Construction Of Dietary Polyphenols Curdlan-based Carriers And Their Probiotic Activities

Curdlan is a microbial-produced β-1,3-glucan,which has been approved by the US Food and Drug Administration as a food additive.Curdlan is a dietary fiber with a variety of biological activities.However,the tight triple-helix structure of curdlan leads to their insolubility in water,which limits their wide application in food and pharmaceutical fields.The modification of curdlan gave them new functional properties,which is of great significance to the construction of curdlan carrier system loaded with functional nutrients.In this study,curdlan-based carrier delivery systems loaded with dietary polyphenols were constructed by non-covalent binding,covalent binding and self-assembled micelles based on pH-driven method,graft copolymerization reaction and hydrophobic modification.In addition,the interactions,structural properties,digestion behavior and fermentation characteristics of curdlan-polyphenol carrier system were evaluated,which could provide strategies for the development of curdlan-polyphenol functional materials or controlled release delivery system of polyphenols.The main research contents are as follows:(1)Preparation of curdlan-polyphenol non-covalent complex by pH-driven method and its prebiotic activityThe non-covalent complex of curdlan and polyphenol was constructed by pH-driven method,the effects of different polyphenols(curcumin,quercetin and chlorogenic acid)on molecular conformation,gel properties,thermodynamic stability,antioxidant activity and prebiotic activity of curdlan were investigated.The results showed that polyphenols were embedded in the molecular structure of curdlan in an amorphous state.The most hydrophobic curcumin showed the higher content of 34.04 ± 1.73 mg/g in the complex,while chlorogenic acid with lower hydrophobicity showed poor binding capacity to curdlan in the complex.The molecular structure of untreated curdlan was linear,while molecular chains could self-assemble to form spherical particles after the interaction between curdlan and polyphenols.The alkalineutralization curdlan(ANCD)without polyphenols exhited a thick flake structure with a rough surface.After the loading of polyphenols,the complexes exhited larger pore structure,thinner lamellar structure and smoother surface with the increase of hydroxyl groups in polyphenols.The viscoelasticity of the curdlan decreased after the addition of the three polyphenols,and the viscoelasticity of the curdlan-curcumin complex was the lowest,indicating that the polyphenol molecules broke the hydrogen bonding between the curdlan molecules.The antioxidant activity of curdlan was significantly increased after combined with polyphenols,and the antioxidant activity was well maintained during in vitro gastrointestinal simulated digestion.The in vitro human fecal fermentation results showed that ANCD and all three curdlan-polyphenol complexes promoted the production of short-chain fatty acids,and the curdlan-quercetin complex(CDQUE)showed the best antioxidant activity and production capacity of short-chain fatty acids.CDQUE could promote the proliferation of Prevotella and inhibit the growth of Escherichia＿Shigella.In conclusion,the curdlan-polyphenol complexes prepared by pH-driven method could effectively deliver polyphenols,improve the gastrointestinal stability of polyphenols.It could be catabolized and metabolized by gut microbiota to produce short-chain fatty acids.with lager molecular weight,such as propionic acid and butyric acid,which have prebiotic activity.(2)Synthesis of curdlan-quercetin covalent complex,and their antioxidant and probiotic propertiesTo improve the water solubility and pH responsiveness of the curdlan-quercetin complex,carboxymethylated thermogel(CMCD)with high carboxymethylated substitution(0.95)was firstly prepared,and then esterified with quercetin to obtain quercetin grafted CMCD copolymers(CMCD-QUE).The apparent morphology and crystal structure of the complexes were analyzed by scanning electron microscopy and X-ray diffraction,repectively.The results showed that the structure of quercetin became more fluffy after compounding with CMCD,appearing as filamentous and lamellar structures,indicating that quercetin was grafted on the CMCD molecular chain in an amorphous state.CMCD-QUE contained 49.856 mg/g of quercetin and exhibited excellent antioxidant activity throughout the simulated gastrointestinal digestion.CMCD-QUE was not degraded