Multi-scale Recombination Of Lotus Seed Starch And Chlorogenic Acid By Microwave Irradiation And Its Retrogradation Behavior

Lotus seeds are the mature seeds from the lotus plant of the waterlily family（Nelumbo nucifera Gaertn.）and are a specialty economic crop in China,ranking first in the world in terms of production.Lotus seeds are typically a highly starchy food rich in phenolic components（chlorogenic acid,CA,is the major phenolic component）,with starch accounting for60%,and more than 40%are amylose,which is very susceptible to retrogradation.In this study,the formation,multi-scale structure,molecular docking pattern and retrogradation behavior of lotus starch-chlorogenic acid complexes were systematically investigated.The research results provide a new idea for the formulation of lotus seed products with tailored quality characteristics and nutritional properties,and are of theoretical significance and practical value in promoting the application of microwave technology in high starchy foods.（1）Dielectric response and combining behavior of lotus seed starch-chlorogenic acid under microwave treatmentThe dielectric response and combining behavior of lotus seed starch-chlorogenic acid complex was analyzed by using modern analysis techniques.The results showed that the dielectric constantε′of the starch system decreases rapidly with decreasing moisture content in the tested frequency range（ε′70-75 for 10%starch solution and 50-40 for 50%starch solution in 10～3000 MHz）.A comparison of the two systems revealed that the addition of chlorogenic acid will result in a more pronounced downward trend in the dielectric constantε′over the same temperature range.The variation of the imaginary dielectric constantε′′and the dielectric loss tangent tanδεof the microwave thermal conversion parameter indicates that the lotus seed starch-chlorogenic acid complex has a stronger microwave-thermal energy conversion efficiency.Compared to the lotus seed starch,the lotus seed starch-chlorogenic acid complex exhibited stronger aggregation behavior,resulting in the re-association of free starch molecules to form large size agglomerates.Accordingly,the dispersion pattern of the lotus seed starch-chlorogenic acid complex changed to a two-component characteristic with coarse particle size predominating in the late pasting stage.The morphological changes indicated that chlorogenic acid and starch formed different types of binding modes at different microwave powers,and only at the high microwave powers can chlorogenic acid actively participate in the reorganization of the internal conformational units of starch.In addition,the excessive microwave power（>300 W）caused the starch chains to agglomerate without sufficient water absorption,which inhibited the cross-linking of the lotus seed starch and chlorogenic acid.The results provide a theoretical basis for regulating the physicochemical properties of lotus seed starch by using phenolic components during microwave processing.（2）Multi-scale structure of lotus seed starch-chlorogenic acid complexThe fine structure of microwave lotus seed starch-chlorogenic acid complexes was further investigated from a multi-scale perspective.The results showed that chlorogenic acid was involved in the rearrangement of starch in various forms（non-inclusion complex,V₇-type inclusion and simply physically entrapped）.When the starch granules are intact,chlorogenic acid was evenly distributed over the starch granule surface,forming a thin film or shell,with increasing power levels,the granular structure of starch was gradually destroyed and formed colloidal agglomerates,facilitating exposure of the starch chains originally buried in the starch granules and allowing more binding sites for chlorogenic acid.The long-range ordered and short-range ordered structural changes indicate that hydrogen bonding is the main force stabilizing the lotus starch-chlorogenic acid complex system,and the amylose is the main binding site for the chlorogenic acid.Helical and aggregated structure studies show that unlike the usually well-arranged starch complexes,the lotus seed starch-chlorogenic acid complexes could not form new periodic molecular organization due to the bulky aromatic ring of chlorogenic acid,a small amount of V7-type polycrystalline peaks（103.5 ppm）can only be formed under microwave power conditions of 300 W.In this regard,these aggregated structural units were more likely to be involved in the formation of a compact amorphous-aggregated conformation.The results provide a theoretical basis for clarifying the interactions between lotus seed starch and guest molecules in microwave fields.