Comparison of extraction solvents and techniques used for the assay of isoflavones from soybean

The impact of extraction solvents and techniques on the assay of isoflavones from soybean was investigated. This systematic study was undertaken to address substantial variations in the solvents and procedures used for the extraction of isoflavones from soybeans by different research groups as described in the recent peer-reviewed published literature. Comparison of four previously optimized and commonly deployed solvent mixtures (acetonitrile:water 58:42 (v/v); ethanol:water 70:30 (v/v); methanol:water 90:10 (v/v); superheated pressurized water) was carried out for the extraction of isoflavones. In addition, we also examined the extraction efficiencies of three additional new solvent mixtures (dimethyl sulphoxide:acetonitrile:water, 5:58:37 (v/v/v); dimethyl sulphoxide:ethanol:water, 5:70:25 (v/v/v); Genapol:water 5:95 (v/v)) for the extraction of isoflavones from soybeans. Assessments of six commonly used extraction techniques (shaking, vortexing, sonication, stirring, Soxhlet, and pressurized liquid extraction (PLE)) with an optimized extraction solvent mixture was also performed. Both, the total isoflavones content and the isoflavones HPLC profile varied significantly with different extraction solvents and techniques. Optimum total isoflavones recoveries from soybean samples were obtained with dimethyl sulphoxide:ethanol:water (5:75:25, v/v/v) solvent mixture using a PLE. Intermediate extraction recoveries of total isoflavones from soybean samples were obtained with the other extraction solvent mixtures and techniques tested. The extraction efficiencies of isoflavones with shaker, vortex, stirring, and Soxhlet were between 65% and 70% as compared to PLE. Total isoflavones extracted by the sonication procedure was 93.3% as compared to PLE.     

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。     

发酵豆粕中大豆异黄酮的超声波提取工艺

为提高大豆异黄酮的提取率，以发酵豆粕为原料，采用超声波提取技术，通过单因素实验及正交实验对大豆异黄酮提取工艺中提取溶剂、料液比、时间、次数等因素进行探讨。结果表明：采用80％乙醇为提取剂，料液比1g：15ml，提取时间20min，提取2次，大豆异黄酮提取率最高，可达到0．548％。超声波提取发酵豆粕中大豆异黄酮是一种较为理想的方法。     

豆粕中大豆总异黄酮的提取工艺优化

主要研究豆粕中大豆总异黄酮的醇提工艺,并用HPLC法测定大豆总异黄酮含量。以大豆总异黄酮为提取目的,并通过对各单因素的考察和正交试验对工艺进行优化。结果表明,大豆异黄酮醇提工艺的最佳条件为:乙醇浓度70%,料液比1∶15(mg/mL),提取温度80℃,提取4 h,分2次提取,第1次提取时间为3 h,第2次提取1 h,总提取量是16.11 mg/g。     

发酵豆粕中大豆异黄酮提取条件的优化

旨在优化发酵豆粕中大豆异黄酮的提取条件,通过单因素试验,确定从发酵豆粕中提取大豆异黄酮的最佳提取条件是:60％乙醇,料液比200g/L,室温下提取60 min,提取2次.此条件下异黄酮提取总量为2855.485 μg/g.     

大豆异黄酮糖苷及其苷元调节免疫功能的比较研究

目的:比较大豆异黄酮糖苷及游离型大豆苷元对小鼠免疫功能能力的影响。方法:将实验室提取大豆异黄酮糖苷和苷元(纯度为41%左右)的产品按照40～100mg/kg,连续灌胃21d后,与对照组相比能显著提高小鼠腹腔巨噬细胞的吞噬能力及小鼠脾重。结果:大豆异黄酮糖苷和苷元具有较强的调节小鼠腹腔巨噬细胞的吞噬能力,能刺激小鼠脾脏和胸腺器官的发育;同一剂量的大豆异黄酮糖苷和苷元雌鼠的效果要好于雄鼠,游离型大豆苷元的吸收效果明显好于糖苷。     

三波长比色法测定大豆异黄酮的研究

大豆胚芽的乙醇提取液，颜色较深，背景吸收较大，以一般常用的单波长比色法测定大豆异黄酮的含量时，色素和醇溶性蛋白等杂质干扰严重。研究建立了三波长比色法，能有效地扣除色素和醇溶性蛋白等杂质的干扰，精密度试验的相对标准偏差为2.14%，平均加样回收率为104.3%，加样回收率的相对标准偏差为3.70%，并用此方法测定6个品种大豆全豆及其胚芽中异黄酮的含量，其异黄酮的含量分别为0.308%-0.447%和1.23%-2.49%。     

大豆异黄酮的提取及其抗氧化稳定性研究

以大豆粉为原料,分别以超声波辅助和微波辅助提取大豆异黄酮。在单因素实验基础上,通过正交试验优化确定最佳提取工艺。结果表明:大豆提取异黄酮工艺方法中超声辅助提取法优于微波辅助提取法,其最佳工艺条件为:乙醇质量分数80%,超声时间20 min,超声温度50℃,料液比1∶20。金属离子除K+和Ca2+外,Zn2+、Cu2+、Mg2+对大豆异黄酮抗氧化活性稳定性影响较为明显。这可为大豆异黄酮的综合利用开发提供依据。     

