油菜籽饼粕中硫苷的酶解条件优化及降解产物分析

doi:10.3864/j.issn.0578-1752.2014.02.018

摘要/Abstract

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。

关键词: 油菜籽饼粕 , 硫苷 , 黑芥子酶 , 异硫氰酸酯 , GC-MS

Abstract: 【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 reaction system. 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℃, amount of ascorbic acid 0.005 mg•g-1, the degradation time was 1h. Under this condition, the isothiocyanates content was 1.799 mg•g-1 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.

Key words: rapeseed meal , glucosinolates , myrosinase , isothiocyanates , GC-MS

丁艳1, 李丽倩1, 曹蓉2, 唐根胜2, 顾振新1, 韩永斌1. 油菜籽饼粕中硫苷的酶解条件优化及降解产物分析[J]. 中国农业科学, 2014, 47(2): 383-393.

DING Yan-1, LI Li-Qian-1, CAO Rong-2, TANG Gen-Sheng-2, GU Zhen-Xin-1, HAN Yong-Bin-1. Effect of Enzymolysis Conditions on Glucosinolates in Rapeseed Meal and Identification of Their Degradation Products[J]. Scientia Agricultura Sinica, 2014, 47(2): 383-393.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2014.02.018

https://www.chinaagrisci.com/CN/Y2014/V47/I2/383

参考文献

[1]岳隆耀, 谯仕彦. 油菜籽饼粕营养价值总结修正[C]. 中国林牧渔业经济学会饲料经济专业委员会第五届学术交流会论文集, 北京: 中国农业出版社, 2008: 111-116.

Yue L Y, Qiao S Y. Nutritional value of rapeseed meal summary correction[C]. Chinese forest and fishery Economic Association Professional Committee of the Fifth Feed Economy Symposium, Beijing: China Agriculture Press 2008: 111-116. (in Chinese)

[2]Berot S, Compoint J P, Larre C. Large scale purification of rapeseed proteins (Brassica napusl L. ). Journal of Chromatogra Phy B, 2005, 818(1): 35-41.

[3]申跃宇, 尹佩辉, 莫放. 菜籽饼(粕)的抗营养因子及其对奶牛生产的影响. 中国草食动物, 2009, 29(2): 49-51.

Shen Y Y, Yin P H, Mo F. Effect of anti-nutritional factors in rapeseed meal on dairy. China Herbivores, 2009, 29(2): 49-51. (in Chinese)

[4]Verhoeven D T, Verhagen H, Goldbohm R A, van den Brandt P A. A review of mechanisms underlying anticarcinogenicity by brassica vegetables. Chemico- Biological Interactions, 1997, 103(2): 79-129.

[5]Moreno R L, Goosen T, Kent U M, Chung F L. Differential effects of naturally occurring isothiocyanates on the activities of cytochrome P450 2E1 and the Mutant P450 2E1 T303A. Archives of Biochemistry and Biophysics, 2001, 391(1): 99-110.

[6]Higdon J V, Delage B, Williams D E, Dashwood R H. Cruciferous vegetables and human cancer risk: epidemiologic evidence and mechanistic basis. Pharmacological Research, 2007, 55(3): 224-236.

[7]张清峰, 姜子涛, 李荣. 辣根等十字花科蔬菜中硫甙水解产物异硫氰酸酯的生物活性. 食品研究与开发, 2005, 26 (3): 83-88.

Zhang Q F, Jiang Z T, Li R. The biological activities of the hydrolytes(isothiocyanates) of glucosinolates in horseradish and cruciferous vegetables. Food Research and Development, 2005, 26(3): 83-88. (in Chinese)

[8]Fernando B L, Richard A H. Enzymatic inhibition by allyl isothiocyanate and factors affecting itsantimicrobial action against Escherichia coli O157: H7. International Journal of Food Microbiology, 2009, 131: 240-245.

[9]Ye L, Zhang Y. Total intracellular accumulation levels of dietary isothiocyanates determine their activity in elevation of cellular glutathione and induction of phase 2 detoxification enzymes. Carcinogenesis, 2001, 22(12): 1987-1992.

[10]Gu X, Dong W, He Y. Detoxification of rapeseed meals by steam explosion. Journal of the American Oil Chemists Society, 2011, 88(11): 1831-1838.

[11]Li J, Wei F, Dong X, Dong X Y, Guo L L, Yuan G Y, Huang F H, Jiang M L, Zhao Y D, Li G M, Chen Hong. Microwave-assisted approach for the rapid enzymatic digestion of rapeseed meal. Food Science Biotechnology, 2010, 19 (2): 463-469.

[12]朱文优, 刘军. 油菜籽粕金属盐脱毒工艺的研究. 饲料工业, 2012, 33(21): 21-22.