（3）Molecular dynamics simulation of lotus seed starch-chlorogenic acid complexes and their rheological propertiesBased on the study of the multi-scale structure of the lotus starch-chlorogenic acid complex,molecular dynamics simulations were introduced to investigate the correlation between the rheological properties and the fine structure of the complex system from the perspective of classical Newtonian mechanics.The results showed that in the starch system,the more structurally flexible single-helical component is more prone to conformational changes,where chlorogenic acid is inserted into the helical binding grooves formed by the sugar chains,forming a good geometrical match and physicochemical bonding,whereas the double-helical structure is unable to form grooves,and chlorogenic acid only attaches to the surface of the double-helical chains.The rheological properties showed that with the increase of the microwave power（200-300W,8 min）,pasting parameters（except gelatinization temperature）,apparent viscosity,hysteresis loop area,elastic modulus and viscous modulus decreased firstly and then increased.Compared with the starch alone,the declining trend of the complex system was more significant,indicating that the cross-linking structure of starch paste was weakened after the addition of chlorogenic acid.Combined with amylose dissolution and molecular weight analysis,chlorogenic acid can effectively intervene in the interaction of amylose molecules,weaken the formation of permanent connection zone in the gel network,accordingly,the polydispersity coefficients（Mw/Mn）of the starch and composite groups increased from 1.67 and 2.77 to 4.47 and 4.64,and make the starch paste system more restorative.As explained above,the formation of the starch-chlorogenic acid complex can enhance the thermal stability and shear stability of starch the paste system.The results provide a theoretical basis for regulating the processing properties of starch-based foods from the molecular level.（4）Retrogradation properties of lotus seed starch-chlorogenic acid complexesBased on the study of the intermolecular interaction pattern between lotus seed starch and chlorogenic acid,the anti-retrogradation mechanism,molecular chain changes,the process of phase changes,water migration pattern,thermodynamic properties and the nano-aggregate of the corresponding long-range ordered structure and short-range ordered structure were discussed.The results showed that microwave treatment of lotus seed starch-chlorogenic acid mixtures resulted in greater cleavage and release of internal B2 and B3 amylopectin polysaccharide chains than in similarly treated lotus seed starch.These short-chain starch components help to stabilize the starch gel and increased the water-holding capacity.In addition,the lotus seed starch-chlorogenic acid complexes appear more inclined to participate in the reorganization of amorphous regions,in the form of random dispersion and as a result,the packing of the amorphous aggregated structure of LS-CA may become more compact through friction-extrusion.The long-range ordered and short-range ordered structural changes suggests that retrogradation of LS-CA is influenced by the inhibitory effect of polyphenols on re-formation of the starch helical conformation and thereby inhibits the periodic long-range helical arrangements during retrogradation,in this instance,the helical structural units could not easily form a crystalline lattice,because of their poor steric compatibility.The results provide a theoretical basis for revealing the retrogradation mechanism of the lotus seed starch-chlorogenic acid complex from the molecular chain level.（5）Digestive properties of lotus seed starch-chlorogenic acid complexesBased on the perspective of in vitro experiments,the slow digestive properties of lotus seed starch-chlorogenic acid complexes and its inhibition ofα-amylase activity were discussed.The results showed that the complexation of chlorogenic acid in the microwave environment significantly improved the slow digestion of lotus seed starch,and there were significant differences in the regulation patterns of starch hydrolysis by different types of complexation sites and chlorogenic acid released behavior.The discrepancy was mainly because starch hydrolysis in a multicomponent system was affected by not only the multi-scale structure of starch but also closely related to the physiological activity of chlorogenic acid.Therefore,it is clear that that complexation with CA modified structural units via the formation of a protective CA shell or the compact amorphous region that directly shielded starch molecules（granular structure）and reduced flexibility and mobility of starch chains through additional hydrogen bonds between the starch hydroxyl groups and the hydroxyl or phenol groups of CA（crystalline structure）.In this regard,a better conformation of the amorphous starch molecules network formed in LS-CA is likely a major structural factor influencing the digestion characteristics of starch.In addition,similar to the microcapsule（encapsulated delivery system）,part of the CAs might be released from the complexes as the hydrolysis proceeded,which would further inhibit hydrolysis via competing with starch for water molecules and enzymes.The results provide a theoretical basis for the formulation of starch-polyphenol complexes with tailored nutritional properties.