摘要
【目的】研究影响油菜籽饼粕中硫代葡萄糖苷在黑芥子酶作用下定向转化为异硫氰酸酯的因素,确定最佳酶解条件,探索外加黑芥子酶对硫代葡萄糖苷降解产物组分和含量的影响,为油菜籽饼粕的综合利用提供基础。【方法】测定4种反应体系中黑芥子酶活性,确定酶解反应缓冲体系。以油菜籽饼粕中异硫氰酸酯总量为指标,单因素结合正交设计研究不同料液比、时间、温度、pH、抗坏血酸添加量对外加黑芥子酶降解油菜籽饼粕中硫代葡萄糖苷转化为异硫氰酸酯的影响,优化工艺参数,获得最佳酶解条件。采用二氯甲烷为提取剂,真空浓缩得到油状降解产物,利用气相色谱-质谱(GC-MS)联用鉴定外加黑芥子酶和内源黑芥子酶酶解产物成分,分析产物相对含量。【结果】4种缓冲体系中,在柠檬酸-磷酸盐缓冲液中测得黑芥子酶活性最高,在相同条件下,外加黑芥子酶的活性显著高于内源黑芥子酶活性;外源黑芥子酶添加量大于油菜籽饼粕量的20%,异硫氰酸酯生成量无显著增加,选择外源黑芥子酶添加量为油菜籽饼粕量的20%;正交优化得到最佳酶解条件为:料液比1﹕15,pH为6.0,反应温度为50℃,抗坏血酸添加量为0.005 mg·g-1,反应时间为1 h,异硫氰酸酯含量达到1.799 mg·g-1,是油菜籽饼粕内源黑芥子酶降解硫代葡萄糖苷生成异硫氰酸酯产物总量的7.6倍。外加黑芥子酶生成异硫氰酸酯含量显著高于内源黑芥子酶降解硫苷生成异硫氰酸酯含量,但两者异硫氰酸酯含量随反应条件变化趋势一致。GC-MS鉴定外加黑芥子酶有7种酶解产物,相对含量在产物的67%以上,含有1种腈类,2种噁唑烷酮和4种异硫氰酸酯。产物中异硫氰酸酯为烯丙基异硫氰酸酯、丁烯基异硫氰酸酯、环戊基异硫氰酸酯和苯乙基异硫氰酸酯;其中丁烯基异硫氰酸酯的含量最高,占酶解产物总量的26.77%。腈类产物为苯丙腈;2种噁唑烷酮为致甲状腺肿素和2,1-苯丙噁唑烷酮。内源黑芥子酶降解硫代葡萄糖苷的产物中检测到4种产物且含量相对较少。【结论】外加黑芥子酶能有效增加油菜籽饼粕中异硫氰酸酯生成量,确定了油菜籽饼粕中硫代葡萄糖苷的最佳降解条件。鉴定出降解产物的主要成分,异硫氰酸酯有4种,其中丁烯基异硫氰酸酯含量最高。
[Objective] The aim of this study was to figure out the optimal factors for directional conversion of glucosinolates to isothiocyanates in rapeseed meal degraded by endogenous and exogenous myrosinase and explore the impact of exogenous myrosinase on the components and content of glucosinolates degradation products. It will provide a basis for the comprehensive utilization of rapeseed meal. [ Method] Myrosinase activity was evaluated in 4 kinds of buffer solutions to establish a better reactionsystem. The effects of different enzymolysis conditions included solid liquid ratio, hydrolysis time, reaction temperature, pH value, and amount of ascorbic acid on the conversion of glucosinolates in rapeseed meal to isothiocyanates by endogenous and exogenous myrosinase were investigated through single factors and orthogonal array design methods. The optimal condition for the conversion was obtained by analyzing the yield of isothiocyanates. Taking methylene chloride as the extraction agent, the degradation products were concentrated in rotary evaporator. The products from endogenous and exogenous myrosinase were detected by gas charomatographic mass spectrometry （GC-MS）. The kind and content of compounds from the products were monitored. [Result] Results showed that among the four reaction systems, the myrosinase had the highest activity under citric acid-phosphate buffer and under the same conditions, exogenous myrosinase activity was significantly higher than that of the endogenous myrosinase. The content of isothiocyanates had no obvious increase when exogenous myrosinase was more than 20% of rapeseed meal, and it was good to add 20% exogenous myrosinase of rapeseed meal to rapeseed meal. The optimal enzymolysis conditions were as follows： ratio of solid to liquid was 1：15, pH was 6.0, reaction temperature was 50~C, amount of ascorbic acid 0.005 mg.g1, the degradation time was lh. Under this condition, the isothiocyanates content was 1.799 mg.g1 which were 7.64 times than these by the autolysis in rapeseed meal. The isothiocyanates content generated by exogenous myrosinase was significantly higher than that by endogenous myrosinase, but both contents had the same trend with the reaction conditions. Seven compounds from exogenous myrosinase were identified by GC-MS from the enzymatic hydrolysates of glucosinolates and accounted for more than 67% of the extracted degradation products which included 1 kind of nitrile and 2 kinds of oxazolidinones and 4 kinds of isothiocyanates. Allyl isothiocyanate, 1-butene,4-isothiocyanate, cyclopentyl isothiocyanate and benzene, and 2-ethyl- isothiocyanate were obtained among glucosinolate degradation products. Besides, 1-butene,4-isothiocyanate had the highest content compared with the other compounds and accounted for 26.77% in the detected products. The nitrile was benzenepropanenitrile. Goitrin and 2,1-benzisoxazole were the two oxazolidinones. Four compounds were analyzed in degradation products from endogenous myrosinase and had lower content than the seven compounds detected in the products from exogenous myrosinase. [ Conclusion ] This study indicated the isothiocyanates content increased significantly by exogenous myrosinase. It can also be concluded from the experiments the optimal enzymolysis condition for glucosinolates and the main compounds of enzymatic hydrolysates of glucosinolates from rapeseed meal. 1-Butene,4-isothiocyanate had the highest content in the 4 kinds of isothiocyanates analyzed in the test.
出处
《中国农业科学》
CAS
CSCD
北大核心
2014年第2期383-393,共11页
Scientia Agricultura Sinica
基金
国家自然科学基金项目(31271912)
江苏省优势学科和六合英才计划项目
