Study On The Substitution Rule And Regulation Of OSA Starch Substituent

As a common modified starch,starch modified with octenyl succinic anhydride（OSA starch）has the advantages of emulsification,film forming and good stability,which had been paid more attentions in fields of food processing,biopharmaceutical,cosmetic development and so on.The degree of substitution（DS）,reflecting the modification degree of OSA starch,plays a key role in the emulsifying properties of OSA starch.However,as a food additive,the US Food and Drug Administration（FDA）stipulates that the amount of OSA added to starch cannot exceed 3%of the weight of starch（DS≈0.02）.Therefore,how to further improve the emulsification property of OSA starch under limited DS is of great significance for the development of OSA starch products with high quality and broadening the application of OSA starch.In this paper,to explore whether the substitution position of OSA groups has the possibility of regulation as the starting point,a continuous mode（CM）was used to control the OSA supply in the esterification reaction between OSA and native waxy maize starch（NS）,the substitution priority and substitution role of OSA groups along starch chains were clarified with the increase of DS.By analyzing the emulsifying properties of OSA starch（OSA-BM and OSA-CM）prepared under batch mode（BM）and CM with different distributions of substituents,the relationship between substitution position of OSA groups and the emulsifying property of OSA starch was established.On this basis,the preparation method of OSA starch combined with BM and CM was designed to achieve the improvement of the emulsifying property of OSA starch.At the same time,the substitution rule of OSA groups along the starch chain was rationalized by analyzing the change of lamellar structure of OSA starch.To further expand the advantage of the OSA substituent position,pretreatment with enzyme was used to conduct directional hydrolysis of NS,achieving the goal of improving the emulsification property of OSA starch by regulating the substitution position of OSA group.The main results are as follows:（1）Considering that the unlimited supply of OSA under BM was probably responsible for the random substitution of OSA groups,CM was used for limited supply of OSA,clarifying the preferential substitution position of OSA groups along the starch chain and the substitution rule with the increase of DS.According to the molecular weight distribution of OSA starch after pullulanase hydrolysis,OSA groups were preferentially substituted at the branching points of the backbone chain（C chain and long B chain）.With the increase of DS,OSA groups were substituted at the branching points between A chain and B chain to the branching points between B chains.Theβ-limit values of OSA starch hydrolyzed by onceβamylase and successiveβamylase-pullulanase-βamylase suggested that when DS increased to 1.4%or above,OSA groups began to be substituted on the branching chain and gradually approached the non-reducing end（NRE）.At low DS（<1.4%）,OSA groups were more substituted at the branching points in OSA-CM,at high DS（>1.4%）,OSA groups were more substituted at branching points in OSA-BM.Combined with structural characteristics such as molecular weight and average chain length of starch,the average substitution position of OSA groups in OSA-CM was closer to the NRE by quantitative analysis.By comparing the emulsifying activity index and creaming index of OSA-CM and OSA-BM,the influence of substitution position of OSA groups on the emulsifying property of OSA starch was preliminarily discussed,when DS was lower than 1.4%,more branch point substitution was beneficial to the improvement of the emulsifying property of OSA starch.（2）By comparing the differences in the structural characteristics and emulsifying properties of OSA-CM and OSA-BM,the relationship between the substitution position of OSA groups and the emulsifying property of OSA starch was established.In terms of emulsifying capacity,emulsion stabilized with OSA-CM showed smaller initial particle size(d₄₃),indicating that more branching-point substitutions of backbone chain was beneficial to improving the emulsifying capacity of OSA starch.For emulsion stability,emulsion stabilized with OSA-BM showed lower change in particle size(Δd₄₃)at low DS（<1.4%）,while at high DS（>1.4%）,theΔd₄₃ of emulsion stabilized with OSA-CM was lower,it indicated that more branching-chain substitutions was beneficial to the improvement of emulsion stability.At the same time,by investigating the surface tension,Zeta potential,interfacial adsorption capacity and adsorption layer thickness of OSA-CM and OSA-BM,it could be speculated that the higher emulsifying capacity probably be attributed to the faster migration rate of OSA starch molecules to the oil-water interface,and the higher emulsion stability was due to the thicker interfacial adsorption layer of emulsion which affected by the substitutent position.The method of preparing OSA starch by combining CM and BM was proposed,which not only further improving the emulsifying property of OSA starch,but also proved that more branching-point substitutions was beneficial to improve the emulsifying property of OSA starch under the condition at 3%OSA level（DS≈1.4%）.（3）Small angle X-ray scattering（SAXS）was used to investigate the changes in the lamellar structure of OSA-CM granules with the increase of DS,in order to verify the rationality of substitution rule of OSA groups along starch chains in the heterogeneous reaction with starch granules.When the DS was less than 1.5%,OSA groups were preferentially substituted in the intermediate region of lamellar structure composed of branches of the backbone chain,which showed an increase in the thickness of the intermediate lamellae（d_B-d）.At the same time,initial substitution position near the crystalline region resulted in a decrease in the crystallinity and compactness of the lamellar structure,which showed a decreased relative crystallinity（RC）and fractal dimension（D_m）.This loosened lamellar structure resulted in an increase in the substitution of branching chain as DS increased to 1.5%,which in turn facilitated OSA substitution of branching chains with higher DS（3.1%and 6.6%）,showing the relatively low D_m and relatively high d_c.In addition,scanning electron microscopy（SEM）,laser particle size analyzer and Raman microscope were used to observe the surface morphology,particle size distribution and overall substituent distribution of OSA-CM granules with different DS,the consistency between the changes in these structural properties and lamellar structure was established by principal component analysis（PCA）.（4）Based on the fact that more branching-point substitutions contribute to the improvement of emulsifying property of OSA starch,NS was pretreated withβamylase（BA）and starch branching enzyme（BE）to achieve the goal of increasing the substitution probability of OSA groups at the branching points via increasing the branching degree（DB）of starch.Due to that the gelatinization of starch resulted in the increase of reaction efficiency,starch was gelatinized before enzyme treatment.After BA pretreatment,OSA groups substituted at the branching points of starch were decreased,which was illustrated by the single distribution of molecular weight after pullulanase hydrolysis and the increasedΔβ-limit value afterβamylase and successiveβamylase-pullulanase-βamylase hydrolysis.The change of substituent position,increase of DS,decrease of molecular weight and molecular density led to the enhancement of emulsifying capacity of OSA starch pretreated with BA,but the emulsion stability did not significantly improve.After being pretreated with BE,more OSA groups were substituted at the branching points of starch,and with the prolong of pretreatment time,more OSA groups were substituted at the branching points,which was illustrated by the decreasedΔβ-limit value afterβamylase and successiveβamylase-pullulanase-βamylase hydrolysis.The change of substituent position,increase of DS and molecular density,decrease of molecular weight resulted in the higher emulsifying capacity and emulsion stability of OSA starch pretreated with BE than those of control OSA starch without BE pretreatment,showing decreased surface tension,lower d₄₃ andΔd₄₃ of emulsion.Pretreatment with BE further improved the emulsifying properties of OSA starch,and the result further proved that more branching-point substitutions was beneficial to improve the emulsifying property of OSA starch at 3%OSA level.