甜叶菊糖基转移酶UGT76G1的克隆表达及其性质研究

目的:利用甜叶菊糖基转移酶UGT76G1特异性催化甜菊糖中含量高并且具有较强后苦味的甜菊甙生成高甜度的莱鲍迪甙A。方法:将合成的UGT76G1编码基因插入pYES2载体的EcoRⅠ和XhoⅠ酶切位点之间,成功构建了pYES2-UGT重组质粒。重组质粒导入表达宿主酿酒酵母YPH499中,利用2%半乳糖对重组菌进行诱导表达。结果:确定了最佳诱导时机为菌体培养后48h,诱导表达时间为12h。并对重组酶粗酶液性质进行了初步研究,确定其最适反应pH为8.0,最适反应温度为40℃,最佳反应时间为36h。结论:为建立经济高效的生物催化法对甜菊糖口味改质奠定了基础。      

重组酿酒酵母全细胞催化合成莱鲍迪苷A

用经密码子优化后合成的甜叶菊UDP糖基转移酶UGT76G1编码基因构建了表达该酶的重组酿酒酵母YPH499(pYES2-UGT)。建立了通过柠檬酸钠调节酿酒酵母细胞内由葡萄糖到尿苷二磷酸葡萄糖(UDPG)的代谢通量,用于催化甜菊苷合成莱鲍迪苷A的全细胞催化方法。优化后的催化体系为:甜菊苷1 g/L,葡萄糖20g/L,普郎尼克F68 10 g/L,MgCl2 6 g/L,柠檬酸钠15 g/L,pH 7.2,细胞密度200 g湿细胞/L反应液,在37℃下经72 h后转化生成莱鲍迪苷A 267.89 mg/L。     

酿酒酵母组成型表达甜叶菊糖基转移酶UGT76G1及相关性质研究

本研究将酿酒酵母穿梭质粒p ESC-Leu的诱导型启动子GAL1和GAL10分别替换为PGK1和TEF1启动子,构建了组成型双启动子表达载体p ESCD,再将甜叶菊糖基转移酶UGT76G1的编码基因亚克隆到p ESCD的Sal I和Xho I酶切位点之间,构建了组成型表达UGT76G1的重组质粒p ESCD-UGT。将p ESCD-UGT转化到酿酒酵母YPH499中进行表达,结果确定该重组酵母在SD-L液体培养基培养24h达到稳定期,菌体培养8h蛋白表达量高,选用1%的甲苯作为重组菌全细胞催化的通透剂时,催化15h产生288mg/L的莱鲍迪甙A,其产量是对照组的5倍。      

环糊精包埋的白藜芦醇对小鼠肝脂代谢相关基因表达的影响

为了研究白藜芦醇(Res)的降血脂功能的分子机制,建立了小鼠高脂模型,在建模的过程中,采用高、中、低3种浓度的白藜芦醇灌胃。实验结果表明,高脂饲料使模型组小鼠血脂水平明显升高(p<0.05);高、中、低剂量组均不同程度降低小鼠的血脂水平,尤以对TC影响最为显著(P<0.01);RT-PCR结果显示高剂量组的白藜芦醇能在一定程度下调ACC1的表达,中剂量能明显上调CPT-1和CYP7A1的表达水平。因此,用环糊精包埋的白藜芦醇能够降低小鼠的血脂水平;可能作为信号分子参与到脂代谢信号转导途径,并且与其参与肝脏内脂代谢基因CYP7A1、ACC1、CPT-1的表达调控有一定相关性。     

微波预处理提取大豆异黄酮的研究

本文以低温脱脂豆粕为原料,利用微波对其进行前处理。通过四个单因素实验选择最佳实验条件。采用四因素三水平的正交实验确定大豆异黄酮提取比较合适的条件是:微波处理15s,65%的乙醇水溶液,1:15的料液比,50℃下提取45min。提取率可达到0.78%。试验结果表明,适当的微波处理可以提高大豆异黄酮的溶出率,是一种有效的处理方法。     

