Microsoft Word - 54catania.docx CHEMICAL ENGINEERING TRANSACTIONS VOL. 44, 2015 A publication of The Italian Association of Chemical Engineering Online at www.aidic.it/cet Guest Editors: Riccardo Guidetti, Luigi Bodria, Stanley Best Copyright © 2015, AIDIC Servizi S.r.l., ISBN 978-88-95608-35-8; ISSN 2283-9216 Variability of Total Carotenoids in C. moschata Genotypes Lucia Maria Jaeger de Carvalho*a, José Luiz Viana de Carvalho b, Rita Mercia Estigarribia B. Faustino c, Ingrid Maroto Kaser a, Victor Gomes Lima a, Danieli Silva Feijó de Sousa a. a Laboratório de Tecnologia e Análise Instrumental de Alimentos – LATAIA. Av. Carlos Chagas Filho, 373, CCS, Bloco L subsolo, Sala 17, Ilha do Fundão. 21.941.902 - Rio de Janeiro, Brazil. b Embrapa Food Technology - Av. das Américas, 29501. Guaratiba. 23.020.470 - Rio de Janeiro, Brazil. c Embrapa Semiarid –. Rodovia BR-428, Km 152, Zona Rural, Petrolina, 56.302.970 - Pernambuco, Brazil - Caixa Postal 23. luciajaegergmail.com Nowadays, the vitamin A deficiency (VAD) is still a public health concern, being one of the most important components of the high rate of morbidity and mortality among children in developing countries. There is a high prevalence of night blindness caused by deficiency of vitamin A. Children and pregnant women are particularly affected. Many pumpkins (C. moschata) genotypes present a great variability of carotenoid content mainly the beta-carotene. The objective of this work is to evaluate the variability of beta-carotene among pumpkins genotypes. Twenty biofortified pumpkins cultivated at Embrapa Table Coastland and Semiarid were evaluated regarding beta-carotene contents.The analyses were carried out by High Performance Liquid Chromatography. The pumkinks showed a large variability in the contents of total carotenoids varying, in fresh pumpkins from 699.06 (genotype 25) to 124.60 µg/g (genotype 26), from 106.96 (genotype 26) to 655.54 µg/g (genotype 25) in samples cooked in boiling water and 117.55 (genotype 19) and 650.54 µg/g (genotype 25) in steamed cooked, respectively. 1. Introduction Pumpkins are fruits of different species of Cucurbita genus, grown around the world for its pulp, seeds and even the flowers serve for human consumption (Provesi, Dias, Lover, 2011). The Food and Agriculture Organization of the United Nations (FAO) reported an estimated world pumpkins production, in 2007, over 20 million tonnes, especially in China, India, Russia, United States, and Egypt ( FAOSTAT, 2008). Although there are no official data in Brazil, the pumpkins production is high, especially of the species Cucurbita moschata and Cucurbita maxima. The great majority of pumpkins are harvested and consumed when fruit is mature (Paris,1994). In several regions of the country, C. moschata are known to contain high carotenoids contents with antioxidant activity (lutein) and pro- vitamin A activity (α and β-carotene) (Rodriguez- Amaya et al., 2008). The β -carotene has 100 % pro- vitamin A activity and α -carotene around 53 % (Boiteux et al., 2007). The processing of food can increase the bioavailability of carotenoids and functional foods have prepared based on it (Mínguez - Mosquera et al., 2002). Khachik, Goli, Beecher (1992) reported that vegetables, after to be cooked, increased the provitamin A carotenoids in relation to the fresh ones. However, it should be noted that the heat treatment as boilingand steam among others promotes carotenoids isomerization in food (trans to cis isomerization), and the degree of isomerization is directly related to the heat intensity and duration (Rock et al., 1998). Nutritionally, the differentiation between the cis and trans isomers of provitamin is important because the cis form exhibits less potential of provitamin A (Rodriguez-Amaya et al., 2008.). Lucia et al (2007) evaluated the effect of processing in some vegetables on the bioavailability of carotenoids concluding that the heat treatment can increase it, and it is possible that, even with significant losses after processing, the