| Phosphorylated oligosaccharides (POs), a new functional oligosaccharide, were composed of maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose, within covalently-bound phosphate groups linked to the glucose residues. As a new, natural and security functional oligosaccharide, POs had the effects of promoting the absorption of alkaline earth metal such as calcium or iron in a living body, preventing carious teeth, suppressing retrogradation of starch, preserving the plants or fruits longer and so on. Accordingly, POs can be widely used in food, health care, medicine, feed additives, etc. Considerable attention has been focused on the capability of promoting the absorption of calcium and iron. As abundantly minerals, the effects of calcium and iron were conspicuous in the living body. At present, situations of reduced calcium and iron absorption were serious, and general inhibitions were less effective. However, Pos had the ability to form a soluble complex with minerals (such as calcium, iron) and had an inhibitory effect on the formation of mineral-phosphate precipitates, to maintain a high concentration of soluble minerals, thus promoted the absorption of minerals alkaline earth metal in human body. As a new inhibitor, the preparation of phosphorylated oligosaccharides has important practical significance. The task of this paper was to optimize the enzymatic preparation of phosphorylated oligosaccharides on the basis of previous researches.1. The structures and functions of POS, methods of preparing and isolating oligosaccharides were reviewed by reading and consulting lots of previous papers. Comparing effects of Pos and homogeneous products,such as promoting the absorption of minerals, preventing carious teeth, suppressing retrogradation of starch. Besides, the different measurements of phosphorus, liquefaction methodology and analytical methods were also compared.2. The total phosphorus and binding-phosphorus of commercial starch and phosphate starch were measured by Spectrophotometry and Inductively Coupled Plasma Emission Spectrometry (ICP-AES). The results showed that Spectrophotometry is more suitable for the analysis of phosphorus in starch than ICP-AES, and the wavelength was 825nm. The binding-phosphrus of commercial potato starch was 440ppm. Furthermore, commercial phosphate starch was not suitable for the preparation of Phosphoryl Oligosaccharides which had a low binding-phosphorus content.3. The POS precursor- maltooligosaccharides with low dextrose equivalent (DE) were prepared by enzymatic methodology and jet liquefaction. Optimization of hydrolysis conditions was investigated by Response Surface Methodology (RSM), and effects of pH, Fungal Amylase, saccharification temperature and time were all tested. The optimal DE of 35 was appeared at the follow conditions:starch concentration at 15 OBé, pH at 5.9, thermostableα-amylase at 35U/g, fungal amylase at 2.3u/g, pullulanase at 1.0U/g, saccharification temperature at 60℃and time at 148min, respectively. The hydrolyte were analyzed by HPAEC-PAD.The results showed that maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaoseas were the main components.4. Separetion and preparetion of POS were done by Chitopearl BCW-2501 column chromatography. Neutral sugars were washed out with a 20mM acetate buffer (pH 4.5), and phosphorylated oligosaccharides were eluted by the same buffer containing 0.1M NaCl. Then, POS were applied to a Sephadex G-10 column to remove the salts. The yield rate of column chromatography was 76.97%.5. Fractions of POS were abtained by HPAEC-PAD. Results demonstrated that retention time of POS was longer than neutral sugars.Comparing the retention time of de-phospharylated oligossacharides with authetic maltooligosaccharides, founded that maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaoseas were the main components. Infrared chromatography showed that stretching vibration absorption peaks of P-O-C and P-O appeared in 1080cm-1 and 928 cm-1 nearby. Thereby, it predicted that POs were carbohydrates combined with phosphate groups. The yield rate of POS was 0.71%, to the total sugar content of indicators.
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