大豆豆脐中异黄酮的提取工艺优化

为了提高大豆豆脐在大豆加工行业的利用率以及更大程度地发挥大豆豆脐的营养价值,本文在国标大豆异黄酮检测方法的基础上,建立了一套适用于测定大豆豆脐中异黄酮含量的高效液相色谱方法。本实验以大豆豆脐为原料,采用乙醇-水溶液作为提取溶剂,通过单因素实验和正交实验确定超声波辅助提取大豆异黄酮的最佳工艺,结果表明:各因素对大豆异黄酮提取率影响大小的顺序为:提取温度(B)>提取时间(C)>料液比(D)>乙醇浓度(A);在乙醇浓度为80%、提取温度为80℃、提取时间为1.5h、料液比为1:35 g/mL时提取三次,大豆异黄酮的提取率可达10.88±0.120 mg/g。本方法准确、高效,能够提取出原料中90%以上的大豆异黄酮,该提取工艺稳定可行,可为大豆豆脐中异黄酮的提取提供理论依据。     

Systematic evaluation of pre-HPLC sample processing methods on total and individual isoflavones in soybeans and soy products

There are mainly two protocols in isoflavone analysis, one that involves hydrolysis prior to HPLC analysis and the other direct HPLC analysis. In this study, three different hydrolysis methods were systematically re-evaluated, and compared with direct HPLC analysis. Acidic hydrolysis (1.2-3 M HCl in ethanol at 80 °C) showed a maximum conversion of ca. 92% from glucosides to aglycones in 2 h; however, longer reaction caused degradation of genistein. Alkaline hydrolysis using 2 M NaOH converted acetylglucosides and malonylglucosides to their respective glucosides within 10 min. Glucuronidase from H. pomatia effectively converted isoflavone glucosides and acetylglucosides to aglycones. Quantification of the total isoflavones in various soy food products showed no significant difference among direct injection and the three hydrolysis methods (P < 0.05). We conclude that direct analysis of isoflavones in crude extracts is a rapid and accurate method to obtain isoflavone profiles and compositions in soybean, soy foods and beverages.     

Chromatographic fingerprints and quantitative analysis of isoflavones in Tofu-type soybeans

Tofu-type soybeans can differ from conventional soy varieties in seed size, seed composition, flavour and nutrition. Using HPLC coupled with ESI-MS and PDA detection, a total of 19 isoflavones were detected and identified from Tofu-type soybean seeds, more than previously reported forms from many other soybeans. An HPLC/UV fingerprint study was performed for qualitative evaluation, which enabled the isoflavone profile of Tofu-type soybeans to be characterised and differentiated from other vegetative soybeans by similarity comparison. To meet the frequent quantitative application for isoflavone contents, a simple, precise and reliable method using HCl hydrolysis during sample extraction and LC/UV for the detection was developed and validated to quantitate total isoflavones in soybeans, and then applied to determine the total isoflavone contents of different Tofu-type soy varieties grown in different field locations over two growing seasons. A range of total isoflavone contents for Tofu-type soybeans was established to confirm the use of this analytical approach for quality control applications.     

碱-低压复合法对大豆苷元转化的影响

以市售大豆异黄酮粉为原料进行碱水解,在碱水解最优条件下探究低压对大豆苷元转化的影响。采用高效液相色谱法测定大豆苷元和葡萄糖苷产量,分别以葡萄糖苷增长率和苷元转化率为指标,以碱水解时间、温度、pH值和低压作用时间、料液比、压力为因素,通过单因素试验考察各个因素参数独立变化时水平对指标的影响,再以响应面法研究各因素及其交互作用对指标影响,优化工艺条件。结果表明：碱水解最优条件为pH?11.5、温度63?℃、时间49?min,葡萄糖苷型大豆异黄酮的增长率可达286.40%。在此基础上进行低压处理,压力0.25?MPa、料液比1∶120（g/mL）、时间10?min,苷元转化率达16.65%。     

Isoflavone profiles,phenol content,and antioxidant activity of soybean seeds as influenced by cultivar and growing location in Ohio