remaining carotenoids are better absorbed. On the other hand, vitamin A deficiency (VAD) is still a major public health concern in developing countries, including Brazil one of the most important components of the high morbidity and mortality among children (Zuorro, 2014). Underwood, Arthur DOI: 10.3303/CET1544042 Please cite this article as: Carvalho L.M.J., Carvalho J.L.V., Faustino R.M., Maroto I., Lima V., Feijo D., 2015, Variability of total carotenoids in c.moschata genotypes, Chemical Engineering Transactions, 44, 247-252 DOI: 10.3303/CET1544042 247 (1996) reported the contribution of vitamin A in public health. The benefits include not only improved health and welfare for individuals and their families, but also improved chances of survival for an estimated 254 million children.For example, there is a high prevalence of night blindness caused by vitamin deficiency A (Tomkins, 2000). High carotenoids content were found, in C. moschata, cv Baianinha (Kandlakunta et al., 2008; Kurz et al., 2008). Azevedo-Meleiro and Rodrigues-Amaya (2007) evaluated the pro-vitamin A carotenoids of C. pepo, C. moschata and C. maxima. The α and β-carotene were most abundant in C.moschata, C.pepo in lutein and β-carotene and, C. maxima in violaxanthin, followed by β-carotene and lutein. The study of pumpkin C. moschata genotypes is part of the project "Biofortification in Brazil: Creating cultures for better nutrition – BioFORT”, which is funded by Embrapa Monsanto Research Fund. The evaluation of brazilian C. moschata genotypes fresh and cooked will provide data on the carotenoid contents and to be recommended for children diet, minimizing nutritional deficiencies of Vitamin A. 2. Material and methods 2.1 Material Twenty genotypes of C. moschata were cultivated in two different experimental locations: Petrolina, state of Pernambuco (samples 3 to 14 and, 21 to 26), Brazil and, at EMBRAPA Table Coastlands, Aracaju, state of Sergipe (samples 15, 16, 17, 18, 19, 20 and 27), Brazil. After maturity of the fruits, 20 pumpkins were used for experiments and analysis. The pumpkins were sent by plane to the Laboratory of Technology and Instrumental Analysis of the Faculty of Pharmacy, Rio de Janeiro Federal University, Rio de Janeiro, Brazil for experiments and analyses. 2.2 Sampling Samples of fresh pumpkin were weighted, peeled and divided into four parts by two longitudinal cuts, from one end to the opposite, resulting in four sections (Figure 1). These four sections, two opposite each other, were discarded and the remaining two were used for analysis and were fragmented and placed in a vertical mixer (IKA - Ultraturrax model T18 basic) to obtain a homogeneous mass (Rodriguez-Amaya, & Kimura, 2004). Figure 1. Quarterization (Rodriguez Amaya, Kimura, 2004). 2.3 Methods 2.3.1 Cooking styles The twenty pumpkin genotypes samples were washed, peeled, cut in small pieces, and divided into 2 different groups. The pumpkin pieces were then, cooked in two different styles: cooked in boiling water (2:1 for 4 minutes) and steamed cooked (five minutes). Additionally, genotypes 15, 16 and 17 were still cooked with cowpea beans (1:1) to verify the carotenoids content and iron and zinc after cooked in boiling water since people from northeast of Brazil still eat pumpkin cooked with cowpea. 2.3.2 Soluble solids The soluble solids were determined using an Abbé refractometer, AR12, Scmidt & Haensch, Germany (AOAC, 2005). 