1 Statistical Optimization of Microwave-assisted Astaxanthin Extraction from Phaffia rhodozym Chuanhe Zhu ∗1 , Wei Han 1 , Zhao Chen 2 1 College of Food Science and Engineering 2 College of Life Sciences Shandong Agricultural University Taian, China Abstract-The statistical experimental design was used in this study to optimize the extraction of astaxanthin with the assistance of microwave. Three factors, including the volume of anhydrous alcohol, the extracting time, and cell wall breaking time, were screened out by Fractional Factorial Design (FFD). Then the stable area was obtained by the steepest ascent method. And finally the extraction process was optimized by the response surface method. The optimal extraction process of astaxanthin was dimethyl sulfoxide (3.0 ml), anhydrous alcohol (12.6 ml), cell wall breaking time (2.6 min), extracting time (15 min), extracting temperature (50 o C) and microwave frequency (416.5 MHz).Under this condition, astaxanthin could be extracted in amounts of 2.9269±0.01 mg/l and the yield of astaxanthin was 21.56% higher than the initial yield. Keywords-Phaffia rhodozyma; Astaxanthin; Extraction; Microwave 1 INTRODUCTION The astaxanthin (3, 3 / -dihydroxy-β, β-carotene-4, 40 / -dione) is one type of pigment widely existing in the salmonoids and crustaceans such as salmon, shrimp, crab, etc [1]. It is reported that the astaxanthin can perform many important pharmacological and biological functions such as antioxidation, antitumor effect, and strengthening the immue system, which make it widely used in food additive, cosmetic, functional food and pharmacological industry [2, 3]. The yeast, Phaffia rhodozyma, can be used in the commercial production of astaxanthin due to its high astaxanthin content [4, 5], but its cell wall is very solid, which becomes the major obstacle in extracting this pigment. Nowadays, the common methods to break the cell wall in the process of extracting astaxanthin include * Corresponding author. Tel.: +86 538 8249157; fax: +86 538 8242623. E-mail address: chhzhu@sdau.edu.cn Chuanhe Zhu and Wei Han contributed equally to this work. This research was supported by the Technology Bureau of Taian, China PR. Ziqiang Han 3 Pingyin Vocational School of Jinan Jinan, China ultrasonic treatment [6], acidic heating [7], dimethyl sulfoxide [8], or alkaline solutions breaking [9]. Taking the breaking effect and astaxanthin protection into account, the best method is dimethyl sulfoxide breaking method [7], which, however, has a low efficiency of cell wall breaking. As a new method of wall breaking, microwave has been widely applied in recent years because of its high thermal efficiency, strong penetrating power [10, 11, 12, 13]. Through optimizing astaxanthin extraction by microwave-assisted dimethyl sulfoxide method to break the cell wall and analyze the results by response surface method, this research provides evidence for further research on astaxanthin production. The purpose of this study is to optimize the microwave-assisted extraction technology in order to maximize astaxanthin yield from Phaffia rhodozyma. This research was based on a statistical experimental design. The following optimization experiments were performed: (a) the factors that significantly affected the extraction were elucidated by using the Fractional Factorial Design (FFD); (b) the stable area was obtained by the steepest ascent method; (c) the extraction technology was optimized by Box–Behnken design (BBD). 2 MATERIALS AND METHODS 2.1 Strain The astaxanthin overproducing mutant, Phaffia rhodozyma TY, was obtained by Microwave irradiation mutation of Phaffia rhodozyma AS2.1557 from Institute of Microbiology, Chinese Academy of Sciences [14]. The strain was maintained in the inclined culture medium containing 20 g/L glucose; 10 g/L yeast powder; 20 g/L peptone; 20 g/L agar. 2.2 Cultivation and production of astaxanthin A Spore suspension of fungal isolates was prepared by washing 3-days old cultures slants with sterile saline solution (0.85% NaCl). Spores were counted by plate count method and the count was adjusted to about 10 6 to 10 7