Studies On Isolation,purification,structural Identification And Properties Of Non-reducing Maltoheptaose (N-G7)

Maltodextrin is a widely used food material.As a mixture,the functional properties of maltodextrin are closely related to the degree of hydrolysis and molecular weight distribution.The wide application of maltodextrin is limited because of the uneven degree of polymerization and the reducing end.Therefore,our group prepared non-reducing maltoheptaose（N-G7）by the double-enzymatic method to provide a new solution.β-cyclodextrin was hydrolyzed by cyclodextrinase to obtain maltoheptaose firstly,and N-G7 was prepared by transforming the terminalα-1,4-glycosidic bond of maltoheptaose into anα-1,1-glycosidic bond by maltooligosyltrehalose Synthase.To provide a theoretical reference for further exploring a series of properties and subsequent applications of N-G7,the crude solution was firstly separated and purified to determine the best purification conditions.Then,a structural analysis of N-G7 with high purity（purity>95%）was carried out to verify the configuration.Finally,the physicochemical properties and in vitro digestive behavior of N-G7 were investigated,and the colonic performance of N-G7 before/after being embedded by calcium alginate was evaluated.Firstly,the purity and content of N-G7 in crude solution were determined by HPLC.The results showed that N-G7 accounted for about 60%～70%of the product after the enzymatic reaction,and there were other maltoligosaccharides（G₁～G₇）impurities,G₃～G₆ was the main component in the impurities.Secondly,ethanol precipitation,cation exchange resin chromatography,and polyacrylamide gel chromatography were used to separate and purify the enzymatic products to remove the excess small molecular sugar.The purity and recovery rate of N-G7 were used as detection indexes to determine the optimal conditions.N-G7 crude solution was precipitated by 70%～90%（v/v）ethanol,which improved the purity of N-G7 in the solution.When the ethanol final concentration was 85%,the purity of N-G7 reached the maximum of 87.43%and the recovery rate was 89.05%,but the small molecular sugar was also precipitated and the effect was not obvious.Compared with the precipitation method,the purity of N-G7 by ion exchange resin chromatography was significantly improved.The purity of N-G7 reached 93.49%and the recovery rate was 65.50%under the most favorable conditions:resin column size was 1.6 cm×70 cm,elution speed was 30 m L/h and maximum sample loading was 5 m L.Ensuring the purity of N-G7 and improving the recovery rate is beneficial to the separation efficiency,so polyacrylamide gel was used for purification.It was found that N-G7 with a purity of 92.37%and recovery rate of 81.19%could be obtained under the conditions of column temperature of45℃,elution rate of 25 m L/h,and the sample loading volume of 5 m L.Subsequently,two chromatographic methods were used to collect high purity N-G7 to explore its structure and properties.Then,the structure of N-G7 was characterized by various means.The molecular ion peak and fragment ion peak of MALDI-TOF-MS indicated that the relative molecular mass of N-G7was 1153,and it was composed of seven hexoses.And it was proved that the monosaccharide of N-G7 was only glucose.Methylation analysis suggested that N-G7 containedα-1,4-glycosidic bonds and anα-1,1-glycosidic bond,and the molar ratio of the two was about 5:1.Infrared analysis showed that N-G7 had typical characteristic absorption peaks of carbohydrates.In contrast to maltose and trehalose,the NMR spectra of N-G7 indicated that the control ectopic proton signal and ectopic carbon signal could be found.Next,the physicochemical properties of N-G7 were explored.The high purity and non-reducibility of N-G7 were confirmed by DNS chromogenic method.The coexistence of N-G7and lysine did not produce brown products,and the low absorbance at 420 nm indicated a successful transition from the reducing end of G₇to the non-reducing end of N-G7.The hydrolysis rate of N-G7 was lower than 10%at different temperatures when the p H value was4～10,and the stability of N-G7 was better than that of fructooligosaccharides even at p H 2.It was found that the hygroscopicity of N-G7 was lower than that of maltodextrin,and the hygroscopicity of maltodextrin with a higher DE value was stronger.The results of thermodynamic analysis illustrated that N-G7 had excellent thermal stability,with a phase transition temperature of 138.38℃and endothermic enthalpy of 91.4 J/g,which were observably different from maltodextrin.The results showed that the N-G7 solution was a pseudoplastic fluid when the shear rate was from 0.01 s^-1to 200 s^-1.The N-G7 solution with low concentration（0.1%,w/v）was close to Newtonian fluid.The dynamic rheological properties of the N-G7 solution at different frequencies and temperatures were studied.The results indicated that G’was always larger than G"in the test range,which appeared typical of weak gel behavior.Finally,N-G7 was completely hydrolyzed to glucose and partial disaccharide in simulated digestion in vitro.N-G7 was embedded with calcium alginate at a concentration of 2%to form microspheres to avoid the loss of N-G7 in the upper digestive tract,and the colonic performance was carried out.During the fermentation process,the OD₆₀₀ of the N-G7 microspheres group was 2.113,which was much higher than the 0.728 of the blank control group.The microorganisms growing on N-G7 microspheres produced the lowest amount of initial gas within 0～6 h,and continued to produce gas during the process,consumed N-G7 in microspheres slowly and continuously,and the carbon source utilization rate was 53.40%,which deduced that N-G7 microspheres could be used as slow fermentation system.After the end of fermentation,the total amount of short-chain fatty acids in the N-G7 microspheres group was53.66 mmol/L,which was better than that in the control group observably,and the content of each short-chain fatty acid almost increased significantly at different fermentation times.The effects of N-G7 microspheres on intestinal flora composition at phylum and genus levels during fecal fermentation were evaluated.Compared to the control group,the presence of N-G7microspheres reduced the relative abundance of pathogenic bacteria Proteobacteria significantly,while the abundance of beneficial bacteria Bacteroides increased dramatically.The abundance of Firmicutes also made a difference,and the F/B value of the N-G7microspheres group was significantly lower than that of other groups,suggesting that N-G7microspheres might be beneficial to obese people.In addition,the growth of Escherichia＿Shigella and the abundance of Bacteroides were significantly increased in the N-G7 microspheres treatment group,which also inhibited the growth of harmful bacteria Enterococcus and Phascolarctobacterium.Therefore,the addition of N-G7 microspheres changed the diversity and richness of intestinal flora.