Study And Application Of Enzymatic Branch Extension Modification Of Different Types Of Rice Starch

Amylosucrase(AS)is an excellent glucosyltransferase in glycoside hydrolase 13 family.When selecting sucrose as donor molecule,the transglycosylation of AS can lengthen the branch chains of starch molecule by modifying their non-reducing ends.This method is currently used in the preparation of low-digestible starches.Compared with amylose(AM),the hyperbranched structure of amylopectin(AP)has more active sites for enzyme-catalyzed reaction.Thus,most of existing researches focus on modification of AP via AS.However,the mechanism of AM during the process of AS modification reaction and the influence on physical and chemical functionality of modified starches are not clear yet.Based on this,this study firstly selected rice starch with different AM content(Liannuo No.1,LN;Longjing 33,LJ;Zhongjiazao 17,ZJ)as receptor to investigate the effect of AM on the reaction process of branch chain extension.Secondly,the enzymatically modified starches with different degrees of grafting were prepared by using the transglycosyl activity of AS.The molecular structure and physical and chemical functionality of modified starches were characterized to reveal the internal contact between structure and in vitro digestibility of modified starches.Finally,modified starches were selected as substitutes of ordinary starches to make rice cakes and its application in traditional rice products was explored.The main results were as follows:The AM content of starches were determined by colorimetry.The results showed that the AM content of LN,LJ and ZJ were 0.94%,17.39%and 27.39%,respectively.Using transglycosyl degree(TD)as evaluation index,the effect of AM on the progress of enzyme reaction was investigated.The results showed that the corresponding TDs of LN,LJ,ZJ at 30min were 47.2%,46.1%,and 42.7%,respectively,with the starch concentration of 1.0%,sucrose concentration of 0.1 mol/L,enzyme activity of 1000 U/L and temperature of 35°C.While after 6 h,the corresponding TDs were 122.8%,109.3%,95.7%,respectively.In addition,the enzyme reaction process of the three starches conformed to the logarithmic function equation,and the fitting result were:LN,Y=-33.127 ln(X+6.199)-68.351,R~2=0.991;LJ,Y=-26.845 ln(X+4.074)-47.282,R~2=0.996;ZJ,Y=-23.770 ln(X+5.689)-44.517,R~2=0.993(where Y and X were reaction time and TD,respectively).The modification of AS resulted in a phase change in the reaction system.The turbidity was measured to characterize the progress of the enzyme reaction.The results showed that the corresponding time points of phase transition of three starches were 56 min,36 min and 26 min,respectively.The above results suggested that the linear molecular structure of AM promoted the retrogradation of starches during the reaction process,causing that most of the non-reducing ends of starches were embedded in the precipitate,which led to a decrease in the efficiency of the enzyme reaction.According to the theoretical reaction equation,modified starches with different grafting degrees(TD=30%,50%,70%,90%)were prepared.The distribution of the chain length showed that with TD increasing from 30%to 90%,the position of the main peak of m LN increased from DP24 to DP40,and the intensity of the main peak gradually increased,which indicated that the branched chains of m LN were significantly elongated.AM in LJ and ZJ promoted the retrogradation(phase transition)of m LJ and mZJ during the reaction process.Before the phase transition,the position of the main peaks of m LJ and mZJ gradually shifted to the high DP region,and the peak intensity increased;after the phase transition,the modification of AS did not change the position of the main peak,while the peak intensity gradually decreased.Besides,new secondary peaks appeared at DP85 and DP211,indicating that AS only extended the non-reducing ends on the surface of starch gel after phase transition and informed some ultra-long chains.The result of scanning electron microscopy showed that the morphology of three native starches were spherical or polygonal particles with a size of about 3?8?m,while the modified starches showed an irregular but densely packed massive structure.Crystal structure analysis showed that the native starch LN,LJ and ZJ all had A-type crystal structure,the relative crystallinity(RC)of which were 37.52%,35.20%,and 32.38%,respectively.The mLN,mLJ and m ZJ all had B-type crystal structure.When TD was constant,R_C(m LN)>R_C(m LJ)>R_C(mZJ),indicating that AM inhibited the formation of starch crystals during the process of modification.With the increase of TD,the gelatinization temperature of modified starches showed an upward trend,indicating that the extended modification on starch branch chains made the starch form crystal structure with better thermal stability.In vitro digestion showed that the higher AM content the native starch possessed,the lower its digestibility was.Among them,the resistant starch(RS)content of ZJ was 37.60%,indicating that the content of AM determined the anti-digestibility of native starches.Compared with the native starches,the modification of AS increased the RS content of modified starches.Taking the phase transition as turning point,the digestibility of modified starches could be divided into two stages:before the phase transition,the content of rapidly digestible starch(RDS)and slowly digestible starch(SDS)of mLN,mLJ and mZJ continued to decrease,while the content of RS increased to64.91%,60.86%,and 57.26%,respectively.The starches with longer branch chains had higher content of RS.After the phase transition,the content of RS in starch decreased slightly with the modification of AS.When TD was constant,starches with higher content of AM tended to have lower content of RS,indicating that the phase transition point of starches with less AM content appeared later.The branch chains of starches were elongated more effectively.The above results indicated that waxy starch was more suitable for the preparation of resistant starch by AS modification.The modified starches with TD=70%were selected and applied to the preparation of rice cake,and the effect of different addition amounts of modified starch(15%and 30%)on the quality of rice cake was researched.The results showed that the digestibility of rice cake with modified starch was reduced compared with the control group.When the amount of modified starch added to 30%,the starch hydrolysis rate of rice cake dropped from 86.01%to 42.83%(hydrolysis time 180 min).The modified starch was easy to retrogradation and it caused the hardness of rice cake increased with the higher addition of modified starch,which led to poor elasticity and reduced the quality of food.When the additive amount was 15%,the starch hydrolysis rate of rice cake reduced slightly,but the quality of rice cake was still within an acceptable range in terms of hardness and elasticity.