TEL: +48-12-622-47-58; FAX: +48-12-622-47-58; EMAIL: rrmichal@cyfkr.edu.pl

Journal of Food Processing and Preservation 33 (2009) 1121. 2009, The Author(s) Journal compilation 2009 Wiley Periodicals, Inc.

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storage. The dynamics of the changes occurring during the storage of both kinds of product are presented in detail.

INTRODUCTION The ability of certain foods to protect human cells from the harmful effects of free radicals has attracted considerable interest and quickened the pace of research into the antioxidant potential of fruits and vegetables. Berry fruits, which have been estimated to provide 27.1% of total plant antioxidants in the Norwegian diet (Halvorsen et al. 2002), have a particular role to play. Strawberries, raspberries and bilberries, all rich in health-promoting properties, are the most popular fruits in this group. Strawberries contain anthocyanins, catechins and quercetin, and kaempferol, as well as being a rich source of ellagic acid (Hannum 2004). Strawberries also contain large amounts of ascorbic acid, about 60 mg/100 g fresh weight (Proteggente et al. 2002), the content in raspberries being about 30 mg/100 g (de Ancos et al. 2000a; Proteggente et al. 2002). The level of ascorbic acid in bilberries is relatively low. The major phenolic compounds identied by Proteggente et al. (2002) in strawberry extracts were pelargonidin-3-glucoside and, in lesser quantities, cyanidin-3-glucoside; in raspberries, however, cyanidin-3-sophoroside was the dominant phenolic compound, with cyanidin-3-glucoside and cyanidin-3rutinoside present in lesser amounts. The content of cyanidins in strawberries is usually very minor (Kalt et al. 1999). Bilberries (Vaccinium myrtillus L.) are exceptionally rich in anthocyanins (about 300530 mg/100 g of fresh fruit) (Prior et al. 1998). Faria et al. (2005) identied 15 of them in this fruit, with cyanidin-3-glucoside, delphinidin-3-galactoside, delphinidin-3-glucoside, cyanidin-3-galactoside, delphinidin-3-arabinoside and petunidin-3-glucoside present in the largest quantities. Considerable research has been devoted to the ability of particular phenol components, fruit extracts and lyophilisates to inhibit the development of neoplasms, coronary disease and other degenerative diseases. A great deal of the documented research carried out on animals has conrmed the efcacy of these substances; however, the ndings were specic to the conditions of the experiments, such as the choice of procancerogen, the species of animals used for testing and the choice of the monitored indicators (Aziz et al. 2002; Roy et al. 2002; Joseph et al. 2003, Hannum 2004; Matchett et al. 2005). Moreover, the anthocyanins in bilberries have been attributed with properties that improve microcirculation and reduce vision impairment (Morazzoni and Bombardelli 1996; Murray 1997). Hence, apart from being traditionally used to regulate the digestive system, bilberries are now used in preparations for improving vision and circulation.

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It is essential to determine what effect processing and storage have on the antioxidant and other health-promoting properties of berry fruits. It is widely known that sublimation drying is much more effective in preserving valuable food compounds than traditional methods of drying. However, the effects are not always long-lasting and the resulting products can be unstable during storage, with the initial high quality rapidly deteriorating (Uddin et al. 2002). The rapidity of this deterioration is sometimes greater than that observed in traditionally dried products of lower initial quality. The phenomenon is clearly seen in, for example, carrots (Tang and Chen 2000; Yen et al. 2008). Therefore, the purpose of this study was to analyze the effect of air-drying, freezedrying and subsequent product storage on the content of polyphenols, anthocyanins and the antioxidant potential of selected berry fruits.

MATERIALS AND METHODS Samples The raw material was raspberry (Rubus ideaus L.), strawberry (Fragaria ananassa Duch.) and bilberry (Vaccinum myrtillus). After stemming, discarding the damaged fruit and cutting the strawberries into halves, the berries were divided into two groups. The rst group was dried using traditional air-drying at a temperature of 40C (raspberry and strawberry for 48 h, bilberry for 72 h). The second group was frozen to a temperature of -23C and then freeze-dried in an OE-950 lyophilizer (Labor MIM, Budapest, Hungary) at a plate temperature of 35C for 24 h (strawberries, raspberries) and 36 h (bilberries). After drying, the fruit was packed into glass jars and stored for 10 months at room temperature in a light-free environment. The analysis was conducted on fresh fruit, 24 h after drying, and every 2 months during storage. Methods Extraction. Material extraction was conducted using acidied methanol (methanol: 2% HCl 95:5 v/v) (Benvenuti et al. 2004). The extractant was added to the dry material and left for 20 min. The samples were then homogenized, stirred with a mechanical stirrer for 5 min and centrifuged for 15 min at 4,000 g. The residue then underwent the same procedure twice more. Finally, a 10% extract of the material was obtained (per weight of fresh raw material), which underwent further investigation. Determination of Total Phenolic Content. Total phenolic content was determined according to the methodology developed by Singleton and Rossi

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(1965) using the FolinCiocalteau reagent. Sample extracts (0.5 mL, three replicates) were mixed with 2.5 mL of FollinCiocalteau reagent and 5 mL sodium carbonate solution (75 g/L). The mixture was brought to 50 mL with distilled water and allowed to stand for 2 h at room temperature before the absorbance was measured. This procedure is described in Benvenuti et al. (2004). The measurements were conducted at a wavelength of 750 nm with a Cecil CE 9500 spectrophotometer (Cecil Instruments, Cambridge, England). The result was expressed as gallic acid equivalents in mg/100 g fresh weight, on the basis of the calibration curve that had been constructed earlier for gallic acid.

