HPLC法测定豆粕中大豆异黄酮的含量

采用索式抽提法从豆粕中提取大豆异黄酮，并用HPLC法对大豆异黄酮各组分进行了定性、定量分析。定性检测出4种异黄酮组分：大豆苷、染料木苷、丙二酰基大豆苷以及丙二酰基染料木苷。测定了各组分在脱脂豆粕中的含量。解决了异黄酮中缺乏标准品的丙二酰基型异黄酮糖苷的定量测定问题，采用水解转化法建立了丙二酰基型异黄酮糖苷的定量测定方法，线性度良好，测量准确，且简便易行。测得河北某油脂厂脱脂豆粕中总异黄酮的含量为3．714mg／g。     

固定化β-葡萄糖苷酶转化糖苷型异黄酮的研究

利用固定化β-葡萄糖苷酶把糖苷型异黄酮水解成苷元型异黄酮,可以提高大豆异黄酮的生理活性.用海藻酸钙包埋富含β-葡萄糖苷酶的黑曲霉孢子,可以方便有效地固定β-葡萄糖苷酶.研究考察了不同底物浓度,pH和温度对固定化β-葡萄糖苷酶酶解作用的影响,以及重复分批酶解条件下固定化酶的稳定性.当固定化酶珠体积占反应总体积的5%,糖苷型异黄酮浓度为1.2mg·mL-1,作用24 h,酶解效果良好.其中,大豆苷比染料木苷易于被酶解.固定化酶适宜的pH范围为3～5,最适pH值为4.8.耐热性比固定化前有所增加,在70℃以下酶较稳定.重复分批酶解糖苷型异黄酮,连续7批的转化率均可保持在90%以上.该研究结果在大豆异黄酮的生物转化方面具有潜在的应用前景.     

纤维素酶协同超声波法提取豆粕异黄酮的研究

采用纤维素酶与超声波相结合的方法研究豆粕异黄酮提取,经纤维素酶和超声波处理后,豆粕异黄酮提取量显著增加,达到8.34 mg·g-1,相对提高51.63%.经分析,超声波、纤维素酶对提取豆粕异黄酮都有明显的促进作用,为超声波、酶生物技术用于提取大豆异黄酮走向工业化提供了一定的理论和技术依据.     

里氏木霉纤维二糖水解酶Ⅱ基因的高表达

纤维二糖水解酶II(CBH II)是纤维素酶的重要组分之一,对纤维素酶的水解性能有着重大影响,而里氏木霉(Trichoderma reesei)纤维素酶制剂中纤维二糖水解酶II明显不足,为了优化其酶系结构,采用了基因重组技术构建CBH II高产菌株:将里氏木霉CBH II基因置于里氏木霉强启动子Pcbh1(及其信号肽)和终止子Tcbh1之间,并进一步以pCAMBIA1300为载体骨架,构建成含潮霉素B抗性标记的重组质粒pCAMBIA1300-hph-PsCT。以里氏木霉ZU-02为宿主,采用根瘤农杆菌介导转化技术将重组质粒转入宿主分生孢子。以潮霉素B为抗性标记初筛到324个阳性转化子,进一步通过复筛,在以微晶纤维素为唯一碳源的筛选培养基上获得8个生长较快的优良转化子。在摇瓶条件下,分别对8个转化子进行产酶试验,培养48 h时,纤维二糖水解酶活力最高可达18.24 U·mL-1,是出发菌株的2.51倍。本结果对于里氏木霉纤维素酶的定向进化、提高其对纤维素的协同糖化效率具有重要意义。     

