Colorant Extraction from Red Prickly Pear (opuntia Lasiacantha) for Food Application

The research on alternative natural colorants is of growing interest as substitutes for synthetic dyes in food. Opuntia lasiacantha Pfeiffer (red prickly pear) pigment was extracted from fruits of three different localities. The extracted pigment was identified as betanin by HPLC analysis comparing to the commercial beet red colorant E-162/1600. Color parameters and betanin content were also determined and no significant differences were found for the betanin content among the studied sites. Pigments extracted from fruits gathered from location L1 were spray dried using maltodextrin as coating material. After 24 weeks of storage color parameters and pigment retention were measured. Pigment retention was 86.2%±3.9 similar to the values found in the red beet pigment. The obtained results suggest that the colorant from red prickly pear can be considered a potential source of natural colorant. INTRODUCTION Betalains are natural pigments that are found in natural form in most families belonging to the Centroespermae order that includes the cactaceae family in which it is found the Opuntia genus (Yizhong et al., 2001a). The chemical structure of these pigments is derived from the betalamic acid and, depending on the united components to this structure, the yellow betaxanthins and the red-violet betacyanins will be present (Piattelli and Minale, 1964). The most-studied betalains are found in red beets (Beta vulgaris) which main betacyanins are betanin and isobetanin. Betalains stability is affected by temperature, pH, oxygen, light, and aqueous activity (Reynoso et al., 1997; Yizhong et al., 1998; Yizhong et al., 2001b). In the food industry, there is a growing tendency to replace synthetic dyes by natural pigments, as the red beet betacyanins which are approved to be used as a food additive in the United States of America (No. 1600), and in the European Union (E-162); and commercially, they are exempt from batch certification and widely used in the world. (Castellar et.al., 2003). Freeze-drying is the best method to dry pigments which are sensitive to high temperatures, similar to the aforementioned. Nevertheless, the freeze-drying is 30 to 50 times more expensive than the spray-drying (method usually chosen due to its economy and suitability). The spray-drying requires capsuling the pigments. In order to do this, maltodextrins are frequent to be used as encapsulating agents for sensitive ingredients, as flavors and dyes, by creating a wall around the pigment in order to isolate it from the environment protecting it from oxidation (Stephane et al., 1997). Díaz et al. EJEAFChe, 5 (2), 2006. [1330-1337] Electron. J. Environ. Agric. Food Chem. ISSN 1579-4377 1331 The use of prickly pears as a source of betalains may be interesting since the plants of the Opuntia genus need minimal requirements from soil and water. This way, they may be a great alternative to agricultural economy in arid and semiarid regions (Castellar et. al., 2003). The purpose to the present work was to obtain and to identify a pigment obtained from the red prickly pear, and their color stability study during storage. MATERIALS AND METHODS The species Opuntia lasiacantha Pfeiffer LS 6813 was chosen to be studied due to its production of red fruits. In the same way, three locations in the state of Hidalgo (México) were chosen because of their O. lasiacantha widespread groups and accessibility. One of these groups is located in San Salvador community, in which some fruits were collected from the plants identified as: LS-6813, FD-11, FD-12 and FD-13 (L1 Location). The second group is located in Pachuca City, in the surroundings of the University of Hidalgo, in which some fruits were collected from the plants: FD-01, FD-02, FD-03, FD-04 (L2). The third group is also located in Pachuca City, in the surroundings of the Healthy Institute in which some fruits were collected from the plants: FD-05, FD-06. FD-07, FD-08 and FD-09 (L3). Among 10 and 20 fruits per plant were collected during July and August, 2002-2003. Pigment extraction The peeled fruits of each plant were homogenized with an equal amount of water. The mixture was heated for 5 minutes at 80°C and quickly cooled on ice bath until it reached a temperature of 8-10°C. Then, the extract was centrifuged at 3400 g and 4°C for 