Preparation,Structure Analysis,in Vitro Digestion And Fermentation Of Blackberry Polysaccharides With Different Molecular Weights

Blackberry,also known as dewberry,is a shrub of the genus Rubus of the Rosaceae family.Polysaccharides are one of the main bioactive ingredients in blackberry fruit.However,the fine structure analysis,the effects of extraction and modifications on polysaccharides,and structure-activity relationship between polysaccharide structures and its hypoglycemic activity are still unclear.In this paper,the effects of extraction solvents on the structures and bioactivities of blackberry polysaccharide were studied.Next,the effect of molecular weight onα-glucosidase inhibitory activity of blackberry polysaccharides,in vitro digestion and fermentation properties were investigated.The fine chemical structure and solution conformation of polysaccharides were also identified by chemical and instrumental analysis.Finally,nanoparticles（RBC@BBP-24-3Se）with core-shell bilayer structure were prepared by selenization modification and erythrocyte membrane coating,and the regulatory effect and mechanism of nano-selenium particles on metabolic disorders of Hep G2 cells induced by palmitic acid were studied,which provided a theoretical basis for developing a new type of selenium supplement for the treatment of diabetes.The main results are as follows:（1）The four polysaccharides were extracted by hot water（Hw）,0.1 M HCl（Ac）,0.1 M Na OH（Al）and 0.1 M Na Cl solution（Na）,respectively.The physicochemical properties,structures and bioactivities of the four polysaccharides were compared.Al had the highest extraction yield with 10.64%,while Ac had the highest uronic acid content（62.76%）.Monosaccharide analysis showed that the monosaccharide compositions of all polysaccharides were different,but they were mainly composed of galacturonic acid and arabinose.The particle size and molecular weight distribution of the four polysaccharides were different,Hw and Na had higher molecular weight with larger particle size.FT-IR,triple helix structure,XRD and SEM analysis showed that all polysaccharide fractions had similar functional groups,but the spatial structure and apparent morphology were significantly different.Rheological analysis shows that Hw and Na with high molecular weight had higher apparent viscosity and better rheological properties.In vitro antioxidant assay,α-glucosidase inhibition assay and anti-glycosylation assay showed that Hw and Na with higher molecular weight had better bioactivity,and Ac with higher content of uronic acid had better anti-glycosylation activity.The results showed that the extraction solvents had great effects on the physicochemical properties,structures and bioactivities of polysaccharides from blackberry.The physicochemical properties and biological activities of Hw and Na were similar.Compared with salt solution extraction,hot water extraction is more economic and environmental friendly.Therefore,hot water extraction of blackberry polysaccharide was used in the following experiments.（2）Four blackberry polysaccharide fractions with different molecular weight were obtained by ultrasonic degradation of the polysaccharide extracted from hot water,which were named BBP（591.39 k Da）,BBP-8（363.93 k Da）,BBP-16（249.51 k Da）and BBP-24（177.42k Da）respectively.The results showed that the sugar content,monosaccharide composition and main functional groups of the four different molecular weight polysaccharides were similar,and all had a triple helix structure,indicating that ultrasonic degradation did not destroy the basic structure of blackberry polysaccharides.The IC₅₀ values of BBP,BBP-8,BBP-16 and BBP-24 againstα-glucosidase were 1.59,2.58,1.96 and 1.11 mg/m L,respectively,indicating that theα-glucosidase inhibitory activity of blackberry polysaccharides was closely related to its molecular weight.In addition,all polysaccharide fractions induced the change ofα-glucosidase internal microenvironment through the mixed type inhibition,that decreased theα-helix,β-corner content and increased the content of random coil ofα-glucosidase,to inhibit theα-glucosidase activity.