12 nd Workshop on the Developments in the Italian PhD Research on Food Science and Technology and Biotechnology University of Reggio Calabria, September, 2007 STABILIZATION OF BLOOD ORANGE JUICE BY HIGH PRESSURE SUPERCRITICAL CARBON DIOXIDE TREATMENT Simona Fabroni C.R.A.-Istituto Sperimentale per l’Agrumicoltura, Acireale (CT) Results and Discussion The objective of this study was to assess the performance of a continuous prototype SC-CO 2 system (Fig.1) on physicochemical parameters of sicilian blood orange juice. Moreover, a study was performed to evaluate the effect of continuous high pressure SC-CO 2 treatment on bacterial growth (mesophilic viable counts-MVC, yeasts and moulds) during storage at different temperatures (0±1 and 10±1 °C). The two temperatures have been chosen to emulate, respectively, refrigerated storage in manufacturing firms and in commercial centres. Aim of the work Blood orange juice immediately after squeezing, was exposed to SC-CO 2 treatment (Temp 33°C, P 380 bar, CO 2 /juice ratio 10/90 %, total flow rate 900 l/h) in a pilot plant developed with Ortogel s.r.l. (Catania, Italy). TSS, pH and TA were determined by standard methods (Kimbal, 1999). Color analysis was evaluated as CIE L*a*b* values. Anthocyanins were determined using the method described by Rapisarda et al., (2000). Carotenoids were determined spectrofotometrically (450 nm) according to Lee et al. (2001). ORAC (Oxigen Radical Absorbance Capacity) units were determined by the Cao et al. (1999) method, modified. Samples were analysed for total phenolics by Folin-Ciocalteu (FC) colorimetric method (Singleton et al., 1999). Vitamin C concentration was determined by HPLC (Rapisarda et al., 1996). PE activity was measured according to Spagna et al., (2003). Cloud was measured by recording absorbance at 650 nm (Corredig et al., 2001). Statistical elaboration of the physicochemical results was carried out with the MSTAT WIN 10 program. The statistical differences between UJ (untreated juice) and TJ (treated juice) were evaluated by variance analysis (ANOVA) and the means separation by Tukey test. Mesophilic viable count (MVC) was performed on Plate Count Agar (Oxoid, CM325), after 48 h incubation at 32 °C while yeasts and moulds counts were determined with Sabouraud Dextrose Agar (Oxoid, CM41) after incubation at 25 °C for 4 days. Materials and methods Parameters Untreated Juice (UJ) Treated juice (TJ) TSS (°Brix)12,22 ± 0,04 A11,88 ± 0,05 B pH3,44 ± 0,02 B3,53 ± 0,01 A TA (% citric Acid)1,43 ± 0,02 A1,28 ± 0,01 B L*27,99 ± 0,19 B32,94 ± 0,12 A a*29,09 ± 0,38 A27,66 ± 0,14 B b*21,44 ± 0,89 B26,34 ± 0,10 A Cloud (λ=650)0,28 ± 0,01 B1,44± 0,02 A PE activity/g of pulp1261,04 ± 15,01 a929,11 ± 48,66 b Table 1 Physicochemical parameters in treated (TJ) and untreated juice (UJ). Means ± SD, n=3 ParametersUntreated Juice (UJ)Treated Juice (UJ) Vitamin C (mg/100 ml)73,22 ± 0,66 a69,94 ± 1,72 b Total anthocyanins (mg/l)127,82 ± 1,66 A122,31 ± 0,42 B Carotenoids (mg/l)10,86 ± 0,27 ns11,36 ± 0,23 ns Total Phenolics (mg/l)986,27 ± 16,48 ns996,93 ± 3,64 ns ORAC (µM trolox equiv/100 ml)2189,33 ± 91,36 ns2406,42 ± 198,90 ns Table 1 shows that TSS and TA decreased significantly (p≤0,01) in TJ. This trend is reflected on pH levels which increased with the treatment. However, these changes are not so drastic to determine a relevant alteration of the quality characteristics of the juice. As regards color parameters, the L* and b* values increased after treatment, while the a* value decreased. PE activity in TJ showed a reduction respect to UJ. Average PE decrease was 26,32 %. This demonstrates that SC-CO 2 improves PE reduction but not completely inhibit its activity. In spite of still active PE, cloud values for TJ were higher than for UJ (average cloud increase 407,95 %). Cloud was not only preserved but also enhanced. It’s probably due to depressurization that leads to homogenization of the juice causing smaller particles of the juice colloid, thereby increasing cloud and stability to PE attack. Table 2 Changes in antioxidant components and ORAC units after SC-CO 2 treatment. Means ± SD, n=3 UJ Pump 1 Mixing chamber Expansion Reactor TJ CO 2 Chiller Pump 2 Average vitamin C content decreased significantly (p≤0,05) after SC-CO 2 treatment (Table 2). Moreover, the vitamin C level in TJ was not such as to lead to an excessive reduction in juice antioxidant protection. Even anthocyanins decreased significantly (p≤0,01) in TJ, this being in accordance to the a* value decrement. However, total anthocyanins level in TJ was still higher than that of thermally processed orange juices (Nicoli et al., 2004). Finally, carotenoids, total phenolics and ORAC units showed no statistical difference between UJ and TJ. This demonstrates that total antioxidant activity of orange juice is not significantly influenced by SC-CO 2 treatment. At time 0 immediately after treatment, there were almost no culturable organisms present in the juice. However, as 10±1 °C storage continued (Table 3), the counts began to increase and by 24 th day the counts reached elevated values. As regards 0±1 °C storage, juice samples increased their counts only by 31 st day. Temperature (°C) Storage days MVC (CFU/mL) Yeast (CFU/mL) Mould (CFU/mL) 0 ± ± Table 3 Bacterial inactivation in TJ stored at 10 ± 1 °C and 0 ± 1 °C. Means ± SD for 3 plates CFU= colony-forming units Selected references Cao G, Prior R L (1999) Measurement of Oxigen Absorbance Capacity in Biological Samples, Methods in Enzymology 299: Corredig M, Kerr W, Wicker L (2001) Particle size distribution of orange juice cloud after addition of sensitized pectin, J. Agric. Food Chem. 49: Kimball D (1999) Citrus Processing. Quality Control and Technology, AVI Books, New York. Nicoli MC, Anese M, Parpinel M (1999) Influence of processing on the antioxidant properties of fruit and vegetables, Trends Food Sci Technol 10: Rapisarda P, Fanella F, Maccarone E (2000) Reliability of analytical method for determining anthocyanins in blood orange juice, J. Agric. Food Chem. 48: Rapisarda P, Intelisano S (1996) Sample preparation for vitamin C analysis of pigmented orange juice, Ital. J. Food Sci. 3: 251. Singleton V L, Orthofer R, Lamuela-Raventos R M (1999) Analysis of total phenols and other oxidation substrates and antioxidant by means of Folin-Ciocalteu reagent, Methods in Enzimology 299: Spagna G, Barbagallo R N, Ingallinera B (2003) A specific method for determination of pectin esterase in blood oranges, Enzyme and microbial Technology 32: Fig.1 Schematic diagram of the continuous prototype SC-CO 2 system UJ=untreated juice TJ=treated juice Bacterial growth during storage Changes in antioxidant capacity Changes in quality parameters Conclusions The results show that high pressure SC-CO 2 treatment is a valid alternative to traditional thermal methods to obtain a good stabilization of freshly-squeezed blood orange juice saving the physicochemical, nutritional, and sensory properties of the product.