Isolation and Identification of Novel Strain Rhizomucor Miehei produces Acid Protease as a Rennet Substitute by Solid State Fermentation and Use Soft Cheese-Making

Abstract

In this study, twenty isolates belonging to the genera Rhizopus and Mucor were isolated from diverse sources and screened for acid protease production as a rennet substitute. All isolates were identified at the genus through morphological and biological testing. Fifteen Rhizopus were named (11, 14, 18, 25, 28, 30, 32, 37, 40, 44, 46, 47, 49, 64, 65) M and five Mucor were named (20, 21, 33, 51, 55) M. The sources of isolates (11, 14, 18) from the air, (20, 21, 25, 28, 30) soil (32, 33, 37, 40, 44) from soybeans (46, 47, 49, 51, 55, 64, 65), and wheat bran. Among these isolates, Mucor sp. 33M has a high-efficiency level in producing acid protease, with a specific activity of 7.8 U/mg and a milk clotting/proteolytic activity of 10.8. This isolate was chosen and identified at the species level by molecular identification and studying the sequences of the large subunit ribosomal RNA gene nitrogenous bases, which included 588 bp, and compared with the information available at the NCBI. It was identified as Rhizomucor miehei and registered with novel strain 33M by accession number PP955195.1 in the NCBI database. The crud enzyme produced in this study was compared with four Rennilase commercial preparations used by estimating the ratio of milk clotting to proteolytic activity, which was highly similar, and by the sensory evaluation analysis of the soft cheesemaking for three replicate samples after storage for 1, 4, and 8 days at 7 °C. At level 0.05, the statistical analysis did not show a significant difference between the sensory evaluations of the cheese product samples.