（3）The effects of molecular weight on the digestive properties of blackberry polysaccharide and lipid digestion were studied by in vitro simulated gastrointestinal digestion experiment.The results showed that the molecular weight of all polysaccharides changed non-significantly after gastric digestion,while decreased significantly after intestinal digestion.The molecular weights of BBP,BBP-8,BBP-16 and BBP-24 decreased 77.48%,69.96%,56.86%and 52.89%,respectively,after intestinal digestion,indicating that gastrointestinal digestion had a greater effect on the higher molecular weight polysaccharides.Furthermore,no free monosaccharides were detected during the whole digestion process,indicating that all polysaccharides could not be digested and absorbed by gastrointestinal tract.The antioxidant activities andα-glucosidase inhibitory activities of all molecular weight polysaccharides decreased with the progress of digestion.In addition,the binding capacities of BBP,BBP-8,BBP-16 and BBP-24 to bile acids were 0.30,0.26,0.23 and 0.16 m M,respectively,and the adsorption capacities to lipid were 265.84,141.37,76.52 and 32.77 mg/g,respectively.In vitro simulated lipid digestion experiments showed that the amount of free fatty acids released（%,w/w）in the emulsion formed by BBP-24,BBP-16,BBP-8 and BBP with corn oil was 65.58%,55.77%,48.11%and 34.88%,respectively.Results showed that higher molecular weight blackberry polysaccharide had stronger binding ability to bile acids and lipids,and more significant inhibition to lipid digestion.（4）The effect of molecular weight on the fermentation properties of blackberry polysaccharide was studied by in vitro fecal flora fermentation model.The results showed that lower molecular weight polysaccharide had higher gas production and carbohydrate consumption rate.During the fermentation,the molecular weight degradation degree of higher molecular weight polysaccharide was greater,and more larger molecular weight oligosaccharides fragments were produced.Furthermore,the monosaccharide utilization sequence of all polysaccharides was:glucose>galactose>arabinose>galacturonic acid.Glucose,galactose and arabinose were almost fully utilized after 24 h in vitro fermentation,and the utilization rate of galacturonic acid was close to 80%.In addition,the fermentation of all polysaccharides significantly promoted the production of short-chain fatty acids（SCFAs）,especially acetic acid.Lower molecular weight polysaccharides had a faster SCFAs production rate,but the molecular weight did not affect the final SCFAs yield of blackberry polysaccharides.At the same time,the fermentation of blackberry polysaccharide promoted the diversity of bacterial community,mainly the increase of Bacteroidetes and the decrease of Firmicutes.The proportion of Bacteroidetes in BBP,BBP-8,BBP-16 and BBP-24 was 45.41%,47.50%,48.08%and 50.09%,respectively.The results showed that lower molecular weight blackberry polysaccharide had a better promotion effect on the proliferation of Bacteroidetes.（5）The BBP-24 was separated and purified by DEAE-52 cellulose column to obtain BBP-24-3 with molecular weight of 145.1 KDa.BBP-24-3 was composed of arabinose,glucose and galacturonic acid with the molar ratio of 5.30%,3.60%and 91.10%,respectively.Methylation and NMR analysis showed that the main chain of BBP-24-3 was composed of→4)-α-D-Gal Ap-(1→repeat unit.Dynamic light diffraction,size exclusion chromatography-multiangle light scattering,TEM and AFM showed that BBP-24-3 exhibited uniform solid sphere conformation in 0.1 M Na Cl solution.In addition,BBP-24-3 showed good inhibitory activity againstα-glucosidase through mixed type inhibition.（6）Nano-selenium particles（BBP-24-3Se）were prepared using BBP-24-3（2 mg/m L）as a surface modifier with a particle size of 167.1 nm.After coated by erythrocyte membrane,the particle size of nano-selenium particles（RBC@BBP-24-3Se）was increased to 239.8 nm,showing an obvious core-shell structure.Furthermore,the absorption rate of RBC@BBP-24-3Se in Hep G2 cells was 4.99 times than that of BBP-24-3Se at a concentration of 10μg/m L.RBC@BBP-24-3Se could effectively protect Hep G2 cells from lipid oxidative damage induced by palmitic acid,improve the activities of hexokinase（HK）and pyruvate kinase（PK）,promote glucose consumption and glycogen synthesis of Hep G2 cells,reduce intracellular total cholesterol（TC）,total triglyceride（TG）and low density lipoprotein cholesterol（LDL-C）content and increase high density lipoprotein cholesterol（HDL-C）content.Moreover,glucose/lipid metabolism disorders of Hep G2 cells induced by palmitic acid were also improved by PI3K/AKT and AMPK signaling pathway.