Study On Preparation,Antioxidant Activity And Structural Characterization Of Carboxymethyl Yeast Glucan

At present,the research on the modification of yeast glucan is mainly focused on carboxymethylation,while the research on the preparation technology of carboxymethyl glucan derivatives of yeast is mainly focused on the preparation of carboxymethyl yeast glucan with the maximum degree of substitution.However,there are few studies on effect of the degree of substitution and molecular weight on the biological activity of carboxymethyl yeast glucan.Therefore,this study using yeast glucan extracted from baker's yeast as raw material,carboxymethyl yeast glucan with different degrees of substitution was prepared by carboxymethylation derivation.The effects of degree of substitution and molecular weight on the antioxidant activity of carboxymethyl yeast glucan?CMG?in vitro were investigated by scavenging hydroxyl radical,ABTS⁺and Fe²⁺chelation experiments,and on the basis of single factor experiments.Box-Behnkn Design in response surface analysis was used to establish a mathematical model to optimize the preparation process of carboxymethyl yeast glucan?CMG?with the best activity.The main experimental results are as follows:?1?By comparing the ability of yeast dextran and carboxymethyl yeast glucan with different degrees of substitution to scavenge·OH,ABTS⁺·and chelate Fe²⁺,the relationship between the degree of substitution and the antioxidant activity of dextran in vitro was found to be dependent on the degree of substitution.Considering the actual production situation and the effect of the degree of substitution on the activity of CMG,it is decided to optimize the CMG with a degree of substitution of 0.8.?2?On the basis of single factor,the response surface method was used to optimize the preparation process of carboxymethyl yeast glucan with a degree of substitution of 0.8.The mass ratio of sodium hydroxide to glucan is 3.2,and the mass ratio of chloroacetic acid to sodium hydroxide is 1.5.Carboxymethylation time is 2.0 h and alkalization time is 1.5 h.Carboxymethyl yeast glucan with a degree of substitution of 0.784 was prepared by this process,and the result was close to the predicted value.?3?The three components CMGB,CMGC and CMGD,of CMGA were separated by gel permeation chromatography and ultrafiltration system.The results showed that the three components were single polysaccharide components with relatively high purity.The number-average molecular weight?Mn?and weight-average molecular weight ?Mw?)of CMGB,CMGC and CMGD were calculated by GPC analysis software to be1.881×10⁶ daltons and 5.403×10⁶ daltons,2.915×10⁶ daltons and 5.990×10⁶ daltons,54294 daltons and 1.300×10⁶ daltons.?4?The effect of molecular weight on the free radical scavenging activity of carboxymethyl glucan from yeast was evaluated by comparing the scavenging ability of CMGB,CMGC,CMGD to scavenging·OH,ABTS⁺·and chelating Fe²⁺.The results showed that the component with lower molecular weight showed better free radical scavenging activity.?5?By the infrared spectrum analysis,the infrared spectra of GLUCAN and CMGB,CMGC and CMGD are basically the same,and all have the characteristic absorption peaks of polysaccharides.The sugar ring configuration of the four polysaccharides samples is that pyran ring contains?-type glycosidic bonds,and the main structures of CMGB,CMGC and CMGD are good.However,CMGB,CMGC and CMGD have new characteristic absorption peaks near 1611cm^-1,1439 cm^-1 and 1379 cm^-1,indicating that all the three samples have carboxymethyl groups.?6?By NMR analysis,Glucan is a kind of D-glucan with?-?1-3?glycosidic bond as the main chain.However,the main chain signals of CMGB,CMGC and CMGD still existed between 60-110ppm,indicating that the main chain structure of polysaccharides did not change,while the chemical shift of part of C-6 signal peak shifted from 61.34ppm to 69.70 ppm?C-6'?,and part of C-4 signal peak shifted from 68.90 to 74.92ppm?C-4'?,and the shift of C-2 signal peak was rarely observed.The results showed that the carboxymethyl derivative reaction mainly occurred in C-6 and C-4 hydroxy position,and the activity order of carboxymethyl derivative reaction was as follows:C-6 position>C-4 position>C-2 position.?7?By X-ray diffraction analysis,in the diffraction pattern of GLUCAN samples,there are wide diffraction peaks of GLUCAN,CMGB,CMGC and CMGD at 20°,indicating that although CMGB,CMGC and CMGD are modified by carboxymethyl,but the main structure remains unchanged.?8?By the analysis of scanning electron microscope,the results show that the whole particle of GLUCAN is neat and compact,and there is no obvious granular structure on the smooth surface.The whole CMGB particles have irregular shape,particle size aggregation,compact structure,rough and uneven surface,and obvious granular structure.The whole CMGC particles are irregular in shape,the overall structure is loose,and the surface is rough and uneven.Many cracks and irregular large pores can be observed on the surface,and there is a large internal space.The surface of CMGD has smooth structure and rough granular structure,and the whole particle connection is closer than that of CMGB and CMGC,and the overall structure is closer.