| ?-carotene,as a fat-soluble antioxidant,is the major source of plant-derived provitamin A.It can be converted into retinoids in vivo and show their benefits in promoting general growth,maintaining visual function and regulating the differentiation of epithelial tissues.It has also been reported that?-carotene could reduce the risk of chronic diseases.However,its low water solubility,poor gastrointestinal stability,low intestinal permeability and high metabolic clearance rate,lead to the low bioavailability after oral administration.Encapsulation has been considered as an effective way to improve the solubility of lipophilic nutrients in the aqueous phase,enhance the gastrointestinal stability and control the release of bioactive compounds in the intestine for effective absorption.Oil-containing nanoemulsion system and oil-free nanoparticle system are two of the most representative delivery systems.In this project,protein-based biopolymer emulsifiers were used to prepare?-carotene loaded nanoemulsion and nanoparticle,using microfluidization and reverse-evaporation method.An in vitro simulated digestion model was used to investigate the digestion behavior of the nanocarriers.Changes in particle size and microstructure during digestion,effects of digestive conditions on the samples,changes in the interfacial composition,lipolysis behavior,and micellization efficiency of nutrients were all studied.Caco-2 cells model and mice animal model were further used to investigate the cellular absorption and metabolism behavior of?-carotene in nanoemulsion and nanoparticle after digestion.The changes in the morphological structure of nanocarriers in vivo,and the distribution of nutrients in tissues were also determined.Subsequently,a high-fat diet-induced obese mice model was carried out to show and compare the anti-obesity and inflammation biological activities of?-carotene loaded in different nanocarriers.Following are the main contents and results:Firstly,the microfluidization method was used to prepare nanoemulsions with whey protein isolate?WPI?,soy protein isolate?SPI?and sodium caseinate?SC?as emulsifiers.The digestion behavior was investigated using an in vitro simulated gastrointestinal digestion model.The results showed that the particle size of WPI,SPI and SC-based nanoemulsions increased from 237.8±5.1,219.9±1.8,484.2±69.4 nm to 273.3±2.8,1168±166.4 and 1554.2±194.5 nm at the end of the gastric digestion.WPI-based sample could keep relatively stable,because?-lactoglobulin,the main component of WPI,has strong resistance to pepsin hydrolysis,which can maintain its emulsifying activity during gastric digestion.By the end of intestinal digestion,the particle sizes of the three protein-based samples reached 274.1±2.5,623.2±32.4,and548.6±21.1 nm,respectively,and the surface charges were-52.6±5.1,-51.37±3.6,and-63.63±1.8 mV.The lipolysis efficiency was in the order of SPI>WPI>SC,and the micellization efficiency of WPI,SPI and SC-based samples were 23.31±2.92%,13.25±1.01%and32.27±1.41%,respectively.It could be found from the results that the lipolytic products and bile salts will quickly adsorb on the oil-water interface of WPI and SC-based samples to inhibit the contact of lipase,resulting in a reduced lipolysis and micellization rate;while for SPI,the interfacial protein hydrolysate can be quickly replaced by the bile salts,providing more binding sites for lipase and can accelerate lipolysis.The above experiments show that the displacement ability of bile salts at the interface will affect the lipolysis efficiency and ultimately the bioavailability of?-carotene.The WPI,SC and SPI-based nanoparticles were prepared by the reverse-evaporation method,and the digestion behavior was investigated using an in vitro simulated gastrointestinal digestion model.The structure of nanoparticles was completely changed in the presence of protein enzymes,and small particles below 100 nm were generated at the end of the digestion.The release rate of?-carotene after digestion reached 94.14±2.7%,99.65±0.97%and92.54±2.18%,indicating that?-carotene was completely exposed to the water phase in the small intestine.The?-potential of WPI,SC,and SPI nanoparticles after digestion reached-42.70±0.65,-36.95±0.21,and-38.90±2.4 mV,respectively,meaning that bile salts were adsorbed on the surface of the hydrophobic core.FTIR spectra showed that the characteristic peak of?-carotene in micelles migrated from 965 cm-1 to 860 cm-1,suggesting that?-carotene in the water phase after digestion was still in an amorphous state,without aggregating and recrystallizing and was encapsulated in micelles made up of bile salts,phospholipids and protein hydrolysates.?-carotene loaded nanoparticle was co-digested with oil to improve the low bioaccessibility of it.The presence of both the coexisting oil and excipient oil phases could promote the bile salt adsorption capacity,lipolysis rate,and the micellization efficiency of nanoparticle systems.The excipient nanoemulsion was more effectively compared with the bulk oil.In order to investigate the cellular absorption and metabolic behavior of?-carotene loaded in nanoparticles and nanoemulsions after digestion,a Caco-2 cells model was constructed.The cell model was completely differentiated after 21 days incubation.The TEER value was greater than 1000?·cm2,the transportation rate of fluorescein sodium in 4 hours was less than 0.67%,and the activity of ALP remained stable.The uptake of?-carotene in WPI,SC and SPI nanoemulsions were increased by 6.5,3 and 1.4 times,compared with samples before digestion,while the nanoparticle samples increased by 1.11,1.56 and 2.03 times,indicating that the absorption efficiency of nutrients loaded in nanocarriers has been improved after digestion.The overall VA absorption,transportation and metabolic behavior of?-carotene for nanoparticles and nanoemulsions were also investigated,and it was found that the overall VA absorption and transportation rate of nanoparticles were better than that of nanoemulsions;while the relative metabolic level of?-carotene in nanoemulsions was higher than nanoparticles.Mice study found that?-carotene nanoemulsions had higher metabolic levels in vivo,while nanoparticles could be absorbed into the blood more efficiently.Nanoemulsions were more likely to convert?-carotene into retinol or retinyl palmitate and carried into the liver for storage,while the nanoparticles can retain more?-carotene into the systemic circulation and be stored in adipose tissues.In order to evaluate the biological activity of?-carotene in nanocarriers,a high-fat diet-induced mice obesity model was constructed.The effects of?-carotene loaded in nanoparticle and nanoemulsion on the body weight,tissue development,and plasma biochemical indexes were explored.It was found that,compared with the HFD control group,the?-carotene nanoparticle,?-carotene nanoemulsion and?-carotene dispersion groups reduced 13.8%,17.8%and 12.7%of the body weight,respectively.The reduction of the fasting blood glucose by17.5%,18.3%,and 10.4%,the weight of adipose tissue by 26.23%,30.52%,and 14.39%,and decreased content of low-density and very-low-density lipoprotein,indicated that?-carotene encapsulated in nanocarriers can effectively inhibit obesity and improve the impaired glucose tolerance caused by a high-fat diet.In addition,it was found that?-carotene nanoparticle can reduce the size of adipose cells more effectively.Both nanocarrier samples can improve NAFLD,?-carotene nanoemulsion can reduce the ROS content in the liver,while nanoparticle can more effectively slow down the liver damage caused by a high-fat diet.It was found that the ingestion of?-carotene increased the permeability of the small intestine and destroyed the structure of the microvilli in the small intestine,but encapsulating?-carotene in nanocarriers can inhibit this damage to a certain extent.The fecal microbiota study revealed that both?-carotene loaded nanocarriers can significantly reduce the F/B ratio and partly reshape the impaired gut microbiome induced by high-fat diets. |
PDF Full Text Request