| β-Carotene is a natural food functional component and has many physiological activities,such as immunoregulation and improvement of intestinal health.However,the poor solubility,small intestinal absorption and bioaccessibility of β-carotene limit its application in functional food.Protein-polysaccharide conjugate-based delivery vehicle has relatively high stability and can encapsulate,deliver and release food functional components for improving their digestive absorption and physiological activity.In this project,oat protein isolate-Pleurotus ostreatus(P.ostreatus)β-glucan conjugate was formed by Maillard reaction.Such conjugate was used to structure delivery vehicle for encapsulating β-carotene,and the stability,in vitro digestion properties and cellular transport were studied for this vehicle.To establish a cyclophosphamide-induced immunosuppressed mouse model,the effect of β-carotene loaded oat protein isolate-P.ostreatus β-glucan conjugate-based delivery vehicle on immunoregulation in mice was studied based on gut microbiota.The main findings of this research were as follows:1.Oat protein isolate was interacted with P.ostreatus β-glucan to form a covalent conjugate with the grafting degree of 26.27%± 1.37%via Maillard reaction under controlled dry-heating conditions.The basic structure and physicochemical properties of oat protein isolate-P.ostreatusβ-glucan conjugates were determined.In this study,an apparent band with high molecular weight appeared on the top of lane in sodium dodecyl sulfate-polyacrylamide gel electrophoresis,suggesting the formation of the covalent bond between oat protein isolate and P.ostreatusβ-glucan.After oat protein isolate was covalently attached to P.ostreatus β-glucan,it caused C-N stretching vibration at the wavelength of 1358.2 cm-1 in the fourier transform infrared spectroscopy.In addition,its cysteine and lysine contents decreased to 0.14%± 0.09%and 3.04%± 0.30%,respectively,while that of histidine content increased to 3.88%±0.28%.Maillard reaction altered the spatial conformation of oat protein isolate,leading to its reduction in tryptophan fluorescence intensity,surface hydrophobicity(1006.533±83.061),α-helix(26.42%± 2.51%)and β-sheet(31.34%±2.46%)contents.Such conjugate presented loose and porous surface microstructure.Changes in structure of conjugates further improved their protein solubility,emulsifying activity index and emulsion stability index at pH 3.0-11.0.At pH 5.0 near the isoelectric point of oat protein isolate,the protein solubility,emulsifying activity index and emulsion stability index of such conjugate increased to 50.85%±1.12%,28.657±1.938 m2/g and 20.084±1.486 min.2.Oat protein isolate-P.ostreatus β-glucan conjugate was used to prepare oil in water emulsion and the stability of emulsion was determined at different pH and NaCl concentrations.The effect of this emulsion-based delivery vehicle on digestive properties of β-carotene was studied in the stimulated oral,gastric and small intestinal environment.The results showed that oat protein isolate-P.ostreatus β-glucan conjugate-based emulsion had higher stability at pH 3.0-7.0 and 50-350 mmol/L NaCl concentrations compared with oat protein isolate-based emulsion and oat protein isolate-P.ostreatus β-glucan mixture-based emulsion.It mainly manifested as uniformly distributed microstructure and lower average particle size of emulsion droplets.Moreover,the encapsulation efficiency and loading efficiency of β-carotene in oat protein isolate-P.ostreatus β-glucan conjugate-based emulsion reached 58.22%± 2.51%and 1.83%±0.05%,respectively.In the in vitro digestion model,β-carotene loaded oat protein isolate-P.ostreatus β-glucan conjugate-based emulsion did not contain obvious discrete aggregations of oil droplets and its average particle size was relatively low.The free fatty acid release and bioaccessibility of β-carotene reached 93.24%± 4.90%and 36.29%± 4.22%after in vitro digestion.3.To further improve the bioaccessibility and small intestinal absorption of β-carotene,β-carotene loaded oat protein isolate-P.ostreatus β-glucan conjugate-based nanoparticles were prepared by emulsion-evaporation.The basic structure,stability,in vitro release and the cellular transport in Caco-2 