里氏木霉QM9414尿嘧啶缺陷型转化体系改进和β-葡萄糖苷酶的过量表达

里氏木霉是广泛应用于纤维素酶生产的工业真菌,但高产突变株遗传操作困难,限制了菌种改良。首先利用定点整合策略敲除里氏木霉突变株QM9414的pyr4基因,成功构建尿嘧啶营养缺陷型菌株QP4,其遗传转化效率显著提高,而且产酶能力不受影响。进一步在QP4中过量表达β-葡萄糖苷酶(BGL)基因bgl1,经大量平板显色筛选获得2株BGL活力明显增强的工程菌QPB4和QPB5,其酶活分别提高10.01倍和8.26倍。利用发酵酶液对2种不同预处理的玉米芯底物进行水解糖化实验,结果显示以酸处理玉米芯为底物时QPB4和QPB5的葡萄糖得率比QP4分别提高60.98%和52.44%,而以脱木素处理玉米芯为底物时其葡萄糖得率分别提高80.01%和86.00%。研究表明改进里氏木霉高产突变株遗传转化体系可以显著促进菌株改良,提高糖化应用效果。     

固定化β-葡萄糖苷酶双相体系中水解大豆异黄酮

采用共价交联的方法将β-葡萄糖苷酶固定到球形壳聚糖上,并对固定化酶的性质进行表征,得出固定化酶的最佳反应条件：pH=5,温度40℃。据此用乙酸乙酯和pH=5缓冲溶液的双相体系,在40℃条件下水解大豆异黄酮。与游离酶相比,固定化酶在此双相体系中起到了稳定酶的作用;与单相体系相比,双相体系中产物的产率显著提高,反应速率也更快,且能有效去除粗品大豆异黄酮的异味。对于30%左右级别的大豆异黄酮,水解的两个主要水解产物（大豆苷元和染料木素）的产率都能达到70%。     

用大孔树脂从大豆乳清中纯化大豆异黄酮

引言 大豆异黄酮是大豆生长中形成的一类次生代谢产物.大豆异黄酮由于具有弱雌激素活性、抗氧化活性、抗溶血活性,能有效地预防和治疗癌症、骨质疏松、妇女更年期综合征等多种疾病,因此在保健食品和医药中有广泛的应用[1-4].大孔吸附树脂是一类新型的高分子分离材料,具有化学性质稳定、选择性吸附、再生简便等许多优点,在天然产物的分离纯化方面其应用日趋广泛[5-6].     

重组里氏木霉粗酶液提取虎杖中白藜芦醇的研究

为从虎杖中提取白藜芦醇并同步实现高效转化,经平板初筛和48 h的试管产酶复筛,从619株里氏木霉的基因重组菌中定向筛选出3株高产β-葡萄糖苷酶的菌株(BG-2,BG-4和BG-5)。摇瓶条件下产酶48h,三者的β-葡萄糖苷酶酶活为1.75,3.50和5.71 IU×m L~(-1),分别是出发菌株的36倍、73倍和119倍。以三者的发酵粗酶液直接处理虎杖粗提物,45℃条件下反应5 h后,与出发菌株相对比,重组菌株对白藜芦醇的提取率可达14.86~16.41 mg×g~(-1),是出发菌株的2.41~2.66倍;转化率可达120.5%~138.4%,是出发菌株的6.0~6.85倍,且反应液中基本无白藜芦醇苷残留。该研究策略从根本上提高了纤维素酶法对苷元型白藜芦醇的提取效率,可推广应用于其他易糖苷化的中草药成分的提取。     

利用葡萄糖转苷酶制备纤维素酶可溶性诱导物的研究

在纤维素酶生产中,常用的诱导物纤维素是不溶性的固体高分子化合物,存在着传质阻力大、不易于流加培养、产酶效率低等问题.今以葡萄糖为原料,利用葡萄糖转苷酶的催化作用,定向合成纤维素酶的可溶性诱导物.经高效液相色谱分析,发现转糖苷产物中含有纤维素酶的强诱导物槐糖.在50℃下,转糖苷反应的适宜葡萄糖浓度为300～500 mg·mL-1,pH 3～4.5,反应100 h,产物中槐糖含量可达40 mg·mL-1.将葡萄糖经转苷酶作用后的复合物用于纤维素酶的生产,与直接采用葡萄糖相比,产酶时间提前25 h,滤纸酶活力提高14倍.该研究结果为酶法制备纤维素酶的高效可溶性诱导物探明了一条新途径,对于提高纤维素酶的生产效率、加速其工业化应用具有重要意义.     

