| Starch and starch derivatives often displayed wide molecular weight distributionand were polydisperse, but the polydispersity restricted their application. Therefore,fractionation of starch hydrolysates into dextrin fractions with narrow molecularweight distribution is one of the most important approaches to stimulate starch intovalue-added products in starch processing. However, the base of research on this fieldwas rather weak in China. Furthermore, the fractionation method used was limitedand difficult to achieve industrialization. This study used the existing fractionationmethod developed abroad and developed a new fractionating-dextrin method tofractionate starch hydrolysate into dextrin fractions with low polydispersity index.Moreover, this study expanded the application of dextrin. Namely, these dextrinfractions were conjugated with uricase to improve its solubility and stability.This study investigated the process of hydrolysing the three starches (rice starch,high-amylose maize starch and cassava starch) by alcohol-HCl into dextrins from thechanges in solubility, starch granule, crystalline, and molecular weight. The presentdata indicated the solubility of1-butanol-HCl-hydrolyzed cassava starch was thehighest and trended to stabilize after hydrolysis for3d. SEM and particle sizeanalysis showed that after hydrolysis by1-butanol-HCl for7d, the granulemorphology of the three starches significantly changed and the granule size decreasedby18.3%,12.3%and44.0%for rice starch, high-amylose maize starch, and cassavastarch, respectively. XRD results indicated the peak of V-type crystallinity wasdetected at around20°in the XRD curves of1-butanol-HCl-hydrolyzed rice starchesand cassava starches, which confirmed the formation of amylose-1-butanol complex.HPSEC-MALLS-RI and iodine staining analysis demonstrated that the molecularweight sharply decreased after hydrolysis for4h, then gradually decreased, andfinally stabilized after hydrolysis for3d (for cassava starch) or4d (for rice starchand high-amylose maize starch). Considering the results of the solubility andmolecular weight, cassava starch hydrolyzed by1-butanol-HCl for3d was chosen asthe raw material for preparing dextrin with narrow molecular weight distribution byfractionation.Gradient alcohol precipitation was used to fractionate the1-butanol-HCl-hydrolyzed cassava starch into dextrin fractions with narrowermolecular weight distribution. This study systematically investigated the effect of the alcohol type, the initial dextrin concentration, the initial pH and the initial KClconcentration on fractionation process of hydrolyzed starch with the polydispersityindex (PI) as index. Adding alcohol into dextrin solution might induce too highalcohol concentration in partial regions, which impeded the fractionation process.Therefore, the rate of adding alcohol was rigorously controlled during fractionation.After fractionation by gradient alcohol precipitation, the1-butanol-HCl-hydrolyzedcassava starch was fractionated into7fractions with the volume ratio of dextrinsolution to alcohol at4:1,2:1,1:1,1:2,1:3,1:4,1:5, respectively. The PI of mostfractions was much smaller than that of the parent sample (For instance, the PI of7dextrin fractions fractionated by methanol was1.309,1.078,1.077,1.133,1.022,1.309and1.025, respectively, while the PI of the parent sample was2.052). The datademonstrated that the fractionation effect of fractionating dextrin by alcohol was inthe order of methanol>ethanol>isopropanol, while the dextrin yield was in the orderof methanol<ethanol<isopropanol. Furthermore, the Mpof each fraction decreasedconsecutively with the increasing of concentration of alcohol at which it wasprecipitated. With the initial dextrin concentration in the range of1.8%~2.7%, the bestfractionation result was obtained, and lower or higher concentration deteriorated thefractionation result. Fractionating dextrin by gradient ethanol precipitation wasapplicable in acidic, neutral, and alkaline environment, but acidic and alkalineenvironment induced the increasing of PI for the first fraction. Salt in solution hadnegative impact on the fractionation result of dextrin, and the higher concentration ofthe salt was, the impact was more evident.It was found that there was incompatibility between dextrin and PEG in aqueoussolution. Based on the incompatibility between dextrin and PEG, a new fractionationmethod, gradient PEG precipitation, was developed. The fractionation process wasaffected by the molecular weight of PEG, the polydispersity of PEG, the initial dextrinconcentration, the initial pH, and the initial salt concentration. The1-butanol-HCl-hydrolyzed cassava starch could be fractionated into several fractionswith narrow molecular weight by PEG2000, PEG4000, PEG6000and PEG10000(Forexample, PEG6000fractionated the hydrolyzed cassava starch into11fractions withPI at1.983,1.295,1.156,1.170,1.102,1.094,1.122,1.067,1.152,1.123,1.442,respectively.), while PEG20000was unsuited for fractionating dextrin, owing to itsexcessively high molecular weight and viscosity. Considering the fractionation result,the dextrin yield, the cost and operational simplicity, PEG6000was the most suitableprecipitant to fractionate dextrin. The polydispersity of PEG was not conducive to fractionating dextrin. Initial dextrin concentration in the range of0.9%~3.6%wassuitable for fractionating dextrin by gradient PEG precipitation, but higherconcentration resulted in bigger PI of the first fraction. This fractionation method wasapplicable in acidic, neutral, and alkaline environment. Salt in solution had negativeinfluence on fractionating dextrin, and the influence was aggravated with theincreasing of the concentration of salt.Based on that dextrin could be hydrolyzed by amylases in vivo, fractionateddextrin and uricase was conjugated to improve the stability of uricase. Firstly, todevelop dextrin as the uricase carrier, the fractionated dextrin was succinylated intodextrin monosuccinate with the functional group, carboxylic acid. The chemicalstructure of dextrin monosuccinate was identified using FT-IR and13C NMR. Thereaction conditions, including reaction solvent, reaction time, reaction temperature,and molar ratio of succinic anhydride (SA) to anhydroglucose units (AGU) in dextrin,were investigated for preparing a series of dextrin monosuccinates with differentdegree of substitution (DS). The optimum conditions were as follows: solvent,dimethyl sulfoxide; reaction temperature,50oC; reaction time,12h. Under theseconditions, a series of dextrin monosuccinates with DS at0.042,0.127,0.186,0.283,0.343,0.410,0.598,0.694,1.099and1.347were prepared by controlling the molarratio of AGU in dextrin to SA. Secondly, dextrin-uricase conjugate was synthesizedby uricase and dextrin monosuccinate and verified by SDS-PAGE electrophoresis,SEC and IEC analysis. It was found that the higher the DS of dextrin monosuccinatewas, the higher the conjugate degree of dextrin-uricase conjugate was, but the activitywas also seriously lost and difficult to recover by α-amylase. Under the condition ofthe molar ratio of dextrin monosuccinate (DS=0.283) to uricase at30:1, the conjugatedegree of dextrin-uricase conjugate reached22.1%, and the activity was decreased to40.4%, but recovered to83.4%after triggering by α-amylase. Finally, the propertiesof dextrin-uricase conjugates, represented by this conjugate, were invesigated andcompared with free uricase. The optimal pH and temperature of this dextrin-uricaseconjugate was9.0and45oC, respectively, whereas the optimal pH and temperature offree uricase was8.5and45oC, respectively. Furthermore, dextrin-uricase conjugatewas more resistant to acid, alkaline and heat. Additionally, free uricase absolutely lostactivity after hydrolysis by trypsin for30min, while the activity of dextrin-uricaseconjugate was reduced to33.6%after hydrolysis by trypsin for60min, whichindicated that dextrin-uricase conjugate was more resistant to trypsin. In conclusion,the properties of uricase were ameliorated by conjugating with dextrin. |
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