Determination of Total Anthocyanins and Anthocyanins Degradation Index. Total anthocyanin content was determined according to the methodology described by Giusti and Wrolstad (2001). The extracts were appropriately diluted to 100 mL volume with two buffers, pH 1.0 (0.025 M potassium chloride) and pH 4.5 (0.4 M sodium acetate). After 20 min incubation at room temperature, the absorption was measured at 510 and 700 nm. The anthocyanin content was calculated using a molar extinction coefcient of cyanidin-3glucoside of 26,900, a molecular weight of 449.2 and absorbance A = (A510 - A700)pH 1.0 - (A510 - A700)pH 4.5. The anthocyanin degradation index (DI) is dened as the ratio of the total (degraded and nondegraded) content of anthocyanins determined with the single pH method, divided by the content of nondegraded anthocyanins determined using the pH-differential method. It was calculated on the basis of the absorbance of the samples diluted with pH 1.0 and 4.5 buffers, DI = ApH1.0/ (ApH 1.0 - ApH 4.5). The anthocyanin DI calculated using this method is useful even if the anthocyanins are unidentied, or the extinction coefcients are unknown (Fuleki and Francis 1968).

Determination of the Scavenging Effect on 2,2-diphenyl1-picrylhydrazyl (DPPH). Antioxidant activity was measured by the colorimetric method, using DPPH free radical in accordance with the procedure described by Brand-Williams et al. (1995). Extracts of different concentrations (0.1 mL) were added to a 3.9 mL methanolic solution of DPPH (0.00236 g/ 100 mL). Absorbances were measured at different time intervals until the reaction reached a plateau at 515 nm against methanol as the blank reference. Antiradical activity was expressed as the amount (in g) of raw material necessary to decrease the initial DPPH concentration by 50% (EC50 [g of fresh material/g of DPPH]) using the procedure described by Brand-Williams et al. (1995).

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500 450 Polyphenols (mg/100g) 400 350 300 250 200 150 100 50 0 raw material 0 2 4 6 8 10 Time of storage (months) strawberry raspberry bilberry strawberry fd raspberry fd bilberry fd

900 800 Polyphenols of bilberry (mg/100g) 700 600 500 400 300 200 100 0

FIG. 1. POLYPHENOLS CONTENT IN RAW MATERIAL AND IN DRIED CONVENTIONALLY AND FREEZE-DRIED (fd) PRODUCTS AS GALLIC ACID EQUIVALENT

Determination of Dry Matter Content. Dry matter content, necessary for calculations, was determined using a vacuum dryer in the presence of a dehydrating agent at a temperature of 70C, drying the samples to a constant mass. Statistical Analysis Experiments were performed in triplicate and results were expressed as mean standard deviation (SD). SD and correlation coefcient were calculated using CSS Statistica software (StatSoft, Tulsa, OK).

RESULTS AND DISCUSSION The total polyphenolic (Fig. 1) and anthocyanin (Fig. 2) content of the analyzed fruit were approximated to the content indicated in the literature, respectively: (1) bilberry 525905 and 280300 mg/100 g fresh weight; (2) raspberry 113228 and 2896 mg/100 g fresh weight; and (3) strawberry 230 530 and 814 mg/100 g fresh weight (Prior et al. 1998; de Ancos et al.

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70 Anthocyanins (mg/100g) 60 50 40

300 Anthocyanins of bilberry (mg/100g) 250 200 150

30 20 10 0 raw material 0 2 4 6 8 10 Time of storage (months) strawberry raspberry bilberry strawberry fd raspberry fd bilberry fd 100 50 0

FIG. 2. ANTHOCYANINS CONTENT IN RAW MATERIAL AND IN DRIED CONVENTIONALLY AND FREEZE-DRIED (fd) PRODUCTS AS CYANIDIN-3-GLUCOSIDE EQUIVALENT