Zhu W Y, Liu J. The research on the metal detoxification technology of rapeseed meal. Feed Industry, 2012, 33(21): 21-22. (in Chinese)

[13]周锦兰, 胡健华, 裘爱泳. 菜籽饼乙醇脱毒脱油工艺研究. 中国油脂, 2004, 29(6): 40-44.

Zhou J L, Hu J H, Qiu AY. Rapeseed oil removal technology of alcohol detoxification. China Oils and Fats, 2004, 29(6): 40-44. (in Chinese)

[14]刘玉兰, 董秀云. 菜籽饼粕的生物化学法脱毒研究. 中国粮油学报, 1994, 9 (3): 49: 56.

Liu Y L, Dong X Y. The detoxification of biochemical methods on rapeseed meal. Journal of the Chinese Cereals and oils association, 1994, 9 (3): 49: 56. (in Chinese)

[15]陆豫, 余勃, 藏超, 杨胜远, 邓丽娟. 发酵菜籽粕脱毒工艺优化研究. 食品科学, 2007, 28(10): 267-270.

Lu Y, Yu B, Zang C, Yang S Y, Deng L J. Optimization of culture medium of multiple strains for reduction of toxicity in rapeseed meals. Food Science, 2007, 28(10): 267-270. (in Chinese)

[16]李燕, 熊巍, 高冰, 陶兴无. 多菌种混合发酵对菜籽饼脱毒效果的初步研究. 饲料工业, 2009, 30(20): 33-35.

Li Y, Xiong W, Gao B, Tao X W. The preliminary study on the multi-strain mixed fermentation detoxification effect of rapeseed meal. Feed Industry, 2009，30(20): 33-35. (in Chinese)

[17]楼洪兴, 卢福庄, 金水仙, 刘敏华, 张国海, 高振川, 姜云侠. 不同菜籽粕含量饲粮喂生长肥育猪效果-----对增重、饲料/增重、胴体品质和甲状腺功能的影响. 中国粮油学报, 1994, 2 (9): 60-64.

Lou X H, Lu F Z, Jin S X, Liu M H, Zhang G H, Gao Z C, Jiang Y X. Content of rapeseed meal fed different diets for growing and finishing pigs effect ----- weight gain, feed / gain, carcass quality and thyroid function. Journal of the Chinese Cereals and oils association, 1994, 2(9): 60-64. (in Chinese)

[18]徐建雄, 叶陈梁, 王晶, 李广良, 彭军. 双低菜籽粕对生长肥育猪生产性能和消化能的影响. 粮食与饲料工业, 2005, 3: 36-38.

Xu J X, Ye C L, Wang J, Li G L, Peng J. The effect of canola meal on productivity and digestibility of growing-fattening pigs. Cereal &Feed Industry, 2005, 3: 36-38. (in Chinese)

[19]Mullan B P, Pluske J R, Allen J, Harris D J. Evaluation of Western Australian canola meal for growing pigs. Australian Journal of Agricultural Research, 2000, 51(5): 547-553.

[20]Drew M D, Borgeson T L, Thiessen D L. A review of processing of feed ingredients to enhance diet digestibility in finfish. Animal Feed Science Technology, 2007, 138(2): 118-136.

[21]Burmeister W P, Cottaz S, Driguez H, Iori R, Palmieri S, Henrissat B. The crystal structures of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active site machinery of an S-glycosidase. Structure, 1997, 5(5): 663-675.

[22]Bones A M, Rossiter J T. The enzymic and chemically induced decomposition of glucosinolates. Phytochemistry, 2006, 67(11): 1053-1067.

[23]汪儆, 雷祖玉, 冯学勤, 姜云侠, 高振川. GB 13087—1991, 饲料中异硫氰酸酯的测定方法. 北京: 中国标准出版社, 1991.

Wang J, Lei Z Y, Feng X Q, Jiang Y X, Gao Z C. GB 13087 – 1991, Method for determination of isothiocyanatesin feeds. Beijing: Standards Press of China, 1991. (in Chinese)

[24]李培武, 张文, 丁小霞, 汪雪芳, 姜俊, 谢丽华. NY/T 1596—2008 油菜饼粕中异硫氰酸酯的测定硫脲比色法[S], 中华人民共和国农业部, 2008.

Li P W, Zhang W, Ding X X, Wang X F, Jiang J, Xie L H. NY/T 1596-2008 Determination of isothiocyanates in rapeseed meal--- Thiourea colorimetry method, Ministry of Agriculture of the People’s Republic of China, 2008. (in Chinese)

[25]李洁, 王清章, 周琦, 孙银, 谢笔钧. 萝卜中芥子苷酶的特性研究. 食品科学, 2008, 29(11): 451-455.

Li J, Wang Q Z, Zhou Q, Sun Y, Xie B J. Study on Properties of Myrosinase from Radish, Food Science, 2008, 29(11): 451-455. (in Chinese)

[26]周锦兰, 胡键华, 裘爱泳. 油菜籽中主要硫甙的分离提纯. 色谱, 2005, 23(4): 411-414.

