Chemical Structure, Chain Conformational Transition, And Self-assembly Behaviors Of Stiff Glucan From Auricularia Auricular-judae

Polysaccharides as an important component in life, receive increasing attention. Exploring the self-assembly process makes us even closer to the most fundamental structural and functional secrets of living organisms. A bound of cellulose molecules self-assemble into nanofibril â… , which further organize into the cell walls. And the entangled chitin molecules assemble into nanofibers, which further interact with protein and minerals to form the exoskeletons of crabs and shrimps. These behaviors strongly depend on the stiff chains. Meanwhile, the triple helical lentinan, polysaccharide from Auricularia (A.) auricula-judae, and the polysaccharide derivatives from Poria cocos mycelia exhibit a high specificity immunopotentiation, which is related with the stiff chain conformation in organism and its unique molecular recognition ability. A. auricula-judae has been a famous tonic food with a rich resource and long history in Asia. However, the chemical structure of the water-soluble β-D-glucan with stiff chain from A. auricula-judae, as well as the functions are seldom reported. In this thesis, a simple and moderate approach for the extraction of the stiff β-D-glucans from A. auricula-judae was first provided. The molecular weight, size and chain conformation transition were investigated. Based on the comb-branched structure and hydrogen bonding interaction, the glucans self-assemble into a series of supramolecular architectures, aiming at revealing the secrets of life phenomenon in nature. Meanwhile, the chain conformational transitions in different conditions including heating treatment and polar solvent, were investigated to understand the multiple hydrogen bonding interaction. Therefore, this work is in a cross field of polymer physics, nanoscience, materials science and biology, and also one of the international research frontiers of polymer science.The innovative points of this thesis are as follows:(1) It has been firstly revealed that a water-soluble β-(1â†'3)-D-glucan (AP1) extracted from the A. auricula-judae, which was determined to show a comb-branched structure. Additonally, AF1exhibited stiff chain conformation in water, whereas a flexible chain in DMSO.(2) It has been firstly revealed the self-orientation and self-assembly behaviors of AF1in a parallel manner. It self-assembled simultaneously to form hollow nanofibers, lamella and hollow fibers in water.(3) An aggregation-induced emission (AIE) probe was used to prove the hydrophobic cavity of the nanofibers. The hollow fibers exhibited excellent tensile strength and good resistance to oil.(4) It was revealed that the AF1fractions underwent a thermal induced conformation transition from stiff chains to flexible chains. The conformation transition also existed in various DMSO/water mixtures, which was an irreversible process.(5) It revealed that the multiple chain conformation transitions and hydrogen bonds could be determined by the ultrasensitive differential scanning calorimetry (US-DSC) combined with viscometry.The main contents and conclusions in this thesis are divided into the following parts. A water-soluble neutral polysaccharide (AF1) was extracted from A. auricula-judae with0.15M aqueous NaCl at80-100℃. Its chemical components and structure were analyzed by GC, GC-MS, and NMR. AF1was identified as a β-(1->3)-D-glucan with two β-(1â†'6)-D-glucosyl residues for every three main chain glucose residues, showing a comb-branched structure. By using static laser scattering (LLS), size-exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS), AF1was proved to existed as stiff chains in water, whereas flexible chains in dimethylsulfoxide (DMSO). The high intrinsic viscosity [η] of1753mL/g and the structure-sensitive parameter p (=Rg/Rh) value of2.3in water also revealed the stiff chain conformation of AF1. Moreover, we directly observed the extending stiff chain conformation by atomic force microscopy (AFM). The branching structure led to the water solubility of AF1, and the intra-molecule hydrogen bonds sustained the stiff chain conformation. The rheological results showed that this polysaccharide aqueous solution had higher viscosity even than xanthan, a pronounced thickening agent. This work provided important information for developing new thickeners in the food field.Hollow fibers with high strength were successfully spun from the AF1aqueous solution at a concentration of0.02g/mL. AFM and transmission electron microscopy (TEM) directly confirmed that AF1existed as stiff chain conformation in water, and displayed parallel self-orientation behavior. AF1could self-assemble into well defined hollow nanofibers with diameters less than100nm and length of tens micrometers in dilute solution, supported by scanning electron microscopy (SEM). Moreover, AF1in the disulfonated tetraphenylethene (TPE-SO3Na) aqueous solution exhibited strong luminescence, indicating that the TPE-SO3Na molecules without luminescence in water were trapped into the cavities of the hollow nanofibers