| Starch is the main carbohydrate in human dietary and has a wide range of applications inindustry. Nutritional properties of modified starch depend on the structure and its associatedconditions. With the increasing pace of life, digestive-related diseases have become a hugethreat to people ’s healthy conditions, such as diabetes, obesity and so on. In previous research,slow digested starch (SDS) and resistant starch (RDS) could effectively alleviate the starchdigestion rate in the body due to its slow digestion characteristics, for example, SDS couldreduce postprandial blood glucose response and relieve related diseases.4-α-glucanotransferase (4αGT) is a multifunctional enzyme, and it has the function ofintramolecular transglycosylation. Branching enzyme (BE) can catalytic the formation of α-1,6glycoside. So both of this two enzymes can change the structure of starch, and caneffectively influence the proportion of starch digestion characteristics. This study was aboutthe modification of starch by using4αGT and BE separately from the viewpoint of enzymedosage and reaction time, and then elucidated its related properties in order to provide atheoretical basis for its application.The first part was about the preparation of4αGT and BE. The single colony-engineeringbacteria E. coli BL21(DE3)/pET22b-4αGT and E. coli BL21(DE3)/pET22b-BE were kept inlaboratory. After fermentation and centrifugation, the precipitations were freezing dried andthen stored at-20centigrade. The enzyme activity of4αGT and BE was487and417U/g,respectively. Then the hydrolysis rate of these two enzymes on normal cornstarch wasmeasured at different time. According to the hydrolysis rate, Group A, B, C, D and E wereprepared by adding different amount of enzyme or modifying at different time.The second part was the characterization of4αGT-modified starch. Normal corn starchmodified using4αGT for4h showed an increase of SDS from9.40%to21.2%. The viscositywas inversely related to the digestibility of the4αGT-treated starch.The4αGT treatmentdecreased the molecular weight from7.03×107g/mol to3.69×106g/mol, and also decreasedamylose content from32.8%to24.9%. The chain length distribution of4αGT-treated starchshowed an increased amount of short (DP<13) and long (DP>30) chains. Both the short andlong chain fractions of modified amylopectin were attributed to the slow digestion property.1H NMR spectra showed an decrease of α-1,4linkages. These results implied that starchestreated with partial4αGT synthesized novel amylopectin clusters containing slowdigestibility.The third part was to study relevant characteristics of BE treated starch (group C andgroup D). Normal corn starch modified by using branching enzyme for4h showed amaximum SDS content of23.90%. The molecular weight distribution of enzyme-treatedstarches showed a larger proportion of low molecular weight fractions appeared in starchescompared to native starch. The branching enzyme hydrolysis decreased the amylose content.The number of shorter chains (DP<13) increased, accompanied by a reduction of longerchains (DP>30).1H NMR spectra showed an increase of α-1,6linkages from6.12%to7.42%in the enzyme treated starches. The increase in the amount of shorter chains and α-1,6 linkages was likely to contribute to their slow digestion property.The last part was to study the properties of group E, the combined-enzyme modifiedstarch. The order of enzyme modification had little influence on starch characteristics.Compared with the single-enzyme modified starch, group E had a higher SDS content by23%.The gelatinization property of E1was only0.371J/g while the E2was almost undetectable,and the rheological curves were very similar with the single-enzyme modified starch. Themolecular weight decreased to2.5×106g/mol In comparison with native starch, group E had alarger decrease in the amylose content. and the chain length distribution showed that theshort-chain portion (DP <13) had been greatly increased. The content of α-1,6glycosidebonds of E1was7.66%, higher than the native starch which indicated that mixed enzymetreatment had a better increment of the proportion of α-1,6-the of glycoside bond.These results suggested that starches treated with4αGT and BE could synthesize novelstructures with slowly digestible character, and the content of DP<13and DP>30had apositive correlation with SDS. After fitting, we found that the relationship between SDS andchain length was y=-6.802x2+32.265x-14.379while x was the weight ratio of long chain toshort chain, and y was the percentage of SDS. |
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