指導教授：鄭光成臺灣大學：食品科技研究所游婷筑Yu, Ting ChuTing ChuYu2014-11-272018-06-292014-11-272018-06-292014http://ntur.lib.ntu.edu.tw//handle/246246/262442麴酸具有良好的美白效果和金屬螯合、抗氧化的能力，由於其特殊的gamma-pyrone結構，可以做為maltol、ethymaltol等食品添加物或藥物的前驅物，在化妝品、食品及化學品合成上一直扮演重要的角色。工業上麴酸是藉由米麴菌 (Aspergillus oryzae) 以深層培養 (submerged culture)批次醱酵生產，但批次醱酵操作繁複不易，又易伴隨培養液黏度過高所造成質傳能力降低的缺點，使麴酸成本居高不下。Plastic composite support (PCS) 是一種聚丙烯及農業廢棄物經擠壓而成的菌體固定化載體，過去研究指出以PCS作為固定化載體可以有效降低作業成本，甚至可以增加產量及產率。本實驗對於A. oryzae在 PCS固定化醱酵系統生產麴酸進行評估與改善，並討論菌體於生物反應器中的形態變化與產量關係。在進行系統評估前，先確認生產最佳pH後，對五種不同組成的PCS (SFY、SFB、SFR、SFYB、SFYBR) 和兩組控制組聚丙烯棒及菜瓜布條進行挑選，其中以添加綜合氮源的SFYBR有最高麴酸產量。然而，以SFYBR固定米麴菌雖然較傳統方法便利，但麴酸產量低落，僅為傳統方法 (35 g/L) 之五分之一。後續配合不同程度的氮源缺乏培養基或麴酸添加培養基培養，可以有效改善固定化系統造成的低麴酸產量問題，最終產量可達26.34 g/L，並且可以重複使用生產菌體達到多批次發酵的成果，產率比傳統方法高，可達2.395 g/L/d。另外菌體型態在使用不同培養基時也有所不同，使用氮源缺乏培養基時，真菌形態形成羽絨狀菌體，使與受質接觸表面積增加，與產量增加相呼應。PCS固定化醱酵系統有效降低作業繁複程度、減少生產成本，在工業生產上具有極大的潛力。Kojic acid is an industrially important compound due to its powerful skin whitening effect, metal chelating ability, and antioxidant capacity. In addition, the gamma-pyrone, which is polyfunctional for synthesizing chemicals, in kojic acid makes it become a crucial precursor of various drugs and food additives such as Aspirin and maltol. Traditionally, kojic acid is produced by the submerged culture of Aspergillus oryzae in the manner of batch fermentation. Considering the high cost of batch replacements, reinoculations in long term fermentation as well as the poor mass transfer in free suspension culture, immobilization fermentation becomes one of the most potent replacements. Plastic composite supports (PCS), which is an extruding product composed of polypropylene and agricultural wastes, could be used as the immobilization scaffolds for kojic acid production. Previous studies show that applying PCS immobilization system increased the production and decreased the cost of some value-added products including bioethanol and pullulan. In the present research, the efficiency of immobilized culture for production of kojic acid, and the effect of changed morphology of A. oryzae on production were evaluated. Before evaluation, the optimal pH and PCS was selected. The optimal pH was verified as pH 3. Based on the kojic acid production, SFYBR, the PCS with the most complex ingredients, was chosen among 5 PCS with different compositions (SFY, SFB, SFR, SFYB, and SFYBR) and two control ( PP sticks and luffa) as the scaffold in following experiments. However, the production in immobilization system was one fifth of traditional free suspension culture (35.02 g/L). By using different degrees of nitrogen deprived medium and kojic acid added medium, the production in immobilization system increased to 26.34 g/L. Moreover, with the immobilization system, repeated-batch fermentation without loss of mycelia could be conducted for several cycles with productivity of 2.395 g/L/d, which is higher than free suspension culture in batch fermentation. It is an economical way in industry. To sum up, PCS immobilized fermentation system, which decrease the inconvenience and cost of batch submerged fermentation, is a potential tool in industrial production of kojic acid.中文摘要…………………………………………………………………………………I Abstract...………………………………………………………………………………..II Content…..……………………………………………………………………………..IV List of Figures………………………………………………………………………....VII List of Tables…………………………………………………………………………....X List of Abbreviations…………………………………………………………………...XI 1. Introduction 1 2. Literature reviews 4 2.1 Kojic acid 4 2.1.1 The structure of kojic acid 4 2.1.2 Applications of kojic acid 7 2.1.3 Toxicology and safety of kojic acid 8 2.2 Aspergillus oryzae 8 2.3 Biosynthesis of kojic acid 9 2.4 Fungal morphology 12 2.5 Factors affecting secondary metabolites production 15 2.6 Fermentation strategies to enhance production 17 2.6.1 Cultural condition optimization 17 2.6.2 Cell immobilization bioreactors 21 2.6.3 Other strategies 22 2.7 Plastic composite supports 22 3. Materials and methods 25 3.1 Chemicals 25 3.2 Microorganisms and cultural maintenance 25 3.3 Preparation of plastic composite supports 26 3.4 Culture condition in flasks 27 3.5 Immobilization and optimal PCS selection 27 3.6 Analysis of kojic acid 28 3.7 Measurement of mycelial formation 29 3.8 Measurement of residual sugar 29 3.9 Morphology observation 31 3.10 Kojic acid extraction 31 3.11 Product verification 31 3.11.1 FT-IR (Fourier transform infrared spectroscopy) 31 3.11.2 Assay of tyrosinase inhibition 32 3.11.3 Assay of antioxidant capacity ( DPPH radical scavenging activity) 32 3.12 Data analysis 33 4. Results and Discussion 34 4.1 Optimal pH selection 34 4.2 Optimal PCS selection 38 4.3 Effect of nitrogen deficient and carbon deficient medium replacement on kojic acid production 46 4.4 The longevity of PCS system with nitrogen deficient medium 54 4.5 Effect of kojic acid added medium on kojic acid production 60 4.6 Morphology observation 64 4.7 Products verifications 71 4.7.1 FTIR 71 4.7.2 Bioactivity verification 71 5. Conclusion and prospective 74 List of references 77 Appendix 923053086 bytesapplication/pdf論文公開時間：2014/08/21論文使用權限：同意有償授權(權利金給回饋學校)麴酸米麴菌Plastic composite support (PCS)菌體形態氮源缺乏培養基[SDGs]SDG11thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/262442/1/ntu-103-R01641027-1.pdf