Optimization Of Extraction Process And Activity Function Of Sulforaphane From Broccoli

Multiple epidemiological studies have shown that high daily consumption of cruciferous vegetables can reduce the risk of cancer.Sulforaphane exists in cruciferous plants in the form of glucoraphane,which is hydrolyzed by myrosinase under certain conditions.Sulforaphane is the main active ingredient in cruciferous vegetables and has a good development prospect.In this study,sulforaphane was extracted from broccoli and cabbage to study its antioxidant and other active functions,providing theoretical basis for its processing and application in food industry.In this study,sulforaphane was extracted from broccoli and cabbage.Response surface methodology was used to optimize the extraction conditions of sulforaphane,and the obtained sulforaphane was enriched and purified.The sulforaphane myrosinase was hydrolyzed to obtain sulforaphane,and the properties of sulforaphane were studied.Concrete research content and the experimental results are as follows:experiment choice of enzymatic hydrolysis time,enzyme solution temperature,solution pH and cellulase add four factors in single factor experiment.According to the single factor test results,the central combination test was designed.The extraction amount of sulforaphane was taken as the response value,and the optimal process parameters of sulforaphane were obtained by response surface analysis.The results showed that the optimal extraction conditions of sulforaphane were as follows:enzymatic hydrolysis time 7.1 h,enzymatic hydrolysis pH 6.0,enzymatic hydrolysis temperature 36.8?,and the amount of cellulase 2.5%.Under these conditions,the extraction amount of sulforaphane can reach 408.74±0.832 g/g.Under scanning electron microscopy,the cellulase treated samples showed a more complete degree of cell fragmentation,which was more conducive to the release of glucosin and myrosinase to produce sulforaphane.The contents of sulforaphane in different broccoli and cabbage varieties from the same batch produced in Liuhe Experimental Base of Jiangsu Academy of Agricultural Sciences were detected.The results showed that the contents of sulforaphane in 9 broccoli germplasm resources varied from 224.36±35.29 to 438.33± 15.63?g/g DW.The content of sulforaphane in germplasm Lvbao 70 was the highest(429.33± 15.63 ? g/g DW),which was 1.9 times that of 18B173.The content of sulforaphane in 7 cabbage germplasm resources varied from 34.25± 12.3 to 481.28± 16.35 ?g/g DW.The content of sulforaphane in germplasm resource GZ-1 was the highest,which was 481.28± 16.35 ?g/g DW,and the content of sulforaphane in germplasm resource GZ-7 was the lowest,which was 34.25±12.3 ?g/g DW.Compared with broccoli,the content of sulforaphane in different varieties of cabbage was significantly different,and the content of sulforaphane in most of cabbage varieties was low.Broccoli and cabbage both have the highest content of sulforaphane in usable parts.The variation trend of ???? content in both broccoli and cabbage was consistent.The content was high at the seedling stage,but decreased sharply during the development of the four-leaf stage.In the subsequent growth stage,???? gradually accumulated,so the content gradually increased,and finally reached the highest content at the heading stage,which was higher than that at the seedling stage.The crude sulforaphane extract was eluted and collected by semi-preparative high performance liquid phase.The structure of sulforaphane was analyzed by UPLC-MS,and it was confirmed that the compound was sulforaphane.In the process of producing sulforaphane by enzymatic hydrolysis,sulforaphane can only reach the highest activity under the optimum conditions.The properties of sulforaphane specific enzyme were studied.The molecular weight of myrosinase in broccoli was 35 kDa by SDS-PAGE gel electrophoresis.The optimum pH value is 6.2,and the pH value is stable between 5.8 and 6.8.Under strong acid or alkaline conditions,the stability of myrosinase decreases rapidly,indicating that broccoli myrosinase is neither acid nor alkali resistant.Secondly,the optimal temperature of the enzyme was about 60?,but the stability of the enzyme showed that broccoli myrosinase had the best stability at about 20?,and then with the increase of temperature,myrosinase activity began to decline.The Km value was 3.43 mmol/L with potassium glucosinolate as substrate.The activity of myrosinase was about 2 times when Immol/L vitamin C was added.The antioxidant properties of carrot sulfur were systematically analyzed by chemical and cellular antioxidant methods.For chemical antioxidant,the total reducing power of carrot sulfur was analyzed and its scavenging effect on three free radicals-ABTS+radical,DPPH-radical scavenging and hydroxyl radical was measured,the results show that sulforaphane total reducing power and vitamin C close,although ABTS+radical scavenging capacity and DPPH radical scavenging ability more vitamin C than slightly,but still to prove that sulforaphane has stronger antioxidant ability,is conducive to the development of antioxidant food.The antioxidant activity of sulforaphane in Hep-G2 cells was determined by CAA method,and the antioxidant activity of sulforaphane increased with the increase of sulforaphane concentration.MTT assay was used to determine the effect of sulforaphane on the growth inhibition rate of Hep-G2 cells,the effects of different concentrations of sulforaphane on Hep-G2 hepatoma cells were quantitatively evaluated by flow cytometry.The results showed that sulforaphane could induce apoptosis obviously,and the rate of apoptosis and necrosis increased with the increase of sulforaphane dose.