<![CDATA[Mozzarella cheese manufacture depends on milk-clotting enzymes (rennet), conventionally obtained from calf abomasum at relatively high cost. This preliminary study evaluated Rhizomucor miehei and Rhizomucor pusillus (formerly Mucor miehei and Mucor pusillus) grown on a wheat bran–molasses (1:1) medium as a source of microbial rennet and assessed their crude extracts in mozzarella cheese making. Enzyme production was evaluated using a full factorial design with incubation temperature (34 and 37 °C), medium pH (5, 6, and 6.5), and incubation time (4, 6, 7, 8, and 10 days) in duplicate. Milk-clotting activity (MCA), proteolytic activity (PA), and MCA/PA ratio were analyzed using three-way ANOVA followed by Tukey's test. Incubation temperature significantly affected MCA of both strains (p < 0.01). The highest MCA was obtained at 37 °C, 6,495.89 ± 241.52 U/mL for R. miehei (pH 6.5, 7 days) and 7,246.84 ± 81.69 U/mL for R. pusillus (pH 6, 8 days), with clotting times of approximately 83–92 s. Incubation at 37 °C doubled PA of R. miehei, resulting in a higher MCA/PA ratio at 34 °C, whereas R. pusillus showed similar ratios at both temperatures. Mozzarella produced with the highest-MCA extracts yielded 3.19%, significantly lower than 3.27% obtained with commercial rennet, although the absolute difference was small. Yields were substantially below typical cow's milk mozzarella yields, suggesting that the low yield was more likely related to the cheese-making procedure, particularly preacidification to pH 5.0, than to the enzyme source. Extract-based cheeses had lower moisture (48.9–49.6%) and meltability than the control. Both strains produced milk-clotting enzymes using low-cost wheat bran–molasses substrate. However, process optimization is needed before economic feasibility can be assessed.]]>
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