Biochemical and colour changes of watercress (Nasturtium officinale R. Br.) during freezing and frozen storage

The effects of water blanching, freezing, and frozen storage during 400 days at three different temperaress (N ntent l on colo l value. es in co degrad and hu cessfull kept c tures ( 7, 15 and 30 C), on waterc chlorophyll degradation, vitamin C co blanching induced significant changes activity was reduced 85% from its initia however, promoted significant differenc age, ascorbic acid (AA) and POD activity (LH aH bH, aH/bH, LH aH/bH, LH/aH bH The storage temperature effect was suc droascorbic acid (DHAA) contents were * Corresponding author. Tel.: +351 22 5580058; fax E-mail address: [email protected] (C.L.M. Silva). 1 Tel.: +351 21 7127153; fax: +351 21 7127162. asturtium officinale R. Br.) colour Hunter Lab parameters, oss and peroxidase (POD) activity were evaluated. The ur values and chlorophylls and vitamin C contents. POD Freezing did not affect chlorophylls and vitamin C levels, lour values and POD residual activity. During frozen storations followed first-order kinetics, and colour parameters e (h0H)) were successfully described by zero-order kinetics. y described by the Arrhenius law. Chlorophylls and dehyonstant during frozen storage. Watercress (Nasturtium officinale R. Br.) is an aquatic perennial herb from Cruciferae family, which is native to Europe and an economically important vegetal in Portugal, with wide applications in local cuisine. Raw watercress leaves are used as salad greens, or can be steamed and consumed as a normal processed vegetable. Watercress contains a relatively large amount of vitamins C and provitamin A, folic acid, iodine, iron, protein, and especially calcium and sulphur compounds, which influence its characteristic odour, but also adds to its nutritional benefits (Rose et al., 2000). In addition, watercress has also a history of medicinal use. This vegetable has been the focus of several studies regarding its anticancer properties, mainly due to its high anti-oxidants content (Murphy et al., 2001). However, in spite of its importance, the application of preservation methods, like freezing, for its shelf-life extension is still very limited, and there is a lack of information on this subject. Freezing is one of the most important methods for retaining vegetables quality during long-term storage. Blanching, as a prefreezing operation, is a thermal treatment applied to raw vegetables that determines largely the final product quality. Its main objective is the inactivation of the enzymes responsible for deterioration reactions during frozen storage. The intensity of this treatment has to assure the enzymatic inactivation, while minimising : +351 22 5090351. the possible negative effects of heat on product quality, such as degradation of texture, and vitamins and colour changes. During frozen storage, the properties of the vegetables are greatly influenced by storage conditions, especially temperature and time, even at low temperatures, suffering important quality attributes modification as a result of the action of biochemical activity, chemical and physical phenomena (Giannakourou and Taoukis, 2003). Colour, is one of the most important attributes which affects the consumer perception, and is also an indicator of the vegetable pigment concentration (Francis, 1995). During freezing and frozen storage, the colour of green vegetables suffers modifications due to chlorophylls changes, which can follow chemical and enzymatic pathways. The chemical pathway involves removal of the Mg ion from the porphyrin ring via: (i) acidic substitution and/or heat, such as in the conversion of chlorophylls into to pheophytins and (ii) or decarbomethoxylation, such as in the conversion of pheophytins or pheophorbide to pyropheophorbide, respectively. Enzymatic changes are usually due to the action of chlorophyllase on chlorophylls, resulting in chlorophyllides and pheophytins, and then pheophorbides (Heaton et al., 1996). However, other enzymes, like peroxidases, lipases and lipoxygenases, are also associated with the chlorophylls conversion into pheophytins (Buckle and Edwards, 1970). Ascorbic acid (AA) (or vitamin C) is another indicator commonly used to evaluate frozen vegetables quality. In spite of its importance for human health (Naidu, 2003), it is generally observed that if this vitamin is well preserved, the other nutrients are also well retained (Lin et al., 1998). In general, a reversible equilibrium occurs between ascorbic acid and dehydroascorbic acid (DHAA), which is then irreversibly hydrolysed to 2,3-diketogulonic acid (DKGA) that does not have vitamin C activity. It seems that vitamin