Plant Foods Hum Nutr (2011) 66:416–423 DOI 10.1007/s11130-011-0249-x
ORIGINAL PAPER
Influence of Fruit Ripening Stage and Harvest Period on the Antioxidant Content of Sweet Pepper Cultivars María C. Martí & Daymi Camejo & Fernando Vallejo & Félix Romojaro & Sierra Bacarizo & José M. Palma & Francisca Sevilla & Ana Jiménez
Published online: 27 July 2011 # Springer Science+Business Media, LLC 2011
Abstract Pepper (Capsicum annuum L.) fruits are highly appreciated by producers and consumers for their economical and nutritional value. Four different cultivars of coloured peppers in immature and mature stages were harvested throughout the spring and examined for their content of phenolic compounds, ascorbic acid and total antioxidant capacity (TAA) as well as for lipid peroxidation and carbonyl proteins as index of oxidative stress. Ripening and harvest period influenced the antioxidants and the development of oxidative processes in the cultivars differently: lipid peroxidation increased in mature peppers except in one cultivar (Y1075), while no changes in protein oxidation or in TAA were produced, except in Y1075 in which both parameters increased. Each cultivar presented differences in antioxidant compounds depending on the harvest period, but we could recommend May as the optimal if all cultivars have to be harvested at the same time, when levels of ascorbate, phenols and TAA were not decreased, fresh weight and proteins were elevated, and M. C. Martí : D. Camejo : F. Romojaro : F. Sevilla : A. Jiménez (*) Department of Stress Biology and Plant Pathology, CEBAS-CSIC, P.O. Box 164, 30100 Murcia, Spain e-mail:
[email protected] F. Vallejo Department of Food Science and Technology, CEBAS-CSIC, P.O. Box 164, 30100 Murcia, Spain S. Bacarizo Syngenta Seeds, S.A., 04710 El Ejido, Almería, Spain J. M. Palma Department of Biochemistry, Cellular and Molecular Biology of Plants, EEZ-CSIC, P.O. Box 419, 18080 Granada, Spain
levels of oxidation were not as high as in June (except for Y1075). A previous study of the response of each cultivar to different environmental conditions results essential to establish a good program of selection of cultivars with high quality and productivity. Keywords Antioxidant . Oxidized ascorbate (DHA) . Phenolic compounds . Pepper . Reduced ascorbate (ASC) . Ripening Abbreviations ASC L-ascorbic acid CO-protein Carbonylated-proteins DHA Dehydroascorbate ROS Reactive oxygen species TAA Total antioxidant activity TBARS Thiobarbituric acid reactive substances
Introduction Fruit and vegetable production comprises approximately half of Spanish agriculture, with great product diversity. Fresh peppers are highly demanded by European markets all year round with almost half the production in the Spanish South-eastern regions. Peppers are considered a good source of most essential nutrients, being rich in natural colours, aromas, antioxidants and vitamins as C, E, A and B complex [1, 2]. The intake of these compounds in food is an important health-protecting factor when they are taken daily in adequate amounts [3]. Also, these fruits are a good source of provitamin A and oxygenated carotenoids, important for the prevention of macular degeneration and cataracts [4] and phytochemicals as flavonoids, that may
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reduce the risk of degenerative disease [5]. During maturation, fruit undergoes transformation of colour, aroma and softening and in this process, production of reactive oxygen species (ROS) plays an important role, among others, in the biosynthesis of carotenoids and in the transformation of chloroplasts to chromoplasts [6, 7]. During fruit senescence in which an oxidative process takes place, cellular structures and enzymes are degraded along with increase of lipid peroxidation [8], which in foods is one of the main causes of deterioration of quality, leading to the appearance of flavours and disagreeable scents and destruction of vitamins. The interaction between ROS and proteins is quite complex, and the formation of carbonyl groups is considered as an irreversible modification being a very valuable marker of oxidative stress [9]. In this context, the function of the cellular antioxidant system is to prevent the offset of chain oxidations, removing ROS. The enzymatic system of defence includes superoxide dismutase (SOD), catalase, peroxidases and the ascorbateglutathione cycle enzymes [10]. In the last years, several reports have provided evidence for the involvement of antioxidants in the fruit physiology, including response at the level of mitochondria, peroxisomes and chloroplasts during pepper ripening and during fruit storage