Enzymatic Synthesis, Purification And Property Research Of L-ascorbyl Octanoate

L-ascorbyl fatty acid esters are one kind of good oil-soluble multifunctional natural antioxidants with surface and antioxidant activity, have been widely applied in the field of foods, medicines, daily chemical products and so on. The current research mainly focus on L-ascorbyl long-chain fatty acid esters, L-ascorbyl medium-chain fatty acid esters are reported rarely. However, because of higher melting point, L-ascorbyl long-chain fatty acid esters are easy to crystallize at low temperature, which would influence their solubility and antioxidant activity in oils, fats and oil-soluble products. This study selects n-octanoic acid as acyl donor, explore the optimal enzymatic synthesis conditions of L-ascorbyl octanoate; with purity and recovery of the product as indexes, optimize the process of separation and purification by extraction-crystallization method and silica gel column chromatography; conflrme the product by structural characterization and research its antioxidant activity, thermal property, oil-solublity, stability, surface activity, antimicrobial activity and preliminary application in edible oils, providing reliable basis for the application of L-ascorbyl octanoate. The main results are as follows:(1) In the synthetic process of L-ascorbyl octanoate through esterification with medium-chain fatty acid - n-octanoic acid as acyl donor catalyzed by immobilized lipase in organic systems, the best enzyme was Novozym 435, reaction medium was tert butyl alcohol. On the basis of single factor experiment, response surface experiment was employed to obtain the optimal synthesis conditions:concentration of L-ascorbic acid 0.24mol/L, lipase dosage 19.2%, substrate molar ratio 6:1, reaction time 11.44h, reaction temperature 55.3 ℃, adding molecular sieve at the beginning of reaction, the amount of 60mg/ml, resulting in a conversion rate of L-ascorbic acid of 88.31%. The test results of repeated use of lipase showed that the conversion rate was highest of 88.3% at second times, it remained above 75% after ten times reuse.(2) With purity and recovery of the product as indexes, the separation and purification effect of L-ascorbyl octanoate by extraction-crystallization method were investigated, suitable conditions as follows:ethyl acetate dosage 8mL/g, the volume of water was twice that of ethyl acetate, crystal solvent — n-hexane dosage 6mL/g, crystallization temperature 15℃, mixing time 5min, recrystallization solvent n-hexane-dichloromethane ratio 3:1, recrystallization temperature 20℃, under this conditions, the purity of product was 95.50%, recovery was 83.41%; the optimal separation and purification conditions of silica gel column chromatography were obtained through orthogonal test:silica gel dosage 24g, sampling amount 5g, the concentration of sample 0.3g/mL, resulting in a purity of 97.41% and recovery of 76.34%. Two purification methods were compared:extraction-crystallization method has the advantages of simple operation, higher recovery; silica gel column chromatography could obtain higher purity product, but time-consuming, material-consuming and lower production.(3) The product was confirmed as L-ascorbyl octanoate by IR, MS,13C NMR and 1H NMR analysis, and the results also displayed that n-octanoic acid was introduced into the sixth hydroxyl of L-ascorbic acid. At the same time, comparing with L-ascorbyl palmitate (L-AP), L-ascorbyl laurate and other types of L-ascorbyl fatty acid esters, L-ascorbyl octanoate has the highest synthesis efficiency, relatively simple separation and purification technology and higher product purity and recovery. Therefore, using n-octanoic acid as acyl donor to synthesize L-ascorbyl fatty acid esters has certain advantages.(4) The antioxidant experiment in vitro showed that, in different free radical systems, the free radical scavenging capacities of L-ascorbyl octanoate and usual antioxidants including VC, TBHQ (tertiary butyl hydroquinone), BHT (butylated hydroxytoluene), BHA (Butylated hydroxyanisole), PG (propyl gallate), L-AP and VE are different from each other. The increasing of scavenging rate with concentration rise basically presented dose-effect relationship. L-ascorbyl octanoate had stronger effect on scavenging superoxide anion, hydroxyl and DPPH free radical generally than BHT, BHA, L-AP and VE, but no as TBHQ/PG and VC. The ABTS free radical scavenging ability of L-ascorbyl octanoate was general and its reducing power was comparable to BHT, BHA and L-AP, after VC, PG and TBHQ, slightly higher than VE. The reducing power of L-ascorbyl octanoate and L-AP were much weaker than VC, which indicated that the introducing of fatty acid into L-ascorbic acid greatly reduced its ability to supply electron.(5) Compared with L-AP, L-ascorbyl octanoate had lower melting temperature, melting enthalpy, crystallization temperature and higher crystallization enthalpy. Its solubilities in all kinds of vegetable oils were far greater than that of L-AP, which showed that introducing medium-chain fatty acid into the structure of L-ascorbic acid was more advantageous to increase oil-solublity. The stability of L-ascorbyl octanoate was better than that of VC and comparable to L-AP. L-ascorbyl octanoate had no cloud point, HLB value of 11.5, critical micelle concentration of 11.3* 10-5mol/L, and good emulsifying and foaming properties.(6) The antibacterial effect of L-ascorbyl octanoate on Escherichia coli, Bacillus subtilis and Staphylococcus aureus were better than the commonly used preservatives such as sorbic acid potassium and sodium benzoate. It could effectively lengthen the growth lag phase of bacteria and reduce the final growth at the minimal inhibitory concentration respectively of 1.4,0.6,0.6 g/L.(7) The antioxidant effect of L-ascorbyl octanoate on common vegetable oils (soybean oil, rapeseed oil etc.), functional vegetable oils (grapeseed oil, wheat germ oil etc.) and lard were studied, the results displayed that the lower polyunsaturated fatty acid content, the better antioxidant effect. L-ascorbyl octanoate could prevent common vegetable oils and lard from oxidation rancidity more effectively than functional vegetable oils, but with the exception of sunflower seed oil, peanut oil and corn oil. Its inhibitory effect were obvious at the temperature of 40℃, better than BHT, BHA, L-AP, VE and other antioxidants, second only to TBHQ, and the oxidation induction time could be extended effectively. Nevertheless, the lipid antioxidant efficiency weakened with the increase of forced oxidation temperature and once grease began to oxidize, it would be worse than the usual antioxidants.