Preparation Of Carboxymethylated Euglena Gracilis Polysaccharide And Its Digestion And Fermentation Characteristics In Vitro

Euglena gracilis polysaccharide（EGP）is a unique linearβ-1,3 glucan in Euglena gracilis,which has many biological activities such as antioxidant,anti-inflammatory and improve immunity.However,the water insolubility of Euglena gracilis polysaccharide limits its application in food and medicine fields,and carboxymethyl modification is an effective way to significantly improve water solubility.Therefore,in this thesis,the EGP was extracted and purified from Euglena gracilis,and then it was carboxymethylated and characterized.In vitro digestion and fermentation models were designed to explore the effects of EGP and carboxy methylated Euglena gracilis polysaccharide（C-EGP）on intestinal flora.The specific research results are as follows:（1）The effects of three different extraction methods,namely enzymatic hydrolysis,alkaline extraction and ultrasonic-assisted sodium dodecyl sulfate（SDS）extraction methods,on the yield of polysaccharide and the removal rate of protein were compared and analyzed.The ultrasonic-assisted SDS extraction method was determined to be the best.Based on the outcom of univariate tests,the optimum extraction conditions of EGP were determined by Box-Behnken central combination experiment:ultrasonic power was100 W,ultrasonic time was 17 min,solid-liquid ratio was 1:20（g/m L）,and SDS concentration was 20 g/L.By these conditions,the maximum yield of EGP was 35.28%.The crude polysaccharides were isolated and purified by DEAE-52 and Sepharose CL-6B.The Raman spectrum and ultraviolet full-band scanning technology confirmed that the polysaccharide was a high-purity EGP.Ion chromatography analysis showed that EGP was mainly composed of glucose.（2）The carboxymethylated derivatives with degree of substitution（DS）of 0.14,0.55and 0.78 were prepared by solvent method under different conditions.The components analysis showed that the total sugar content of the sample decreased from 55.06%to38.91%,and the uronic acid content also decreased to some extent with the increase of DS.FTIR and HPGPC proved that carboxymethyl was successfully introduced into EGP.The NMR confirmed that carboxymethyl substitution occurred at C4 and C6 positions.The structure and physicochemical properties of the modified polysaccharide were characterized by SEM,XRD,CR and TG.The results showed the surface of C-EGP became rough,but the original triple helix structure was not destroyed,and its thermal stability and crystallinity debased with the increase of DS.In addition,through the water solubility and adsorption experiments,it was found that the water solubility of C-EGP with three different degrees of substitution increased by 53.31%,75.52%and 80.96%respectively,and the adsorption of peanut oil,methylene blue dye and metal ions(Fe²⁺,Cd²⁺)was also improved after carboxymethyl modification.（3）The total reducing sugar content and triple helix structure of EGP and C-EGP did not change during digestion,which showed that both of them could not be digested in upper digestive tract.In vitro fermentation results showed that EGP and C-EGP could reduce the p H of fermentation broth.However,the level of acetic acid,propionic acid and butyrate acid augmented.The total level of SCFA was 23.48±0.92 mmol/L in C-EGP group and that was 20.25±0.46 mmol/L in EGP group after 48 h fermentation,which was significantly larger than that in Blank group（15.09±0.42 mmol/L）.The results of 16S r RNA sequencing showed that EGP and C-EGP could significantly improve the relative abundance of good bacteria,for example Firmicutes and Bacteroides.It was indicated that both EGP and C-EGP would change the composition of intestinal flora,and had potential probiotic effects.