Biosynthesis Of L-Ascorbyl Oleate Catalyzed By Lipase In Non-Aqueous Phase And Study On Its Structure And Properties

L-ascorbyl fatty acid esters, as one kind of fat-soluble derivatives of L-ascorbic acid,have been widely used in food, medicine and cosmetics industries for their good biologicalactivity and antioxidant properties. However, the melting point of saturated L-ascorbyl fattyacid esters was so high that they could be crystal separation from fats and oils and lipophilicproducts when temperature decreased, which would affect their antioxidant effect. Therefore,searching for alternatives with low melting point, high solubility and high oxidation resistancehas become a research hotspot.At present, saturated L-ascorbyl fatty acid esters in the market are mostly synthesized bychemical methods. However, there are many disadvantages of chemical synthesis methods,such as lots of side reactions, environmental pollution et al. Compared with chemicalsynthesis methods, enzyme catalysis has been widely used for its advantages as highstereoselectivity, fewer side reactions, mild reaction conditions et al. In this research,unsaturated fatty acid–oleic acid which has one double bond, was used as acyl donor toprepare L-ascorbyl oleate in non-aqueous phase–organic solvents catalyzed by immobilizedlipases to improve the solubility in lipophilic products and antioxidant activity.Organic solvents not only greatly affect the enzyme active site, but also affect thedistribution ratio of substrates between solvents and enzyme active centers, which wouldaffect the reaction yield finally. Therefore, reaction mediums were firstly selected accordingto the lgP of organic solvents, the results showed that lipase lipozyme435performancedbetter catalytic activity when tert-amyl alcohol was used as reaction media, and the highestyield was obtained. When the reaction temperature was51℃, reaction time was24h,substrate molar ratio of O/L-AC was3.67:1, enzyme amount（relative to the quality of oleicacid） was5.13%, molar concentration of substrate （oleic acid） was0.4mol/L, molecularsieves amount was60mg/mL, the highest yield52.43%was obtained.In this research, the separation and purification of the L-ascorbyl oleate was alsodiscussed by extraction and crystallization with organic solvent and silica gel columnchromatography. Two purification methods were compared, each had its advantages.Extraction and crystallization method was easily treated, and the recovery was relativelyhigher. However, high purity product could be obtained by silica gel column chromatography.Therefore, in practice, the method of separation and purification should be selected accordingto the requirements of product. L-ascorbyl oleate was characterized by fourier transform infrared（FT-IR） and massspectrometry（ESI-MS）. Result of FT-IR showed that characteristic absorption peak of the fivemember cyclical lactone C=C double bond of L-ascorbic acid was found near1676cm^-1,characteristic absorption peak of the C=C double bond of oleic acid appeared near1610cm^-1,stretch vibration absorption spectra of carbonyl of carboxylic ester appeared near1749cm^-1,and stretch vibration absorption spectra of C–O appeared near1128cm-1besides stretchingvibration absorption peak outside of C-H. Then L-ascorbyl oleate was furtherly proved byESI-MS. The signal m/z439.2（M-1） appeared in the primary ionization mass spectramatched with the molecular weight of L-ascorbyl oleate, in the secondary ionization massspectra, ion fragments m/z156.8was C₆H₆O₅⁺, ion fragments m/z281.1was C₁₈H₃₃O₂⁺.Therfore, the product could be confirmed as L-ascorbyl oleate.In applied research experiments, fat solubility of L-ascorbyl oleate was studied bysilico-molybdenum blue spectrophotometry. Results showed that solubility of L-ascorbyloleate in peanut oil and rapeseed oil were greatly improved compared with L-ascorbylpalmitate（L-AP） with an increase about10times; Result of differential scanning calorimetrycalorimeter （DSC） was that crystallization peak of L-ascorbyl oleate was observed at54.31℃,crystallization enthalpy was25.53J/g; and its melting peak appeared at63.18℃, and meltingenthalpy was22.64J/g, Lower melting enthalpy could make L-ascorbyl oleate has goodsolubility in oil. Schaal oven test and Rancimat method were used to study the oxidationresistance of the L-ascorbyl oleate, the results showed that the antioxidant effect of L-ascorbyloleate in corn oil and sunflower oil was better than other antioxidants.