Accumulation And Extraction Of Isothiocyanates In Rapeseed Meal By Myrosinase And Its Bacteriostatic Acivity

Rapeseed meals were byproducts produced by oil extraction,which were rich in proteins,amino acids and other nutrients.However,anti-nutrients in the meals such as glucosinolates,phytic acid and polyphenols limit the application of rapeseed meal in food and sector.Many papers reported that isothiocyanates were the degradation products of glucosinolates,were not only spicy flavor contained in cruciferous vegetables and mustard condiments,but had biological activities in plant defence,auxin homeostasis and human cancer prevention.The research showed the effect of glucosinolate hydrolysis by myrosinase to promote the formation of isothiocyanates.Comparison of myrosinases from Brassicaceae seeds brought out different glucosinolate degradation components in rapeseed meal,and the source of exogenous myrosinase was got.The process conditions of glucosinolates degrading to isothiocyanates and extraction of isothiocyanates were optimized.Compared with different elutions,the butenyl isothiocyanates were separated from glucosinolates degradation products.A preliminary study on the bacteriostatic action and mechanism of butenyl isothiocyanates were carried out in this work.The results were as follow:1.The components and relative content of glucosinolates in the rapeseed meal degraded by these different myrosinases from 7 kinds of cruciferous seeds were determined with the method of gas chromatography-mass spectrometry(GC-MS).Butane,1-isothiocyanate was as the new compound found in the products of White cabbage seeds myrosinase,Kale seeds myrosinase,Mustard seeds myrosinase and Cabbage seeds myrosinase,which relative content were similar to each other.The highest content(33.22%)of 1-Butene,4-isothiocyanate was found in the products by Cabbage seeds myrosinase,which was more than the content of 32.20%by Rape seeds myrosinase.Besides,this compound in the same experimental conditions was not detected in the degraded products by Mustard seeds myrosinase and Radish seeds myrosinase.Comparing with the products by Rape seeds myrosinase,the other six myrosinases added to rapeseed meal increased four new kinds of oxazoles and three new kinds of nitriles.The oxazoles were 2,4-dimethyl-Thiazole,2-Ethyl-4-methylthiazole,5-Methoxy-4-methyl-2,1,3-benzothiadiazo le,2,1-Benzisoxazole,2-Cyanomethyl-1,3-benzothiazole.Three nitriles were Butanenitrile,5-Cyano-1-pentene,Thiolane-3,4-dicarbonitrile,1H-Indole-3-acetonitrile.Under same condition,the glucosinolates degradation products genereted by Rape seeds myrosinase and other myrosinases were significantly different.It can be concluded that Rape seeds myrosinase was the appropriate myrosinase source to degrade the glucosinolates in the rapeseed meal.2.The isothiocyanates content were increased significantly by exogenous myrosinase,which was 5.5-8.2 times than the isothiocyanates content by endogenous myrosinase in rapeseed meal.Results showed that the myrosinase had the highest activity under the reaction system of citric acid-phosphate buffer,and by adding 20%of exogenous myrosinase to rapeseed meal,the optimal enzymolysis conditions were as follows:ratio of solid to liquid was 1:15,pH was 6.0,the reaction temperature was 50 ?,amount of ascorbic acid 0.005 mg/g,the degradation time was 1h.In this condition,the isothiocyanates content was 1.799 mg/g,which were 7.64 times than these by the autolysis in rapeseed meal.Seven compounds 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.Besides,1-Butene,4-isothiocyanate had the highest content compared with the other compounds and accounted for 26.77%in the detected products.3.The ultrasonic-assisted solvent method was applied to extract of isothiocyanates which were the products of glucosinolates degraded by myrosinases from rape seed.The orthogonal array was adapted to the experiment according to the single factor experiments.The optimum extracting conditions of total isothiocyanates were as follows:ultrasonic temperature 45 ?,ultrasonic time 40 min,solid-liquid ratio 1:12.5,ultrasonic power 60 W,with of the total isothiocyanates content 2.812mg/g.Three eluents were analyzed to comfirm the eluents to silica gel column chromatography.Butenyl isothiocyanates were separated from extract of the products of glucosinolates by using immiscible solvent extraction with petroleum ether-ethyl acetate,hexane-ethyl acetate.Butenyl isothiocyanates separated with petroleum ether-ethyl acetate were puried than separated with hexane-ethyl acetate.Volume ratio of petroleum ether-ethyl acetate was 30:1,20:1,10:1.The purity of butenyl isothiocyanates could be reached 93.1%.The structure of butenyl isothiocyanates was determined by GC-MS.4.The result showed that butenyl isothiocyanates had obvious bacteriostatic action upon Escherichia coli,Staphylococcus aureus and Lactobacillus.Gram-positive Staphylococus aureus was more sensitive to butenyl isothiocyanates than the others.The results of bacteriostatic experiment of butenyl isothiocyanates indicated that their minimum inhibitory concentration(MIC)on Staphylococcus aureus was 16?g/mL.The other compounds sepatated by silica gel column chromatography did not have antimicrobial activity against tested bacterial.It was indicated that the butenyl isothiocyanates might change the permeability of cell membrane,inhibited the activity of of succinate dehydrogenase and malate dehydrogenase in TCA cycle,and had some effects on synthesized quantities of some proteins.