1. OPTIMISATION OF MONOOLEIN CONCENTRATION IN A LIPASE CATALYSED REACTION ESTERIFICATION 1 Department of Chemistry and Biochemistry, Medical Faculty of Rijeka, Braće Branchetta 20, HR-51000 Rijeka; 2 Adria-PI, HR-51513 Omišalj; 3 Department of Reaction Engineering and Catalysis, Faculty of Chemical Engineering & Technology, University of Zagreb, Savska cesta 16, HR-10000 Zagreb, Croatia. *jasminka@mamed.medri.hr Jasminka Giacometti 1*, Fabio Giacometti 2, Čedomila Milin 1, Đurđa Vasić-Rački 3 Introduction Monoglycerides and mixture of the mono- and diglycerides of long chain fatty acids are food additives (E471) in products such as bread, ice cream, and margarine. On an industrial scale, these food-grade surfactants are normally manufactured by chemical glycerolysis of fats and oils 1. An important application of enzyme catalysis in low-water organic media is the synthesis of esters catalysed by lipase 2. Regarding of the 1,3-specificity, immobilised lipase from Mucor miehei was use for production of monoolein (MO) and diolein (DO) mixtures. The effect of hydrophobic organic solvents, the presence of molecular sieves 3 and reaction temperatures were investigated for the synthesis optimisation of MO and DO mixture in a batch stirrer tank reactor (BSTR). Monoglycerides and mixture of the mono- and diglycerides of long chain fatty acids are food additives (E471) in products such as bread, ice cream, and margarine. On an industrial scale, these food-grade surfactants are normally manufactured by chemical glycerolysis of fats and oils 1. An important application of enzyme catalysis in low-water organic media is the synthesis of esters catalysed by lipase 2. Regarding of the 1,3-specificity, immobilised lipase from Mucor miehei was use for production of monoolein (MO) and diolein (DO) mixtures. The effect of hydrophobic organic solvents, the presence of molecular sieves 3 and reaction temperatures were investigated for the synthesis optimisation of MO and DO mixture in a batch stirrer tank reactor (BSTR). Experimental Lipase catalyzed esterification of glycerol with oleic acid in a solvent system was carried out with 6.67 mg ml -1 immobilized 1,3-specific Mucor miehei lipase at 25, 37 and 50 o C, using n-hexane, cyclohexane and isooctane, without and by adding 40 mg ml -1 of 5 Å molecular sieves at the start of esterification, at 100 rpm, in a batch stirrer- tank reactor (BSTR). The reactions were followed and simultaneously monitored by the determination of free oleic acid and monoolein by the gas chromatographic method during 45 hrs. Diolein was calculated in the mass balance. Lipase catalyzed esterification of glycerol with oleic acid in a solvent system was carried out with 6.67 mg ml -1 immobilized 1,3-specific Mucor miehei lipase at 25, 37 and 50 o C, using n-hexane, cyclohexane and isooctane, without and by adding 40 mg ml -1 of 5 Å molecular sieves at the start of esterification, at 100 rpm, in a batch stirrer- tank reactor (BSTR). The reactions were followed and simultaneously monitored by the determination of free oleic acid and monoolein by the gas chromatographic method during 45 hrs. Diolein was calculated in the mass balance. Figure 1. Final concentrations of monoolein in the solvent system with and without (  ) molecular sieves in the presence of various organic solvents. Figure 2. Concentrations of monoolein after 45 hrs followed reaction esterification, in the solvent system with n-hexane in the presence of molecular sieves at various temperatures: red symbols – 25 o C; yellow symbols – 37 o C; green symbols – 50 o C. Results After 45 hrs followed reaction esterification, the amounts of MO were higher in the presence of n-hexane in the system without molecular sieves. In the system with molecular sieves, the amount of MO was higher in the presence of isooctane. A higher amount of MO was found at 37 o C and DO at 50 o C in the presence of molecular sieves and n-hexane. The enzyme activity, as function of hydrophobicity of organic solvents, affected DO/MO ratio. In the system without molecular sieves, the solvent hydrophobicity did not affect DO/MO ratio, but by adding of molecular sieves in the system, the ratio depended on solvent hydrophobicity. The all examined conditions show the different initial reaction rates. The presence of molecular sieves and examined reaction temperatures affected on final MO and DO concentrations. In the examined conditions, DO concentration did not exceed 69% (w/w) and MO 46% (w/w). After 45 hrs followed reaction esterification, the amounts of MO were higher in the presence of n-hexane in the system without molecular sieves. In the system with molecular sieves, the amount of MO was higher in the presence of isooctane. A higher amount of MO was found at 37 o C and DO at 50 o C in the presence of molecular sieves and n-hexane. The enzyme activity, as function of hydrophobicity of organic solvents, affected DO/MO ratio. In the system without molecular sieves, the solvent hydrophobicity did not affect DO/MO ratio, but by adding of molecular sieves in the system, the ratio depended on solvent hydrophobicity. The all examined conditions show the different initial reaction rates. The presence of molecular sieves and examined reaction temperatures affected on final MO and DO concentrations. In the examined conditions, DO concentration did not exceed 69% (w/w) and MO 46% (w/w). Figure 3. Concentrations of monoolein after 45 hrs followed reaction esterification, in the solvent system with molecular sieves in the presence of organic solvents: red symbols – isooctane; yellow symbols – n- hexane; blue symbols – cyclohexane.  1  Stevenson, D.E., Stanley, R.A. & Furneaux, R.H., Biotechnol.Lett. 1993, 15, 1043-1048.  2  A. Zaks, A. M. Klibanov, J. Biol. Chem.,1988, 263, 3194-3201.  3  J.Giacometti, F. Giacometti, Č. Milin, Đ. Vasić-Rački, J. Mol. Catal. B-Enzym. 2001, 11, 921-928. References Figure 4. Effect of hydrophobicity (versus log P value) on DO/MO ratio after 45 hrs followed reaction esterification, in the solvent system with n-hexane, with ( ▲ ) and without (  ) molecular sieves at 37 o C. Acknowledgements This work was supported by the Primorsko-Goranska County and the Croatian Ministry of Science, Project No. 125042.