Construction and Evaluation of Polysaccharide and Whey Protein Based Delivery System for Citrus Flavonoids

Author:Hu Yan

Supervisor:zhou zhi qin li yan


Degree Year:2019





Citrus has abundant germplasm resources,mainly including mandarin(Citrus reticulata),pummelo(Citrus grandis),lemon(Citrus limon)and orange(Citrus sinensis).Citrus fruit are widely used for medicine and food,containing a large number of active compounds,including dietary fiber,flavonoids,vitamin,mineral,limonins,carotenoids,which have many functions including anti-oxidation,anti-inflammatory,anti-cancer and regulating lipid metabolism.Among them,flavonoids have attracted much attention owing to their excellent physiological functions,pharmacologic and health actions.Flavonoids are mainly from citrus fruit and accumulated in the peels.Therefore,it is of great significance to extract flavonoids from citrus peels and develop their nutritional and pharmaceutical values.Many flavonoids exhibit excellent bioactivity in vitro,while the bioavailability is relatively low in vivo because of their poor solubility and absorption efficiency.Flavonoids are sensitive to environmental stresses(light,heat,oxidation,pH,etc.)during processing or storage.In addition,flavonoids are unstable in gastrointestinal environment,which limit their physiological activity and potential health promotion value.Thus,it is necessary to improve the bioavailability of flavonoids by some technical means.Nanotechnology has been applied in pharmaceutical and food industries.Nano-encapsulation can be used to encapsulate and protect the physiological activity of bioactive substances.In addition,encapsulation can control their release and improve their bioavailability.In this reserach,natural polysaccharide and protein were used to construct micro/nano-carriers,which were used to encapsulate citrus peel extracts(CPE).The basic physical and chemical properties of each carrier were evaluated.We hope to construct an excellent delivery system with high encapsulation efficiency and good stability in gastrointestinal environment.We aim to innovate the application form of citrus flavonoids and improve their bioavailability and bioactivity.Firstly,we selected four loose skin mandarin,namely Guangxihongpiju(GX),Wulongsuanju(WL),Penggansanhao(PS)and Taiwanpenggan(TP).The main flavonoids in these CPE were analyzed by HPLC,and the flavonoids content and antioxidant activity as a function of pH value and storage time were investigated.Then whey protein concentrate nanoparticles(WPC NPs),gum arabic(GA)based emulsion and GA/WPC based emulsion and their microcapsules were prepared,which were used to encapsulate CPE.The basic physicochemical properties and in vitro release characteristics were determined.Furthermore,the antioxidant activity was explored.Finally,WPC emulsion was used to delivery TAN,a polymethoxyflavone riched in citrus peel.CA,GA,HPMC were added to modify WPC emulsion,the in vitro digestive characteristics and plasma concentration and tissue distribution of TAN in rats were expolored.1.Qualitative and quantitative analysis of flavonoids and antioxidant activity of citrus peel extracts by HPLCResults showed that the main flavonoids in CPE were HES,TAN,NOB,SIN and 5-DEM.HES content was the highest.The flavonoid composition of four CPE was the same,but the content was slightly different.WL contained more PMFs than others.The TPC,TFC and antioxidant activity of GX were the highest.In addition,the effects of pH value and storage time on flavonoids and antioxidant activity of CPE were also studied.The results showed that flavonoids were sensitive to pH,and easily degraded at strong acid environment(pH 2-3),and antioxidant activity was also lower at pH 2-3 than those at pH 4-7.With the prolongation of storage time,TFC and antioxidant activity decreased gradually,and the degradation rate accelerated in strong acid environment.2.Preparation of WPC NPs and their basic physical and chemical propertiesWPC NPs was prepared by Ca2+-crosslinking.Different CPE loaded WPC NPs lead to the difference in particle size and zeta potential.It was proved that the interaction between flavonoids and WPC was mainly through H-bond and hydrophobic interaction,which were confirmed by FTIR,fluorescence spectra and CD spectra.CPE addition could change the secondary structure and enhanced the hydrophobicity of WPC.WPC NPs showed good stability in intestinal tract,they also could control the release of flavonoids.The antioxidant activity of CPE in intestinal tract was improved.The maximum encapsulation efficiency of CPE by WPC NPs was 27.9±0.9%.3.GA and GA/WPC emulsions and their microencapsulesGA emulsions had extensive pH stability and were relatively stable at pH 2-7.When WPC was added to modify the interface of GA emulsion,the particle size was increased,while the encapsulation effeciency of flavonoids was improved.In order to make it more convenient for storage and application,spray drying technology was used to produce microcapsules.In general,microcapsules showed good encapsulation efficiency for flavonoids,and the GA/WPC microcapsules was better with high encapsulation efficiency(97.60±0.99%).During storage,GA/WPC microcapsules showed better protective effect.After 3 months,the retention rate of GA/WPC microcapsules was 63.39±2.10%,while that in unencapsulated CPE was only 19.29±0.57%.Futhermore,microcapsules also had promising antioxidant activity after storage,they could be used as natural nutritional supplements.4.Influence WPC emulsions on in vitro digestion and distribution of TAN in vivoGA,CA and HPMC were added to modify the interface of WPC emulsion and the encapsulation efficiency of TAN in these emulsion systems was significantly increased.During the in vitro digestion,the oil digestion rate of different emulsion systems was different.After adding Ca2+to form WPC/HPMC emulsion gel,the oil digestion was inhibited,and it was not completely digested after 2 h.The bioaccessibility of TAN encapsulated into emulsion systems were greatly improved compared with the MCT suspension,and the bioaccessibility of emulsion gels was higher than that of emulsions.These results showed that interface composition and structure of emulsion would affect the digestion behavior,leading to different bioaccessibility.Similar to the results of in vitro digestion,the concentration of TAN in rat plasma was much higher than that of MCT suspension,which indicated that the emulsion delivery system was a good carrier for improving the bioavailability of hydrophobic nutrients.HPMC further increased the plasma concentration of TAN in rat and prolonged its circulation time in vivo(22 h).Different emulsion systems had effects on the tissue distribution of TAN.Generally speaking,WPC/HPMC complex improved the concentation of TAN in tissues,and TAN was mainly accumulated in liver and kidney.In conclusion,the flavonoids composition of four CPE was the same,while the content of flavonoids was slightly different,and the flavonoids were sensitive to pH value.In acidic conditions,they had poor solubility and were easy to be degraded.During storage,the flavonoids content and antioxidant activity decreased.Flavonoids degraded fatser in strong acid environment.WPC NPs and GA/WPC microcapsules could protect the antioxidant activity of flavonoids.However,the encapsulation efficiency of GA/WPC microcapsule on CPE was better than that of WPC NPs.The emulsion systems significantly improved the bioaccessibility,plasma concentration of TAN,and prolonged its circulating time in rat.Especially,the peak concentration of TAN in WPC/HPMC emulsion/gel was 20 times that of MCT suspension.The metabolic time of TAN in rats was prolonged to 22 h.Tissue distribution showed that TAN was mainly accumulated in liver and kidney.