紫外诱变选育高活性蛋白酶枯草芽孢杆菌及其降解饲料能力评价

以凡纳滨对虾(Litopenaeus vannamei)养殖池塘底泥中分离的枯草芽孢杆菌(Bacillus subtilis)BC2为出发菌株,利用紫外诱变的方法培育蛋白酶活性高的枯草芽孢杆菌菌株,并评价其降解饲料的能力。结果表明:(1)经6次紫外诱变,突变菌株B38的透明圈直径(H)与菌落直径(C)之比(H/C值)达到6.42,提高了2.13倍;(2)福林酚法测得B38的蛋白酶活性为86.82 U/m L,是出发菌株BC2的3.14倍,但紫外诱变对B38纤维素酶活性影响不显著;(3)连续传代培养10代后发现B38产蛋白酶和纤维素酶能力保持稳定,证明诱变菌株具有较好的遗传稳定性;(4)利用凯氏定氮法评价B38降解饲料蛋白的能力,发现与BC2相比,B38降解饲料中可溶性蛋白的能力提高了2.57倍,而降解不溶性蛋白的能力变化不大。本研究诱变选育的枯草芽孢杆菌B38为开发优良的水产微生态制剂产品提供了重要的前提和基础。

UV-mutated breeding of Bacillus subtilis strain with high protease ac-tivity and evaluation of its substrate degradation ability

Modern high-intensity aquaculture has seen a rapid increase in annual cultured production over recent decades, and it is essential to deal with the amount of waste produced. Aquaculture effluent is typically characterized by increased nitrogen species (ammonia, nitrites and nitrates), organic carbon, phosphates, suspended solids, and high biological oxygen demand and chemical oxygen demand. Probiotics are defined as live microbial or cultured product feed supplements that beneficially affect water quality or the host. For example, some Bacillus subtilis strains can degrade ammonia, nitrites or nitrates and are important for water manipulation in intensive aquaculture. As a result, the use of probiotics in aquaculture is gaining increasing scientific and commercial interest worldwide. A strain with high protease activity, which can degrade protein or nitrogen, is key to its commercial application in probiotics production. To breed a B. subtilis strain with high protease activity, an original strain (B. subtilis BC2) was mutated using different doses of ultraviolet (UV) radiation, and a preliminary study determined the optimal UV radiation conditions. The value of flat transparent circle to colony diameter (H/C) was used to estimate the UV mutation effect on B. subtilis. The results showed that the H/C value of mutated strain B38 increased from 3.01 to 6.42. A spectrophotometry method with Forint phenol (Lowry) was used to measure the protease activity of the mutated strain. The protease activity of B38 was 86.82 U/mL, which was increased by 3.14 times compared with the original strain. After ten successive generations, the protease activity of B38 was 77.01 U/mL and did not change significantly, showing its good genetic stability. Cellulase activity analysis was used to determine whether the UV mutagenesis also affected other enzyme activity. The result showed that the cellulase activity of B38 was not significantly different from BC2. Subsequently, we evaluated the ability of mutated strain B38 to degrade feed protein. Protein content in the feed medium was assayed by the Kjeldahl method. The liquid protein degradation ability of B38 was an increase of 2.57 times compared with original strain BC2. However, the ability of B38 to degrade solid protein did not increase significantly. The UV-mutated high protease-producing B38 strain provides an important foundation for the development and application of aquatic probiotics.