Plant Foods Hum Nutr (2011) 66:238–244 DOI 10.1007/s11130-011-0241-5
ORIGINAL PAPER
Evaluation of the Biochemical Components and Chromatic Properties of the Juice of Vaccinium macrocarpon Aiton and Vaccinium oxycoccos L. Laima Česonienė & Remigijus Daubaras & Ina Jasutienė & Jonė Venclovienė & Inga Miliauskienė
Published online: 7 July 2011 # Springer Science+Business Media, LLC 2011
Abstract Benzoic acid, total anthocyanins, soluble solids, titratable acidity, and colour properties in juice of the American cranberry Vaccinium macrocarpon and the European cranberry Vaccinium oxycoccos were investigated. Berry juices of V. macrocarpon cultivars were distinguished by their higher total anthocyanin and benzoic acid amounts. These cultivars accumulated on average 43.11 mg/l of benzoic acid and 92.45 mg/l of total anthocyanins. The levels of benzoic acid and total anthocyanins in V. oxycoccos cultivars were 17.52 mg/l and 42.54 mg/l, respectively. The V. macrocarpon cultivars ‘Franklin’, ‘Le Munyon’, ‘Searles’, and ‘Early Richard’ were selected as the best according to the enhanced total anthocyanins and benzoic acid amounts. The separation of anthocyanins by HPLC-UV-VIS revealed the presence of six anthocyanins, with peonidin-3-galactoside being the most prevalent. Galactoside together with glucoside conjugates comprised the largest percentage of total anthocyanins in the juices of V. macrocarpon and V. oxycoccos cultivars.
L. Česonienė (*) : R. Daubaras Kaunas Botanical Garden, Vytautas Magnus University, Žilibero 6, Kaunas 46324, Lithuania e-mail:
[email protected] I. Jasutienė : I. Miliauskienė Food Institute of Kaunas University of Technology, Taikos pr. 92, 51180 Kaunas, Lithuania J. Venclovienė Faculty of Natural Sciences, Vytautas Magnus University, Vileikos 8, Kaunas 44404, Lithuania
Keywords Anthocyanins . Benzoic acid . Cranberry . Cultivar . Juice
Introduction The consumption of fresh berries and different berry products is an important factor in the maintenance of human health and in disease prevention. In recent years, various research studies have focussed on bioactive compounds in berries and the use of the compounds in functional foods [1–3]. Interest in the biochemical composition of cranberries has been intensified because of the increased awareness of their healthful properties. Cranberries are a particularly rich source of phenolic phytochemicals, including phenolic acids (benzoic, hydroxycinnamic, and ellagic) and flavonoids. Cranberry juice is characterized by considerable amounts of benzoic acid and a very low pH, which is advantageous in controlling the growth of human pathogens [4]. Of the secondary metabolites found in cranberries, anthocyanins have received exceptional interest [5], and considerable attention has been paid to the potential antioxidant effect of cranberry anthocyanins. As reported by other authors [6, 7], anthocyanins have positive effects on blood vessel walls. Cranberries have also been shown to possess antibacterial [8, 9], anticarcinogenic [10], and antioxidant activities [5]. Dietary and therapeutic properties of cranberries have been widely reported by different authors. Cranberry juice is used in the treatment of urinary system infections [11, 12], as well as in the treatment of periodontitis [13] and other diseases. Cranberry juice can inhibit the colonization of teeth by streptococci and can slow the development of dental plaque [14].
Plant Foods Hum Nutr (2011) 66:238–244
There have been many studies regarding the biochemical components of the American cranberry Vaccinium macrocarpon Aiton, which is commercially grown in North America. Only few reports have focussed on the European cranberry V. oxycoccos L. More comprehensive evaluations of V. oxycoccos have been completed in Russia, Lithuania and Finland [15–18]. In addition, increasing interest in natural pharmaceuticals and functional foods has led plant breeders to initiate the selection of cranberries cultivars with higher contents of biologically active substances [19]. The objectives of this study were: (1) to evaluate the accumulation of titratable acidity, total anthocyanins, benzoic acid, and soluble solids in fresh berry juice of V. macrocarpon and V. oxycoccos cultivars, (2) to measure chromatic characteristics, and (3) to select cultivars which produce the most valuable berries.
