Screening and isolation of substitute-rennet producing thermophilic phycomycetes, by modified Warcup method and improved selective medium

Abstract

Fungal milk-clotting enzymes are as valuable as bovine Chymosin in the cheese industry. Members of the Mucoraceae family, have important biotechnological potential as some species are enzyme producers at industrial scale. In this study, 31 isolates of thermophilic Phycomycetes of different taxa were selected from different sample sites and were studied for their aspartic protease producing capabilities. For primary isolation, modified Warcup method using improved Phycomycetes selective solid media along with high temperature selective for thermophilic Mucorals were applied. The purpose of this study was to collect the local biotechnologically important strains of thermophilic phycomycetes capable of producing rennin-like proteases. Based on a high relative enzyme activity (REA), the majority (83.7%) of the 31 species of isolated thermophilic Phycomycetes were selected for next isolation steps. By trial production of acid proteases using shake flask fermentation, Milk clotting activity (MCA), and Protease activity (PA) of each fermentation broth of primary isolates were measured. Later, based on their high MCA/PA ratio, three isolates were chosen for macroscopic and microscopic examinations of each isolate determining their taxonomy identification and classification. The results showed that two of final isolates (HPA-01 and ZDO-16) belong to Rhizomucor family (R. nainitalensis and R. pusillus) and the third thermophilic Zygomycetes (ZDP-03) is a Rhizopus (R. rhizopodiformis). Of the 3 isolates, Rhizomucor nainitalensis was chosen for further studies and could be introduced as a commercially important isolate in future.