氧化石墨烯的制备及其催化水解大豆异黄酮

采用改良的Hummers法制备了负载有-SO3H、-COOH和-OH的氧化石墨烯,对其进行了结构表征。并首次以氧化石墨烯为催化剂,对天然产物大豆异黄酮糖苷水解反应进行了研究。实验结果显示氧化石墨烯在异黄酮糖苷水解反应中具有较好的催化活性,其最佳反应温度为105℃,此时3种大豆异黄酮糖苷的转化率分别达到94.3%,92.1%和88.8%,苷元的收率分别达到69.6%、60.6%和58.8%。其催化活性明显优于其他固体酸催化剂,如沸石HZSM-5和大孔树脂NKA-9,而与0.02 mol.L.1的硫酸相当。     

大豆异黄酮的纯化工艺研究

采用大孔吸附树脂、硅胶、葡聚糖凝胶及反相硅胶RP-18等4种填料的柱层析法纯化大豆异黄酮。结果表明,四种层析填料的纯化效果均较好,大豆异黄酮纯度达30％以上,其中以葡聚糖凝胶为填料的柱层析法分离纯化效果最佳,纯度可达55．45％,提取率为90％。     

超声波提取大豆异黄酮的工艺研究

大豆异黄酮是一种从大豆种子中分离出的具有异黄酮类化合物结构的主要活性成分.广泛应用于食品及医药行业.利用超声波法提取大豆种子中大豆异黄酮,摸索了各种工艺参数对大豆异黄酮提取效果的影响.结果表明,乙醇浓度为75%,料液比为1∶20,提取时间为40 min,为大豆异黄酮的最佳提取工艺.     

大豆异黄酮纯化研究现状

大豆异黄酮(SIF)是大豆中的一类活性物质,具有较显著的生理功能,因此引起了广泛关注.基于色谱法、萃取法、大孔树脂吸附法、膜分离法及几种技术联用的纯化精制方法,对近年来SIF的分离纯化方法进行了归纳,为进一步合理高效的分离纯化SIF提供参考.     

MATE-Type Proteins Are Responsible for Isoflavone Transportation and Accumulation in Soybean Seeds

Soybeans are nutritionally important as human food and animal feed. Apart from the macronutrients such as proteins and oils, soybeans are also high in health-beneficial secondary metabolites and are uniquely enriched in isoflavones among food crops. Isoflavone biosynthesis has been relatively well characterized, but the mechanism of their transportation in soybean cells is largely unknown. Using the yeast model, we showed that GmMATE1 and GmMATE2 promoted the accumulation of isoflavones, mainly in the aglycone forms. Using the tobacco BrightYellow-2 (BY-2) cell model, GmMATE1 and GmMATE2 were found to be localized in the vacuolar membrane. Such subcellular localization supports the notion that GmMATE1 and GmMATE2 function by compartmentalizing isoflavones in the vacuole. Expression analyses showed that GmMATE1 was mainly expressed in the developing soybean pod. Soybean mutants defective in GmMATE1 had significantly reduced total seed isoflavone contents, whereas the overexpression of GmMATE1 in transgenic soybean promoted the accumulation of seed isoflavones. Our results showed that GmMATE1, and possibly also GmMATE2, are bona fide isoflavone transporters that promote the accumulation of isoflavones in soybean seeds.     

