| Diacylglycerols (DAGs) are well known as bio-surfactants. Their excellent biodegradability as well as the behaviors of tasteless, odorless, nontoxic, non-irritant and non-ionic has been paid more and more attraction in numerous industrial fields. It is found that, DAGs are a minor natural component in vegetable oils and fats, as well as an endogenous product of triglyceride metabolism. DAGs are generally recognized as safe as direct human food ingredients and are approved for use in food as dough strengtheners, formulation acid, lubricant and release agents, vehicles, surface finishing agents, stabilizers and thickeners, texturizers (FDA, 2000). In addition, the long-term ingestion of dietary DAG, in contrast to TAG, prevented the accumulation of visceral fat and body weight gain in obesity-prone mice, and decreased postprandial blood lipid levels of rats administration with lipid emulsion. On the other hand, for a long time, large scale's production of DAG remained mainly in the realms of organic chemistry and chemical processing. Chemical methods are mainly performed at high temperatures in the presence of alkaline catalysts. High energy's consumption, coloring of products and low selectivity are major disadvantages of these methods. Moreover, some chemicals are toxic and not readily biodegradable, thus causing their limited application in cosmetics, food industry and pharmaceutics. In this case, the enzymatic synthesis offers an alternative way.In the present work, a novel and effective enzymatic method which could eliminate most of the above-mentioned problems, was developed for the production of DAGs. Non-adsorptive glycerol and free fatty acids were directly used as starting materials in order to increase production yield. The selection of biocatalyst is very crucial. It turned out that Rhizopusmucor miehei lipase (Lipozyme RMIM) and Candida antarictica lipase (Novo 435) are the only suitable biocatalysts for the production of 1,3-DAG, not only for their higher esterification activities, but also for the easy reuse. In view of regiospecificity (1,3-specific) and economics, Rhizopusmucor miehei lipase is more suitable than Candida antarictica lipase. Studies on esterification characteristics of Lipozyme RMIM suggested that the selectivity for LCFA is higher than for MCFA and SCFA at 60 degree, but the situation is reversed at 40 degree. The optimum alcoholic substrates for Lipozyme RMIM are primary alcohols, followed by the secondary alcohols, and then tertiary alcohols. However, Rhizopusmucor miehei lipase hardly catalyst phenol hydroxyl group. The optimum water activity is 0.11. When water activity is more than 0.54, the enzymatic activity decrease significantly. Studies on non-aqueous phase suggested that isooctane is the optimum medium, followed by heptane and hexane, and then EMK and acetone, as their polarities are strong enough to peel off essential water for enzyme activity.For monitoring of intermediate process and analysis of products, thin-layer chromatography, normal-, reverse-phase high-performance liquid chromatography were adopted to isolate neutral lipid. Mono-, di-, and tri-acylglycerol were determined with density colorimetric, evaporative light-scatting detector (ELSD) and refractive index detector (RID) individually. It was found that the 1,3-DAGs were evidently resolved from the 1,2-DAG position isomers. Intermediate and final product can be assayed according to methods mentioned above. Esterification reaction of linoleic acid and glycerol catalyzed by Lipozyme RMIM in solvent-free batch system were studied in detail. The conversion rate of fatty acid was obviously affected by reaction conditions such temperature, molar ratio of substrate, enzyme load, emulsifier, agitation and water-removal methods, and so was the composition of esterification products. In order to get high conversion yield and concentration of 1,3-DAG, some important parameters such as substrate ratio (R, acyl donor to glycerol), reaction temperature (T), enzyme load ([E], based on s...
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