Supercritical Fluid Extraction of Wheat Bran Oil and Characterization and Formulation of the Extracts

Supercritical fluid extraction (SFE) of wheat bran oil with pure supercritical carbon dioxide (SC-CO2) at different extraction pressures (25, 40 and 55 MPa) and temperatures (40, 70 and 95 oC) has been studied. Since wheat bran is characterized by having an important content of antioxidant compounds, the content of such compounds in the extracted oil and its antioxidant activity were evaluated. The highest extraction yield was obtained when 55 MPa and 95oC were used as extraction conditions. These conditions also provided the highest oil content in alkylresorcinols (AR), which are phenolic lipids characteristic of cereal bran. Characterization of wheat bran oil and evaluation of its antioxidant activity was carried out. Significant content of AR, steryl pherulates, tocopherols and polyunsaturated fatty acids (PUFA) was found in the extracted oil, which could be related to the antioxidant activity of wheat bran oil, as determined by DPPH, FRAP and ABTS methods. Searching suitability of wheat bran oil for aqueous-based matrix applications, its formulation as oil in water nanoemulsions was studied. The nanoemulsions obtained showed good stability during 60 days storage at 4 and 25oC and darkness. INTRODUCTION Wheat bran is an important source of bioactive compounds, which are related to the healthprotective mechanisms of whole-grain cereals [1]. Some of these bioactive compounds, such as alkylresorcinols (AR) and tocopherols, and to a lesser extent phenolic compounds, can be extracted by supercritical fluid extraction processes [2] However, wheat bran oil extracted with SC-CO2 has not been widely characterized [3-5] and it is known that the solvent system used in the extraction process influences the oil composition and quality [6] therefore, new studies on the levels of the different bioactive compounds and the antioxidant activity of supercritical extracted wheat bran oil are necessary in order to evaluate its potential uses in the food industry. There has been growing interest in the utilization of natural antioxidants in the food, beverage and pharmaceutical industries due to the increasing consumer ́s demand for substituting synthetic compounds by natural substances. Due to its lipophilic character, wheat bran oil must be formulated before it can be used for aqueous-based matrix applications. The high stability and low turbidity of nanoemulsions (10-200 nm) make them suitable for incorporating lipophilic active ingredients in aqueous-based food and beverages [7, 8]. The aim of the present work was to study the supercritical fluid extraction of wheat bran oil and to evaluate the oil composition and antioxidant activity. Additionally wheat bran oil-inwater nanoemulsions have been formulated for the application of the wheat bran oil in aqueous systems. MATERIALS AND METHODS Supercritical fluid extraction equipment and procedure The extraction experiments were carried out in a semi-pilot SFE-plant whose P&I diagram has been presented elsewhere [9]. In a SFE experiment, 300 g of wheat bran (Triticum aestivum L.) were placed in the extractor (2 L capacity) that was later pressurized with CO2 up to the extraction pressure. Then, the solvent was circulated at the desired extraction temperature, T, with a solvent flow of 9 ± 1 kg CO2/h and during an extraction time of 120 min. The solvent was continuously recycled to the extractor after removing the solute in the separator that was kept at 4.9 ± 0.6 MPa and 24 ± 2 oC. Nine runs were carried out by triplicate in order to study the influence of extraction pressure (25, 40 and 55 MPa) and temperature (40, 70 and 95 oC). Determination of AR content and profile Wheat bran oil AR content and profile were evaluated by using previously reported colorimetric and HPLC-DAD methods, respectively [10]. Determination of fatty acids content and profile Fatty acids content and profile was evaluated by GC-FID by the AOAC method, as previously reported by Rebolleda et al. [10]. Determination of steryl ferulates content and profile Steryl ferulates were analyzed by HPLC-DAD according to a previously reported method, with some modifications [11]. Separation was carried out in a Zorbax XDB C18 column (150 x 4.6 mm, 5 μm) using isocratic elution with acetonitrile/methanol/isopropanol (50:40:10) at 1 mL/min. Methanolic solutions of wheat bran oil (10 mg/mL) were injected (30 μL). Steryl ferulates were monitored at 330 nm and identified using a standard mixture of steryl ferulates and literature data [12]. Determination of tocopherol content and profile Tocopherol content and profile were evaluate in wheat bran oil by solid phase extraction followed by HPLC-DAD [13]. Determination of phenolic content and profile Phenolic compounds were evaluated by HPLC-DAD according to a previously reported method [14]. Evaluation of antioxidant activity DPPH, FRAP and ABTS methods previously reported were used