本研究藉由改良之米麴菌進行麴酸發酵之探討，以提高麴酸生產效率。在培養基篩選方面，以sucrose 100 g/L為碳源，yeast extract 2.5 g/L或rice bran 15 g/L為氮源時，麴酸產量最高分別可達42.1 g/L及41 g/L。另外在氮源種類對於菌體形態影響的研究中，以yeast extract為氮源時，菌體會形成pellet，而此形態適合於重複批次發酵的操作。在批次發酵培養方面，實驗結果顯示各項參數與搖瓶實驗並無太大差異，productivity最高可達5.1 g kojic acid/L/day，證實利用發酵槽大量生產麴酸的可行性。在重複批次發酵實驗中，利用改良式發酵槽進行麴酸發酵研究，經過3次的培養基替換後，改良之米麴菌仍具有高麴酸生產能力。

In the present study, kojic acid fermentation by mutated Aspergillus oryzae strain M3B9 was investigated with the aim of improving productivity. When 2.5 g/L yeast extract or 15 g/L rice bran was used as nitrogen source along with 100 g/L sucrose as carbon source, the highest kojic acid production of 42.1 g/L and 41 g/L, respectively, were obtained. In addition, the nature of the nitrogen source had a great influence on the fungal morphology. Yeast extract as nitrogen source can induce pellet formation, which is favorable for repeated batch operation. Similar performances in the fermentor as well as in shake flask with the highest productivity(up to 5.1 g kojic acid/L/day) revealed the practicability of using the mutated strain for large scale kojic acid production. In repeated batch experiments using cell-retention fermentor, the mutated strain maintained high kojic acid production capability throughout the fermentation with three times of medium exchange.

Arenstein HR, Bentley R. The biosynthesis of kojic acid. I. Production from (1-14C) and (3:4-14C2) glucose and (2-14C)-1:3-dihydroxyacetone. Biochemical Journal 54:493-508. 1953.

Ariff AB, Rosfarizan M, Herng LS, Madihah S and Karim MIA. Kinetics and modelling of kojic acid production by Aspergillus flavus link in batch fermentation and resuspended mycelial system. World Journal of Microbiology & Biotechnology 13:195-201. 1997.

Ariff AB, Salleh MS, Ghani B, Hassan MA, Rusul G and Karim MIA. Aeration and yeast extract requirements for kojic acid production by Aspergillus flavus Link. Enzyme and Microbial Technology 19:545-550. 1996.

Bajpai P, Agrawal PK and Vishwanathan L. Kojic Acid - Synthesis and Properties. Journal of Scientific & Industrial Research 41:185-194. 1982a.

Bajpai P, Agrawala PK and Vishwanathan L. Enzymes Relevant to Kojic Acid Biosynthesis in Aspergillus-flavus. Journal of General Microbiology 127:131-136. 1981.

Bajpai P, Agrawala PK and Vishwanathan L. Production of kojic acid by resuspended mycelia of Aspergillus flavus. Canadian Journal of Microbiology 28:1340-1346. 1982b.

Balaz S, Uher M, Brtko J, Veverka M, Bransova J, Dobias J, Podova M and Buchvald J. Relationship between antifungal activity and hydrophobicity of kojic acid derivatives. Folia Microbiology 38:387-391. 1993.

Beelik A. Kojic acid. Advances in Carbohydrate Chemistry 48:145-183. 1956.

Bentley R. Preparation and analysis of kojic acid. Methods in Enzymology 3:238-241. 1957.

Bhat R, Hadi SM. Photoinactivation of bacteriophage lambda by kojic acid and Fe(III): role of oxygen radical intermediates in the reaction. Biochemistry and Molecular Biology International 32:731-735. 1994.

Bon E and Webb C. Glucomylase production and nitrogen nutrition in Aspergillus- awamori. Applied Biochemistry and Biotechnology 39:349-369. 1993.

Briganti S, Camera E and Picardo M. Chemical and instrumental approaches to treat hyperpigmentation. Pigment Cell Research 16:101-110. 2003.

Budavari S, O'Neil MJ, Smith A, and Heckelman PE. The Merck Index. 11th . Rahway, NJ. 1989

Burdock GA, Soni MG and Carabin IG. Evaluation of health aspects of kojic acid in food. Regulatory Toxicology and Pharmacology 33:80-101. 2001.

Futamura T, Ishihara H, Tamura T, Yasutake T, Huang GW, Kojima M and Okabe M. Kojic acid production in an airlift bioreactor using partially hydrolyzed raw corn starch. Journal of Bioscience and Bioengineering 92:360-365. 2001a.