in simulated saliva and gastric fluids,while the molecular weight of CMCD-QUE gradually decreased in simulated small intestinal fluid.The particle size of the complex gradually became larger and the zeta potential value gradually became smaller with the increase of pH,which was conducive to the aggregation of the complex in the stomach to reduce the destruction,and the expansion in the colon to facilitate the breakdown,thus improving the bioavailability of polyphenols.In vitro human fecal fermentation of CMCD-QUE showed that CMCD-QUE could specifically promote the proliferation of Akkermansia.These results suggest that CMCD-QUE exhibited pH-responsive,which was beneficial to maintain the antioxidant activity of polyphenols during digestion and had the potential application for colonic delivery of quercetin.(3)pH responsive amphiphilic self-assembled curdlan micelles for the delivery of curcuminTo ensure the structural integrity and improve the utilization of polyphenols,a novel amphiphilic curdlan micelle was prepared for the encapsulation polyphenols.Firstly,carboxymethylated curdlan with suitable carboxymethyl substitution degree(0.498)were synthesized,and then and then amphiphilic octenyl succinic anhydride(OSA)modified carboxymethyl curdlan(OSA-CMCD)was obtained by modification with OSA.Curcumin with highest hydrophobic used as a hydrophobic polyphenol model.The effect of amphiphilic polymers with different OSA substitution degrees on the self-assembling behavior of micelles and polyphenol loading ability was investigated.The critical micelle concentration was decreased from 0.0960 to 0.0455 mg/m L with OSA substitution degree increased from 0.0306 to 0.6375.The amphiphilic copolymers self-assembled micelles were monodispersed and spherical with a particle size of 157.95 - 200.57 nm.The maximum loading capacity(7.77%)were observed when the the OSA substitution of CMCD-OSA polymer was 0.047.The curcumin loaded micelles displayed a slow release profile in PBS at pH 1.2 or 6.8,whereas68.00% of curcumin was released when micelles were dissolved in pH 7.4,suggesting a sustained pH-dependent release of curcumin.These results suggest that OSA-CMCD micelles can be used for the encapsulation of hydrophobic polyphenols and have potential applications in specific release and colon-targeted delivery.(4)Encapsulation of polyphenols in pH-responsive micelles self-assembled from octenyl-succinylated curdlan oligosaccharide and its effect on the gut microbiotaTo improve the bioavailability and colonic targeting of polyphenols,low molecular weight curdlan oligosaccharides were first obtained by oxidative degradation by hydrogen peroxide to achieve the release of polyphenols triggered by microorganisms.Then,OSA-modified curdlan oligosaccharide(MCOS)was synthesized to impart pH-responsiveness and the ability to encapsulate hydrophobic polyphenols.MCOS could self-assemble into spherical micelles with a particle size of 230.1 nm and a zeta potential of-37.9 m V.When used for polyphenol encapsulation,the loading capacity of curcumin and quercetin co-encapsulated micelles was higher than that of single-polyphenol encapsulated micelles.In vitro gastrointestinal release test showed that the MCOS micelle presented a pH-dependent release,released a little polyphenol in simulated gastric fluid,but presented sustained release in the simulated intestinal fluid.The gastrointestinal-digested polyphenol-loaded micelles exhibited excellent antioxidant ability.In vitro human fecal fermentation indicated that the MCOS carrier could promote the production of short-chain fatty acids by gut microbiota and exhibited the highest relative abundance of Megamonas.In addition,the supplementation of curcumin and quercetin-co-loaded MCOS micelles increased the relative abundance of Bifidobacterium and inhibited the growth of Escherichia＿Shigella.The above results indicate that MCOS carriers were pH-responsive and fermentable by gut microbiota,which can improve the encapsulation efficiency,gastrointestinal stability and specific release of polyphenols,and have the potential to be applied to the colontargeted delivery of hydrophobic polyphenols.In summary,the curdlan-based delivery system could improve the gastrointestinal stability and bioavailability of dietary polyphenols.Meanwhile,the curdlan carrier could be decomposed and utilized by gut microbiota to improve the intestinal environment,which has potential applications in specific release and colon-targeted delivery.