Formation And Stability Mechanism Of Starchy Complexes Based On Molecular Dynamics Simulation Study

Starch,as a cheap,easily obtained and widely used natural polysaccharide,can form a complex with ligands to transfer and protect them,which has been attracted much attention on delivering food bioactive compounds and drugs due to its good biocompatibility and degradability.Fatty acids and starch can form V₆ complexes,which enhance the digestion resistance of starch-based carriers to the digestive enzymes,while the binding of fatty acids does not affect the encapsulation of small bioactive compounds or drugs in the spiral cavity of amylose.This kind of starch-based complex,however,is insoluble,offering great limits in its application.The preparation of the starch-protein-fatty acid complex can effectively solve the problem,i.e.,starch-fatty acid complex is hydrophobic,and improve the transportation capacity of starch-based complex for the hydrophobic small active substances.This expands the application of the starch-based carrier,such as applying to the functional drinks and cosmetics.In this work,common corn starch?NCS?-linoleic acid?LA?complex and NCS-bovine serum albumin?BSA?-LA complex were prepared,and their properties were tested and compared.Meanwhile,the formation process,interaction mechanism and stability of amylose-LA complex and amylose-BSA-LA complex were studied by the molecular dynamics simulation.The main research contents are as follows:The NCS-LA complex was prepared by NCS and LA,and then its properties were determined.The results revealed that,when NCS complexed with LA,the granule morphology of NCS were destroyed and they formed a more compact structure.Some of the crystalline structures were embedded in it and formed a new lamellar structure.The NCS were located at the outside.The complex displayed a V type structure.And it was a kind of type II polymorphs,which was thermally stable.The complex exhibited an obvious shear thinning behavior?non-newtonian fluid?.It was observed that the-C=O-group of the LA chain was blue shifted when complexing with NCS.Compared with gelatinized NCS,the NCS-LA complex was resistant to the enzymic hydrolysis to some extent.Then,NCS-BSA-LA complex were prepared by NCS,BSA and LA.The gelatinization property,molecular weight,microstructure and crystal structure of the complex were investigated.The pasting and high performance size exclusion chromatography curves confirmed the formation of NCS-BSA-LA complex,which was spherical with the molecular weight of 1?3×10⁷ g/mol.The NCS-BSA-LA complex was elliptical,scattered and no obvious aggregation was observed.The structure of the complex was more compact than that of NCS-LA complex.Some proportions of the BSA were located outside of the complex,so some NCS were encased.At the same time,the crystal structure of starch also changed.NCS showed an obvious A type structure,while NCS-BSA-LA complex showed a V type structure.It was also observed that the thickness of lamellar structure of NCS became thinner when it complexed with BSA and LA,indicating that BSA and LA were involved in the complex.When the complex formed,the sulfhydryl and disulfide bonds of BSA were exposed;the absorption of aliphatic hydrocarbon chain of LA decreased,while a blue shift of-C=O-in the carboxyl head group occurred,suggesting that LA was located in the hydrophobic cavity of amylose.The average particle size of NCS-BSA-LA complex was 136 nm,and the Zeta potential was-18.6 mV,indicating that the structure of the complex was stable.Besides,it is more resistant to enzymic hydrolysis compared with the NCS-LA complex.5-fluorouracil?5-FU?was used to test the encapsulation effect of the NCS-BSA-LA complex on the small hydrophobic bioactive components/drugs.The results showed that the mean particle size of the complex did not change significantly after the 5-FU was encapsulated,and the Zeta potential changed to-22.8 mV.These indicated that the complex had a good effect on the encapsulation of 5-FU,and that the basic morphology of the carrier was maintained.The stability of amylose-LA complex was studied by molecular dynamics simulation,and a single amylose fragment was used as a reference.The initial conformation was:a typical V type amylose fragment was composed by 26 glucose units;the tail of LA molecule was located in the cavity of the amylose fragment,while the head of carboxyl group was outside.The simulation results showed that,when there was no LA molecule,the V type structure of the amylose was destroyed quickly,owing to the number of intramolecular hydrogen bonds decreased sharply,especially for intra-turn O₂-O₃ hydrogen bonds.When there was a LA molecule,LA complexed with amylose.The LA molecule was mostly located in the middle parts of the amylose,maintain some of the V helical