Screening of thermophilic bacterial isolates for protease production and its application to food processing

A total of 416 thermophilic isolates were obtained from soil and mud samples of Mudsprings, Mt. Makiling, Los Banos, Laguna [Philippines] (206) and NAPOCOR [National Power Corporation] Geothermal Site, Tiwi, Albay [Philippines] (210). Out of this number, 272 isolates showed proteolytic activity using a rapid screening assay method on skimmilk agar test plates for 24 hours at incubation temperatures of 45, 55 and 65 deg C. A 99.3% of the total isolates showed proteolytic activity at 45 deg C while 73.5% and 23.4% retained its activity at 55 and 65 deg C, respectively. Six thermophilic isolates (3SM-23, 4MM-2, 4MM-22, 5-SM-6, 7MM-8 and 7MM-16) were selected for extracellular neutral and acid protease production using modified soybean cake extract broth. Crude enzymes were assayed using 0.6% casein and results showed that three isolates, 3SM-23, 7MM-8 and 7MM-16, have high neutral protease activity (NPU) of 183.9, 128.7 and 154.1, respectively. Crude neutral proteases (CNP) from these isolates have maximum proteolytic activity at pH7 and temperature of 55 deg C for 3SM-23 and 7MM-16 are at 40 deg C for 7MM-8, CNP were stable over a pH range of pH4 to 7 for 3SM-23 and 7MM-16 and 4 to 8 for 7MM-8. On one hand, the enzymes were stable at temperatures 20-60 deg C for 3SM-23, 20-40 deg C for 7MM-8 and 20-55 deg C for 7MM-16. CNP preparation from 3SM-23 was then tested in making pan de sal, using different concentration of 0.00, 0.20, 0.40, 0.60 and 0.80% with the dough without the enzyme as the control. Based on the sensory evaluation of pan de sal using a 10-member panel, pan de sal treated with 0.20% CNP was the most acceptable. The proteolytic activity of the crude acid protease (CAP) produced by the 6 isolates was assayed using 2% casein as substrates. Results showed that acid protease from 4MM-22 and 7MM-16 yielded activities of 277.5 and 247.0 APU, respectively. Characterization of the two isolates showed that 4MM-22 was active and stable at pH4 and 55 deg C and pH 2.0 to 4.0 and 20 to 50 deg C, respectively. Isolate, 7MM-16, was active and stable at pH4 and 55 deg C and pH 2.0-5.0 and 20 to 50 deg C, respectively. CAP produced by two isolates was then used to clarify calumpit wine. High clarifying values of 80.5% and 79.5% were obtained for 1.5% (4MM-22) activated at 45 deg C and 2.5% (7MM-16) at 45 deg C, respectively. Cultural and morphological characteristics and physiological and biochemical tests showed the identity of the isolates as Bacillus subtilis (7MM-8) and (7MM-16), B. brevis (4MM-22 and 5SM-6), B. pumilus (3SM-23) and B. polymyxa for 4MM-2. Results obtained showed great potential of these thermophilic bacteria to produce both neutral and acid proteases for possible use in the food industry and other applications