Study On Antioxidant Activities And Lipase-catalyzed Transesterification Of Lard To Produce L-ascorbyl Fatty Acid Esters

L-ascorbyl fatty acid esters contain ascorbic acid with water-soluble, fatty acid with its lipophilic, so act both as antioxidants and surfactants. L-ascorbyl fatty acid esters are the new multi-functional food additives. This paper studied cheap lard and L-ascorbic acid as raw materials, microbial source of lipase as a catalyst, enzymatic synthesis of L-ascorbyl fatty acid esters in organic phase, its objective to develop a nutritional antioxidant, and provide a theoretical basis for the development of green food additives. The main results are as follows:(1) Enzymatic synthesis of L-ascorbyl fatty acid esters was studied. The catalytic properties of Novozyme 435, Lipozyme TL IM, Lipozyme RM IM and Pseudomonas cepacia lipase S31 were compared, as result, Novozyme435 presented higher selectivity and activity for synthesis of L-ascorbyl fatty acid esters.The synthesis of L-ascorbyl fatty acid esters through transesterification of ascorbic acid and lard through immobilized-lipase-catalyzed was described. The reaction conditions of molar ratio of lard to ascorbic acid, enzyme loading, temperature, addition of molecular sieve and time were optimized and conversion of 65.1% could be obtained under the optimum condition:the favorable reaction medium was tert-amyl alcohol,40% of enzyme dosage (W/W, immobilized enzyme/ascorbic acid),2.5:1 of mole ratio of lard to L-ascorbic acid, at 55℃with 15 g/L molecular sieves after reaction time 6 h for 24 h reaction time. At the same time, Enzyme was stable after six batches with enzyme activity no significantly, and the conversion rate was still above 50%. On the base of single-factor experiment, the effect of the substrate molar ratio, temperature, enzyme concentration, reaction time on synthesis of L-ascorbyl fatty acid esters were evaluated by orthogonal design L9(34). The result showed that highest conversion (79.1%) was determined after 24 h reaction at 55℃using 30% of enzyme dosage with lard to ascorbic acid mole ratio of 3:1. It was higher 14 points than single factor experiments (65.1%).(2) Continuous production of L-ascorbyl fatty acid esters in a packed-bed reactor with immobilized lipase was studied. The results showed that at 55℃and with 0.4 nm molecular sieves, lard concentration of 100 mmol/L(lard to ascorbic acid molar ratio 1.4:1), flow rate of 1.07 mL/min, under above conditions, the conversion of L-ascorbic acid reached 42.5% and the concentration of L-ascorbyl fatty acid esters was 13.5 g/L after 48 h. On the base of single-factor experiment, response surface methodology (RSM) was applied to optimize the effect of the transesterification three crucial conditions, namely flow rate of substrate, temperature and lard to ascorbic acid molar ratio, on the conversion of L-ascorbic acid in the PBR. The results indicated that the optimum process conditions for L-ascorbyl fatty acid esters production in the PBR were as follows:flow rate of 1.07, reaction temperature of 56.44℃, lard:ascorbic acid molar ratio of 2.24. In the optimum condition, the conversion was 50.83%. The stability of the immobilized lipase in the PBR was also studied, and the conversion remained above 50% after the third batch.(3) The purification process of L-ascorbyl fatty acid esters was studied. The ratio of crude product:n-hexane 1:6, washing temperature at 40-50℃were determined. As result, product purity and conversion remained above 55%. when the volume ratio of ethyl acetate and water was 1:4 after washing ascorbic acid, products recrystallization temperature was 15℃, the volume ratio of solvent n-hexane and ethanol wasl:0.5, as result, the product of the crystallization rate and crystal morphology were ideal, recovery rate was up to 70%, and purifity was above 96%.(4) The antioxidation activities of L-ascorbyl fatty acid esters were studied. The purified product was further characterized by ESI-MS and the mixture of ascorbyl monoesters obtained were identified as L-ascorbyl myristate (1.81%), L-ascorbyl laurate (0.75%), L-ascorbyl palmitoleate (2.66%), L-ascorbyl palmitate (46.45%), L-ascorbyl oleate (14.29%) and ascorbyl stearate (34.04%). In vitro antioxidant activities of L-ascorbyl fatty acid esters were evaluated by several methods:hydroxyl radical system, superoxide anion free radical system, system of DPPH·and reducing power. Subsequently, L-ascorbyl fatty acid esters were used in lard and bean oil, and its antioxidant effects were evaluated. The results showed that significant antioxidant activities of L-ascorbyl fatty acid esters in these radical systems were observed, besides the antioxidant activities become stronger with the increase of the concentration and were almost the same to AP. When adding amount of L-ascorbyl fatty acid esters in lard was 0.2%, with forced oxidation temperature 100℃, the POV of lard was 42.8 meq/kg after 20 h. It had obvious antioxidant activities. When adding amount of L-ascorbyl fatty acid esters in soybean oil was 0.2%, with forced oxidation, the POV of soybean oil was 11.9 meq/kg after 14 h. The activity of L-ascorbyl fatty acid esters for soybean oil was higher than TBHQ, was similar to AP. L-ascorbyl fatty acid esters were potential food antioxidant.