World Applied Sciences Journal 4 (4): 578-585, 2008 ISSN 1818-4952 © IDOSI Publications, 2008 Corresponding Author: J. Contiero, Department of Biochemistry and Microbiology, São Paulo State University-Unesp, Rio Claro, São Paulo, Brazil 578 Production of Rennet in Submerged Fermentation with the Filamentous Fungus Mucor miehei NRRL 3420 C.J.B. De Lima, Mariana Cortezi, Roberta B. Lovaglio, 1 1 1 E.J. Ribeiro, J. Contiero and E.H. De Araújo 2 1 2 Department of Biochemistry and Microbiology, 1 São Paulo State University-Unesp, Rio Claro, São Paulo, Brazil Department of Chemical Engineering, Federal University of Uberlândia, 2 Campus Santa Mônica, 38400-902, Uberlândia-MG, Brazil Abstract:Mucor miehei NRRL - 3420 was cultured to produce microbial rennet using two types of media, both with micronutrients: one with glucose and another with lactose from deproteinized cheese whey as carbon sources. A factorial experimental design was used to evaluate the results, studying the following variables: pH, glucose, lactose and casein concentrations. The experiments were carried out in a bioreactor for up to a 40-h fermentation period at 380 rpm, aeration of 1 vvm and temperature of 30±1°C. The response parameters were cell growth and enzyme activity. Maximum enzyme activity was 1200 Soxhlet Units (S.U.) and 631 S.U., respectively, when glucose and lactose were used as carbon sources. Key words: Mucor miehei Microbial rennet Milk clotting Submerged fermentation Whey Experimental factorial design INTRODUCTION Among these microorganisms, it was discovered that Cheese production using rennet is the oldest use of produce a coagulant with characteristics that are very milk [1]. The production of countless different types of similar to commercial rennet, producing high coagulation cheese use proteolytic enzymes, of which rennin is the and production rates [5] and [6]. most widely used. Commercial preparations based on the filamentous Rennin is extracted from the gastric mucous fungus Mucor miehei are currently replacing animal membrane of bovines. Milk coagulation is irreversible and rennin in the production of more popular cheeses and has two phases. In the first phase or enzymatic phase, the already represent about 33% of the production process colloid that protects the casein (K-casein) dissociates the [7]. glycomacropeptides (part of K-casein, integrated by non- The aim of the present study was to investigate proteinic nitrogen), thereby making the protective effect growth conditions of the microorganism Mucor miehei disappear. The second stage or coagulation phase NRRL 3420 in submerged fermentation and the production involves the formation of saline bridges at favorable of the milk clotting enzyme using a lactose concentration temperatures between the calcium-sensitive casein from cheese whey and glucose as carbon sources. The micelles, thereby quickly producing coagulation [2]. study was carried out using a factorial experimental The increasing global cheese production (4% a year) design in order to provide an appropriate tool to in the last twenty years, in association with a decrease in determine the effects of the independent variables on bovine rennet production, has raised the price of the enzyme synthesis. traditional rennet. This situation has stimulated the search for milk clotting enzymes from alternative sources [3] and MATERIALS AND METHODS [4]. Microorganisms such as Endothia parasitica, Mucor pussilus, Cryptococcus albidus, Baccilus cereus and Microorganism and Culture: The microorganism used for Mucor miehei are known to produce rennet, such as the production of the clotting enzyme was Mucor miehei proteinases, which can substitute calf rennet [3] and [5]. NRRL 3420, obtained from the Fundação André Tosello the zygomicetes Mucor miehei and Mucor pussilus