Extraction is primary unit operation for processing of vegetable oils and fats. The selected extraction technique should give the maximum possible amount of oil with desired quality. Generally, oils and fats are extracted using hexane, a solvent obtained from petroleum source. It has been identified and enlisted as hazardous air pollutant by US EPA as per Clean Air Amendments of 1990. Hexane may react with other pollutants and create health hazards (Wan et al., 1995; Hanmoungjai et al.

, 2000). The traces of hexane present in the extracted oil will reduce the food and nutritional value of the oil. All these factors increased the interest for finding out alternatives to hexane. Carbon dioxide exhibits supercritical fluid properties above 31 °C and 73.97 bar and is chemically stable, non-toxic and non-flammable. The other main advantages of the SC-CO2 are its higher solubility, higher efficiency of extraction and better selectivity. SC-CO2 extraction shows supercritical properties at near to ambient temperature and it helps in processing heat sensible products. Because of all these reasons, in recent years, SC-CO2 extraction has gained huge importance as a green solvent.

This technique was used to extract oil from various oil bearing materials (Friedrich and List, 1982; Bozan and Temelli, 2002; Roy et al., 2006; Han et al., 2009). However, the process is not properly understood as studies on process optimization are yet to be done.

Wheat germ is a by-product during the processing of wheat grain. It is the reproductive part of the grain and contains large amount of vitamins. It contains around 8-14 % of oil (Sonntag, 1979; Dunford and Zhang, 2003). Wheat germ oil contains up to 2500 ppm of tocopherols and tocotrienols, inclusively known as tocols (vitamin E). The presence of large quantities of tocols gives antioxidant properties (Tracy et al.

, 1944; Saleh et al., 2010). This oil was reported to have anti-cancer and anti-inflammatory properties (Zalatnai et al., 2001; Reddy et al., 2000;  Janthachotikun et al., 2015). One very significant observation was noted that, though conventional hexane extracted oil contain considerable amount of phospholipids (>600 ppm of phosphorous content) (Taniguchi et al.

, 1985). The SC-CO2 extracted had significantly lower amount of phospholipids (<50 ppm of phosphorous content). The presence of higher amount of phospholipids leads to a unit operation called degumming where water/phosphoric acid etc. are used to reduce the phosphorous content for further refining. This will result in oil losses and also reduction of tocols. Moreover, this process generates considerable amount of effluents that are to be treated. SC-CO2 extraction does not require this step of refining if the phosphorous content is kept low.

It was, therefore, decided to optimize the SC-CO2 extraction of wheat germ oil yield with respect of phosphorous content and tocols content.Literature reports the application of Box Behnken design to optimize the supercritical parameters on oil yield (Li et al., 2010; Tao et al.

, 2014; Aladic et al., 2016). This design was preferred because relatively few experimental combinations of variables are adequate to estimate complex response function. Some researchers carried out studies on supercritical fluid extraction of wheat germ (Zacchi et al., 2006; Gmez and Ossa, 2000) and optimization of process conditions either for oil yield (Shao et al., 2008; Jiang and Niu.,  2011) or for tocols content (Ge et al., 2002) using response surface methodology.

These data only give limited information and are not giving any input on phosphorous content.  Since, phosphorous content is one of the major aspects of processing and it has direct impact on ultimate refined oil yield and quality, in the present study, Box Behnken design was adopted for optimization of SC-CO2 extraction for phosphorous content also. Accordingly, the effects of process parameters like pressure, temperature and CO2 flow rate on yield of oil, phosphorous content and tocols contents of the extracted oils were studied and the optimized conditions were predicted using Box Behnken design. At the optimized conditions, experiments were done and the results obtained were compared with the predictions by the design. 

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