at 20 °C [7, 11, 12]. Among the non-enzymatic antioxidants, the phenolic compounds, ascorbic acid, glutathione and carotenoids are important in vegetables including peppers. Polyphenols are widely distributed in plants contributing to the colour and flavour, and among them, flavonoids protect the organism from the damage produced by oxidative agents being a good indicative of the antioxidant capacity of peppers [13], with numerous epidemiological studies indicating a possible association between their uptake and the risk of coronary disorders and cancer [14]. The human organism cannot produce these protective chemical substances, so they must be obtained through food. Most L-ascorbic acid (ASC, vitamin C) in the human diet is provided by vegetables and fruits [15] and because of its antioxidant activity, it has attracted the attention of researchers, due to possible links with the prevention of some important diseases, including certain types of cancer [16]. Due to the importance of the antioxidants in fruit quality and senescence, and the influence of ripening and environmental conditions in which fruits are developed, in this work we analyzed possible markers for optimal fruit stage and harvesting periods based on antioxidant content and oxidative status of the fruits, for the selection of cultivars with a high nutritional quality as demanded by the industrial market and consumers. For this, two different approaches have been carried out, one using four sweet commercial pepper spring cultivars in two ripening stages, immature (green) and mature (red and yellow), and in the other, mature
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fruits of the previous cultivars collected through two additional spring periods.
Materials and Methods Plant Material Pepper (C. annuum L.) plants were grown under plastic-roofed greenhouses (Syngenta Seeds S.A., Torrepacheco, Murcia, Spain) with optimal nutrient levels applied on rockwood as soil substrate and according to the usual crop programme designed by the company for early cycle cultivars: planting seeds and germination in OctoberNovember; flowering starts in late December-January and pollination and fruit setting takes places from middle March to late April. Meteorological data were collected from a station close to the experimental greenhouses. Fresh fruits were collected at the end of April at two distinct maturation stages, fully green and fully red phenotypes from cultivars Almuden, Cabezo and Quito and fully green and fully yellow phenotypes from cultivar Y1075. Ten different fruits from different plants in the same developing stage were collected from each cultivar. Fully red and fully yellow fruits from those plants were also collected in middle May and middle June, when only mature fruits were available. All were frozen in liquid N2 on the same day of collection, and maintained at −80 °C until analysis. Sampling Three fruits per cultivar were chosen for each extract, and at least three different extracts were taken for each independent experiment. The experiment was repeated a second consecutive year. Protein Content Protein was measured by the protein dyebinding method of Bradford [17] using bovine serum albumin as a standard. Lipid Peroxidation and Protein Oxidation The extent of lipid peroxidation in peppers fruit extracts was estimated by determining the concentration of thiobarbituric acid reactive substances (TBARS) [18], and the spectrophotometric dinitrophenyl hydrazine method was followed for the determination of protein oxidation (carbonyl protein content) [19]. Analysis of Total Antioxidant Activity The total antioxidant activity (TAA) in fruit pericarp was measured using the ABTS (ferrylmyoglobin/2,2′-azinobis-(3-ethylbenzthiazoline-6sulphonic acid) method as described in Jiménez et al. [8]. Analysis of Phenolic Compounds Lyophilized pepper (2 g) was homogenized with 50 ml of 70% methanol. After extraction, phenolic compounds were analyzed by HPLCdiode array detection (DAD). The identification of the main
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peaks was done by HPLC-DAD-Tandem mass spectrometry (MS-MS) as described by Marín et al. [20]. Determination of Total Ascorbate Content Ascorbate was extracted from 1 g of pepper fruit tissue homogenized in 2 ml of 5% metaphosphoric acid. ASC and dehydroascorbate (DHA) were determined by HPLC as described in Jiménez et al. [8]. Statistical Analysis Fruit harvesting was conducted using a completely randomized design. The significance of any differences between mean values was determined by one-way analysis of variance; the comparative analysis used was the Student t-test (p
