Carbohydrate Polymers 86 (2011) 1573–1577 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Optimization of medium components for hyaluronic acid production by Streptococcus zooepidemicus MTCC 3523 using a statistical approach Kanchankumar P. Patil a , Kirtee K. Kamalja b , Bhushan L. Chaudhari a,∗ a School of Life Sciences, North Maharashtra University, PO Box 80, Jalgaon 425001, India b Dept. of Statistics, North Maharashtra University, PO Box 80, Jalgaon 425001, India article info Article history: Received 31 October 2010 Received in revised form 17 June 2011 Accepted 23 June 2011 Available online 1 July 2011 Keywords: Streptococcus zooepidemicus MTCC 3523 Hyaluronic acid Response surface methodology abstract Hyaluronic acid (HA), produced from Streptococcus sp., has raised interest in the medical and cosmetics industries because of the various biological functions of HA. Optimization of medium components used for HA production in Streptococcus zooepidemicus MTCC 3523 performed by two-step optimization. A 2 4 full factorial design was employed to study the effect of crucial factors on HA production. To get optimized levels of these factors further, a central composite design was conducted. Through these two phase experiments carried out for optimization of HA production, the medium that yielded maximum average HA comprises 4.05% of glucose, 5.12% of soyapeptone, 0.075% of MgSO 4 ·7H 2 O, and 0.25% of K 2 HPO 4 approximately. The maximum average productivity of HA for this composed medium was 0.798 g/L on fermentation in shake flask. The yield of HA was increased by about 65% using these statistical techniques of media optimization. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Hyaluronic acid (HA) is a uniformly repetitive, linear, high- molecular-weight glycosaminoglycan composed of 2000–25,000 disaccharides of glucuronic acid and N-acetylglucosamine joined alternately by -1-3 and -1-4 glycosidic bonds (Chong, Blank, Mclaughlin, & Nielsen, 2005). Owing to its variety of biological functions, HA has a wide range of applications in the fields of medicine and cosmetics, including osteoarthritis treatment, oph- thalmic surgery, plastic surgery, drug delivery, skin moisturizers, and wound healing (Chong et al., 2005; Goa & BenWeld, 1994; Kogan, Soltes, Stern, & Gemeiner, 2007). Conventionally, HA was extracted from animal tissues like rooster combs and is now increasingly produced by microbial fer- mentation with a lower production cost. Currently, the commonly used strain for microbial HA production on an industrial scale is Streptococcus zooepidemicus, which synthesizes HA as the extracel- lular capsule (Duan, Yang, Zhang, & Tan, 2008). Much work, such as improving the fermentation process (Armstrong & Johns, 1997; Blank, McLaughlin, & Nielsen, 2005; Duan et al., 2008; Hasegawa, Nagatsuru, Shibutani, Yamamoto, & Hasebe, 1999; Huang, Chen, & Chen, 2006; Huang, Chen, & Chen, 2008; Johns, Goh, & Oeggerli, 1994; Kim, Yoo, Oh, & Kweon, 1996; Kim, Park, & Kim, 2006; Liu, Wang, Du, & Chen, 2008b), adding lysozyme (Kim et al., 1996; ∗ Corresponding author. Tel.: +91 257 2257421; fax: +91 257 2258403. E-mail address: blchaudhari@hotmail.com (B.L. Chaudhari). Ogrodowski, Hokka, & Santana, 2005), the alkaline-stress strategy (Liu, Wang, Du, & Chen, 2008a), adding hydrogen peroxide and ascorbate (Liu et al., 2009), and changing the medium composi- tion (Rangaswamy & Jain, 2008; Zhang, Ding, Yang, & Kong, 2006), has been done to increase the production yield of hyaluronic acid in Streptococcus sp. Although many studies have been performed on HA production, there are very few reports on general medium optimization higher HA production and on economically efficient conditions for HA production. In this study, we carried out optimization of medium compo- nents to produce higher amount of HA in S. zooepidemicus MTCC 3523. In the first step, the effects of medium component levels, such as carbon source, nitrogen source, phosphate source, and min- eral sources, on HA production were investigated by full factorial design. In the second step, concentrations of selected medium com- ponents were optimized using central composite design to improve the productivity of HA. 2. Materials and methods 2.1. Microorganism and culture medium Streptococcus equi subsp. zooepidemicus MTCC 3523 was obtained from the Microbial Type Culture Collection (Chandigarh, India) as a freeze-dried culture in ampoules. The stock culture pre- served in 20% glycerol solution at -86 ◦ C was cultivated on slants of streptococcus agar containing (g/L): glucose 20, pancreatic digest of casein 20, K 2 HPO 4 2, MgSO 4 ·7H 2 O 0.1, agar 15, final pH adjusted 0144-8617/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbpol.2011.06.065