Extraction, Purification, Determination And Enzyme Hydrolysis Of Rapeseed Meal Sinapine

Rapeseed meal are the second most widely traded protein ingredients after soybean meal, there are about 600-700 million tons of rapeseed meal in china each year. With the advance of double-low rapeseed, rapeseed meal phenolics become one of the most important anti-nutritional factors. Sinapine is one of the most important polyphenols in rapeseed meal, rapeseed meal content 1.2%-2.3% sinapine,it's prone to dissolved in water, sinapine acid and choline are easy formed by hydrolysis.Sinapine is the main reasons of bitterness, but also can produce brown-shell eggs, fish smell. I studied the extraction process, not only can solve the rapeseed meal feed, the problem of poor palatability, but also for deep processing and utilization of rapeseed meal, the biological activity of rapeseed meal sinapine provide the basis for research and application.(1) Ultraviolet spectrophotometry(326nm) method and visible light spectrophotometry (388nm) method were used to determine the content of rapeseed-meal-sinapine. Through the comparison of precision and recovery, the visible light spectrophotometry (388nm) was selected.(2)I conducted a preliminary study through rapeseed meal sinapine extraction process, by single-factor test and Box-Behnken optimization tests to determine the hot alcohol extract optimum combination of reflux as follows:extraction time is 3.9h, extraction temperature is 85.5℃, solvent ratio is 3:5, solid-liquid ratio is 1:26, under those conditions, rapeseed meal sinapine extraction rate is 1.59%.Through the single factor and orthogonal tests, I determined the optimal extraction process of the accelerated solvent extraction instrument (Accelerated Solvent Extractor, for short ASE) as follows: extraction temperature is 140℃, static extraction time is 15min, extraction times are 3 times,the sample amount is 3g,under those conditions, rapeseed meal sinapine extraction rate is 1.88%.The quality of rapeseed meal that extracted by two different post-extraction was tested, results showed that:The main anti-nutritional factors greatly reduced, while the protein and amino acid content was significantly increased.The ASE extraction of rapeseed meal quality is best, in which the crude protein content of raw materials increased by 18.46% compared with the crude fiber increased by 26.65%, glucosinolate, total phenols were not detected, phytic acid reduced 17.05%, sinapine content only10.14% of raw materials.Soxhlet extracted by the total amino acid content of rapeseed meal than the raw materials increased 22.60%, after the total amino acid content after ASE extraction increased 26.72%; by Soxhlet extracted by the total essential amino acid content of rapeseed meal 17.70%, extracted by the ASEThe total amino acid content of rapeseed meal increased 21.25%.(3) By D-101, DK-16, NKA-9, AB-8 macroporous resin adsorption rate of four and desorption rate measurement, I filtered out the DK-16 is more suitable for purification of rapeseed meal sinapine resin.Selected the DK-16 purified rapeseed meal sinapine the optimum:70%(v/v) ethanol solution to do eluant, pH value is 7.0, the sample flow rate is 2BV/h, elution concentration of 90%(v/v), flow rate is 2BV/h, the best adsorption time is 120min, the best elution time is 90min.The concentration of product after vacuum drying up to 49.89%.Thiocyanate precipitate through the dregs mustard base, I obtained purity sinapine thiocyanate samples, re-crystallization of the more the number the higher the purity, the best crystallization of the frequency of three times, the purity is 94.54%.(4) Using UV, IR, HPLC, HPLC-ESI/MS and other means of modern analytical testing purified rapeseed meal sinapine conducted in-depth study of the identification.(5) From the polyphenol oxidase, laccase,β-glucosidase, lipase enzymes, I found that laccase have a higher degradation rate of rapeseed meal sinapine.by a single factor and Box-Behnken optimization tests, I got the optimum parameters were:enzymolysis time 25min, pH5.10, enzymes adding amount 2mL, hydrolysis temperature is 28℃, at this time laccase degradation of rapeseed meal sinapine degradation rate of 81.93%.We have studied the laccase enzyme Michaelis-Menten constant of rapeseed meal sinapine:laccase at 30℃, the optimum pH, under the condition of rapeseed meal sinapine substrate KM values were 1.16mol/L, maximum enzyme reaction rateVmax= 2.38A/(U* min).