20 minutes in a Centra GP8R centrifuge (IEC, USA). The supernatant was stored at -20°C. Betanin identification Betanin identification was performed comparing by means of HPLC the pigment obtained from red prickly pear against a red beet’s betanin commercial standard (No. 1600/E-162) provided by CHR Hansen de Mexico. The analyses were carried out by means of the equipment Perkin Elmer-200 series (USA), using an analytic column spheri-5 RP-C18, 5μm, 220 x 4.6mm i.d. (Perkin Elmer-USA). The following solvent and gradient system were used: A: 1.5% aq. H3PO4 (Merk de México); B: Acetonitrile (Merk de México); linear gradient from 100% A to 76% A in (A+B) within 40 minutes; the flow rate was 1 ml min . The injection volume was 10μL, and the detection was performed at 536nm (Kobayashi et al., 2000). UV-VIS Analysis and Betanin quantization The betanin content and the UV-Vis absorption spectra were determined on a lambda 40 UV-Vis spectrophotometer (Perkin Elmer, USA). The Betanin content was spectrophotometrically evaluated by the absorbance at 536 nm, using a molar extinction coefficient of 62 x 10 cm mol (Kobayashi et. al., 2000). Colour parameters Color parameters (CIELab) L*,a*,b*,C* and h° were determined by using the computer program CCC3, kindly provided by the University of La Rioja, Spain,, in order to calculate the tristimulus values and color coordinates parting from absorption spectrums. The samples were standardized by dilution to an Díaz et al. EJEAFChe, 5 (2), 2006. [1330-1337] Electron. J. Environ. Agric. Food Chem. ISSN 1579-4377 1332 absorbance of 1.0 at גmax ~535nm (Cai et. al., 1998), performing an absorbance spectra from 380 to 780nm in a 1.0cm quartz cell. Drying method For the drying process, the extract from location L1 was homogenized with maltodextrin 10 ED (Equivalent Dextrose), provided by Industrializadora de Maiz (Mexico), which was used as protective agent adding 14% in order to adjust the solids final percentage to 22%. The feed mixture was spray-dried in a mini spray dryer B-191 (Büchi, Switzerland), under the following operating conditions: initial temperature 150°C, final temperature 90°C, feeding 14% (Cai and Corke, 2000). Freeze drying was conducted for comparison with spray drying. The feed mixture was frozen in liquid N2 and freeze-dried in a freeze dryer Freezone 4.5 (Labconco, USA), for 24hrs. Scanning electron microscopy (SEM) The spray-dried pigment particles were observed in a SEM JSM-6300 (JEOL, Japan). The samples were coated with gold and observed to 2.5kV. Pigment powder storage Pigment powders were stored in crystal glasses sealed and stored at 25 oC in absence of light for 24 weeks. Characteristics at zero storage time were analyzed within 1 day after drying. Storage stability was evaluated comparing their HPLC chromatogram and their absorption spectrum. The pigment retention percentage was calculated using this formula: (betanin content at 24wk. storage time) x 10 / (betanin content at zero storage time). Color parameters were determined by diluting 1g of dried pigment in 100mL of water using the aforesaid absorbance spectra and program. Statistical analysis Data were analyzed using the statistical program Statgraphics Plus (Statistical Graphics Corp., 19941999). RESULTS AND DISCUSSION The visible absorption spectrum (380-780nm), from the obtained extractions shows only one peak of maximum absorption at 536nm. This peak is a characteristic of the red-violet betalains group known as betacyanins which are optical active because they have two chiral carbons: C-2 and C-15. Due to the chiral center in C-15, betacyanins exist in two epimeric forms, One of the most widely studied is the betanin in red beet root and its epimer C-15, isobetanin (Kanner et. al., 2001; Slawomir and Mizrahi, 2002). The obtained colorant was compared under the same conditions in HPLC against a commercial beet-red standard (E-162/1600). Chromatograms are shown in Figure 1. In both chromatograms (beet-red colorant and prickly pear colorant), peaks 23.1 and 24.5 min corresponding to betanin and isobetanin are observed. The epimerization of betanin to isobetanin may be produced due to heat or acid when food containing betanin is heated since the balance of isobetanin to betanin grows up (Fennema, 2000). Díaz et al. EJEAFChe, 5 (2), 2006. [1330-1337] Electron. J. Environ. Agric. Food Chem. ISSN 1579-4377 1333 A