2.3.3 Carotenoids extraction and determination The total carotenoids in pumpkin genotypes were determined using a spectrophotometer (Thermo Scientific Evolution 60) set at 450 nm. To determine the total carotenoids in the fresh and cooked pumpkin genotypes, approximately 0.3 g of the samples and 3.0 g of celite 454 (Tedia, Ohio, USA) were weighed in a mortar on a digital balance (Bel Engineering, model MA0434/05). For the carotenoids extraction, 25 mL of acetone were, successively, added until a paste was obtained. The paste was transferred to a sintered funnel (5 μm) coupled to a 250 mL Buchner flask and filtered under vacuum. This procedure was repeated, at least, three times until the sample was colorless. The obtained extract was transferred to a 500 mL separatory funnel containing 40 248 mL of petroleum ether. The acetone was removed through the slow addition of ultrapure water (Milli – Q - Millipore) to prevent the formation of emulsion. The aqueous phase was discarded, and this procedure was repeated four times until no residual solvent remained. The extract was transferred with a funnel to a 50 mL volumetric flask containing 15 g of anhydrous sodium sulfate. The final volume was adjusted with petroleum ether, and the samples were analyzed at 450 nm. The total carotenoid content was calculated using the equation 1: Carotenoid content (µg/g) = A x V (mL) x 104 [1] A1% x P (g) 1cm Where: A = absorbance; V = total extract volume; P = sample weight; and =%11cmA 2592 (i.e., the β-carotene extinction coefficient in petroleum ether). 2.3.4 - Statistical analyses The data were analyzed using ANOVA and Tukey tests at a significance level of 0.05. The statistical analyses were performed using the Statistica software, version 5.1. 3. Results and Discussion 3.1 Soluble solids and weight The soluble solids contents varied from 4.97 to 17.20 o Brix and, 13 genotypes presented values above 9 o Brix showing a large variability among the samples and, significant differences (P < 0.05) were found. XinZheng et al. (2009) reported soluble solids in C. moschata cultivars ranging from 3.3 to 12.7%. Zinash, Workneh & Woldetsadik (2013) still reported a great variation in soluble solids contents in pumpkins cultivars (4.10 to 10.03 oBrix) for fresh fruit quality assessment. On the other hand, Gajewski et al. (2008) found in the cultivar C. moschata variety “Zemcuzina”, 6.6 o Brix and Valenzuela-Jacobo et al. (2011) 6.42 Brix in C. moschata “Cehualca”. The weight of the pumpkins genotypes ranged from 1,464 g to 7,824 g. However, the pumpkins genotypes did not show correlation between soluble solids versus weight (Table 1). Table 1. Soluble solids and weight of the 20 fresh C. moschata genotypes Identification Soluble solids (g/100g) Weight (g) 3* 10.12 6.685 6* 04.97 5.702 8* 10.07 7.824 9* 08.43 4.240 10* 10.33 5.660 11* 09.87 5.692 14* 10.50 3.858 15* 09.00 4.812 16* 10.10 4.075 17* 09.83 5.356 18* 10.16 4.840 19* 05.83 3.930 20* 05.08 4.162 21** 12.75 1.738 22** 08.10 2.146 23** 07.50 1.464 24** 10.66 2.104 25** 11.00 1.618 26** 17.20 2.042 27* 10.50 5.736 * Genotypes from experimental location at Embrapa Table Coastlands, Aracaju, Sergipe, Brazil. ** Genotypes from experimental location at Embrapa Semiarid, Petrolina, Pernambuco, Brazil. 3.2 Total carotenoids in fresh C. moschata genotypes Among genotypes studied, significant differences (P ≤ 0.05) were observed for total carotenoids suggesting that a genetic variation in carotenoid accumulation is present. Itle and Kabelka (2009) observed the same 249 differences evaluating twenty Cucurbita spp. genotypes. They correlated the L (lightness or darkness), a (color direction in red or green) and, b (color direction in yellow or blue) color space values with the carotenoid contents. According to them these close associations will also assure that breeding for enhanced carotenoid content within pumpkins can be achieved using an easy-to-use and, inexpensive method. Gajewski et al. (2008) found in the cultivar C. moschata variety “Zemcuzina”, ex Poir, total carotenoids contents of 64.5 µg.g-1 in fresh samples. In our study the total carotenoids contents in fresh pumpkin genotypes varied from 699.06 (genotype 25) to 124.60 µg.g-1 (genotype 26). Significant differences in the total carotenoids contents among all fresh samples (P < 0.05) were observed. Carvalho et al. (2012) found total carotenoids in two fresh C. moschata genotypes varying from 234.21 to 404.98 µg.g-1. These values are similar to those found by Ramos et al. (2009) in pumpkin (C. moschata) genotype, which ranged from 100.50 to 365.40 μg.g-1. On the other hand, forty-eight accesses of C. moschata from Embrapa Semiarid bank were evaluate by Souza et al. (2012) and the total carotenoids, in β-carotene, in samples were lower than our results 11.81 to 290.62 µg.g-1. 