Isoflavone levels and isoflavone chemical composition in soybeans vary between planting locations although the exact factors which control isoflavone biosynthesis are unclear. We compared levels of 12 isoflavones in soybean seeds of six cultivars grown in four different locations in Ohio in 2002 as determined by high‐performance liquid chromatography. Antioxidant activity contained in plant‐based foods can improve food oxidative stability and phenolics and isoflavones have proven active in food systems. Radical scavenging activity was assessed using the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical. Total phenolics (TPCs) were determined by using Folin–Ciocalteu reagent. Total isoflavones (TIs) varied five‐fold (1573–7710 nmol g^?1) between seeds from the various location–cultivar combinations. One location (Wooster, Ohio) produced seeds containing half the isoflavones as the other locations tested apparently due to poor growing conditions. The cultivars could be divided into two groups based on TI, one having approximately 50% more isoflavones. Surprisingly, across the entire data set, with increasing TI, the proportion of isoflavones accounted for by the daidzein family increased due primarily to malonyl daidzin. DPPH scavenging did not differ significantly by location or cultivar (P > 0.05) and did not correlate with TPC or TI. Profiling soybean isoflavones could help elucidate how isoflavone biosynthesis is regulated and lead to better disease resistance of soybean crops and soy foods with greater health benefits. Copyright © 2007 Society of Chemical Industry     

Determination of optimal acid hydrolysis time of soybean isoflavones using drying oven and microwave assisted methods

The aim of the present research was to determine an optimal acid hydrolysis condition using drying oven and microwave assisted methods to estimate isoflavone contents by RP-HPLC in soybean. All isoflavone glucosides were completely converted to their aglycones at 120 min for drying oven and 50 min for microwave. Optimal extraction time of the highest isoflavone aglycone content after acid hydrolysis was achieved in 3 h. These results indicated that the optimised hydrolysis and extraction conditions of isoflavones in soybean were: soybean (1 g) hydrolysed by 10 ml of 1 N HCl at 100 °C for 50 min using microwave assisted acid hydrolysis method, and then 15 ml of EtOH was added to the mixture which leaved alone for 3 h at room temperature for complete extraction. Thus, microwave is an easy, consumed less time, and reliable acid hydrolysis method to estimate soybean isoflavones in comparison with drying oven method.     

不同豆类中4种大豆异黄酮的HPLC法测定

建立同时测定豆类中4种大豆异黄酮成分含量的高效液相色谱法,探讨了该方法的最佳测定条件,结果显示,70％的乙醇在60℃超声提取40min时,效果最佳。该方法简便、灵敏、准确,适用于豆类中4种大豆异黄酮成分的含量测定和产品质量控制。     

Effects of riboflavin photosensitization on the changes of isoflavones in soymilk

Effects of riboflavin photosensitization on the distribution of isoflavones in commercially available soymilk were analyzed using high-performance liquid chromatography (HPLC). Total isoflavones (TI) in soymilk with riboflavin (1000 ppm, w/v) under light were significantly different from those stored in the dark for 24 h (P < 0.05), while TI in samples with 0 and 1000 ppm added riboflavin were not significant from each other in dark conditions (P > 0.05). To test the effects of the concentration of riboflavin on TI, soymilk was mixed with riboflavin to make 0, 100, 250, and 500 ppm (w/v) and stored under light at 25 degrees C for 24 h. TI in soymilk with 100 ppm riboflavin under light significantly decreased by 13.5% for 24 h (P < 0.05) compared to control samples and were not significantly different from those with 250 or 500 ppm samples (P > 0.05). Daidzin and genistin were predominant isoflavones in soymilk, and the rate of photo degradation of genistin was higher than that of daidzin for 24 h in soymilk under riboflavin photosensitization.     

Identification and determination of isoflavones in germinated black soybean sprouts by UHPLC−Q-TOF-MS mass spectrometry and HPLC-DAD

Ultra-performance liquid chromatography coupled with electrospray ionization tandem quadrupole time-of-flight mass spectrometry (UHPLC?Q-TOF-MS) and HPLC-DAD with an ultrasonic extraction method was developed for qualitative and quantitative compositions and contents in black soybean sprouts. Operational conditions of ultrasonic extraction were optimized by single-factor experiment. Phytochemical compositions of black soybean sprouts were identified by UHPLC?Q-TOF-MS in positive ion mode. A total of 23 compounds were tentatively characterized and identified by means of accurate mass and characteristic fragment ions. Among of them, the six isoflavones in different germinated black soybean sprouts were further quantified by HPLC-DAD. The results indicated that the calibration curves showed good linearity (r² > 0.9994). The intra-day and inter-day precision variations were less than 1.65% and 1.73%, respectively. The recoveries were in the range of 94.95–106.45%. The results indicated that the maximum amounts of isoflavones were produced on the fourth to fifth day germinated black soybean sprouts.     

