Content Determination And Structure Characterization Of Potato Resistant Starch Based On Asymmetrical Flow Field-Flow Fractionation

Resistant starch(RS)has dietary fiber properties and health effects such as lowering blood sugar,preventing gastrointestinal and cardiovascular diseases,improving immunity,and promoting mineral absorption.RS has been widely used in food industry,medical industry,and materials science.It is reported that the contant of RS in raw potato starch(RPS)is approximateively 80%.The digestion process of starch is a complex,which is influenced by the structure and conformation of starch.The structural and conformational characterization of RS at the molecular level is important to better understand its anti-digestive properties.It is challenge to study the molecular structure and conformation of RS due to its large molecular weight and wide molecular weight distribution.The structure characterization and content deterimation of RS simultaneously over the entire molecular weight distribution remain a challenge.In addition,the relationship between the structure and resistance to enzymatic digestion of starch needs to be further explored.Therefore,it is important to develop accurate methods for the structure characterization and content determination of RS.Asymmetrical flow field-flow fractionation(AF4)is a gentle separation technique that has been used in combination with a multi-angle light scattering(MALS)and a differential refractive index(d RI)detector(AF4-MALS-d RI)to characterize the molecular structure of starch.In this thesis,in order to accurately characterize the content and molecular structure of potato resistant starch(PRS),the PRS recovery was improved by optimizing the separation conditions of AF4,and the structure and conformation of RPS were characterized at the molecular leve.AF4-MALS-d RI was used to monitor the structural changes of potato starch during digestion process.The loss of samples lower than the AF4 ultrafiltration membrance molecular weight cutoff(MWCO)on affects the accuracy of RS quantitative characterization.Thus,a new quantitative method combining AF4 with liquid chromatography(LC)was further developed for the quantitative content determination and structural characterization of RS in the entire molecular weight distribution.The content of RS can be affected by debranching,gelatinization,and retrogradation treatment of potato starch.Therefore,the structural changes of potato starch modified by pullulanase(PUL,a starch debranching enzyme)were investigated,the retrogradation behaviors of pullulanase modified potato starch(PULMPS)after low temperature storage were monitored.The relationships sturcute and function of enzyme modified potato starch were investigated.The specific research work of this thesis is as follows.(1)AF4-MALS-d RI method for the characterization of RPS was developed.The solubilization behavior of RPS was investigated.The effects of the ionic strength of the carrier liquid,injection mass,cross-flow rate,and focusing time on the RPS recovery were evaluated.The repeatability of this method was examined.The results showed that the highest recovery of RPS(97.3% ± 1.5%)was achieved with a carrier liquid containing 5 m M Na NO3 and 3 m M Na N3,p H 7.00;an injection mass of 15 μg,a cross-flow rate of 1.0 m L/min and an focusing time of 2.5 min.The relative standard deviations of the radius of gyration(Rg)and weight average molar mass(Mw)were 1.2% and 5.7%,respectively.The results indicated that the repeatability of the AF4-MALS-d RI method developed in this study was good.(2)The ability of AF4-MALS-d RI to monitor the structural and conformational changes during RPS enzymatic digestion was investigated.The solubilization behavior of type 2 potato resistant starch(PRS)was investigated.The effects of the action time of mixed enzymes(α-amylase and amyloglucosidase)and the concentration of amyloglucosidase on the structure and conformation of RPS were investigated.The Rg and Mw distributions of PRS were determined to be 10-180 nm and 9×105-4×108 g/mol,respectively.The apparent density(ρapp)and the ratio of Rg to hydrodynamic radius(Rh)(Rg/Rh)obtained by AF4-MALS-d RI were used to study the conformation of PRS.The results indicated that the pasting process resulted in a loose,irregularly curled conformation of potato starch molecules,which could contribute to the enzymatic digestion of RPS.In addition,an intermediate with a long branching conformation was identified between the straight-chain starch(amylose,AM)and branched-chain starch(amylopectin,AP)fractions,which may play a role in the digestive properties of potato starch.The results suggest that AF4-MALS-d RI is contribute to better understanding of the PRS conformation.(3)The ability of AF4 combined with liquid chromatography(LC)method for structural characterization and quantification of RS was evaluated.AF4-MALS-d RI was used to characterize the structural information and content of RS with Mw greater than 5 k Da.Meanwhile,RS was characterized by scanning electron microscopy(SEM),X-ray diffractometer(XRD)and Fourier transform infrared spectroscopy(FTIR).LC-d RI was used to quantify the glucose content(180 Da)in the supernatant of the digested starch solution.The subtraction method was used to calculate the RS content with the Mw range of 180 Da-5 k Da.It was found that the content of RS varied widely among different plant origins.The results show that AF4-LC is a feasible method for the structural characterization and quantitative analysis of RS over the entire Mw distribution range.(4)The structural properties and retrogradation behavior of PULMPS were investigated based on AF4-MALS-d RI combined with several detection techniques.To avoid the possible retrogradation of starch samples during alkali solubilization process,PULMPS was solubilized using dimethyl sulfoxide(DMSO)in this chapter.The effects of p H,enzyme treatment time,enzyme concentration,and temperature on the structure of PULMPS were studied.Meanwhile,the effects of enzyme modification on the morphology,crystallinity,AM content,thermodynamic properties,and retrogradation behavior of RPS were investigated.The results showed that the p H 5.0,enzyme treatment time of 12 h,enzyme concentration of 45 U/g and temperature of 55°C were the optimal preparation conditions for PULMPS.After the modification by pullulanase,the RPS granules were broken and the surface of granules had some pores.And the crystallinity of PULMPS decreased,while pasting temperature and AM content of PULMPS increased.The retrogradation of PULMPS was investigated.The results showed that increasing AM content by PUL modification facilitated starch retrogradation.The informations obtained from AF4-MALS-d RI are useful for better understanding of the mechanism of enzymatic modification of starch and the development of functional foods.