for the evaluation of the antioxidant activity in wheat bran oil [10, 15]. Emulsification procedure To prepare an emulsion, 1% of wheat bran oil was mixed with 7.3% of a surfactant mixture formed by Span 80 (37.4%) and Tween 80 (62.6%), before water milli-Q was added. Then, emulsification was carried out by using high speed blender (Miccra D9, 29000 rpm, 5 min) followed by ultrasonic processor at 20% of amplitude (Sonics VCX 500, 50 s). Evaluation of nanoemulsion stability Stability of wheat bran oil nanoemulsions was measured in terms of their droplet growth ratio during 60 days storage at 4 oC and darkness (Zetasizer Nano ZS). Also, optical characterization of creaming stability (Turbiscan Lab Expert equipment) was made for nanoemulsions storage during 60 days at 25oC and darkness. RESULTS Effect of process variables on oil extraction yield and AR content The effect of temperature on the extraction yield and oil quality was evaluated at 40, 70 and 95 oC at three different pressures (25, 40 and 55 MPa) and a constant flow of 9 ± 1 kg CO2/h. The results of the extraction yield are shown in Figure 1. At the lowest pressure used in this work, 25 MPa, it appears not to be a significant effect of temperature on the total amount of oil obtained, while at the two other pressures evaluated, 40 and 55 MPa, the extraction yield increases with temperature, which may indicate that, at these pressures, the increase of oil vapor-pressure with temperature is more important than the decrease of SC-CO2 density. These results suggest a crossover behavior of the isotherms around 25 MPa. This behavior has not been described in the literature for wheat bran oil but it has been generally observed for different oils [16]. Due to the possibility of a crossover region around 25 MPa, influence of a pressure increase seems to be stronger at the highest temperature evaluated in this work. Figure 1: Influence of extraction pressure and temperature on total oil yield Figure 2: Influence of extraction pressure and temperature on total AR content of wheat bran oil The total AR content of the oils obtained by SFE under the different extraction pressures and temperatures are shown in Figure 2, where it can be observed that the amount of AR in oil slightly increased with extraction temperature when extraction pressure was 40 and 55 MPa. However, at 25 MPa, there was not significantly effect of the extraction temperature on the AR oil content. This suggests a crossover behavior around 25 MPa for AR extraction, similar to that found for the extraction yield. Higher temperatures seem to provide not only higher extraction yields but also oil with higher AR content. Evaluation of oil composition and antioxidant activity Analysis of the composition and antioxidant activity of wheat bran oil extracted under specific conditions (25 MPa and 40oC) was evaluated. Table 1 shows the total amount and profile of some bioactive compounds such as alkylresorcinols, tocopherols, steryl ferulates, phenolic compounds and fatty acids. The fatty acid profile reveals that most of the fatty acids are polyunsaturated fatty acids (PUFA), around 63%, with a low amount of saturated fatty acids (around 18%). Linoleic acid (LA, C18:2ω6) was the major PUFA detected (around 58%), and significant quantities of α-linolenic acid (ALA, C18:3ω3) were also quantified. Both compounds are essential PUFA, precursors of the omega-6 and omega-3 families respectively, and therefore, very important in the human diet. Its large PUFA content makes wheat bran oil to be considered of higher quality than some of the most commonly used oils, which usually have very low levels of PUFA (e.g. palm oil) and often show very low levels of ALA (e.g. sunflower oil, sesame oil, and palm oil) [17]. Alkylresorcinols have been described to have a wide range of biological activities such as antibacterial, antifungical, anticancer and enzyme inhibitor activities, among others [18]. The intensity of these activities is different for each AR homologue [19] which is probably due to the different length of the alkyl chain. The AR profile of the bran oil under study (Table 1) is similar to that previously reported for wheat bran [20], being C19 and C21 homologues the major ones (around 30 and 48% respectively). Steryl ferulates, which are esters of ferulic acid with sterols, have been widely described for rice bran oil and they are considered to be potential antioxidants because of the hydrogen-donating ability of the phenolic group of ferulic acid [21]. Data in Table 1 show significant levels of steryl ferulates, higher than those described for hexane extracted oils [22]. The steryl ferulate profile obtained (Table 1) was similar to that reported in acetone Table 1. Bioactive compounds evaluated in supercritical extracted wheat bran oil Fatty acids (mg/g oil) 712 ± 19 Palmitic acid (C16:0) 118 ± 2 Stearic acid (C18:0) 7.9 ± 0.1 Oleic acid (C18:1) 114 ± 3 Linoleic acid (C18:2) 410 ± 10 αlinolenic acid (C18:3) 37.3 ± 0.8