Enzymatic kinetic analysis of lotus seed and characterization of enzymatic hydrolysis of products polysaccharides

Here the mild and simple enzymatic hydrolysis was developed to prepare the plant polysaccharides with the high purity. The pepsin (endopeptidase) and koplase (telopeptidase) were used as the complex enzymes. Kinetic models were established for the enzymatic hydrolysis. Water extraction was used to prepare the polysaccharide at 45℃ and 90℃. Also included are pepsin extraction and double enzyme extraction. Nutritional components of polysaccharides extracted by these four methods were characterized by their structure (monosaccharide composition, infrared spectrum, thermal features, low field nuclear magnetic field, and dynamic thermomechanical properties). The results showed that the kinetic models of single (pepsin)/double enzyme segmenting hydrolysis were D_H=2.101ln1+(0.6133(E0/S0)+0.1441)t and D′_H-DH1=2.439ln1+(3.923(E0/S0)+1.1756)t. The highest contents of polyphenols（0.42±0.008mg/mL） and uronic acid（15.65%±0.98%） were obtained in the polysaccharide of lotus seed that extracted by double enzyme. While the lowest protein content（0.73%±0.24%） was found. Among them, uronic acid was a derivative of sugar. The high contents of uronic acid and galactose were detected in all the polysaccharides that extracted from lotus seed, indicating the acidic polysaccharide. Glc, Ara, Man and Rha were contained in the four kinds of lotus seed polysaccharides, among which more Gal-UA and Man were found in the polysaccharides that extracted by double enzyme, which were 10.700 and 10.752% respectively. This acidic polysaccharide was benefit to the dissolution of active polysaccharide. The four samples of lotus seed polysaccharide also shared the similar infrared spectral characteristic peaks, α-type pyranose. The wider characteristic peaks of 1654 cm-1 were observed in 45℃ water extraction, indicating the more bound water. The double enzyme was also used to break the binding between polysaccharide and protein. The highest content of bound water was detected with the less fixed and free water in the double enzyme after water distribution analysis. It infers that the double enzyme extraction was more beneficial to the stability of polysaccharide structure. In addition, only the first-order relaxation was found in the polysaccharide of lotus seed that treated by double-enzyme. The energy storage modulus and loss modulus were also higher than before. The glass transition temperature（Tg ）was dropped to about 60℃ (close to that of plant polysaccharide). In summary, the double enzyme extraction can be expected to effectively reduce the protein content and Tg of lotus seed polysaccharide, compared with conventional methods. The content of bound water and hydrophilic ability were improved to extract the active components. The specific gravity values of polysaccharide aldehyde and rehydration were also improved for the purity of polysaccharide. The kinetic model of enzymatic hydrolysis can also provide the theoretical support to the purification of lotus seed polysaccharides, in order to understand the degradation of lotus seed protein. The two enzyme extractions can share the stronger specificity and simple steps, compared with the high-temperature ones. The mannan polysaccharide components can be extracted to improve the polysaccharide purity. Therefore, the enzymatic hydrolysis of lotus seed polysaccharide can ensure the protein to release from the polysaccharide. The effective and moderate hydrolysis can be achieved for the synchronous separation of protein and polysaccharide in the step of polysaccharide alcohol precipitation. The mathematical models were further simulated and validated, according to the mechanism of enzymolysis reaction. The kinetic models of enzymatic hydrolysis with single and double enzymes were also established to determine and characterize the nutritional structure of polysaccharide products between the low/high temperature extraction and low temperature enzymolysis. This finding can also provide the theoretical guidance for the further development and utilization of animal and plant polysaccharides.