Enzymatic Determination Of L-lactic Acid

L-lactic acid, also called 2-hydroxy propanoic acid, is a kind of organic acid fermented from starch.It is colorless viscous liquid, acidic in aqueous solution. It is completely miscible with water,ethanol,aether,but insoluble in chloroform. It yields lacto-lactic acid in acid catalyzed condensation when boiled. Lacto-lactic acid hydrolyze to lactic acid when heated. Because of its L-conformation, it has good biocompatibility,thus can directly participate in metabolism without any side-effect. It is widely used in food,medicine and other industries. L-lactic acid is used mainly for sweets, drinks (such as beer, wine and lactic acid drinks)in the food industry,as the acid condiment,it is called secure food additive.It can also be used in cool drink and vegetable processing and storage. In medicine industry L-lactic acid is an important intermediate. In clinical test,the concentration of lactic acid can show the degree of tissue blood Hemoperfusion, as well as the quantitative index of anoxia.Therefore, quantitative detection of lactic acid is of high significance in the clinical and food science. In recent years , enzymatic analysis have been widely used in bioanalytical chemistry as the basic research method of life science.The thesis is devided into four parts:In chapter one, some major quantitative analysis methods of lactic acid is summarized. Futhermore, the theory and researches of enzymatic analysis is described.In chapter two, the determination of L-lactic acid is studied using enzymatic kinetic UV Spectrophotometry. The reaction of L-lactic acid and nicotinamide adenine dinucleotide oxidized form (NAD) catalyzed by L-lactate dehydrogenase (L-LDH) in Tris-HCl-NH2NH2 buffer solution (pH9.2). The changing rate of the absorbance of nicotinamide adenine dinucleotide reduced form (NADH) was determined at 340nm for calculating the velocity of the enzymatic reaction and the calibration curve of L-lactic acid was drawn. The L-lactic acid in samples was determined by the calibration curve.The effects of the amount of reagents,pH value,temperature of reaction and coexisted ions in the solution were discussed. The method has been used to determine the concentration of L-lactic acid in calf serum, beer and vinegar.In chapyer three, the determination of L-lactic acid is studied by enzymatic kinetic fluorescent spectrophotometry. Base on the reaction: L-lactic acid + NAD L-LDH pyruvate + NADH. By the fluorescence character of NADH the changing rate of fluorescence intensity of NADH was determined for calculatin the speed of the enzymatic reaction and the calibration curve of L-lactic acid was drawn.The L-lactic acid in samples was determined by the calibration curve. The method has been used to determine the concentration of L-lactic acid in calf serum, beer and yoghurt with satisfactory.In chaptre four, the kinetic properties of LDH in reversed micelles and application of this system to determine L-lactic acid were researched. The effect for the immobilization of LDH of w0, pH value, surfactant concentration, the amount of cosolvent were discussed. It is confirmed that the molecular conformation of LDH has changed in reversed micelles, but activity remains still. The research shows that in reversed micelles thermal stability and heat resistance were improved compared to aqueous phase.The system has been used to determine the concentration of L-lactic acid in samples.