C degradation rate is affected by the oxidation–reduction potential of the reaction medium (Serpen and Gökmen, 2007). The oxidizing and reducing agents are naturally present, while some additional species may also occur during thermal treatments. Accordingly, the main mechanisms of ascorbic acid losses during the blanching operation are thermal induced degradation or by leaching (Garrote et al., 1986). During frozen storage the degradation is susceptible to conditions, such as temperature and storage length, being the chemical and enzymatic oxidation the major reasons for AA degradation. The actions of enzymes like AA oxidase, phenolase or peroxidase are also directly or indirectly responsible for vitamin C loss, as referred by Lee and Kader (2000). As previously referred, enzymes are involved in multiple degradation processes. Consequently, their inactivation is important to reduce colour and some nutrients degradation. Due to its higher thermal resistance, many studies used peroxidase enzyme as an indicator of the thermal treatment applied before freezing vegetables (Anthon and Barret, 2002). It was not found in published works, studies concerning the effect of frozen storage conditions on vegetables’ peroxidase kinetics. Normally, researchers discuss the possible reactivation of peroxidase activity after thermal inactivation, under certain frozen storage conditions. However, as Bahçeci et al. (2005) referred, the results are contradictory and peroxidase regeneration normally occurs in purified enzymes. The objective of this study was to evaluate the effect of blanching and freezing processes on watercress (N. officinale R. Br.) colour changes, chlorophylls and ascorbic acid contents, and peroxidase activity, and to evaluate the degradation kinetic parameters for these quality factors during frozen storage at three different temperatures ( 7, 15 and 30 C). This study contributes for the industrial development of a new, nutritive, functional and attractive frozen vegetable product. Fresh watercress (N. officinale) was gently supplied by Vitacress, a company that grows watercress in Almancil, Algarve, one day after harvesting. Firstly leaves were sorted, washed and blanched for 20 s at 95 C, as it was recommended by Cruz et al. (2006), in a thermostatic water bath (±1 C) with 45 l capacity. During the heat treatment, the temperature of the water was monitored with a digital thermometer (Ellab ctd 87) and a thermocouple (1.2 mm needle dia; constantan-type T). After cooling and drying, watercress was moulded in a parallelepiped form (4.6 cm 3.3 cm 1.8 cm) and frozen in an air blast freezer, Armfield FT 36 (Armfield Ltd., Ringwood, England). Samples were frozen at an average air temperature of 40 C, until the temperature of 25 C was reached in the sample geometric centre (thermocouple – 1.2 mm needle dia; constantan-type T). Frozen watercress blocks (approximately 90 g) were packed in sealed polyethylene bags and stored in three laboratory chambers (Fitotherm, model S550 BT, Aralab, Portugal) at temperatures of 7, 15 and 30 ± 1 C, for a total of 13 months, using the same methodology as in Martins and Silva (2003). Samples were randomly taken and analysed raw, after blanching and prior to frozen storage (t = 0). Frozen samples were also randomly collected and analysed weekly in the first 5 months, fortnightly in the following trimester and monthly after the 8th month for each storage temperature. All frozen samples were firstly thawed at room temperature during approximately 1 h before analysis. The Hunter Lab co-ordinates were measured by a tristimulus colorimeter (model CR 300, Minolta Corporation, Japan) and a CIE standard illuminant C. The colorimeter was calibrated with a standard white tile (Y = 95.3, x = 0.3133, y = 0.3197). Colour was expressed using the LH, aH and bH Hunter scale parameters. Samples were homogenised and placed in a Petri dish for the evaluation. Measurements were taken in triplicates with nine readings. Combinations of colour parameters may be more effective to evaluate the overall colour changes of processed vegetables than the individual Lab parameters. Therefore, to describe watercress storage colour modifications, the values of LH aH bH, aH/bH, LH aH/bH, and LH/aH bH, as well as the hue parameter (h0H), expressed as h0H= tan bH/aH, that gives the chromatic tonality, were evaluated. A spectrophotometric method adapted by Vernon (1960) was used to quantify chlorophylls a and b. Acetone (Merck) was added with pure water to give a final solution which was 80% in acetone. Watercress samples (2.5 g) were diced and homogenized into a Waring Blendor and 25 ml of acetone solution were added. The samples were