| Ferulic acid (4-hydroxy-3-methoxy cinnamic acid, FA) is a phenolic component of the cinnamic acid family occurring in most higher plants. FA is a component acting as antioxidant, UV absorbant as well as being anti-inflammatory, anticarcinogenic, and antimutagenic. However, FA is less effective in hydrophobic media as a result of its low solubility, which limited its applications in oil-food processing, cosmetics, medicines, and other corresponding industries. It is necessary to improve its hydrophobic properties. Feruloylated acylglycerols (feruloylated mono- and di-acylglycerols, FMAGs and FDAGs) are lipophilic derivatives of FA. However, previous reports regarding the enzymatic synthesis of FMAGs and FDAGs have many disadvantages including low yields, long reaction time, solvent requirements, etc. For developing an efficient and green synthesis route, the different enzymatic synthetic routes, and the purification and performance characteristics of products were mainly studied in the paper.The novel solvent-free two-step route established in this study provides a much efficient means to synthesize FMAGs and FDAGs with a combined yield of 96%, which were consisted of two consecutive steps, namely, transesterification of ethyl ferulate (EF) and glycerol to synthesize glyceryl ferulate (FG), followed by esterification of oleic acid with FG obtained in the previous step. The biocatalysts of both steps were Novozym 435. And vacuum-rotary evaporation reactor was very efficient for water removal to shift the reaction in the desired direction kinetically. The effects of various reaction parameters, and kinetics on the yields of FG, FMAGs, and FDAGs were studied. A FG yield of 96% was obtained using the vacuum-rotary evaporation reactor under optimized conditions: EF/glycerol molar ratio 1:10, enzyme load 10% (relative to all substrates), reaction temperature 60 oC, reaction time 10 h, and vacuum pressure 10 mm Hg. The immobilized lipase in the present work can be used at least 10 times. The activation energy (Ea) value of this reaction was 83.1 KJ/mol. The reaction kinetic values for Km, Vm, and kI were 4.02×10-2 mol/L, 10.05 mol/(L·min), and 1.33×102 mol/L, respectively. Response surface methodology (RSM) was used to optimize the effects of the reaction temperature (55-65 oC), enzyme load (8-14%; relative to the weight of total substrates), oleic acid/(FG + glycerol) (6:1-9:1; w/w), and the reaction time (1-2 h) on the conversion of FG and yield of FMAGs. The optimum preparation conditions were as follows: reaction temperature, 60 oC; enzyme load, 8.2%; substrate ratio, 8.65:1 (oleic acid/(FG + glycerol), w/w); and reaction time, 1.8 h. Under these conditions, the conversion of FG and yield of FMAGs are 96.7±1.0% and 87.6±1.2%, respectively. The Ea value of this esterification was 42.1 KJ/mol. The esterification of FG and oleic acid satisfied the"pseudo-first-order"reaction conditions. The reaction kinetic values for Km, Vm, and kI were 4.25×10-1 mol/L, 7.35×10-2 mol/(L·min), and 1.62×10-2 mol/L, respectively. The optimum synthesis conditions for FDAGs were as follows: reaction temperature, 65 oC; enzyme load, 7.5%; substrate ratio, 7.5:1 (oleic acid/(FG + glycerol), w/w); and reaction time, 12 h. Under the optimum conditions, the conversion of FG and yield of FDAGs reached 98.8±1.0% and 82.6±2.2%, respectively. Novozym 435 in the present work can be used 18 times under the optimum conditions without essential losses in activity. The influence of external mass transfer limitations on the reaction could be eliminated. The Ea value of this esterification was 67.4 KJ/mol. The reaction kinetics agrees with the Ping-Pong Bi-Bi mechanism characterized by Vm and Km values of 5.26×10-4 mol/(L·min) and 0.26 mol/L, respectively, which can be calculated by Lineweaver-Burk plot.Short-path molecular distillation (SPD), which was a novel green separation technology, was applied for separation and purification of FMAGs and FDAGs, respectively. RSM was applied for optimization of FMAGs and FDAGs separation. The optimum purification conditions for FDAGs were as follows: the evaporator temperature, 213℃; the feed flow rate, 1 mL/min. Under these conditions, the FDAGs content and the ratio of FDAGs product to the initial materials were 93.3±1.5% and 75.5±2.5%, respectively. The purification conditions for FMAGs were as follows: the FMAGs material was firstly separated at 178℃and 1.45 mL/min to remove EF and FG in the FMAGs product; then the residual of FDAGs and FMAGs was purified at 213℃and 1 mL/min. Under these conditions, the FMAGs content and the ratio of FMAGs product to the initial material were 92.1±1.2% and 30.1±1.9%, respectively.The maximum UV absorption wavelengths of FMAGs and FDAGs were 326nm and 329nm, respectively, which is consistent with that of FA (322nm). These maximum falls between the UVB and UVA regions, and thus FMAGs and FDAGs can be used potentially as all-natural UV-absorbing ingredients. FMAGs and FDAGs also showed good antioxidant by lard test. FMAGs and FDAGs have good hydrophobic properties, UV absorbency, and antioxidant, which opened up a novel path for their uses in oil-food processing, cosmetics, medicines, and other corresponding industries.
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