through hydrophobic interactions, leading to the aggregation-induced emission (AIE). The nanofibers were composed of relatively hydrophobic inner-walls and hydrophilic shells in water. Interestingly, SEM and the polarized light microscopy verified that the nanofibers fused to form an ordered architecture of lamella and then tended to curl into hollow fibers in the relatively concentrated solution. The hollow fibers exhibited excellent tensile strength, organic solvent resistance and birefringence. A schematic model was proposed to describe the construction of the hollow fibers via the hierarchical self-assembly process.The thermal-stability of polysaccharides under heat treatment is an important factor to their functionality in food and pharmaceutical fields. A glucan fraction with moderate mole mass was prepared by ultrasonication, coded as AF1-1. The solution properties of AF1-1was investigated with viscometer, dynamic light scattering (DLS) and SEC-MALLS in water at25～170℃. The chain conformation of AF1-1in the aqueous solution exhibited a sharp decrease in viscosity, hydrodynamic radius (Rh), and Mw at elevated temperature in a narrow range of140～160℃, revealing that the conformation transitions of the AF1-1chains from rod-like chains to flexible occurred at140～160℃. Meanwhile, the double peaks suggested the coexistence of the flexible chains and stiff chains at155℃, as a result of the breaking of the intra-and inter-molecular hydrogen bonds of AF1-1. The results from SEM and AFM further directly proved that the AF1-1nanofibers in water were destructed into flexible coils at temperature higher than155℃. The conformational transition from stiff to flexible chains at140～160℃was irreversible. However, the chain shape and stiffness of AF1-1was stable below140℃and hardly changed with an increase of temperature. This was important for its application in the fields of food and pharmaceutical.The conformation transition of AF1in DMSO/H2O mixtures were investigated by viscometry and laser light scattering (DLS/SLS). It has been proven that AF1displayed as stiff chains in water, whereas flexible chains in DMSO. By being dissolved directly in the mixtures, AF1experienced a conformational transition in the vDMSO (volume ratio of DMSO) range of0.8～1.0. However, after a long dialysis, the transition moved to vDMSO,d=0.9～1.0. The more narrow transition range indicated that the dialysis treatment promoted the three phase system to achieve a thermodynamic balance, which made the AF1chains well dispersed in mixtures. By adding a small amount of water, the viscosity and chain size increased immediately, suggesting that the ability of hydrogen bonding formation for AF1chains was very strong. It could quickly form hydrogen bonds with water to make extension of chains. When the content of DMSO was over the range of transition, there was a consistent drop in [η], Rh and Rg. Meanwhile, the values of Rh exhibited an obvious angle dependence. The results indicated that the intra-and inter-hydrogen bonding of AF1were interrupted simultaneously, leading to a flexible chain. The TEM images further proved the occurrence of the conformational transition. Further more, by adding water with content up to50%to the interrupt AF1solution, the viscosity and chain size hardly changed. It indicated that the conformation transition for AF1in DMSO/H2O mixtures was irreversible. Meanwhile, with a longer storage time, the hydrogen bonding formed immediately in the renatured system was prone to achieve the dynamic balance with a broader distribution.The hydrogen bonding interaction and multiple conformation transition of AF1in DMSO/H2O mixtures after heating treatment were investigated by13C NMR, viscometry and US-DSC. The results from NMR and DSC indicated that the weak hydrogen bonding interaction formed between the side chains of AF1and water clusters was disrupt after heating in the lower temperature range of0～70.6℃. It was a reversible transition. When vDMSO<0.7, as a result of the competiton between DMSO and water as well as solvation of the solution, the transition temperature increased with an increase of vDMSO in the low temperature range. When vDMSO>0.7, the transition temperature decreased with an increase of vDMSO-It indicated that when the content of DMSO was high enough, the main contribution of DMSO was to form new and relatively stable hydrogen bonds with AFl, leading to the destruction of the associated water layer. On the other hand, in the higher temperature range of95～160℃, the results of viscosity suggested a conformation transition of AF1from stiff chains to flexible coils, which resulted from the destruction of the intra-and inter-hydrogen bonds.This foundation research mentioned above focused on the chemical structure, molecular size and chain conformation of polysaccharides extracted from A. auricula-judae. We revealed the mechanism of the unique self-assembly behaviors for stiff glucans in water. It not only shows scientific significance, but also provides important scientific data for the exploration and application of A. auricula-judae as Chinese medicine resources.