Study Of Purification Technology And Mechanisms Of Soy Oligosaccharides From Soy Molasses

Soy molasses is the by-product of soy concentrate protein, which shows brown color and smells sweet. Soy molasses is a kind of extensive and inexpensive resource which is usually used for feed supplement or fermentation substrate since its viscous properties impede the utilization, and that resulted in a waste of resources because soy molasses consist of soy oligaosaccharides and other phytochemicals. After diluted and adjusted to acidic p H, the supernatant of soy molasses become a good resource of soy oligosaccharides and the precipitate can be used to extract functional components such as phosphatide, soy saponins and soy isoflavones. Nowadays, there are many of studies focus on the purification technics of soy oligosaccharides but few reports about purification mechanisms. This research identified and analyzed the main contains in soy molasses supernatant, and then studied the removal of impurities except soy oligosaccharides accompanied the purification mechanism. The main contents and results of the research are as follows:First, soy molasses was diluted with distilled water to the solid content of 10%, and then adjusted to p H 2.5 with HCl. Clear and brown supernatant was obtained after the mixture was centrifuged with 3200×g by 20 min. Analysis result with HPLC-RI showed that the total carbohydrates content was about 77.76% of the solids content. Soy oligosaccharides consist of sucrose, raffinose and stachyose took 80.33% of the total sugar content, and the concentration was 31.150±0.716, 5.938±0.106 and 25.378±0.591 g/L, respectively. The ash of the soy molasses supernatant was about 6.08% of the solids. The result of ion chromatography suggested that the supernatant mainly contained four kinds of cation including K+, Na+, Ca2+, Mg2+ and two kinds of anion including Cl- and PO43-, with the concentration of 4061.67±65.49、541.55±12.90、246.81±4.16、161.94±3.90、3889.43±73.90 and 494.57±10.35 mg/L, respectively. Combined the results of LC-MS/MS and RP-HPLC, it was considered that there was no soy saponins but five kinds of soy isoflavones including daidzin, glycitin, genistin, 6’’-O-malonyldaidzin and 6’’-O-malonylgenistin in the supernatant with the concentration was 73.21±1.31、11.26±0.25、93.86±0.78、107.41±2.29 and 138.43±2.41 mg/L, respectively. And the total soy isoflavones content was about 0.42% of the solids. The protein content analyzed by the Kjeldahl method(N×5.71) was 4.487±0.086 g/L in the supernatant, which took 4.49% of the solids. Protein with high molecular weight from soy molasses supernatant was identified to be the Bowman-Birk trypsin inhibitor on the basis of Tricine-SDS-PAGE analysis and its trypsin inhibitor activity. Other nitrogen-containing components with lower molecular weight were analyzed by LC-MS/MS and LC-MS/MS with OPA precolumn derivatization, and the contents were identified as five kinds of free amino acid including Aspartic acid, Glutamic acid, Asparagine, Alanine and Tyrosine and one kind of dipeptide Glu-Tyr. HPLC-FLD with OPA precolumn derivatization analysis showed the concentration of the above nitrogen-containing components was 238.13±4.75、380.22±7.49、212.67±4.68、134.25±2.79、167.36±3.26 and 432.56±7.92 mg/L, respectively. GC-MS analysis of the soy molasses supernatant revealed the main organic acid was citric acid with the concentration of 2.892±0.097 g/L, which took about 2.89% of the solids.Then the soy molasses supernatant was decolorized by activated carbon. The content changes of soy isoflavones was chosen to study the mechanism of decolorization after data analysis. The adsorption of isoflavones by activated carbon fitted to pseudo-second order kinetic model which illustrated the equilibrium was arrived at 1.5 h and the adsorption was limited by micro-pore diffusion. Isotherm of the adsorption could be described by Langmuir model illustrated the adsorption of isoflavones by activated carbon was single layer adsorption. There were specific adsorption sites for isoflavones with different aglucaons, while the influence of oligosaccharides on the adsorption of isoflavones was related to the derivative groups conjucted by the aglucons. Thermodynamic analysis revealed that the adsorption was an entropy increased, spontaneous and endothermic process.Ultrafiltration membrane with molecular cut off 10 k Da was used to retain trypsin inhibitors in the discolored supernatant and so permeation showed no inhibitor activity was obtained. The initial p H of the supernatant had low effect on the membrane flux, while the decolorization operation by activated carbon improved the membrane flux obviously. Ultrafiltration membrane was fouled mainly by inorganic deposition and organic occlusion. Membrane flux would be recovered by washed with dilute acid and alkali.The permeation was desalinated by ion-exchange resins and electrodialysis respectively. Results suggested that the cation exchange barely adsorbed Na+ but performed better on adsorbing K+. The adsorption of Cl- by anion exchange resin performed not well either since the exist of citric acid. The desalination rate was barely affected by the initial p H of feed liquid at the earlier stage, but decreased obviously for higher p H at the later stage. That meant the ion membrane was fouled more seriously by feed liquid with higher p H. The fouling of ion membranes were cause by deposition of inorganic salt and blocking of organic components, and circling clean by 1-2% HCl successively 0.1 mol/L of Na OH recovered the membrane. Chromogenic components such as isoflavones seriously fouled the membrane and the damage was hard to be recovered, hence the feed liquid was better to be decolorized before eletrodialysis.Finally the discolored and desalinated permeation was treated by ion-exchange resins to removing nitrogen-containing components and citric acid. The static adsorption suggested that the best adsorption for nitrogen-containing components and citric acid by cation resin and anion resin was 001×7 and D201 respectively. Both the isotherm of two adsorptions could be described by Langmuir model, illustrated that the adsorption by resins were single layer adsorption. Fixed-bed column adsorption showed that the adsorption capacity of nitrogen-containing components by cation resin were Aspartic acid < Glutamic acid ≈ Asparagine < Alanine < Tyrosine < Glu-Tyr. When resin volume was 40 m L and flow rate was 2.00 m L/min, the elution volume for cation resin column was 37.5 BV when the Aspartic acid was breakthrough, and the elution volume for anion resin column was 23.3 BV when citric acid breakthrough.By decolorization by activated carbon, removal of trypsin by ultrafiltrition, desalination by electrodialysis as well as removal of nitrogen-containing components and citric acid by inseries cation and anion exchange resins, soy oligosacchairdes with light color and high purities could be got from soy molasses supernatant. The contents of raffinose and stachyose were about 40% of the total sugar content in the soy oligosaccharides, which was fitted to the national standard that functional oligosaccharides shouled take more than 35% of total sugar content. The ash content(dry basis) was 0.43%, which was lower than the provison in national standard that the ash content(dry basis) should not more than 3.0%. This research provides technical route and theroretical principle for the purification of soy oligosaccharides, and also can be a reference to other purification process.