homogenized for 3 min. The homogenate was filtered through filter paper Whatman no. 1, with a Büchner funnel under vacuum. The filter cake residues were washed with 80% acetone and the filtrate brought to a final volume of 50 ml with the same solution. For each sample extract solution an unconverted and a converted sample were required for spectroscopic measurements. The unconverted was prepared by adding 3.0 ml of 80% acetone to a volumetric flask and diluting to 10 ml with the filtered extract. The converted sample was prepared by placing 1.5 ml of saturated oxalic acid in 1.5 ml of 80% acetone in a volumetric flask and diluting to 10 ml with the same filtered extract. Both the control and converted sample were kept in the dark at room temperature for 3 h, after which the absorbances of both samples were determined using 10 mm-path-length glass cells (Amersham Biosciences) in a spectrophotometer (Shimadzu UV 1601, Japan). The solution of 80% acetone and a solution of oxalic acid with 80% acetone with a ratio of 1:1 (v:v) were used as a blank to zero the instrument, respectively, for unconverted and converted samples. All samples were run in three replicates. Chlorophylls a and b concentrations were calculated using the following equations: Chlorophyll a ðmg=100gÞ 1⁄4 25:38ðDA662Þ þ 3:64ðDA645Þ ð1Þ Chlorophyll b ðmg=100gÞ 1⁄4 30:38ðDA645Þ þ 6:58ðDA662Þ ð2Þ The abbreviations DA645 and DA662 stand for change in absorbance of unconverted and converted sample at 645 and 662 nm, respectively. Total chlorophyll content was obtained as the sum of chlorophylls a and b contents. The results of chlorophyll concentration are expressed in mg per 100 g of product. Ascorbic acid determination was carried out by an adaptation of the method developed by Zapata and Dufour (1992). After treatments each sample was homogenized with a Moulinex blender in 20 ml of methanol–ultrapure water (5:95, v/v) during 5 min. Each sample (5 g) was transferred to a 20 ml volumetric flask and 1 ml of isoascorbic acid (IA) standard solution (0.03 g/50 ml) was added. The pH was adjusted with HCl (Merk) to obtain final values between 2.20 and 2.45. The volume was completed to 20 ml with methanol–ultrapure water (5:95, v/v). The content was centrifuged (Sorval Instruments RC5 C) for 5 min at 10,000 rpm and 4 C. Afterwards, 3 ml were transferred to another tube with 1 ml of 1,2-phenylenediamine (OPDA) from Sigma (0.03 g/50 ml), daily prepared and maintained in dark. The mixture was then vortexed and placed in dark at room temperature for 40 min. Then the mixture was filtered in a 0.22 lm membrane (Millipore – GS Filter), the first milliliter was discarded and 20 ll were injected in the HPLC. The wavelength detector was set to 348 nm, and after elution of DHAA the wavelength was shifted to 262 nm for AA and IA detection. For the mobile phase, 13.61 g of potassium dihydrogen phosphate (Merk) and 3.64 g of cetrimide (Fluka) were added to 2 l of methanol–ultrapure water (5:95, v/v). The eluent was filtered in a 0.45 lm membrane (Macherey-Nagel, Porafil) and degassed in an ultrasonic bath for 15 min. HPLC chromatograms were analysed with the software Gold Neuveau Chromatography Data System, Version 1.7 . The results of AA and DHAA were expressed in mg/100 g of sample. Total vitamin C content was obtained as the sum of AA and DHAA contents, quantified by a HPLC technique. All samples were run in six replicates. Peroxidase activity was determined by a spectrophotometric method adapted from Hemeda and Kleind (1990). The enzyme extract was obtained with 20 g of vegetable sample and 50 ml of distilled water, blended for 2 min in a mixer (Moulinex, France) at room temperature. The homogenate was filtered with membrane filters (Whatman no. 1). The filtrate (100 ll) was mixed to 2.9 ml of substrate solution (prepared daily), (which contained 10 ml of guaiacol (1%, Sigma–Aldrich, G-5502), 10 ml of H2O2 (0.3%, Pancreac) and 100 ml of 0.05 M potassium phosphate buffer (pH 6.5)) in a 10 mm-path-length glass cuvettes (Amersham Biosciences). The assay was carried out with a spectrophotometer (Shimadzu UV 1601, Japan). Increase in absorbance at 470 nmwas measured with 10 s intervals at 25 C, during 2 min. Peroxidase activity was defined as a change in 0:001 in absorbance per minute, in the linear region of the curve. Enzyme specific activity was expressed as U/ g min. Three replicates per sample, raw, blanched, prior to frozen storage and at each storage time–temperature, were measured. In general, the studies presented in literature on quality parameters changes during frozen storage of vegetables report zero-order (Eq. (3)) or first-order (Eq. (4)) degradation reaction kinetics,