Screening and identification of amylase-producing strain and optimization of the fermentation conditions

Abstract:

[Background] amylase hydrolyses starch and has a wide range of applications in the food fermentation industry, including starch-based sugar, white wine, yellow wine, beer, and vinegar. [Objective] In order to provide excellent amylase resources, an amylase-producing Bacillus strain was screened from high-temperature Jiuqu, and its classification and identification were carried out, and the fermentation conditions of amylase production were optimized. [Methods] The Bacillus strain was identified by colony morphology, Gram staining, and 16S rRNA gene sequence analysis. The effects of medium composition (carbon source, nitrogen source, and metal ions) and fermentation conditions (temperature, pH, and inoculum size) on amylase production was evaluated by single factor experiment, Plackett-Burman experiment, steepest climb experiment, and Box-Behnken design experiment. The optimum medium and fermentation conditions were applied to amylase production using starch hydrolysis. [Results] A high amylase producing strain LT-2 was obtained and identified as Bacillus velezensis LT-2. The optimum fermentation parameters for amylase production by B. velezensis LT-2 are maltose 8.00 g/L, soy protein isolate 8.69 g/L, ammonium chloride 3.00 g/L, calcium chloride 13.94 mmol/L, initial fermentation pH 7.5, temperature 34.08℃, and inoculum size 6%. B. velezensis LT-2 had an extracellular amylase activity of 972.88 U/mL in 33 h incubation under optimal fermentation parameters, which was 5.01 times higher than that before optimization; it had an amylase production of (1 198.40±4.56) U/mL in 51 h incubation in a 5 L fermenter, which was 1.23 times higher than in shake flask level and 6.17 times higher than in shake flask level (before optimization). The amylase produced by B. velezensis LT-2 had a good starch hydrolysis ability, and the hydrolysis rate was 6.17×10^-5 g/(U·h). [Conclusion] B. velezensis LT-2 has promising applications in amylase secretion and starch hydrolysis, which provides theoretical support for the improvement of saccharification capacity and liquefaction power of Jiuqu.