2000a,b; Proteggente et al. 2002; Faria et al. 2005; Rababach et al. 2005). Air-drying caused different percentage losses in the analyzed components of the various fruits. The relatively large decrease in the polyphenol and anthocyanin content of bilberries may have been due to the relatively long drying period (72 h), causing a naturally occurring waxy layer on the fruits. Moreover, the degradation of anthocyanins depends, among other factors, on polyphenol oxidase activity, organic acid content, sugar concentration, pH and the content of more reactive anthocyanins, e.g., cyaniding-3-glucoside (de Ancos et al. 2000b). Lyophilization either slightly lowered the total polyphenolic (Fig. 1) and anthocyanin (Fig. 2) content, or slightly increased it, depending on the species. This slight increase could have resulted from improved extraction of the lyophilized material. Kwok et al. (2004), comparing the effects of freezedrying and air-drying on serviceberry fruit, found considerably greater losses of the abovementioned compounds in air-dried fruit. During the storage of air-dried fruit, total polyphenolic content fell dramatically, with the largest percentage decrease in bilberries. There was a similar fall in anthocyanin content. The changes in the anthocyanin DI (Fig. 3),

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2,6 Anthocyanins degradation index . 2,4 2,2 2,0 1,8 1,6 1,4 1,2 1,0 raw material 0 2 4 6 8 10

Time of storage (months) strawberry raspberry bilberry strawberry fd raspberry fd bilberry fd

FIG. 3. ANTHOCYANINS DEGRADATION INDEX IN RAW MATERIAL AND IN DRIED CONVENTIONALLY AND FREEZE-DRIED (fd) PRODUCTS

which indicates the proportion of degraded anthocyanins in the sample, were most signicant for raspberries. According to Fuleki and Francis (1968), the DI is a better indicator of sample color than anthocyanin content. It is possible to establish an index limit beyond which the degradation becomes so noticeable or so signicant that the product should be disqualied. The values of this indicator for fresh raw material were also highest in the case of raspberries. In light of these changes, the analysis of EC50 (Fig. 4) changes brought about some surprising results. The value of this parameter increased considerably for stored dried strawberries and raspberries; however, despite very large losses in polyphenols, there was a relatively insignicant (less than twofold) increase in the EC50 value of stored bilberries. The storage of lyophilisates resulted in considerably smaller losses in polyphenols compared with traditionally dried fruits (Fig. 1), especially in the case of bilberries and raspberries. Anthocyanins were also very well preserved (Fig. 2). No anthocyanin loss was observed in strawberries. In raspberries, the loss was slight, while in bilberries it was 50%. There was also very little change in the anthocyanin DI during the storage of lyophilisates (Fig. 3). Garzn and Wrolstad (2001) indicate that the chemical structure of anthocyanins (e.g., glycosylation and acylation by different sugars and acids) is

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M. MICHALCZYK, R. MACURA and I. MATUSZAK

70 60 EC50 (g fw/g DPPH) 50 40 30 20 10 0 raw material 0 2 4 6 8 10

Time of storage (months) strawberry raspberry bilberry strawberry fd raspberry fd bilberry fd

FIG. 4. EC50 VALUE FOR RAW MATERIAL AND DRIED CONVENTIONALLY AND FREEZE-DRIED (fd) PRODUCTS

believed to be a major factor affecting their stability. In their experiments, they also found a direct relationship between increasing water activity and the anthocyanin degradation rate. During the 10-month storage of the freeze-dried products, the EC50 increased only slightly, and in bilberries, despite this fruit showing the largest losses in polyphenols and anthocyanins, it hardly changed (Fig. 4). Some authors have observed a strong correlation between antioxidant properties, and phenolic (R = 0.83R = 0.95) or anthocyanin content (R = 0.77R = 0.92), but this has been reported mainly for the raw material (Prior et al. 1998; Kalt et al. 1999; Kalt et al. 2000). The present work investigated the correlation coefcients for processed and stored products. For air-dried berries, a strong inverse correlation was found between the EC50 and phenolic or anthocyanin content (from -0.86 to -0.95 phenolic; from -0.72 to -0.87 anthocyanins). This was not so clear in the case of lyophilisates. Among freeze-dried products, a similarly strong correlation was found only for bilberries (-0.83 and -0.90), while no correlation was observed for strawberries and raspberries. Mastrocola et al. (1997) noticed that partially processed fruits are not readily available to the food industry for desired end uses; despite the high cost of obtaining them, lyophilisates can satisfy these specic needs. Moreover, products dehydrated in this way are commonly used in most Asian countries as

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ingredients in soups and are usually included in products such as instant noodles (Nindo et al. 2003). It is widely accepted that freeze-drying is the best dehydration method for preserving product quality (George and Datta 2002). The results of the present study indicate that, even after long-term storage, freeze-dried products retain the antioxidant properties found in the raw material to a very high degree despite being exposed to atmospheric oxygen.

CONCLUSIONS Sublimation drying was far more effective in preserving valuable food compounds than traditional air-drying. Stored lyophilisates of fruits maintained the properties of the raw material much better than air-dried products, in spite of exposure to atmospheric oxygen. There were considerable differences between the three species examined in maintaining total polyphenolic content, total anthocyanin content and antioxidant properties. The highest percentage losses of polyphenols and anthocyanins were found in stored bilberries, with very little accompanying change in antioxidant potential. Air-dried strawberries and raspberries showed reduced antioxidant properties after storage despite relatively high levels of polyphenols and anthocyanins.

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