Study On The Mechanism Of Determination Of Oat β-glucan By Congo Red

Oat β-glucan is a linear polysaccharide consists of glucose residues linked by β-(1→3)andβ-(1→4)glycosidic bonds.It acts as a functional polysaccharide which has increasingly attracted people’s interest.The development of methods for determination of β-glucan plays an important role in further study on stucture and physiological function of oat β-glucan.In this paper,the effects of various experimental factors on quantification of oat β-glucan by Congo red were studied,and optimum determination conditions were proposed.In order to verify the feasibility of the Congo red method,standard enzymatic assay were compared to Congo red method.On this basis,a variety of instrumental analysis methods were used to explore the mechanism of interaction,binding mode and conformational change of the combination ofbetween Congo red and oat β-glucan.Moreover,the combination of Congo red and certain macromolecules were also studied in order to reveal the general rule of conjugation of Congo red and polysaccharide.The optimal conditions of Congo red method were as follows:the final concentration of Congo red was 15 μg/mL,the ion concentration of phosphate buffer was 0.1 mol/L and proper pH was 7.5,the experiment was carried out at 25 ℃.The absorbance measured at 550 nm had a linear relationship with β-glucan in the concentration from 0 to 10 μg/mL.The co-existence of starch and protein at low concentration had little effect on results of determination.Under certain conditions,Congo red method had good precision and high correlation between enzymatic method on quantification of β-glucan,therefore it can determine the content of β-glucan accurately.A series of analysis methods such as UV-visible spectroscopy,dynamic light scattering,nuclera magnetic resonance,scanning electron microscopy and atomic force microscopy were used to invest the aggregation state of Congo red solution.The results indicated that Congo red in aquous solution at low concetration formed aggregates,which may related to the hydrophobic interaction.Besides,salts can promote aggregation of Congo red,however elevated temperature was not conducive to the formation of aggregates.The mechanism of Congo red binding with oat β-glucan is complicated.Electrostatic interaction、hydrogen bonds and hydrophobic interactions were involved in the binding processThere were two critical concentration of ions that affected the composite of Congo red and oatβ-glucan.The first critical concentration C,was the lowest concentration which caused aggregation of Congo red to form micelles,while at the second critical concentration C2,the complex of Congo red and oat β-glucan could form more large supramolecular by aggregation of Congo red which resulting in red shift of absorption wavelength.Different ions had different critical concentration,and the law was in line with Hofmeister series.The generation of the induced circular dichroism signal revealed that Congo red was combined to β-glucan in the manner of polymer.The results of dynamic light scattering-autosizer,atomic force microscope and scanning electron microscope indicated that the particles of complex became larger and the scattering intensity enhanced as well as the surface morphology changed when oat β-glucan was combined with Congo red.Studies showed that Congo red could form complex with a variety of polysaccharides with different glycosidic bonds.To some extent,The binding of Congo red and polyol compounds validated the important role of hydrogen bonds in the binding process.Moreover,the combination of Congo red with methyl cellulose and carboxymethyl cellulose revealed the electrostatic and hydrophobic interaction between oat β-glucan and Congo red.