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Study On The Production Of Vanillin By Bioconversion

Posted on:2005-09-13Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y H LiFull Text:PDF
GTID:1101360125960727Subject:Fermentation engineering
Abstract/Summary:PDF Full Text Request
Vanillin is an important flavour widely used in foods, beverages, cosmetics and pharmaceuticals. Now it is produced mainly through chemical synthesis, which means the vanillin obtained is not "natural". According to European regulations, natural products are those obtained from materials of plant or animal organisms through physical, enzymatic or microbiological processes. The increasing concern for health and nutrition stimulates a worldwide demand for natural vanillin that is very expensive and can no longer be satisfied only by extracting from vanilla pods which can not be planted widely for its requiring manpower pollination. Therefore, alternative processes for the production of natural vanillin should be studied. Approaches based on biotransformation, which is current hotspot in biotechnology field, become more and more attractive. Investigation in this field is very significant. Our main achivements on the preparation of biological vanillin are as follows. Some analytical methods were established for detecting the production and substrate in bioconversion. The spectrometric method is fast and economical, but it may be interfered by many factors, so it can only be used for half quantitative analysis. The TLC method, detecting product and substrate simultaneously, is also economical, but the thickness of plates may influence the determination results, and it can only be used for crude quantitative analysis, too. The HPLC method, capable of detecting product and substrate simultaneously, is proved sensitive, precise and accurate. Its cost is high, however. During different periods of our research, different methods were adopted according to realistic demand. The spectrometric method or TLC method was utilized for samples requiring fast detection so as to save time and cost, and the HPLC method was adopted for samples needed accurate results so as to guarantee the results' reliability. Ferulic acid, with low toxicity to microbs, is the most widely used substrate for microbial conversion. The microbial one-step conversion method for preparing vanillin from ferulic acid was investigated, and it was found that Nocardia mediterranei CoSr10 could convert ferulic acid to vanillin. The preliminary optimization on slope agar incubation condition, shaking culture condition and converting condition were carried out. But this strain, only producing a little vanillin (22 mg/L), can not be used for industrial production directly, but it may be changed to a high-yield one after metabolic interdiction mutation.Eugenol may be extracted from clove oil on industrial scale, and isoeugenol is usually obtained from eugenol by isomerization. They are much cheaper than ferulic acid, the substrate for two-step technology. Crude soybean lipoxygenase is also cheap and easy to obtain. Therefore, the technology to prepare vanillin from isoeugenol catalysed by crude soybean lipoxygenase is very promising and was investigated systematically.The preparation method of crude soybean enzyme was investigated, and its properties were studied. It was found that crude enzyme, with the optimum pH of 9, showed little lipoxygenase activity under acidic or neutral condition. Enzyme solution is unsteady, so the lyophilized enzyme was studied and found very stable with the same bioconversion characteristics as freshly-extracted crude enzyme. Finally, different breeds of soybean were studied and the best for practical production was obtained. In addition, we tried to separate lipoxygenase isoenzymes and obtained several different ones through DEAE-Cellulose DE-52 column. But our investigation results did not tally with previous literature. The technology for preparing vanillin from isoeugenol catalysed by crude soybean lipoxygenase was investigated. Main operation parameters of conversion (such as temperature, pH, liquid amount in flask, amount of enzyme added, amount of substrate added and substrate adding mode) were optimised, and some measures were taken to solve the realistic problems met in the process of conversion. Fo...
Keywords/Search Tags:Vanillin, enzymatic conversion, microbial conversion, isoeugenol, ferulic acid, TLC, HPLC, extraction, purification, LOX pathway
PDF Full Text Request
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