Abstract:

Response surface methodology (RSM) was employed to optimize ultrasonic power, irradiation time andlysozyme dose for the release of holoenzyme from Lactobacillus delbrueckii subsp. bulgaricus. Experiments were conductedto investigate the hydrolysis of skim milk and casein with the holoenzyme. Results indicated that the enzyme activity roseat first and then declined with increasing ultrasonic power and extended irradiation time. From the proposed regressionmodel, the optimal conditions for holoenzyme release were determined as ultrasonication for 7.5 min at a power of 71%and hydrolysis with lysozyme at a dose of 290 μL/30 mL of cell suspension. The whole fermentation broth of Lactobacillusdelbrueckii subsp. bulgaricus was treated under these conditions to obtain holoenzyme 1 or centrifugally separated into cellsand supernatant. The cells were individually treated under the same conditions and combined with the supernatant to obtainholoenzyme 2. The content of free amino nitrogen in hydrolyates of skim milk and casein by holoenzyme 2 was increasedby 223.86% and 324.11%, respectively, when compared with holoenzyme 1. This may be attributed to losses of extracellularenzyme activities caused by ultasonication. HPLC analysis showed that polypeptides with diverse molecular weightdistribution were generated in higher amounts by using the whole enzymes than extracellular enzymes alone.

Key words: Lactobacillus delbrueckii subsp. bulgaricus, holoenzyme, hydrolysis, milk, casein