While the pumpkin slices were still hot, their peel was removed and their remaining pulp was crushed and homogenised in an industrial blender (Metvisa, Brusque, Brazil). The pulp samples were put into 260 ml glass bottles and then heat-treated in an autoclave at selleck compound 121 °C for 20 min for commercial sterilisation. A headspace was left in all the bottles so that a partial vacuum was generated inside them. Besides the analysis that was performed on the final product (pumpkin puree), aliquots were removed before and after cooking (raw pumpkin and cooked pumpkin) for analysis of carotenoids. The collection of the aliquots was performed with a special care regarding the uniformity and the quantity

of the samples so that they high throughput screening compounds were representative of the batch as a whole. To prevent any modification of carotenoids after collecting the samples, the aliquots were frozen and kept at −20 °C until required for analysis on the following day. The puree samples were stored in a ventilated environment that was protected from light and had its temperature and relative humidity monitored for 6 months. After specific periods of storage (0, 15, 30, 60, 90, 120, and 180 days), the samples were randomly picked for analysis of the changes in the carotenoids in the pumpkin puree samples. The method used for carotenoid

analysis was proposed by Kimura and Rodriguez-Amaya (2002) and used by Azevedo-Meleiro and Rodriguez-Amaya (2007) for carotenoid analysis on pumpkins. The extraction was performed with acetone (previously refrigerated for 2 h) on 10–20 g of sample, using a pestle and mortar until the residue became colourless, and after that the extract

was partitioned with petroleum ether. In the case of the C. maxima ‘Exposição’ samples, the extract was submitted to overnight saponification with methanolic KOH (10%, w/v), while in the case of C. moschata ‘Menina Brasileira’, where xanthophylls, which are oxy-carotenes, are in lower concentrations, saponification was not performed in order to Vasopressin Receptor minimise the loss which can occur in this step. The extracts were washed with distilled water and concentrated at low pressure in a rotoevaporator (Tecnal, TE-211, Piracicaba, Brazil), always at a temperature below 35 °C and using glass pearl for optimisation of the recovery in the re-dissolving process. In order to avoid errors during the carotenoid analysis, all the necessary precautions were taken as recommended by Rodriguez-Amaya (1999). The carotenoids were analysed in a liquid chromatograph, consisting of a pump and a degasser (LC-20AT), an autosampler injector (SIL-10 A), a column oven (CTO-20A) and a photodiode array (DAD) (SPD-M20A) controlled by a system controller (CBM-20A), all manufactured by Shimadzu Corporation, Kyoto, Japan. Detection with DAD was at the wavelengths of maximum absorption.