structure of the amylose.And the two end parts of amylose changed into a randomly coiled conformation.Most of the intramolecular hydrogen bonds in amylose still existed,which indicated that intramolecular hydrogen bonds were the main force to maintain the V helical structure of amylose.For the amylose-LA complex,the middle parts of amylose were stable,while the two end parts fluctuated greatly.The middle parts of amylose interacted with the non-polar part of the LA molecule,limiting the entry of water molecules into the helical cavity of the amylose so that fewer water molecules interacted with the amylose.There was a significant difference between the single amylose and the complexed amylose for principal component analysis,suggesting a significant difference in the conformation between them After the formation of the complex,the conformation of glucose ring in the amylose slightly changed,but the main glucose ring conformation was chair ⁴C1.Similarly,molecular dynamics simulation was used to investigate the complex process of amylose with different numbers of linoleic acid molecules.The initial conformation was:amylose consisted of 26 glucose units with a randomly coiled conformation,and LA molecules of different numbers?1,2,3,4?were located around the amylose molecules at a distance of about 15?.The results showed that amylose could form complexes with different numbers of LA molecules,and LA molecules preferred to locate in the middle part of amylose.The head of the carboxyl group of LA molecule was more likely to be exposed to the solvent,while the tail of aliphatic hydrocarbon chain was located in the helical hydrophobic cavity formed by amylose.With the increase of the number of LA molecules,the steric hindrance between amylose and LA molecules increased,and a longer time was required for amylose to form complex with all LA molecules.The helical diameter of the amylose was represented by the distance between O_3n and C_5n+3 atoms,which increased with the increasing of LA molecules,but it was still a V₆ type.When more than one LA molecule presented,the LA molecules tended to be located in the helical cavity of amylose in a head-to-tail orientation.As the number of LA molecules increased,the proportion of chair ⁴C₁ conformation of the glucose ring in amylose increased,but chair ⁴C₁ still accounted for a large proportion.Using the same force field as to the above,a random coiled amylose consisting of 26glucose units and a different number of linoleic acid molecular simulations were used to simulate the formation of complexes formed by amylose and different concentrations of linoleic acid molecules.The results showed that amylose could form a complex with one,two,three and four linoleic acid molecules in the system,and the linoleic acid molecule was located in the middle part of the amylose.From the snapshots of the trajectory,it shows that the carboxyl head group of the linoleic acid molecule tended to be located in the solvent,while the aliphatic hydrocarbon tail was located in the hydrophobic helical cavity of the amylose;as the number of linoleic acid molecules increased,the steric hindrance between the amylose and linoleic acid molecules increased,and the time required to form a complex with amylose lasted longer.From the diameter of the structure forming the helix,represented by the distance between the two atoms of O_3n and C_5n+3,helix diameter of the amylose increased with the increasing number of linoleic acid molecules,but they still displayed a V₆ type structure.From the analysis of the linoleic acid vector direction,the linoleic acid molecules tended to be located in the helical cavity of the amylose in a head-to-tail manner.The chair⁴C₁ glucose ring conformation increased as the number of linoleic acid molecules increased.Finally,56 amino acid residues selected from the A-chain of the BSA crystal structure were added to the simulation system,which consisted of a randomly coiled amylose containing 26 glucose units and a LA molecule,to simulate the complex formation between starch,BSA and LA.Amylose,BSA,and LA formed a complex.In addition,LA first complexed with amylose,which was located inside amylose,and then bound to BSA.LA acted as a bridge between amylose and protein fragments.The principal component analysis diagrams between amylose-BSA-LA complex and amylose-LA complex were both round,indicating they formed a similar conformation.The distance between the atoms of O_3n and C_5n+3 was smaller than that of the amylose-LA complex.Compared with the amylose-LA complex,an increasing solvent accessible surface area were observed around the amylose for amylose-BSA-LA complex,and the number of water molecules in the first hydration shell also increased,both of which,to some extent,indicated the water-soluble mechanism of NCS-BSA-LA complex.