Effect of soybean varieties on the content and composition of isoflavone in rice-koji miso

To clarify the effect of soybean varieties on isoflavone, a useful component for human health, in soybean products, we investigated changes in the isoflavone content and composition in rice-koji miso, after fermentation/aging for 6 or 12 months using varieties of soybeans (Tohoku-126, Tohoku-135, Tohoku-139, Suzuyutaka and Chinese soybeans), by high performance liquid chromatography. In soybeans, the total isoflavone content in Tohoku-126 was 444 mg/100 g, which was 1.2–2.0 times the content in the other soybean varieties. The malonyl glycosides and aglycones in soybeans accounted for more than 60% and only a few percent, respectively. As for rice-koji miso, the total isoflavone and aglycone contents were the highest in miso prepared from Tohoku-126. The ratios of glycosides to aglycones (80.1–92.6%) in miso were higher than those in the original soybeans. The time course of the isoflavone composition during the fermentation/aging process of rice-koji miso indicated that glycosides decreased from 86.4% to 44.9% after 6 months but aglycones increased from 9.6% to 53.3%.     

脱脂在异黄酮分析中的作用

以染料木素(Genistein)和黄豆苷原(Daidzein)为代表分析了在提取过程中除去脂类过程对大豆异黄酮分析的影响。用染料木素来表征大豆制品中的异黄酮较为合适,用正己烷除油的效果好。     

功能性成分异黄酮的来源与生理功能

本文从异黄酮的来源、分布与结构、生理功能等几方面简述了异黄酮研究的概况。     

Skim milk powder supplementation affects lactose utilization, microbial survival and biotransformation of isoflavone glycosides to isoflavone aglycones in soymilk by Lactobacillus

Four probiotic bacteria, Lactobacillus acidophilus 4461, L. acidophilus 4962, Lactobacillus casei 290 and L. casei 2607, were used for fermentation of soymilk (SM) prepared from soy protein isolate (SPI) supplemented with skim milk powder (SMP) (SSM). Soymilk and reconstituted skim milk (RSM) were used as controls. Lactose utilization in SSM by these probiotic organisms ranged from 14.97 to 18.15mg/ml, compared to 14.12-16.06mg/ml for RSM. The pH in SSM dropped to 4.07-4.29 compared to 6.15-6.36 for SM and 4.10-4.96 for RSM. The microbial viable counts were also significantly enhanced by up to 0.98logCFU/ml by the supplementation of SMP to SM. The biotransformation level of isoflavone glycosides (IG) to isoflavone aglycones (IA) in SSM ranged from 81.4% to 85.1%, which was 13.9-19.0% higher than that for SM, after 24h of incubation. Most of IG in SSM was completely converted to IA, except malonyl glycitin and malonyl genistin. At the end of the incubation, IA comprised up to 76.8% of total isoflavones in SSM.     

Isoflavones reduce inflammation in 3T3-L1 adipocytes

Isoflavones are widely consumed and they have been attributed beneficial effects. We have explored how genistein, daidzein and equol affect the adipocyte functions of glucose uptake and the secretion of inflammatory molecules in 3T3-L1 adipocytes inflamed with TNF-α.     

维生素A的毒理学研究进展

维生素A作为人体必需的维生素具有很多生理功能。维生素A作为一种预防疾病的手段,伴随其产生的问题越来越多,维生素A摄入过多造成急性或慢性中毒、肝脏毒性、生殖毒性、发育毒性及致畸性等。维生素A的毒性作用应引起人们的关注。本文总结了维生素A的功能及代谢,重点关注了过量维生素A及其代谢物对细胞或个体造成的毒害作用。     

DNJ对糖脂代谢机理的影响研究进展

DNJ是一种哌啶类多羟基生物碱,具有多种生理活性,是目前功能食品、医药等行业的研究热点。本文对DNJ的基本性质及其对糖脂代谢机理的影响等研究进展进行综述,并提出未来的发展方向,以期为广大科研工作者的进一步研究提供一定的理论参考。      

