22 The supernatant was ready for analysis. All the samples preparation and analysis were repeated three times. Analyses of oligosaccharides Soybean sheet Soymilk slurry Okara first filtrating Raw soymilk boiling second filtrating water bath Sweet slurry grinding with water Soybean sorting soaking Figure 1. General flow of soybean sheet production.
Q Wang, L Ke, D Yang, B Bao, J Jiang and T Ying 90 High Performance Liquid Chromatograph method (HPLC) was used to analyze the oligosaccharides. The method
91 Ol ig o saccharide losses in soybean sheet production o lig o saccharide losses in soybean sheet production used in this research was described by Martínez-Villalu- enga et al . 23 with slight modification. HPLC system (Wa- ters 2695, Marlborough, MA) consists of multi solvent delivery system, 717 plus auto sampler, 2414 refractive index detector. An amino-bonded column (Hypersil, 250 mm × 4.6 mm i.d.) connected with a guard column (Hy- persil, 4 mm×50 mm) packed with C 18 Porasil was used to analyze the oligosaccharides. The samples were filtrated through millipore membrane (0.45 μm) prior to HPLC analysis. The mobile phase acetonitrile-water (75:25 v/v, HPLC grade) was ultrasonicated for 30 min before chro- matographic analyses. Chromatogram conditions were 40 ℃ column temperature, 10 μL injection volume and 1.5 mL/min flow rate. Calibration curves and recovery Standard sugars were dried at 70 ℃ in vacuum oven and stored in a desiccator at room temperature prior to testing. Sugars were first dissolved in purified water and then di- luted with 75% acetonitrile to get a similar condition with the mobile phase. The concentration of the standards so- lutions was 10 mg/mL. The standard curves were gener- ated from eight different concentrations of sugars. The regression coefficients of the curves for sucrose, raffinose and stachyose as well as fructose and glucose were greater than 0.990. Quantification of individual sugars was achieved by comparison of the peak areas with stan- dards. The HPLC graph and retention time of sucrose, raffinose and stachyose as well as fructose and glucose is illustrated in Figure 2. Statistical analyses All experimental treatments were repeated 3 times. Val- ues shown in this paper were the average of three repli- cates ± standard deviation (SD) in all the results tables. Statistical significance was analyzed using the Statistical Package for Social Science,Version 11.5 (SPSS for Win- dows, SPSS Inc., Chi a cago, IL, USA). Results Oligosaccharide levels in raw soybeans The oligosaccharides and monosaccharides contained in the raw soybean were identified mainly as sucrose, raffi- nose and stachyose as well as fructose and glucose (Fig- ure 2). Sucrose (4.34%), raffinose (0.75%) and stachyose (4.13%) are the predominant sugars in soybean (Table 1). Effect of soaking on content of oligosaccharides The concentration of oligosaccharides left in the soaked soybeans was calculated by the initial oligosaccharides amount in the raw soybeans minus those lost in the soak- ing water. Soaking the soybean for 12 to 14 hours at 20 ℃ did not reduce the sugar content too much. The to-
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