Futamura T, Okabe M, Tamura T, Toda K, Matsunobu T and Park YS. Improvement of production of kojic acid by a mutant strain Aspergillus oryzae, MK107-39. Journal of Bioscience and Bioengineering 91:272-276. 2001b.

Horiuchi JI, Yamauchi N, Osugi M, Kanno T, Kobayashi M and Kuriyama H. Onion alcohol production by repeated batch process using a flocculating yeast. Bioresource Technology 75:153-156. 2000.

Katz ME, Rice RN, Cheetham BF. Isolation and characterization of an Aspergillus nidulans gene encoding an alkaline protease. Gene 150:287-292. 1994.

Kitada M and Fukimbara T. Studies on kojic acid fermentation (Ⅳ). Production and recovery of kojic acid fermentation on an industrial scale. Journal of Fermentation Technology 49:343-349. 1971.

Kwak MY, Rhee JS. Cultivation Characteristics of Immobilized Aspergillus-oryzae for Kojic Acid Production. Biotechnology and Bioengineering 39:903-906. 1992.

Lee HF, Boltjes B and Eisenmann WM. Kojic acid as an inhibitor of tubercle bacilli. American Review of Tuberculosis 61:738-741. 1950.

Lin YC, Wu JY and Chen TL. Production of Acinetobacter radioresistens lipase with repeated batch culture in presence of nonwoven fabric. Biotechnology and Bioengineering 76:214-218. 2001.

Marwaha SS, Kaur J and Sodhi GS. Organomercury(II) complexes of kojic acid and maltol: synthesis, characterization, and biological studies. Journal of Inorganic Biochemistry 54:67-74. 1994.

Mohamad R, Ariff A, Ali Hassan M, Karim MIA, Shimizu H and Shioya S. Importance of carbon source feeding and pH control strategies for maximum kojic acid production from sago starch by Aspergillus flavus. Journal of Bioscience and Bioengineering 94:99-105. 2002.

Morton HE, Kocholaty W, Kocholaty RJ and Kelner A. Toxicity and antibiotic activity of kojic acid produced by Aspergillus luteovirescens. Journal of Bacteriology 50:579-584. 1945.

Novotny L, Rauko P, Abdel-Hamid M and Vachalkova A. Kojic acid - a new leading molecule for a preparation of compounds with an anti-neoplastic potential. Neoplasma 46:89-92. 1999.

Parekh S, Vinci VA and Strobel RJ. Improvement of microbial strains and fermentation processes. Applied Microbiology and Biotechnology 54:287-301. 2000.

Piccoli-Valle RH, Brandi IV, Silva DO and Passos FJV. Pectin lyase production by Penicillium griseoroseum grown in sugar cane juice in repeated batch cultures. World Journal of Microbiology & Biotechnology 17:433-437. 2001.

Rosfarizan M, Ariff AB. Kinetics of kojic acid fermentation by Aspergillus flavus using different types and concentrations of carbon and nitrogen sources. Journal of Industrial Microbiology & Biotechnology 25:20-24. 2000.

Sanchez S and Demain AL. Metabolic regulation of fermentation processes. Enzyme and Microbial Technology 31:895-906. 2002.

vanLier FLJ, vandenHombergh JPTW, deGooijer CD, denBoer MM, Vlak JM and Tramper J. Long-term semi-continuous production of recombinant baculovirus protein in a repeated (fed-)batch two-stage reactor system. Enzyme and Microbial Technology 18:460-466. 1996.

Wakisaka Y, Segawa T, Imamura K, Sakiyama T and Nakanishi K. Development of a cylindrical apparatus for membrane-surface liquid culture and production of kojic acid using Aspergillus oryzae NRRL484. Journal of Fermentation and Bioengineering 85:488-494. 1998.

Wan HM, Chen CC, Chang TS, Giridhar R and Wu WT. Combining induced mutation and protoplasting for strain improvement of Aspergillus oryzae for kojic acid production. Biotechnology Letters. (in press)

Werner A, Lutkemeyer D, Poggendorf I, Lehmann J and Muthing J. Serum-free production of a chimeric E-selectin-IgG protein from 1 to 100 l scale: Repeated batch cultivation versus continuous spin filter perfusion. Cytotechnology 38:47-56. 2002.

Woodhead S, Walker JR. The effects of aeration on glucose catabolism in Penicillium expansum. Journal of General Microbiology 89:327-336. 1975.

Zhang W, Jin MF, Yu XJ and Yuan Q. Enhanced anthocyanin production by repeated-batch culture of strawberry cells with medium shift. Applied Microbiology and Biotechnology 55:164-169. 2001.