Key Laboratory of Green Process and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, People's Republic of China.
Appl Biochem Biotechnol. 2012 Dec;168(7):1753-64. doi: 10.1007/s12010-012-9894-2. Epub 2012 Sep 18.
This study presents a novel and integrated preparation technology for nattokinase functional food, including strain screening, fermentation, separation, and encapsulation. To rapidly screen a nattokinase-productive strain, PCR-based screening method was combined with fibrinolytic activity-based method, and a high productive strain, Bacillus subtilis LSSE-22, was isolated from Chinese soybean paste. Reduction of poly-γ-glutamic acid (γ-PGA) concentration may contribute to separation of nattokinase and reduction of late-onset anaphylaxis risk. Chickpeas were confirmed as the favorable substrate for enhancement of nattokinase production and reduction of γ-PGA yield. Using cracked chickpeas, the nattokinase activity reached 356.25 ± 17.18 FU/g (dry weight), which is much higher than previous reports. To further reduce γ-PGA concentration, ethanol fractional extraction and precipitation were applied for separation of nattokinase. By extraction with 50 % and precipitation with 75 % ethanol solution, 4,000.58 ± 192.98 FU/g of nattokinase powders were obtained, and the activity recovery reached 89 ± 1 %, while γ-PGA recovery was reduced to 21 ± 2 %. To improve the nattokinase stability at acidic pH condition, the nattokinase powders were encapsulated, and then coated with methacrylic acid-ethyl acrylate copolymer. After encapsulation, the nattokinase was protected from being denatured under various acid conditions, and pH-responsible controlled release at simulated intestinal fluid was realized.
本研究提出了一种新颖的纳豆激酶功能食品的综合制备技术，包括菌株筛选、发酵、分离和包封。为了快速筛选纳豆激酶产生菌，本研究将基于 PCR 的筛选方法与纤维蛋白溶解活性筛选方法相结合，从中国豆酱中分离出一株高产纳豆激酶的枯草芽孢杆菌 LSSE-22。降低聚γ-谷氨酸（γ-PGA）浓度有助于纳豆激酶的分离和降低迟发性过敏反应的风险。鹰嘴豆被证实是提高纳豆激酶产量和降低γ-PGA产量的理想底物。使用破碎的鹰嘴豆，纳豆激酶的活力达到 356.25 ± 17.18 FU/g（干重），明显高于以往的报道。为了进一步降低 γ-PGA 浓度，采用乙醇分步萃取和沉淀法分离纳豆激酶。通过 50%乙醇萃取和 75%乙醇溶液沉淀，得到 4000.58 ± 192.98 FU/g的纳豆激酶粉末，活力回收率达到 89±1%，而 γ-PGA 的回收率降低至 21±2%。为了提高纳豆激酶在酸性 pH 条件下的稳定性，将纳豆激酶粉末进行包封，然后用甲基丙烯酸乙酯共聚物进行涂层。包封后，纳豆激酶在各种酸条件下不会变性，在模拟肠液中实现了 pH 响应的控制释放。
