Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, P.O. Box 1177, Sfax 3018, Tunisia.
Laboratory of Microorganisms and Biomolecules (LMB), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, P.O. Box 1177, Sfax 3018, Tunisia.
Biomed Res Int. 2020 Jan 22;2020:3707804. doi: 10.1155/2020/3707804. eCollection 2020.
This study was designed with the aim to produce microbial proteases in presence of speckled shrimp by-product. For this reason, three strains belonging to genus, namely, VP3, C250R, and M1V were studied under co-culture procedure. A Taguchi L27 experimental design was applied to optimize the co-culture parameters. The experimental design was built with 9 factors (by-product powder concentration, the pH of the medium, the temperature, the sucrose concentration, the agitation speed, the inoculum sizes of VP3, M1V, and C250R strains, and the culture volume) at three different levels. The obtained results showed that a total protease activity of 8,182 U/mL could be achieved after 24 h of incubation in presence of 20 g/L shrimp by-product and 10 g/L sucrose, at an initial pH of 7, a 40°C temperature and absorbance, at 600 nm, of inoculum sizes of 0.1, 0.3, and 0.1 for VP3, M1V, and C250R strains, respectively. The agitation was set at 200 rpm, and the final volume was 25 mL. Taguchi's design allowed the identification of temperature, the inoculum size for strain VP3, the inoculum size for strain M1V, and the final culture volume as the most influencing variables. A Box-Behnken design with 27 experiments was carried out for the optimization of these four selected factors. Following such design, the highest protease production reached was 11,300 U/mL. This yield was obtained in a final culture volume of 15 mL containing 20 g/L shrimp by-product powder and 10 g/L sucrose and inoculated with VP3, C250R, and M1V strains at 0.05, 0.1, and 0.2, respectively. The flasks were incubated at 45°C for 24 h with shaking at 200 rpm. The efficiency of chitin extraction by co-cultivation was investigated under the latter conditions. The chitin yield from shells by-product was 16.7%. Fourier-Transform Infrared (FTIR) analysis of the obtained chitin displayed characteristic profiles similar to that of the commercial -chitin.
本研究旨在利用斑节对虾副产物生产微生物蛋白酶。为此，研究了属于 VP3、C250R 和 M1V 属的三种菌株在共培养过程中的情况。应用 Taguchi L27 实验设计优化共培养参数。该实验设计由 9 个因素（副产物粉末浓度、培养基 pH 值、温度、蔗糖浓度、搅拌速度、VP3、M1V 和 C250R 菌株的接种量以及培养体积）在三个不同水平下构建。结果表明，在 20 g/L 虾副产物和 10 g/L 蔗糖存在下，初始 pH 值为 7，温度为 40°C，接种量为 0.1、0.3 和 0.1（VP3、M1V 和 C250R 菌株），培养 24 小时后可获得 8182 U/mL 的总蛋白酶活性。搅拌速度设置为 200 rpm，最终体积为 25 mL。Taguchi 的设计确定了温度、VP3 菌株的接种量、M1V 菌株的接种量和最终培养体积为最具影响力的变量。进行了 27 次实验的 Box-Behnken 设计，以优化这四个选定因素。根据该设计，最高的蛋白酶产量达到 11300 U/mL。该产量是在最终培养体积为 15 mL 的情况下获得的，其中含有 20 g/L 虾副产物粉末和 10 g/L 蔗糖，VP3、C250R 和 M1V 菌株的接种量分别为 0.05、0.1 和 0.2。在 200 rpm 摇床中于 45°C 下孵育 24 小时。在后者的条件下，研究了共培养提取壳聚糖的效率。从虾壳副产物中提取的壳聚糖产量为 16.7%。对获得的壳聚糖进行傅里叶变换红外（FTIR）分析，显示出与商业壳聚糖相似的特征谱。