发芽大豆中异黄酮的富集及其组成特征的研究

目的确定发芽大豆中异黄酮的最佳富集条件,并分析其组成特征。方法用紫外分光光度法测定大豆异黄酮含量;分析固定适宜的芽长条件,以浸泡时间、培养温度及培养湿度3个因素,设计3个水平进行正交试验,确定最佳富集条件;用高效液相色谱法分析最佳富集条件下大豆异黄酮的组成特征。结果发芽大豆芽长30 mm时,异黄酮最佳富集条件为浸泡时间16 h,培养温度25℃,培养湿度90%;最佳富集条件下大豆异黄酮主要由金雀异黄酮、黄豆苷元与黄豆黄素等单体成分组成。结论大豆通过优化发芽培养条件可以富集异黄酮,尤其可提高功能因子金雀异黄酮与黄豆苷元等单体含量,为开发富含大豆异黄酮的功能性大豆食品提供了一定的理论指导。     

Functional attributes of soybean seeds and products,with reference to isoflavone content and antioxidant activity

The concentrations of isoflavones, especially daidzein and genistein, were determined by high performance liquid chromatography in 4 soybean cultivars and 26 soybean products. The total isoflavone content of the soybean cultivars was in the range of 525–986 mg per kg, and for soy products it was 32.9–795 mg per kg. Amongst the soybean products, the isoflavone content decreased in the order: soy sprouts, soy seeds, soy flour, soy milk, soy meals and soy sauce. Significant differences in the concentration of genistein and daidzein were observed between the commercial soy products and also within the soybean cultivars. The antioxidant activity of soybean and soy products correlated well with total phenolic content (TPC) and total isoflavones (TI), whereas TPC showed higher correlation with TI.     

高效液相色谱法测定大豆提取物中大豆异黄酮的含量

本文建立了大豆提取物中大豆异黄酮的高效液相色谱测定方法,通过正交试验了样品水解最佳条件为１．０ｍｏｌ／ｌ ＨＣｌ－ＭｅＯＨ溶解,８０℃下,回流水解０．５ｈ,采用Ｎｏｖａ－Ｐａｋ Ｃ１８ ３．９＊１０ｍｍ４μｍ色谱柱ＭｅＯＨ－０．４％Ｈ３ＰＯ４（４７：５３Ｖ／Ｖ）为流动相,流速０．７ｍｌ／ｍｉｎ检测波长２６０ｎｍ等色谱条件下测定甙元含量,通过换算因子计算大豆异黄酮的含量。该方法快速,灵敏、重理性好,     

Effects of oven-drying,roasting, and explosive puffing process on isoflavone distributions in soybeans

Distributions of isoflavones in soybeans treated with oven-drying, roasting, or explosive puffing were analysed using high-performance liquid chromatography (HPLC). As oven-drying time increased from 0 to 120 min at 100 °C, concentration (μmol/g) of malonyl derivatives of isoflavones decreased and β-glucosides increased significantly with over 0.99 coefficient of determination (R²) (P < 0.05). Roasting at 200 °C for 7, 14, and 21 min and explosive puffing at 490, 588, and 686 kPa decreased malonyl derivatives significantly and increased acetyl-β-glucosides and β-glucosides significantly (P < 0.05). Total isoflavones (TI) in 21 min roasted and 686 kPa puffed soybeans decreased by 25.46% and 10.42%, respectively, while TI in 120 min oven-dried soybeans was not significantly different (P > 0.05) compared to untreated samples. Regression analysis showed that malonyl-β-genistin had higher slopes of decreases (μmol/g/min) than malonyl-β-daidzin in oven-dried soybeans. This is the first report on the effects of explosive puffing and the changes of isoflavone profiles in soybeans.     

微胶囊大豆异黄酮有效成分的HPLC含量测定

目的:为微胶囊大豆异黄酮的质量研究建立含量测定方法。方法:采用InertsilODS-3(150mm×4.6mm,5μm)色谱柱,1.5%乙酸-甲醇-乙腈(62:18:20)为流动相,检测波长260nm,柱温40℃,流速1.0ml/min。样品用70%的乙醇超声提取,经0.45μm滤膜过滤后进样。结果:HPLC过程于13min内完成,三种组分的分离度均大于1.5。对三批样品中大豆苷元、黄豆苷元和染料木黄酮作了分析,并与原料作了比较,微囊化前后含量呈比例关系。结论:方法简便、快速、准确,为本品以及仅含大豆异黄酮苷元类的保健食品质量控制提供了较为理想的方法。     

高温下丹贝异黄酮的抗氧化效应研究

丹贝异黄酮 (TI)是从大豆发酵食品丹贝 (tempeh)中分离到的一组异黄酮混合物。具有多种生物活性。文章中研究了TI的热稳定抗氧化活性 ,结果表明TI具有很好的热稳定性。在 180℃高温下 ,TI对猪油和菜籽油的抗氧化效果均显著优于其他抗氧化剂 ,在室温下 (37℃ )TI抗氧化作用与BHT和茶多酚 (TH)相当 ,但明显优于VE。TI还对由过渡金属离子 (Cu2 +、Fe2 +)引起的促氧化作用有明显的抑制效应 (p <0 .0 5 )。此外 ,TI还被作为抗化剂用于鸡腿油炸试验。     