Simultaneous saccharification and fermentation of alkaline-pretreated corn stover to ethanol using a recombinant yeast strain

Bio-ethanol converted from cheap and abundant lignocellulosic materials is a potential renewable resource to replace depleting fossil fuels. Simultaneous saccharification and fermentation (SSF) of alkaline-pretreated corn stover for the production of ethanol was investigated using a recombinant yeast strain Saccharomyces cerevisiae ZU-10. Low cellobiase activity in Trichoderma reesei cellulase resulted in cellobiose accumulation. Supplementing the simultaneous saccharification and fermentation system with cellobiase greatly reduced feedback inhibition caused by cellobiose to the cellulase reaction, thereby increased the ethanol yield. 12 h of enzymatic prehydrolysis at 50 °C prior to simultaneous saccharification and fermentation was found to have a negative effect on the overall ethanol yield. Glucose and xylose produced from alkaline-pretreated corn stover could be co-fermented to ethanol effectively by S. cerevisiae ZU-10. An ethanol concentration of 27.8 g/L and the corresponding ethanol yield on carbohydrate in substrate of 0.350 g/g were achieved within 72 h at 33 °C with 80 g/L of substrate and enzyme loadings of 20 filter paper activity units (FPU)/g substrate and 10 cellobiase units (CBU)/g substrate. The results are meaningful in co-conversion of cellulose and hemicellulose fraction of lignocellulosic materials to fuel ethanol.     

Anti-photoaging effects of soy isoflavone extract (aglycone and acetylglucoside form) from soybean cake

Soy isoflavones, found in soybean and soybean products, have been reported to possess many physiological activities such as antioxidant activity, inhibition of cancer cell proliferation, reduction of cardiovascular risk, prevention of osteoporosis and alleviation of postmenopausal syndrome. In our previous study, soy isoflavone extract ISO-1 (containing 12 soy isoflavones) from soybean cake was demonstrated to prevent skin damage caused by UVB exposure. In this study, soy isoflavone extract from soybean cake was further purified and evaluated for the protective effects on UVB-induced damage. The results revealed that Fraction 3, which contains the aglycone group (daidzein, genistein and glycitein) and acetylglucoside group (acetyldaidzin, acetylgenistin and acetylglycitin) of soy isoflavones, could inhibit UVB-induced death of human keratinocytes and reduce the level of desquamation, transepidermal water loss (TEWL), erythema and epidermal thickness in mouse skin. Furthermore, topical application of Fraction 3 increased the activity of catalase and suppressed cyclooxygenase-2 (COX-2) and proliferating cell nuclear antigen (PCNA) expression in mice exposed to UVB. In addition, in comparison with ISO-1 and genistein, the Fraction 3 possessed much greater protective effects on both UVB-induced oxidative stress and keratinocyte death than other fractions. Therefore, the soy isoflavone extract Fraction 3 from soybean cake is a desirable anti-photoaging agent for skin care.     

Enhanced extraction of isoflavones from Korean soybean by ultrasonic wave

The amounts of isoflavones extracted from Korean soybean by various ultrasonic waves were compared using 60% aqueous ethanol solution. The effect on extraction yield of variations in solvent composition, temperature, and extraction time was investigated. The experimental results confirmed that ultrasonic waves are a desirable method to extract isoflavones from Korean soybean. The highest yield of aglycone isoflavones was obtained by ultrasonic waves with a frequency of 20 KHz and an extraction time of 10 min, which produced yields of glycoside and aglycone isoflavones three-fold greater than those by dipping method.     

Isoflavone content among maturity group 0 to II soybeans

This study reports the isoflavone contents of 210 soybean cultivars grown in South Dakota and explores possible relations between isoflavone contents and agronomic characteristics. Total isoflavone contents (normalized) ranged from 1161 to 2743 μg/g. A number of agronomic characteristics were documented for each variety including maturity group, hilum color, disease resistance, seed weight, yield, maturity (in days), and plant height. Varieties in maturity group I had significantly higher total isoflavones when compared to maturity group 0. Hilum color was related to differences in genistin, daidzein, and genistein content. No differences in isoflavone content were observed based on disease resistance profiles. Genistein content was found to be negatively correlated with yield, days of maturity, and plant height. Weak but significant correlations also existed between these agronomic characteristics and other isoflavones.