Isolation And Characterization Of Sinapine Degrading Microbes And Their Degrading Mechanism

Rapeseed meal are the second most widely traded protein ingredients after soybean meal, there are about 600-700 million tons of rapeseed meal produced in china per year. With the promoting of double-low rapeseed (low sulfur glycosides, low erucic acid) process, rapeseed meal phenolics become one of the most important anti-nutritional factors. Sinapine is one of the most important phenolic compounds in rapeseed meal, belongs to simple phenols,rapeseed meal contains 1.2%-2.3% sinapine, prone to hydrolysis reaction when dissolved in water, generate sinapic acid and choline. Sinapine is the main reason for the bitter taste of rapeseed meal, but also can produce fish smell in brown-shell eggs, combined with protein or enzyme may reduce protein digestibility. Therefor, select the strain that can degradate rapeseed sinapine, study the enzyme of sinapine and it’s degradation, not only can solve the poor palatability of rapeseed meal. but also for deep processing and utilization of rapeseed meal, provide the basis for research and application of the biological activity in rapeseed sinapine.The main contents and the results of this paper are as follows:1. Thesis apply buried method, selected12 strains with the ability of to degrade sinapine from the rich rapeseed meal soil, Including 6 strains of mold,3 strains of yeast,3 strains of bacteria.Use the 12 strains with the 15 saved laboratory strains, solid-state fermentation of rapeseed meal under the same condations.4 strains were measured a higher degrading ability by the determination of phenols and sinapine content, for safety and convenience, mainly on one J-9 (enriched by the yeast culture medium, and rapeseed sinapine alternate culture medium) were studied, the strains were identified at first. J-9 strains wsa identified as saccharomyces cerevisiae by morphological observation and 18SrDNA sequence analysis.2. Determine the activity of several enzymes in saccharomyces cerevisiae yeast fermentation broth, the results are as follows:β-glucosidase activity was 7.68 IU (mean), laccase activity was 0.65 U, ethanol dehydrogenase 193.3U, aldehyde dehydrogenase 26.6U, Tyrosinase to 19.1U. Determine the enzymes produced by yeast were as follows:β-glucosidase, Tyrosinase, ethanol dehydrogenase, aldehyde dehydrogenase.3. Established the UPLC method to determine the content of sinapine, sinapic acid, through the precision test, stability test, repeatability test, recovery test, and linear relationship study, show that the method has good stability and reproducibility, and the sinapine,sinapine acid has a good resolution.4. useβ-glucosidase, Tyrosinase, ethanol dehydrogenase, aldehyde dehydrogenase 4 pure enzymes to degrde sinapine, by the sinapine content determine the enzymes that degrade the sinapine are:Tyrosinaseβ-glucosidase, the degradation rate reached 79.6%,17.65%.5. By examining the reaction temperature, time, substrate concentration, pH and other factors, sinapine degradations as investigate indicators, selected Box-Behnken experimental design to optimize the Tyrosinase optimal parameters to degrade sinapine are as follows:hydrolysis time 30.56min, Ph5.50, enzyme addition level 0.33mL, hydrolysis temperature is 57.27℃, then Tyrosinase enzymed sinapine degradation of rapeseed sinapine reached 84.93%.6.Optimized the fermentation conditions of polyphenol oxidase and P-glucosidase produced by selected saccharomyces cerevisiae. First, throughe the single factor and orthogonal experiment, received the optimal culture medium ratio, that is, molasses 3%, ammonium sulfate 0.6%, rapeseed addition amount 8%, ferrous sulfate 0.1%; optimized culture conditions:medium volume 80ml (300ml erlenmeyer flask), rotation speed 200r/min, culture temperature 30℃. The optimized medium and culture conditions encreased Tyrosinase activity nearly 5 times than before, at the second day of fermentation, Tyrosinase activity reached 93.31IU/mL,β-glucosidase activity reached 20.93IU/mL.7.Study the relationship between degradation rate and enzyme activity of sinapine by orthogonal, and found that enzyme activity is proportional to the degradation rate, using the best medium under the optimal culture conditions in fermentation, the sinapine degradation rate is significantly improved than before,increase from the previous 50.23% to 76.75%.