Preparation And Structural Characterization Of Polysaccharides From Auricularia Cornea Var.Li. And Their Interactions With Whey Protein

Polysaccharides from edible fungi were known as“biological response modifiers”（BRMs）because of their biological activities,such as antitumor,antioxidative,antivirus,anti-aging,hypoglycemic and immunomodulatory activities.Due to their great nutrition value and desirable biological activities,more and more studies are focusing on edible fungi polysaccharides in the world.Auricularia cornea var.Li.（“Yu Mu Er”in Chinese）is a novel edible fungus which has the potential health benefits.Auricularia cornea var.Li.is widely cultivated in China,including Jilin and Liaoning province.In order to further enhance the application of Auricularia cornea var.Li.and Auricularia cornea var.Li.polysaccharides（ACP）in food applications,we mainly studied the extraction process and the effects of different extraction methods on polysaccharides from Auricularia cornea var.Li.,including extraction yield,physicochemical properties,preliminary structure and rheological properties.Meanwhile polysaccharides from Auricularia cornea var.Li.were isolated and identified the structural characteristic of polysaccharides fractions.Finally,the interactions of ACP with whey protein were investigated.The main results are as follows:1.Polysaccharides from Auricularia cornea var.Li.were extracted by hot water extraction（HWE）and ultrasound-assisted extraction（UAE）methods,respectively.Take extraction yield and extraction rate as index,optimum conditions for HWE（particle size of 150-200 mesh,water to raw material ratio of 60:1（v/w）,extraction temperature at 90°C for 3.0 h）and UAE（particle size of 150-200 mesh,water to raw material ratio of 70:1（v/w）,extraction temperature at 70°C for 40 min,ultrasonic amplitude of 40%）were obtained via single-factor experiment.Under optimum conditions,extraction yield of polysaccharides by UAE was 30.99±1.93%which showed no significant difference with that by HWE（30.35±1.67%）（P>0.05）.Extraction rate（29.29±1.41%）and purify（88.62±2.80%）of polysaccharides by UAE were higher than those by HWE（extraction rate of 24.95±2.78%and purity of75.33±6.15%）（P<0.05）.Scanning Electron Microscopy（SEM）microphotograph of residues by UAE showed more broken cells than those by HWE.Ultrasonic assisted extraction made the cell walls of auricularia auriculata appear more rod-like structures,which greatly damaged the cell walls of raw materials and accelerated the diffusion and dissolution of polysaccharides.The results indicated that ultrasound-assisted extraction may be a quick and high-efficient method to extract polysaccharides from Auricularia cornea var.Li.2.The effects of UAE on physicochemical properties and primary structure of ACP were investigated by determining the particle size,zeta-potential,molecular weight（Mw）,monosaccharide composition and analyzing Fourier Transform Infrared spectra（FTIR）and SEM microphotograph.Effects of UAE on rheological properties of ACP were studied including steady-shear flow property and dynamic viscoelastic property.The results indicated that UAE significantly reduced the particle size,potential value and molecular weight of ACP,which resulted in wider particle size and molecular weight distributions.Meanwhile,SEM microphotograph also showed the microstructure of ACP by UAE had more small particles than that by HWE.UAE had no significant effect on monosaccharide composition of ACP.FTIR spectra showed that ACP extracted by UAE and HWE all had virtually identical and typical absorption peaks associated with polysaccharides,which indicated that UAE had no significant effect on the primary structure of ACP.ACP extracted by UAE and HWE all showed typical shear thinning and temperature-independent behaviors（25-90°C）.Moreover,the viscoelastic of ACP had a certain concentration-dependent behavior.UAE resulted in polysaccharides with remarkably lower viscosity and exhibiting more liquid-like state.3.Four main fractions,two neutral polysaccharide fractions（ACPN-1a,ACPN-1b）and two acidic polysaccharide fractions（ACPA-1a,ACPA-2a）,were isolated from ACP using DEAE-Cellulose DE-52 anion exchange column and Sepharose CL-6B column.The results of physicochemical properties and UV spectra showed that the purity of four polysaccharides fractions were all more than 90%and contained no nucleic acid and protein.The results of High Performance Gel Permeation Chromatography（HPGPC）showed that the two neutral polysaccharides fractions（ACPN-1a,ACPN-1b）had the higher average molecular weight（².18×10⁶ Da and².07×10⁶ Da）.ACPA-1a exhibited a broad Mw distribution from⁵×10⁵ Da to²×10⁶ Da and the average Mw of ACPA-2a was estimated to be 8.5×10⁵Da.The results of monosaccharide compositions showed that two neutral polysaccharide fractions（ACPN-1a,ACPN-1b）all mainly composed of glucose（96.17%,95.79%）;two acidic polysaccharide fractions（ACPA-1a,ACPA-2a）were heteropolysaccharides and composed of mannose（44.85%,56.40%）,glucose（16.17%,1.06%）,xylose（24.26%,28.52%）,glucuronic acid（9.35%,10.89%）and other minor monosaccharides.SEM microphotographs showed ACPN-1a and ACPN-1b all had the chain conformation expressed by fibrous filaments and ribbons with branches,but ACPA-1a had the complex microstructure with some small particles and aggregates.ACPA-2a had more uniform particles.FTIR spectra revealed that ACPN-1a and ACPN-1b were all neutral polysaccharides and mainly containβ-glycosidic bonds of sugar units.ACPA-1a and ACPA-2a were all acidic polysaccharides with two glycosidic linkage ofɑ-andβ–configuration.1D NMR(¹H,¹³C)and 2D NMR spectra showed that ACPN-1a and ACPN-1b all wereβ-?-glucan consisting of（1→3）-β-?-Glcp,（1→6）-β-?-Glcp,（1→3,6）-β-?-Glcp residues.ACPA-1a was a glucuronoxyloglucomannan with（1→3）-linkedβ-Glcp and（1→6）-linkedɑ-Manp as backbone andβ-Xylp orβ-GlcAp residues as side chains.ACPA-2a was a glucuronoxylomannan and composed of backbone of（1→6）-linkedɑ-Manp and side chains withβ-Xylp orβ-GlcAp residues.4.Take turbidity as index,we studied the effects of pH and protein/polysaccharides ratio on the interactions between ACP and whey protein isolate（WPI）.The increased ratio of ACP in the mixed system could significantly reduce the pH region of complex coacervation.When the ratio of WPI and ACP was1:1 and pH was at 3-5,ACP and WPI may formed the complex through strong electrostatic interaction.In order to investigate the interaction of ACP and WPI at the neutral pH,particle size,Zeta-potential and viscosity of ACP-WPI mix solutions with different ratios were determinted.The results showed that the particle size and viscosity of ACP-WPI were increased as the increased ratio of ACP in the mixed system.ACP-WPI mixed solution all exhibited shear thinning and pseudoplasitic fluid behavior.When the ratio of WPI and ACP were 3:2 and 1:1,there might be weak electrostatic interaction between ACP and WPI at neutral pH.The FTIR spectra revealed possible weak interactions in ACP-WPI mixed system at neutral pH.In summary,polysaccharides from Auricularia cornea var.Li.were isolated and characterized for physicochemical properties and structural characteristics.The results could provide useful information for the future studies on biological activities or structure-activity relationship of ACP.The data of interactions between ACP and whey proteins may be helpful for the applications of polysaccharides from Auricularia cornea var.Li.in food applications.