Study On Enzymatic Synthesis Of Monoglyceride Citrates

Citric acid esters of monoglyceride of fatty acids (monoglyceride citrates, CITREM) are obtained by the esterification of citric acid and edible fatty acids, with glycerol. As a new type of anionic surfactant, CITREM are used as emulsifiers, thickening agents, anti-oxidants and synergistic components for antioxidants, which are widely used in food, pharmacy, cosmetics and other related fields. Chemical synthesis was the common method for manufacturing CITREM presently, which has many drawbacks including severe reaction conditions, reaction randomness and formation of undesirable byproducts. Lipase catalysed esterification in non-aqueous phase for synthesis of CITREM is a specific reaction under mild conditions, which could effectively overcome the shortcomings of chemical processes. The aim of the present work was to establish an optimized route for CITREM synthesis via lipase catalysed esterification in organic solvent, to evaluate the thermodynamics and kinetics of the reaction involved therein, and to characterize the resulting CITREM in regard to its physicochemical properties and synergistic effects towards oil antioxidants.Factorial test results indicated that synthesis of CITREM catalyzed by Novozym 435 in anhydrous t-butyl alcohol was the best model, and the optimum conditions for the corresponding enzymatic synthetic reaction were: citric acid concentration 0.12 mol/L, monoglyceride to citric acid ratio 2:1, and 4? molecular sieves concentration 120g/L. Based on the results of factorial tests, response surface methodology was employed in order to further investigate the effects of Novozym 435 dosage, reaction temperature, and reaction time on the conversion of citric acid to CITREM. Under the optimized conditions, i.e. Novozym 435 dosage 9.02% (w/w), reaction temperature 54.18 oC and reaction time 47.5 h, the conversion rate of citric acid reached its maximum value of 77.37%. Significance of the three factors to affect conversion rate of citric acid was in the order: Novozym 435 dosage > reaction time > reaction temperature. The interaction of the three factors was not significant (P<0.05). Repeated tests indicated that Novozym 435 could be used 8 times under the optimum conditions with 92% of its original catalytic activity still retained.Qualitative analysis of the reaction products was investigated by thin layer chromatography, electron spray ionization-mass spectrometry, and infrared spectrometry. Results of electron spray ionization-mass spectrum indicated that citric-fatty acid esters of glycerol were the majority components, in addition to minor amounts of glyceryl citrate, diglyceryl citrate and glyceryl citric- and fatty acid esters of glycerol, fatty acids as byproducts. The infrared spectra implied thatα-citric acid esters of monoglyceride were the majority form of CITREM. Results of qualitative analysis indicated that CITREM was synthesized by lipase in an organic medium, and enzymatic synthesis of CITREM was relatively a specific reaction with limited quantity of by-products formed.The mechanism of enzymatic synthesis of CITREM was studied by thermodynamic and kinetic analyses. Thermodynamics analysis using the Arrhenius model indicated that the activation energy (Ea) of the enzymatic reaction was 57.72 kJ/mol, which was higher than that of fatty acids esterification catalyzed by lipase. Kinetics analysis implied that the esterification reaction could be inhibited by high concentration of citric acid and was in accordance with double-substrate Ping-Pong Bi-Bi mechanism. The reaction kinetic values for Vmax, KB, KA, and KiA were 0.7142 mmol/(g·min), 0.0557 mol/L, 0.0137 mol/L , and 0.1945 mol/L, respectively.Surface activity and antioxidation properties of CITREM were also studied. The hydrophile-lipophile balance value was estimated at 8, and the critical micell concentration was 23.45μmol/L. CITREM has better emulsifying capacity and poorer emulsifying stability than monoglyceride, and it was not suitable to be used as vesicant because of poor foaming ability and stability. In vitro anti-oxidation tests indicated that CITREM could chelate traces of metal ions directly and scavenge hydroxyl radical indirectly, but CITREM could not scavenge DPPH radical and superoxide anion radical. In addition, CITREM could be used as antioxidants for chelating traces of metal ions in lard and soy oil, or as synergistic components for TBHQ in preventing oil oxidation.