Materials and Methods
239
vegetable juices—Determination of titratable acidity”. Juice was diluted 1-to-9 with water, and 25 ml of this solution titrated by 0.1 mol/l NaOH until the pH reached 8.1±0.2. The results were expressed as g/100 g of citric acid. The juice pH was determined according to the method ISO 1842:1991 “Fruits and vegetable products—Determination of pH” using a Denver Instrument pH meter (USA). The determination of the soluble solids (SS) content was carried out according to ISO 2173:2003 “Fruit and vegetable products-Determination of soluble solids— Refractometric method” using an Atago RX-5000CX refractometer (Japan). The total anthocyanin content (TAC) in cranberry juice was determined using a pH differential method. The absorbance was measured using a Genesys 5 spectrophotometer (USA) at 510 nm and at 700 nm in buffers with pH levels of 1.0 and 4.5. Results were expressed as mg/l of cyanidin-3-galactoside [21]. The anthocyanin pigment concentration expressed as cyanidin-3-galactoside mg/l was calculated as follows:
Plant Material Nine American cranberry V. macrocarpon cultivars, ‘Le Munyon’, ‘Searles’, ‘Pilgrim’, ’Franklin’, ‘Early Richard’, ‘Stevens’, ‘Washington’, ‘Black Veil’, and ‘Howes’, and thirteen V. oxycoccos cultivars, ‘Vaiva’, ‘Reda’, ‘Žuvinta’, ‘Vita’, ‘Amalva’, ‘Krasa Severa’, ‘Dar Kostromy’, ‘Sazonovskaja’, ‘Soontagana’, ‘Kuressoo’, ‘Nigula’, ‘Virussaare’, and ‘Maima’, were investigated. These genotypes were previously selected from the experimental collection of the Kaunas Botanical Garden of VMU based on high yield, large berries, and disease resistance. The Vaccinium genetic resources collection is located in the Central region of Lithuania. This region is typical of an average temperature −6 °C in January and +16 °C in July with an average annual rainfall of 600 mm. The growing season lasts for 180 days approximately. The cranberry collection is located 76 m above the sea level. These cultivars were propagated using cuttings that were planted in acid peat (pH 4.0–5.0) beds. Collection and Preparation of Berry Samples The berries used in the biochemical investigations were harvested at the stage of full ripening. The stage was determined by the visual assessment of the cultivar’s characteristic berry colour and brown seeds [20]. Berries were lightly crushed and pressed in a hand press to obtain fresh cranberry juice. For each cultivar, three replicate samples were prepared. Biochemical Evaluation The titratable acidity (TA) of cranberry juice was determined according to the procedure of EN 12147 “Fruit and
ðA510 A700 ÞpH1:0 ðA510 A700 ÞpH4:5 "L
MW DF ε L
MW DF 103
molecular weight, for cyanidin-3-galactoside 445.2 g/mol dilution factor molar extinction coefficient for cyaniding-3galactoside 41,700 pathlength, cm
For HPLC-UV/VIS analysis of individual anthocyanins, berry cake (the residue after juice pressing) was homogenized, and 3 g of the homogenate was extracted for 1 h at room temperature with 10 ml of acidified ethanol (95% [v/v] food-grade ethanol containing 0.1 M HCl). The obtained extracts were analyzed by HPLC using a reversed-phase C18 LiChrospher®100 RP 18e column (125×4 mm, 5 μm) (Merck, Darmstadt, Germany). The eluents were (A) 4% H3PO4 in water and (B) 100% HPLC-grade acetonitrile (Merck) [22]. The chromatographic conditions were as follows: 10% B in A at the time of injection, 14% B in A (4 min), 16% B in A (10 min), 30% B in A (25 min), initial conditions (26 min). The flow rate was 0.8 ml/min, and 20μl was injected. The samples were filtered through a 0.45-μm cellulose syringe filter before analysis. Detection was performed using an L-7400 LaChrom Merck Hitachi UV detection system (Merck KGaA) at 520 nm. Mass spectra were registered by the Hewlett Packard 1100 MS (Agilent Technologies). The voltage in the capillary was 4500 V, the voltage of fragmentation was 100 V, and the temperature was
240
Plant Foods Hum Nutr (2011) 66:238–244
250 °C. The scanning range was 100–1000 m/z with the interval of 0.1 m/z. Mass spectra showed elution sequence on the C18 column to be cyanidin-3-galactoside, cyanidin-3glucoside, cyanidin-3-arabinoside, peonidin-3-galactoside, peonidin-3-glucoside, and peonidin-3-arabinoside. The benzoic acid (BA) concentration was determined according to ISO 22855:2008 “Fruit and vegetable products—Determination of benzoic acid and sorbic acid concentrations—High-performance liquid chromatography method”. Juices (10 ml) were diluted with 75 ml of extraction solution (60 volume parts ammonium acetate/acetic acid buffer solution and 40 volume parts of methanol). The flasks were put into an ultrasonic bath for 10 min. The samples were clarified with Carrez I and Carrez II solutions and diluted to 100 ml with extraction solution. The samples were filtered through a paper filter and a 0.45-μm cellulose syringe filter before analysis. The chromatographic conditions were as follows: the eluent was ammonium acetate 50 v/v with methanol 40 v/v (pH adjusted to 4.5–4.6), the flow rate was 0.8 ml/min, and 20μl was injected. The reversed-phase ZORBAX Eclipse XDB-C18 (5 μm) 150×4.6 mm (Agilent Technologies, USA) column was used. Detection was performed using an Agilent 1200 diode-array detection system (Agilent Technologies, USA) at 235 nm. Determination of Juice Chromatic Characteristics Colour was measured using duplicate samples in a test cell. The L*—juice lightness, a*—redness, and b*— yellowness values were measured using a Cintra 202 spectrophotometer (GBC Scientific Equipment, Australia) with the Cintral software package in reflectance mode with illuminant C and a 2° observer angle. The chroma » » 1=2 C¼ a 2þb 2 and hue angle ho = arctan(b*/a*) were calculated [23].
Statistical Analysis The statistical program package STATISTICA 6 was used for statistical analysis. Associations between the biochemical components were analyzed using Spearman’s rank correlation coefficients. Statistical differences were declared at p