Isoflavone profiles of soymilk as affected by high-pressure treatments of soymilk and soybeans

High hydrostatic pressure was applied to hydrated soybeans (100–700 MPa, 25 °C) and soymilk (400–750 MPa; 25 and 75 °C) to assess its effect on isoflavone content, profile and water-extractability. Neither pressure level nor initial treatment temperature affected soymilk isoflavone content. However, combined pressure and mild thermal treatment modified the isoflavone distribution. At 75 °C, the isoflavone profile shifted from malonylglucosides toward β-glucosides, which was correlated to the effect of adiabatic heating. When pressure was applied to the hydrated soybeans, the soymilk isoflavone concentration varied between 4.32 and 6.06 μmol/g. The content of protein decreased and fat increased in soymilks prepared from pressurized soybeans with increasing pressure level.     

超声波对蛋清蛋白酶解的研究

以蛋清蛋白和Alcalase为原料,研究了超声波功率对蛋清蛋白酶解的水解度的影响,进行了有、无超声波作用下蛋清蛋白酶解的优化实验。结果表明,40kHz、125W的超声波处理,能有效地促进蛋清蛋白的酶解,使其水解度提高13%～35.48%。超声波作用不影响蛋清蛋白的其他酶解条件,优化工艺参数为pH是9.15,加酶量(E/S,即酶相对于底物蛋白质质量)是4.85%,温度是70℃,且各因素对蛋清蛋白水解度影响的大小顺序一致,为pH>T>E/S。     

Mushroom tyrosinase inhibitory effects of isoflavones isolated from soygerm koji fermented with Aspergillus oryzae BCRC 32288

The inhibition of mushroom tyrosinase in soygerm koji, fermented with Aspergillus oryzae BCRC 32288, was investigated. A methanol extract of the soygerm koji was partitioned into hexane, ethyl acetate and water. The ethyl acetate extract showed potent anti-tyrosinase activity with an IC₅₀ value of 0.19 mg/ml. The active compounds were isolated by activity-guided silica gel column chromatography and high-performance liquid chromatography (HPLC) methods. Seven tyrosinase inhibitors were purified and identified as 6,7,4′-trihydroxyisoflavone, 7,8,4′-trihydroxyisoflavone, 5,7,8,4′-tetrahydroxyisoflavone, 7,4′-dihydroxyisoflavone (daidzein), 6-methoxy-7,4′-dihydroxyisoflavone (glycitein), 4′-hydroxyisoflavone-7-O-glucoside (daidzin), and 5,4′-dihydroxyisoflavone-7-O-glucoside (genistin) by comparing their mass, ¹H NMR, and ¹³C NMR spectral data with those in the literature. The purified seven isoflavones from fermented soygerm koji were divided into two groups, based on their inhibitory effects on mushroom tyrosinase. Five isolated isoflavones showed inhibitory activity against monophenolase activity of mushroom tyrosinase only, with IC₅₀ values of 0.009 ± 0.001 (6,7,4′-trihydroxyisoflavone), 0.203 ± 0.018 (daidzein), 0.218 ± 0.007 (glycitein), 0.267 ± 0.008 (daidzin), and 0.343 ± 0.013 (genistin) mM. The kinetic study indicated that the five inhibitors significantly lengthened the lag time of the monophenolase activity of tyrosinase and acted competitively for the l-tyrosine binding site of the enzyme. So, the five isoflavones were competitive inhibitors for the monophenolase activity of tyrosinase. The other two isoflavones, 7,8,4′-trihydroxyisoflavone and 5,7,8,4′-tetrahydroxyisoflavone, inhibited both monophenolase and diphenolase activities of tyrosinase. Moreover, pre-incubation of each of the two isoflavones with tyrosinase resulted in total irreversible inhibition of the enzyme activity, even at concentrations as low as of 10 μM. Hence, 7,8,4′-trihydroxyisoflavone and 5,7,8,4′-tetrahydroxyisoflavone were irreversible inhibitors of mushroom tyrosinase.