| With the increase in people’s health care awareness,soy isoflavones have become a highly soughtafter natural substitute for estrogen.As a major soybean resource country,China has a strong advantage in the development and utilization of soy isoflavones,as they are a by-product of the soybean production industry.Soy isoflavones can be classified as glycosidic and aglycone types,with the biological activity mainly reflected by the aglycone type,while the aglycone type has a much lower content.Therefore,in this experiment,β-glucosidase was firstly used to transform the glycosidic soy isoflavones into more active ones(aglycone type),and ultrasonic technology was applied to further increase the enzyme hydrolysis rate.The obtained aglycone soy isoflavones were then purified and enriched by macroporous resin.Finally,the anti-solvent precipitation micronization process was used to resolve the issue of aglycone soy isoflavones’ poor solubility and low bioavailability,resulting in the production of a fine powder with good dispersibility and bioavailability.The physicochemical property and biological activities of the aglycone soy isoflavones were also characterized.The results were as follows:(1)The optimum conditions for the enzymatic hydrolysis of glycosidic soy isoflavones by β-glucosidase with ultrasonic technology were obtained by single-factor as well as response surface tests with the enzymatic rate as the index: reaction temperature was 55 ℃,ultrasonic power was 200 W,p H =5.2,enzyme addition was 8%,reaction time was 50 min,and the enzymatic yield was 90.03±0.81%.Meanwhile,the content of soy isoflavone components before and after enzymatic digestion was analyzed by High Performance Liquid Chromatography(HPLC),and the results showed that a large amount of glycosidic soy isoflavones were transformed into aglycone soy isoflavones,which demonstrated the effect of enzymatic digestion was significant.(2)The adsorption and desorption performances of three macroporous resins with different polarities(D-101,AB-8,and S-8)on aglycone soy isoflavones was investigated,and the AB-8 resin with the highest saturation adsorption,adsorption,and desorption rates on aglycone soy isoflavones was selected for the subsequent purification.The effects of different ethanol concentrations and column flow rates on the purification efficiency were studied,and the results showed that 80% ethanol was favorable for the desorption of aglycone soy isoflavones,while a column flow rate of 1 m L/min contributed to the delay of the breakthrough point and an increase in adsorption capacity.The purity of the purified glycosidic soy isoflavones was 82.42±1.15%,which was 2.01 times higher than the purity before purification(40.88%).(3)The aglycone soy isoflavone micropowder was prepared by the anti-solvent precipitation.Firstly,the solvent selection was carried out,and five aglycone soy isoflavone solvents were selected.Deionized water was used as the anti-solvent to prepare the aglycone soy isoflavone micropowder.The microstructures of the obtained particles were observed by scanning electron microscopy(SEM).Microparticles prepared with dimethyl sulfoxide(DMSO)as the solvent were found to be more uniform in size and smaller in dimension,and thus it was decided to utilize dimethyl sulfoxide as the solvent and deionized water as the anti-solvent for the aglycone soy isoflavone micronization.The optimal conditions for the preparation of micropowder with the smallest particle size were obtained through single-factor and response surface experiments: sample concentration of 20 mg/m L,anti-solvent-to-solvent volume ratio of 12.5,flow rate of 8.5 m L/min,injection hole size of 0.6 mm,surface active concentration of 3 mg/m L,stirring speed of 1500 rpm,and deposition time of 0 min.The smallest particle size was 238.03±11.81 nm.(4)The physicochemical properties of micro aglycone soy isoflavones were characterized.The SEM results showed that the prepared microparticles were very small and shaped like needles.The Fourier transform infrared spectroscopy(FT-IR)results showed that the micronization process did not cause any significant changes in the structure of the aglycone soy isoflavones.X-ray diffraction(XRD)and differential scanning calorimetry(DSC)results indicated that the reduction in particle size led to a decrease in the crystallinity and enthalpy of the aglycone soy isoflavones.The residual solvent analysis showed that the residual DMSO was 0.2%,which is lower than the maximum limit(0.5%)of the toxic reagent dimethyl sulfoxide set by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use(ICH).(5)The results of four in vitro antioxidant activity determinations showed that the antioxidant capacity of the raw aglycone soy isoflavone powder was affected by its poor water solubility but performed well in an ethanol solution.In addition,the antioxidant capacity of the micropowder was significantly higher compared to the raw powder.The in vitro dissolution analysis indicated that the aglycone soy isoflavone micropowder had a smaller particle size and a larger surface area as a result,which led to an increase in the solubility in water and a significant improvement in dissolution in simulated gastric and intestinal fluids.At 360 minutes,the dissolution rates of the micropowder were 2.56times(for simulated gastric fluid)and 3.27 times(for simulated intestinal fluid)higher than those of the original powder.In summary,high-purity aglycone soy isoflavones were obtained after enzymatic hydrolysis and purification by AB-8 resin.The microproduct of aglycone soy isoflavones was prepared by the antisolvent precipitation method,which has good water solubility and does not contain reagent toxicity,broadening the application field of soy isoflavones and providing a theoretical basis and production guidance for the research on the development and utilization of more natural insoluble plant components. |
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