3.3 Total carotenoids in cooked C. moschata genotypes The total carotenoid contents in boiled pumpkin genotypes ranged from 655.54 (genotype 25) to 106.96 µg.g-1 (genotype 26) and, in steamed cooked pumpkins varied from 133.63 (genotype 6) to 650.54 µg.g-1 (genotype 25). Samples cooked with cowpea beans showed the lowest contents among all (83.60 µg.g-1 – genotype 17 to 117.04 µg.g-1 – genotype 18), as expected. By comparing the initial total carotenoid contents with α- carotene in both cooking styles, the genotype 25 presented the lowest percentage (11.57 % - 75.85 µg.g-1 - boiled and 11.48 % - 74.71 µg.g-1 - steamed). Eight cooked pumpkin genotypes showed significant differences (P < 0.05) in total carotenoids between the two cooking styles (boiled versus steamed). Comparing the total carotenoids between fresh and boiled pumpkins genotypes it was observed that 17 of them did not revealed significant differences (P < 0.05). On the other hand, 12 steamed cooked pumpkins showed significant differences (P < 0.05) compared to fresh samples. Table 2. Total carotenoids in pumpkin genotypes of C. moschata cooked in boiling water, steamed and cooked with cowpea beans. Total carotenoids Identification Cowpea Boiled Steamed 3 - 218.77 c,d,e,f 242,80 c,d 6 - 124.20 a,b,c 133.63 a 8 - 32.66 g,h 257,93 d 9 - 224.02 d,e,f 239.23 c,d 10 - 149,23 a,b,c,d 154,60 a 11 - 428,20 i,j 492.76 f 14 - 325,49 g,h 340.85 e 15 - 308,03 f,g,h 356,80 e 16 110.02 a 237.92 d,e,f,g 211.75 b,c,d 17 83.60 a 206.62 b,c,d,e 224.71 b,c,d 18 117.04 a 198.47 a,b,c,d,e 148.49 a 19 - 117.55 a,b 177.93 a,b 20 - 166.90 a,b,c,d,e 159.42 a 21 - 343.76 h,i 458.25 f 22 - 151.29 a,b,c,d 152.50 a 23 - 178.71 a,b,c,d,e 208.68 b,c 24 - 485.67 j 502.08 f 25 - 655.54 k 650.54 g 26 - 106.96 a 140.83 a 27 - 261.38 e,f,g,h 332.96 e Different letters in the same column means significant difference at 0.05%. Is well known that β-carotene isomers, mainly the 13 and 9-Z- β-carotene, are formed by isomerization or decomposition after heat treatments as boiling and steaming, light exposure, singlet oxygen, enzymes, free radicals and high oxygen concentrations (Rios et al., 2009). Provesi et al., (2011) evaluated the carotenoids losses in C.moschata and C. maxima purees as well as the β-carotene isomers after during 180 days. They observed a slight degree of isomerisation of β-carotene in the puree samples, but with low concentrations of cis-isomers. Okpalamma et al., (2013) studying T. occidentalis (fluted pumpkin) leaves observed that the lutein and β-carotene concentrations were much higher than typical contents in conventional edible leafy vegetables. Meals prepared with pumpkin cooked with cowpea beans presented zinc contents ranging from 250 11.52 to 13.87 and iron from 13.69 to 15.68 mg.kg-1. Comparing the iron and zinc contents in the other preparations (boiled and steamed), as expected, these contents were highest. 4, Conclusions Taking into account that, usually, the proportion of all-trans-β-carotene,is the most abundant carotenoid found in C.moschata genotypes, varying from 37-85% of the total carotenoid content in the cultivars tested in our study, they are promising for consumption, corresponding of the 101 % of DRI (intake) for children of preschool age (1-3 y) in a daily serving of 120g of food, considering the minimum concentration of total carotenoids found in the samples. The C. moschata genotypes can be recommended for cultivation given its high levels of total carotenoids. Acknowledgements We would like to thank the HarvestPlus Program and the Embrapa/Monsanto Research Fund for financial support (BioFORT), Embrapa Table Coastlands and Embrapa Semiarid for providing the pumpkin genotypes and to FAPERJ for financial support and scholarships. 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