**9.4.1 Preparation of liposome**

Liposomes are non-toxic (mostly) and effective in encapsulation (Mortazavi et al. 2007) and controlled release in food industry (Mozafari and Khosravi-Darani 2008). Manufacturing of liposome by SCF covers three separate methods including: (i) phospholipids solvation in a near critical fluid, mixture with a protein containing buffered solution (ii) decompression of solvated phospholipids prior to injection to solution, (iii) the critical fluid decompression technique in which phospholipids are first hydrated in an aqueous buffer, mixed with SCF, with the mixture being then submitted to decompression. Several parameters can improve the characteristics of the liposomes prepared with SCF ethane. Optimization studies would be necessary to examine whether liposomes of higher quality can be made using SCF technology. Also, other SCF should be tested (Frederiksen et al. 1997).

### **9.5 Production of different morphologies of biocompatible polymers**

SC antisolvent method has great potential for processing of pharmaceuticals (Mosqueira et al. 1981; Steckel et al. 1997) and labile compounds such as proteins (Debenedetti et al 1993; Winters et al. 1999; Yeo et al. 1994; Yeo et al. 1993) and to obtain various morphologies of biopolymers (Bleich et al. 1996; Debenedetti et al. 1993; Dixon and Johnstone 1993;

Supercritical Fluid Application in Food and Bioprocess Technology 565

• Manipulating crystal size of solid compounds produced from SCFs by change in P and T • Separating of compounds that cannot be distilled, owing to their thermal instability. • Increased enhancement factors (ratio of actual solubility to ideal gas solubility) • Low reactivity and toxicity of SC-CO2 or ethane, and their gaseous state

The main disadvantages of SCE processes include low solubility of biomolecules in SCF and high capital costs. Furthermore, insufficient data exist on the physical properties of many bio-molecules, making prediction of phase behavior difficult. The addition of co-solvents

There are only a few reports using SFE on bacterial cell. SCFs are found to be useful in extracting desired materials from animal tissues, cells, and organs (Kamarei and Arlington, 1988). By varying the choice of SCF, experimental conditions, and biological source materials, one may obtain lipids, proteins, nucleotides, saccharides, and other desirable components or remove undesirable components (Kamarei and Arlington, 1988). Processing of lipid natural products by SCF has been reviewed (King, 2004). SCF can be applied for obtaining aromatic and lipid components from plant tissues (Kamarei and Arlington, 1988), lignin conversion (Avedesian, 1986), carotenoids extraction from carrots (Bath et al., 1995), tomato paste waste (Baysal et al., 2000) and microalgae (Mendes et al., 1995). The CO2

Moreover, there are some reports which describe the SFE of bacterial (Gharaibeh and Voorhees, 1996) and fungal lipids (Cygnarowicz et al., 1992) for use in the classification of them by fatty acid profiles. A simple two–step process was developed to extract and purify medium chain length polyhydroxyalkanoates (MCL-PHA) from bacterial cells (*Pseudomonas resinovorans*) grown on lard and tallow (Hampson and Ashby, 1999). The process consists of SCE of the lyophilized cells with CO2 to remove lipid impurities, followed by chloroform extraction of the cells to recover the MCL–PHA. SFE conditions were varied as to T 40 – 100°C, P (13.78 – 62.05 MPa), and CO2 flow rate (0.5 – 1.5 L/min, expanded gas). The results show that the two- step process saves time, uses much less organic solvent, and produces a purer MCL-PHA biopolymer than previous extraction and purification methods. Khosravi-Darani et al. (2003) have reported the equilibrium solubility of poly(hydroxybutyrate) (PHB) in SC-CO2. The effects of the main parameters such as P, T, and solvent density on solubility were determined at different T (35 – 75°C) and P (12.2 – 35.5) MPa. Hejazi et al. (2003) reported the effects of process variables such as exposure time, P, T, volume of methanol as a modifier, and culture history on PHB recovery from suspended *R. eutropha* in buffer solution. In another report, Khosravi-Darani et al. extended this work to obtain maximum recovery with minimum energy consumption (2004). In this work PHB recovery was examined using a combination of supercritical disruption and chemical (salt and alkaline) pretreatments. Bacterial cells, treated in growth phase, exhibited less resistance to disruption than nutrient limited cells in the stationary phase. It was also found that the wet cells could be utilized to recover PHB, but purity of the product was lower than that obtained from freeze-dried cells. Pretreatment with a minimum of 0.4% wt NaOH was necessary to enable complete disruption with two repetitions of P release. Salt pretreatment was less effective;

may obviate the advantage of minimal solvent residues in the final product.

**10.2 Supercritical extraction (SFE) from biomass 10.2.1 Post fermentation extraction of products** 

extraction process is selective in the presence of chlorophyll a.

however, disruption was improved by the application of alkaline shock.

The use of SCE of biologically active compounds (chaetoglobosin A, mycolutein, luteoreticulin, 7,8–dihydro–7,8–epoxy–1–hydroxy–3–hydroxymethylxanthone–8–carboxylic

Reverchon 1999; Subramanian et al. 1997), such as microspheres (Falk et al. 1997) threads, fibers, networks (Dixon and Johnstone 1993), sponges, foams, and films. One of the advantages of using SCF in polymer processing is the possibility of producing different solid shapes and structures at low temperature with a minimum amount of residual organic solvents. Also the process is environmentally safe and economic (Elvassore et al. 2001). A basic description of these techniques is reported by Bertucco and Pallado (2000).

The conformation of monomeric enzyme trypsin has been reported in SC-CO2 (Zagrobelny and Bright 1992). To follow in situ conformation of trypsin (as a function of CO2 density), steady state fluorescence spectroscopy was used. Zagrobelny showed that protein denaturation can occur during the fluid compression step and that the native trypsin is only slightly more stable (1.2 kcal/mol) than the unfolded form.
