Journal of Biotechnology 170 (2014) 28–34 Contents lists available at ScienceDirect Journal of Biotechnology jou rn al hom ep age: www.elsevier.com/locate/jbiotec Innovative use of Mucuna monosperma (Wight) callus cultures for continuous production of melanin by using statistically optimized biotransformation medium Shrirang Inamdar, Swati Joshi, Vishwas Bapat, Jyoti Jadhav ∗ Department of Biotechnology, Shivaji University, Kolhapur 416 004, India a r t i c l e i n f o Article history: Received 25 July 2013 Received in revised form 22 October 2013 Accepted 19 November 2013 Available online 27 November 2013 Keywords: Biotransformation Continuous production Melanin Mucuna monosperma Response surface methodology a b s t r a c t Melanins are predominantly indolic polymers which are having extensive applications in cosmetics, agri- culture and medicine. In the present study, optimization of nutritional parameters influencing melanin production by Mucuna monosperma callus cultures was attempted using the response surface methodol- ogy (RSM). Standardization of four factors was carried out using the Box–Behnken design. The optimized levels of factors predicted by the model include tyrosine 0.978 g L -1 , pH 5.85, SDS 34.55 mg L -1 and copper sulphate 21.14 mg L -1 tyrosine, which resulted in highest melanin yield of 0.887 g L -1 . The optimization of medium using RSM resulted in a 3.06-fold increase in the yield of melanin. The ANOVA analysis showed a significant R 2 -value (0.9995), model F-value (1917.72) and probability (0.0001), with insignificant lack of fit. Optimized medium was used in the laboratory scale column reactor for the continuous production of melanin. Uninterrupted flow column exhibited maximum melanin production rate of 250 mg L -1 h -1 which is the highest value ever reported using plant as a biotransformation source. Melanin produc- tion was confirmed by spectrophotometric and chemical analysis. Thus, this study demonstrates the production of melanin by M. monosperma callus, using a laboratory scale column reactor. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Melanins is a group of high molecular weight, black and brown pigments that are produced from oxidation and polymerization of polyphenols and are widely distributed in plant and animal king- dom and are also reported to be synthesized by microorganisms (Plonka and Grabacka, 2006). Coloration of seeds, berries, flowers, human skin, or hair is essentially due to the presence of melanin (Nicolaus, 1968). Melanins are among the most stable, insoluble and resistant to biochemical materials and could enhance the sur- vival and competitive abilities of organisms in environments (Bell and Wheeler, 1986). Melanins are widely employed in cosmetics, photo protec- tive creams, eyeglasses as well as protective agents in Bacillus thuringenesis insecticidal crystals (Zhang et al., 2007). Microorgan- isms producing melanin have been used for the immobilization of radioactive waste of uranium (Turick et al., 2008), for screening recombinant bacterial strains (Adham et al., 2003), for AIDS treat- ment (Montefiori and Zhou, 1991), and for the treatment of human metastatic melanoma (Dadachova et al., 2008). In addition to phar- maceutical applications, melanins also have high commercial value ∗ Corresponding author. Tel.: +91 9673517771; fax: +91 231 2692333. E-mail address: jpjbiochem@gmail.com (J. Jadhav). in food industries as natural additives and colorants. Synthetic col- orants are frequently perceived as undesirable or harmful which uphold the craze for natural colorants and food additives among consumers (Kannan and Ganjewala, 2009). Plants are always considered to be the best source of natural pig- ments such as melanin and others. Wang et al. (2006) have isolated and characterized melanins from seeds of Osmanthus fragrans and studied its biological properties to be used as food colorants. Exten- sive studies have been carried out regarding biological properties of melanin pigment from different plant sources (Nicolaus, 1968; Zherebin et al., 1982). However, to fulfill the ever-growing demand of the melanin, these sources are not sufficient and necessitated development of a cost effective process such as biotransformation of l-tyrosine to melanin. The plant under present study is Mucuna monosperma; seeds of which are proved to be a good source of l-DOPA and possess sufficient level of tyrosinase, an enzyme required for biotransfor- mation of l-tyrosine to melanin (Inamdar et al., 2012). Considering this background, callus cultures raised from seeds are expected to provide a continuous source for biotransformation which could be further augmented using fermentation process. Medium opti- mization and physical conditions in fermentation have been traditionally performed using one-factor-at-a time method which is time consuming, laborious and expensive; in addition, it fails to determine the combined effect of different factors on the contrary, 0168-1656/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2013.11.012