cells for such β-carotene loaded conjugate-based nanoparticles were determined.The results showed that the average particle size and ζ-potential of β-carotene loaded conjugate-based nanoparticles were 169.868±5.261 nm and-26.018±1.221 mV,respectively.In addition,their microstructure was regular spherical in shape and β-carotene was encapsulated in conjugate-based nanoparticles in the form of amorphism.The encapsulation efficiency and loading efficiency of β-carotene in conjugate-based nanoparticles reached 86.21%±1.29%and 5.43%±0.12%,respectively.Compared with β-carotene loaded oat protein isolate-based nanoparticles and β-carotene loaded oat protein isolate-P.ostreatus β-glucan mixture-based nanoparticles,β-carotene loaded conjugate-based nanoparticles had higher stability at pH 3.0-7.0 and 50-350 mmol/L NaCl concentrations.The cumulative release rate of β-carotene in such conjugate-based nanoparticles was lower in the stimulated gastric juice,falling to 25.16%±3.10%,and the cumulative release rate of β-carotene reached 72.30%±2.31%after stimulated small intestinal digestion,exhibiting higher bioaccessibility of β-carotene.β-Carotene loaded conjugate-based nanoparticles had relatively low cytotoxicity in Caco-2 cells,exhibiting good biocompatibility.The cellular uptake of such conjugate-based nanoparticles may rely on clathrin-mediated and caveolae-mediated endocytosis.The apparent permeability coefficients and transport accumulation of β-carotene across Caco-2 cell monolayers increased to(3.982±0.218)× 10-6 cm/s and 3.276±0.438 μg,respectively in β-carotene loaded conjugates-based nanoparticles.It suggested that such conjugate-based nanoparticles could significantly enhance the small intestinal absorption of β-carotene.4.To establish a cyclophosphamide-induced immunosuppressed mouse model,effects ofβ-carotene-loaded oat protein isolate-P.ostreatus β-glucan conjugate-based nanoparticles on the regulation of immune function and gut microbiota were studied in mice.Compared withβ-carotene and oat protein isolate-P.ostreatus β-glucan conjugate-based nanoparticles,β-carotene loaded oat protein isolate-P.ostreatus β-glucan conjugate-based nanoparticles increased the contents of red blood cells,white blood cells,blood platelet and hemoglobin,the concentrations of tumor necrosis factor-α,interleukin-2,interleukin-4 and interferon-y in spleen,the levels of immunoglobulin A,immunoglobulin G and immunoglobulin M in serum,thereby enhancing immune response in mice.In addition,β-carotene loaded oat protein isolate-P.ostreatus β-glucan conjugate-based nanoparticles could relieve spleen and thymus damage in mice and maintain their normal physiological structure.High-throughput sequencing was used to analyze the diversity and community composition of gut microbiota in immunosuppressed mice.The results showed that β-carotene loaded oat protein isolate-P.ostreatus β-glucan conjugate-based nanoparticles increased the diversity of gut microbiota and regulated gut microbiota composition.Thereinto,the relative abundances of Erysipelotrichaceae,Rikenellaceae,Ruminococcaceae,Lachnospiraceae and Porphyromonadaceae at the family level decreased but the relative abundances of Bacteroidaceae and Lactobacillaceae increased.Moreover,β-carotene loaded oat protein isolate-P.ostreatus β-glucan conjugate-based nanoparticles increased short-chain fatty acids concentrations in the feces of immunosuppressed mice,including acetic,propionic,n-butyric and n-valeric acids.Spearman’s correlation analysis further indicated that β-carotene loaded oat protein isolate-P.ostreatus β-glucan conjugate-based nanoparticles reversed 7 OTUs related to immunoregulation and short-chain fatty acids concentrations.These OTUs included the increase in relative abundances of positively related Clostridium_ⅩⅣa(OTU018),Akkermansia(OTU052),Bifidobacterium(OTU055),Roseburia(OTU436),the decrease in relative abundances of negatively related Parasutterella(OTU064),Erysipelotrichaceae(OTU011)and Alistipes(OTU097).It indicated that β-carotene loaded oat protein isolate-P.ostreatus β-glucan conjugate-based nanoparticles could probably improve immunoregulation by regulating gut microbiota. |
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