淀粉酶产生菌的筛选、鉴定及其发酵条件优化

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

摘要:

[背景] 淀粉酶可以水解淀粉，在淀粉制糖、白酒、黄酒、啤酒和食醋等食品发酵行业有着广泛的应用。[目的] 从高温酒曲中筛选获得产淀粉酶的芽孢杆菌属菌株，并对其进行分类鉴定和产淀粉酶发酵条件优化，为发酵过程提供优良的淀粉酶资源。[方法] 取高温酒曲，经过富集培养和可溶性淀粉平板筛选培养基初筛，摇瓶复筛得到高产淀粉酶的菌株；通过菌落形态、革兰氏染色观察和16S rRNA基因序列比对进行菌株鉴定；通过单因素试验、Plackett-Burman试验、最陡爬坡试验和Box-Behnken design试验考察培养基成分（碳源、氮源和金属离子）和发酵条件（温度、pH和接种量）对淀粉酶产量的影响，确定最佳产淀粉酶培养基和发酵条件，并将淀粉酶应用于淀粉水解。[结果] 筛选获得一株高产淀粉酶的菌株LT-2，经鉴定为Bacillus velezensis LT-2； B. velezensis LT-2产淀粉酶的最佳发酵参数为：麦芽糖8.00 g/L，大豆分离蛋白8.69 g/L，氯化铵3.00 g/L，氯化钙13.94 mmol/L，发酵初始pH 7.5，温度34.08 ℃，接种量6%。在最佳发酵参数下，B. velezensis LT-2经33 h发酵淀粉酶产量为972.88 U/mL，是优化前的5.01倍；5 L发酵罐中发酵，B. velezensis LT-2发酵51 h淀粉酶产量可达（1 198.40±4.56） U/mL，是摇瓶发酵的1.23倍，是发酵参数优化前摇瓶发酵的6.17倍；B. velezensis LT-2所产淀粉酶具有较好的淀粉水解能力，水解速率为6.17×10^-5 g/（U·h）。[结论] B. velezensis LT-2在淀粉酶分泌和淀粉水解方面具有良好的应用前景，研究结果为酒曲糖化能力和液化力的改善提供了理论支持。

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.