Comparison of microbiological and HPLC – fluorescence detection methods for determination of niacin in fortified food products

A comparison has been made between results obtained by a recently published HPLC method, involving post-column reaction and fluorescence detection, with a microbiological assay using Lactobacillus plantarum. The HPLC method includes a modified acid hydrolysis extraction step (0.1 M HCl) and yields niacin values from fortified foods somewhat lower than by the microbiological assay. The most significant differences were observed for the cereal-based products. These differences arise principally from the lack of specificity of L. plantarum and from the stronger acid hydrolysis extraction conditions (1 M HCl) of the microbiological assay, which probably releases a part of the non-bioavailable niacin. Moreover by HPLC, excellent recoveries of added nicotinic acid and nicotinamide (95–100%) were obtained and better precision (RSDr=0.3–0.8%) was observed than from the micobiological assay.     

半化学酱油新工艺的研究

本文对多菌种半化学法生产酱油的新工艺进行了研究。采用响应曲面分析法(RSA),对豆饼的水解条件进行了优化,得到最佳水解条件为:液固比LR=3,温度T=110℃,时间t=30h,盐酸浓度为9.0％。 用GC-Head Space法结合其它方法对4种酵母进行了试验,结果认为Zygosaccharomyces SRO66性能优良。 酿造试验的结果显示最佳的发酵条件为:混合曲的添加量为所用豆饼量的30％,发酵3天后将发酵温度由50℃降30℃,随后以4％的接种量接入SRO66酵母,再经12天发酵得到产品。 感官鉴定的结果认为,产品优于所选的3种市售高档酱油。产品的含盐量为9.8％,氨基氮(AN)=1.10g/100ml,总氮(TN)=1.79g/100ml。蛋白质利用率和氨基酸游离率分别达到90.5％和61.50％。     

Quercetin content in some food and herbal samples

Quercetin is a typical flavonoid ubiquitously present in vegetables and fruits, and its antioxidant effect is implied to be helpful for human health. The efficiency of extraction process and acidic hydrolysis parameters for HPLC analysis of quercetin present in glycosides and aglycone forms was investigated. Hydrolysis for 5 min in the presence of 2.8 M HCl as well as for 10 min with 1.1 M HCl efficiently released quercetin from rutin. The method developed in this study was applied for quantitative determination of quercetin in some food (onion, apple) and herbal (Hypericum perforatum and Sambucus nigra) products.     

大豆异黄酮提取及其生物转化的研究进展

大豆异黄酮是大豆生长过程中形成的一种次级代谢产物,主要以糖苷和游离苷元的形式分布于大豆的子叶和胚轴中。研究表明,游离型大豆异黄酮具有许多重要的生理功能,诸如抗氧化、抗癌抑癌、保护心血管、预防骨质疏松及女性更年期综合症等。随着科学技术的进步,大豆异黄酮的应用日趋广泛。对国内外大豆中异黄酮的提取方法及其优缺点,以及由糖苷型大豆异黄酮转化为游离苷元的方法进行综述,以期为大豆异黄酮的应用研究提供帮助。     

A new ultra-high pressure liquid chromatography method for the determination of total isoflavone aglycones after enzymatic hydrolysis: Application to analyze isoflavone levels in soybean cultivars

Since only isoflavone aglycones are considered to be bioactive, the determination of total aglycones that are released from conjugated isoflavones after hydrolytic treatment may facilitate an objective alternative for quantifying isoflavone contents in soy products. Given this major benefit, a new ultra-high pressure liquid chromatography (UV-UPLC?) method was developed for the fast and reliable determination of total aglycones in soybeans (daidzein, glycitein, and genistein) after enzymatic hydrolysis applying helix pomatia digestive juice. Capitalizing on the enhanced performance of UPLC?, aglycones were separated within 3 min only, with a total runtime of 8 min till the next injection. Thus, especially compared to HPLC protocols, UPLC? proved to be superior due to significantly shorter runtimes and accordingly increasing sample throughput. Additionally, regarding the performed validation (linearity, precision, recovery, selectivity, and robustness), the established method proved to be suitable for quantifying total aglycones in soybeans. Moreover, method applicability was demonstrated by analyzing 23 commercial soybean cultivars for their isoflavone contents. Cumulative aglycone levels ranged from 100 to 255 mg per 100 g, hence implying an average ratio of 52%, 41%, and 7% of total isoflavones for genistein, daidzein and glycitein, respectively. However, for some soybeans, other distinct aglycone distributions were observed as well.