Enzymatic hydrolysis of conjugated linoleic acid-enriched anhydrous milk fat in supercritical carbon dioxide

Consumption of conjugated linoleic acid (CLA) has been associated with numerous health benefits. CLA in its free form can be obtained by enzymatic hydrolysis of milk fat. In this study, enzymatic hydrolysis of CLA-enriched anhydrous milk fat (AMF) using supercritical CO2 and Lipozyme TL IM were carried out to maximize the production of free fatty acids (FFAs). The effects of pressure (20–30 MPa), fat:water ratio (1:5–1:30 mol/mol) and their interaction in the FFA production were studied using response surface methodology. After performing the optimization, reactions at two different temperatures (45 and 65 °C) and then with three different enzymes (Lipozyme RM IM, Novozyme 435, and immobilized enzyme from Candida rugosa) were carried out to assess the optimum FFA production. CLA content in its free form and major lipid classes (FFA, monoglycerides (MG), diglycerides (DG), and triglycerides (TG)) in the reaction products were quantified by gas chromatography. An increase in FFA and intermediate reaction products (MG and DG), together with a decrease in TG compared to the starting material was observed at all experimental conditions studied. The maximum level of FFA (86.79%, w/w) was achieved using Lipozyme TL IM at 23 MPa, 1:5 fat:water ratio (mol/mol) and 55 °C. The maximum CLA content in FFA form (6.81 mg/g fat) was obtained using Lipozyme TL IM at 30 MPa, 1:30 fat:water ratio (mol/mol) and 55 °C that corresponds to 98% conversion of CLA in TG form to FFA form.

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► Enzymatic hydrolysis of CLA-enriched AMF in supercritical CO2 media was carried out to maximize the production of free fatty acids.
► Fractions with considerable amounts of FFA, followed by DG and MG were obtained for possible food and personal care applications.
► Approximately 98% of CLA in TG form was converted to FFA form at 55 °C, 30 MPa and fat:water ratio of 1:30 mol/mol using Lipozyme TL IM and supercritical CO2.