Cell immobilization for enhanced milk clotting enzyme production from Bacillus amyloliquefacien and cheese quality.

Background: Milk clotting enzymes, essential for milk coagulation in cheese production, are obtained from the stomach of young ruminants, an expensive and limited source. This study was accomplished by finding a suitable alternative. Bacterial isolates recovered from honey were screened for milk clotting enzyme activity. and further, by immobilization of the microorganisms to enhance stability and facilitate their repeated use.

Result: The most effective enzyme was produced by a microbe identified as Bacillus amyloliquefaciens based on 16 S rRNA sequencing. The cells were encapsulated in Ca²⁺ alginate beads. These beads retained complete enzyme production after being used five times. Glucose and Soybean were selected as the most favorable carbon and nitrogen sources, respectively. The optimum temperature for activity was 35 ℃ for both free and immobilized cells but as the temperature was increased to 55 °C and above, the encapsulated form retained more activity than the free cells. The pH optimum shifted from 6.5 to 7 for the free cells to 7-7.5 for the immobilized cells. The immobilization process decreased the activation energy for enzyme production and activity, prolonged the enzyme half-life, and increased the deactivation energy. Enzyme produced by immobilized cells generated a more compact cheese.

Conclusions: The finding of this study was to identify a less expensive source of milk-clotting enzymes and confirm the success of cell immobilization in improving cell rigidity and stability. Also, immobilization of this B. amyloliquefaciens strain offers an enzyme source of value for industrial production of cheese.