豆粕中大豆异黄酮的超声辅助提取工艺研究

以大豆异黄酮提取率为指标,豆粕为原料,采用超声波辅助提取大豆异黄酮。在单因素的基础上,通过正交试验确定提取豆粕中大豆异黄酮的最佳工艺条件。结果表明,从豆粕中提取大豆异黄酮的最佳工艺条件为:料液比为1∶20,乙醇浓度为80%,超声时间为30 min,超声温度为60℃。在此工艺条件下,大豆异黄酮的平均提取率为0.3522%。     

High-power ultrasonication-assisted extraction of soybean isoflavones and effect of toasting

The effect of high-power ultrasonication (HPU) on extraction of soybean isoflavones and components, including total phenolic and DPPH radical scavenging activities (RSA) were evaluated. Isoflavones from defatted soybean flakes were extracted using a bench scale HPU system at 20 kHz and varying amplitudes (18–54 μm) for 1 and 3 min. Aqueous acetonitrile, and aqueous ethanol based solvents were evaluated for extraction at samples-to-solvent ratios of 0.1:1 and 0.2:1. The non-sonication control extraction was with mixing with magnetic stirrer for 2 h. Preliminary data indicated 1.2–1.5 times more genistein recovery at 1, and 3 min sonication compared to control, however, total phenolic decreased. The genistein recovery was lower at higher sonication levels. RSA also decreased to 40% for HPU-assisted extractions. Total isoflavones recovery in HPU-assisted extractions increased from 600 to 5813 μg/g. The concentration of major isoflavone components, genistein, daidzein, and glycitein also increased by 10-fold. HPU for 3 min reduced the average particle size of treated soy flakes from 530 μm to 60 μm. Toasting at 150 °C of defatted soy flake for longer period of time led to higher aglycone concentration in extract; however, 2 h toasting was sufficient prior to HPU-assisted extraction.     

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.     

浏阳豆豉发酵过程中抗氧化成分及活性变化研究

通过测定大豆异黄酮、总黄酮、总多酚含量的变化,结合发酵过程中浏阳豆豉的抗氧化活性的变化,来探讨浏阳豆豉抗氧化活性变化的机理。结果表明:虽然总黄酮与总大豆异黄酮的含量逐步减少,但发酵初期原料中大豆异黄酮主要为糖苷型(占85.75%),而发酵末期则主要为更具活性的苷元型大豆异黄酮(占81.41%),且总多酚经发酵从2.78 mg/g提升至5.76 mg/g,发酵最后一天(L20)豆豉的总抗氧化能力、DPPH·、·OH、O₂-·清除率分别增加至1088.91 U/g、93.67%、90.24%、88.38%,此外相关性结果表明苷元型大豆异黄酮要比总多酚对豆豉抗氧化活性的影响更大。实验证明发酵有助于大豆中总多酚的增加以及糖苷型大豆异黄酮向苷元型大豆异黄酮转换,这是提高豆豉抗氧化能力及功能性的关键因素之一。     

Optimization of Ultrasound-Assisted Extraction Procedure to Determine Total Isoflavones in Chinese Soybean Cheese by Box–Behnken Design

Isoflavones from Chinese soybean cheese were extracted with aqueous ethanol. Single-factor experiment design was employed to optimize the solid-to-liquid ratio (in grams per milliliter), ethanol concentration (in percent), extraction time (in hours), and extraction temperature (in degrees Celsius). The solid-to-liquid ratio was 1.5:10 (g/mL), and ethanol concentration (50 %–90 %), extraction time (2–3 h), extraction temperature (50–70 °C) were used for further optimization of extraction conditions. The optimal conditions for the ultrasound-assisted extraction of total isoflavones from Chinese soybean cheese were determined using response surface methodology (RSM) by Box–Behnken design. Three variables “ethanol concentration, extraction time and extraction temperature” were regarded as factors in the optimization study. The optimal conditions for total isoflavone extraction in Chinese soybean cheese were: ethanol concentration of 65.43 %, extraction temperature of 65.38 °C, and extraction time of 2.51 h. The verification experimental OD value was 0.534, which agreed with the predicted value, thus indicating suitability of the model employed and the suitability of RSM in optimizing the extraction conditions.     