Zhou J L, Hu J H, Qiu A Y. Separation and purification of the main glucosinolate from rapeseeds, Chinese Journal of Chromatography, 2005, 23(4): 411-414. (in Chinese)

[27]李合生. 植物生理生化实验原理和技术. 北京: 高等教育出版社，2001: 197-199.

Li H S. Plant Physiology and Biochemistry Principles and Techniques of Experimental., Beiing: Higher Education Press, 2001: 197-199. (in Chinese)

[28]刘月萍. 黑芥子酶提取分离、性质及固定化研究[D]. 杭州：浙江工商大学, 2007.

Liu Y P. The research on the extraction, characterization and immobilization of myrosinase[D]. Hangzhou: Zhejiang Gongshang University, 2007. (in Chinese)

[29]Cejpek K, Urban J, Velisek J, Hrabcova H. Effect of sulphite treatment on allyl isothiocyanate in mustard paste. Food Chemistry, 1998, 62(1): 53-57.

[30]Eridsson S, Ek B, Xue J P, Rask L, Meijer J. Identification and characterization of soluble and insoluble myrosinase isoenzymes in different organs of Sinapis alba. Physiologia Plantarum, 2001, 111(3): 353-364.

[31]Eridsson S, Andreasson E, Ekbom B, Graner G, Pontoppidan B, Taipalensuu J, Zhang J M, Rask L, Meijer J. Complex formation of myrosinase isoenzymes in oilseed rape seeds are dependent on the presence of myrosinase-binding proteins. Plant Physiology, 2002, 129(4): 1592-1599.

[32]Ghawi S K, Methven L, Rastall R A, Niranjan K . Thermal and high hydrostatic pressure inactivation of myrosinase from green cabbage: A kinetic study. Food Chemistry, 2012, 131 (4): 1240-1247.

[33]Jiang Z T, Zhang Q F, Tian H L, Li R. The reaction of allyl isothiocyanate with hydroxyl/water and β-cyclodextrin using ultraviolet spectrometry. Food Technology, 2006, 44(3): 423-427.

[34]Zhang Y, Wade K L, Prestera T, Talalay P. Quantitative determination of isothiocyanates, dithiocarbamates, carbon disulfide, and related Thiocarbonyl Compounds by cyclocondensation with 1, 2- benzenedithiol. Ananlytical Biochemistry, 1996, 239(2): 160-167.

[35]Pechacek R, Velisek J, Dacidek J. Decomposition of sinigrin by methanol/ammonia/water treatment in model systems and mustard (Brassica nigra L)seed meal. European Food Research Technology, 2000, 210: 196-201.

[36]Bjergegaard C, Møller, P, Sørensen H, Sørensen J C, Sørensen S. Micellar electrokinetic capillary chromatography of thiocarbamoyl derivatives produced in reactions between isothiocyanates and amino acids. Journal of Chromatography A, 1999, 836(1): 115-127.

[37]苏光耀, 沈莲清, 王向阳, 王奎武. 西兰花籽中硫代葡萄糖苷酶解条件的研究. 中国粮油学报, 2008, 23(2): 178-181.

Su G Y, Shen L Q, Wang X Y, Wang K W. Study on enzymatie hydrolysis conditions of glueosinolate in broccolini seeds. Journal of the Chinese Cereals and oils association, 2008, 23(2): 178-181. (in Chinese)

[38]陈虹霞. 辣根中异硫氰酸酯的制备和活性研究[D]. 北京: 中国林业科学研究院, 2009.

Chen H X. Study on preparation and bioactivity of isothiocyanates from horseradish[D]. Beijing: Chinese Academy of Forestry, 2009. (in Chinese)

[39]Rask L, Andreasson E, Ekbom B, Eddsson S, Pontoppidan B, Meijer J. Myrosinase: Gene family evolution and herbivore defense in Brassicaceae. Plant Molecular Biology, 2000, 42: 93-113.

[40]Ohta Y, Takatani K, Kawakishi S. Decomposition rate of allyl isothiocyanate in aqueous solution. Bioscience Biotechnology, 1995, 59(1): 102-103．

[41]Shikita M, Fahey J W, Golden T R, Holtzclaw D H, Talalay P. An unusual case of uncompetitive activation by ascorbic acid: purification and kinetic properties of a myrosinase from Raphanus sativus seedlings. Biochemical Journal of London, 1999, 341(3): 725-732.

[42]Burmeister W P, Cottaz S, Rollin P, Vasella A H B. High resolution X-ray crystallography shows that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic base. Journal of Biology Chemistry, 2000, 275(50): 39385-39393.

[43]Shen L Q, Su G Y, Wang X Y, Du Q Z, Wang K W. Endogenous and exogenous enzymolysis of vegetable-sourced glucosinolates and influencing factors. Food Chemistry, 2010, 199(3): 987-994.