Optimization of ethanol extraction and further purification of isoflavones from soybean sprout cotyledon

Isoflavones from cotyledons of soybean sprouts were extracted with aqueous ethanol and further concentrated to obtain a product with a high concentration of isoflavone. The ethanol concentration, extraction time and reaction temperature were optimized by using response surface methodology (RSM). Isoflavones in aqueous ethanol were concentrated by a three-step procedure comprised of solid phase extraction (SPE) with Diaion HP-20 and Amberlite-XAD-2 adsorption columns, acid hydrolysis, and liquid–liquid extraction. The maximum amount of isoflavone in aqueous ethanol extracts (11.6 mg/g solid) was obtained when isoflavones in cotyledons (2.18 mg/ g solid) were extracted with 80–90% (v/v) aqueous ethanol above 90 °C for more than 100 min. The isoflavone extracts, obtained by SPE with a Diaion HP-20 column contained 100 mg/g solid. The liquid–liquid extraction (LLE) with ethyl ether further concentrated the extracts up to 229 mg/g solid, retaining 63% of the initial isoflavones.     

萌芽-低温打浆技术制备高苷元类异黄酮豆乳粉

在传统湿法工艺技术制备豆乳粉的基础上,以萌芽大豆为原料,对热烫后的大豆进行低温打浆处理,提高豆乳粉苷元类异黄酮含量。在单因素试验基础上,采用响应面法对萌芽-低温打浆制备高苷元型异黄酮豆乳粉工艺进行优化,确定最优萌芽-低温打浆的工艺参数为萌芽温度25?℃、萌芽时间75?h、浆液pH?6.0、打浆温度57?℃、打浆时间3.5?h,响应值苷元类异黄酮含量有最优值,为6.43?mg/g。利用高效液相色谱测定豆乳粉中异黄酮种类及含量,结果发现萌芽-低温打浆可显著提高豆乳粉中总异黄酮含量及苷元类异黄酮含量（P＜0.05）,与传统湿法工艺相比,其异黄酮总含量及苷元类异黄酮含量分别增加了1.09?倍和9.37?倍。     

永川豆豉传统发酵过程中的大豆异黄酮变化

为了探明永川豆豉在传统发酵过程中大豆异黄酮总含量和构成变化,采用高效液相色谱技术对永川豆豉传统发酵过程中大豆异黄酮总量和构成进行测定。结果表明：永川豆豉在发酵加工过程中大豆异黄酮总含量由发酵起始的3117mg/kg下降到豆豉成熟时的1754mg/kg;大豆异黄酮构成变化显著,游离苷元型大豆异黄酮含量迅速增加由发酵起始的3.2%上升到豆豉成熟时的95.7%,同时结合糖苷型大豆异黄酮含量迅速降低。     

大豆异黄酮提取条件的研究

本文主要研究了乙醇-水体系提取大豆异黄酮的工艺条件,经单因子实验与L9（3^4)正交实验,最终得到了该体系的最佳提取条件,如以总异黄酮作为提取目的,最佳条件为：80%的乙醇浓度,不小于18：1的物料比（溶剂：原料）,时间1h,温度不超过50℃;如以genistin作为目的产物,则温度宜升高至70℃。     

Improved functionality of soft soybean curd containing Monascus fermented soybean ethanol extract

Monascus fermented soybeans (MFS) were produced by solid state fermentation and ethanol extractions were carried out to recover monacolin K (MK) and isoflavones from the MFS. About 99 and 87% of monacolin K (891 mg/kg) and isoflavones (895 mg/kg) were recovered by 80%(v/v) ethanol extraction. The 80% ethanol extract also showed significantly higher antioxidant activities (DPPH, ABTS radical scavenging activity, and ferric reducing antioxidant power) than the other ethanol extracts prepared by either 40 or 60% ethanol. The 80% Monascus fermented soybeans extract (MFSE) also contained significant α-glucosidase inhibitory activity (2.7 acarbose g equivalents/g MFS). Based on the results, MFSE can be used to enrich bioactive MK and isoflavone aglycones in soft soybean curd products.     

大豆异黄酮分离与精制工艺研究

本文通过对比试验研究大豆异黄酮制品的分离与精制条件,提出了其工艺路线,即提取液经等电点沉淀、离心与超滤处理,初步去杂,大孔树脂吸附,水洗,50%～70%乙醇洗脱大豆异黄酮,减压浓缩而制得大豆异黄酮制品,制品最终纯度大于65%。采用梯度洗脱可得到更高纯度的大豆异黄酮。     

D301阴离子交换树脂对大豆异黄酮分离特性的影响

为更好地将离子交换树脂应用于大豆异黄酮的分离纯化,研究了乙醇含量、pH、温度、初始浓度等条件对D301阴离子交换树脂吸附分离大豆异黄酮的影响。静态吸附实验结果表明,该树脂在以水为溶剂、pH 8左右、40℃时对大豆异黄酮有最好的吸附效果。穿透曲线实验表明,动态饱和吸附量受温度影响较大,当柱温为50℃、初始浓度C07.03 mg/mL时,饱和吸附量为105.0 mg/g干树脂。洗脱曲线实验表明,采用体积分数75%乙醇洗脱,异黄酮回收率为89.3%,产品中大豆异黄酮含量高达56.0%,含量比原料提高了十几倍。表明D301阴离子交换树脂对大豆异黄酮的分离纯化具有较好的选择性和应用前景。