| Edible protein not only has high safety and nutritional value,but also possess the functional characteristics of embedding hydrophobic nutrients.Casein(Cas)as the highest content in milk has a wide range of sources,low price,safe and non-toxic and reasonable essential amino acids.However,any single protein is difficult to meet all the needs of food processing,Therefore,protein modification is very necessary.Maillard reaction is a covalent modification technology for protein glycosylation.The reaction process does not require the addition of any organic solvent.Nevertheless,the reaction time is long,the reaction conditions are difficult to control,and the safety of the products is difficult to evaluate.In recent years,some scholars have used the properties of Transglutaminase(TGase)to conjugate polysaccharides containing amino groups into proteins to prepare glycosylated proteins in order to improve the properties of the protein and broaden its application range.Quercetin(Quercetin,Que)is a natural flavonoid with anti-inflammatory,anti-oxidant,anti-cancer,weight loss and other effects.However,Que has poor water solubility and is easily degraded by environmental factors.Therefore,it is quite urgent to improve the water solubility and stability of quercetin for the purpose of better application of Que.In this study,enzymatic glycosylation and ultrasonic self-assembly techniques were used to prepare quercetin-encapsulated glycosylated casein nanoparticles,and their properties were studied.Firstly,enzymatic glycosylation was used to conjugate Chito-oligosaccharides(Cos)into Cas,and the preparation process,structure and properties of casein-chigo-oligosaccharide(Cas-Cos)covalent complex were characterized;secondly,the preparation process of Cas-Cos nanoparticle(_n-Cas-Cos)and stability were used by ultrasonic self-assembly;Finally,_n-Cas-Cos was used as the carrier to encapsulate quercetin to improve the water solubility,stability and biological activity of Que.The main research contents and results are as follows:(1)Firstly,the effects of Cas concentration,mass ratio of Cos to Cas,reaction time,and enzyme addition on the conjugated amount of Cos of Cas were investigated to determine the preparation process of glycosylated products;secondly,the structure of glycosylated product was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),fourier transform infrared spectroscopy(FTIR),circular dichroism chromatography(CD),fluorescence spectroscopy(FS);finally,the surface hydrophobicity and emulsification of glycosylated Cas were measured.The results showed that when the concentration of Cas was 30 g/L,the mass ratio of Cos to Cas was 3:1,the reaction time was 4 h,and the enzyme addition amount was 20 U/g,the Cos attachment of Cas reaches the maximum value of 43.4 g/kg protein;SDS-PAGE indicated that Cos and Cas formed glycosylated copolymer;FTIR indicated that the side chain structure of casein had changed after glycosylation,and the enhanced absorption peak of O-H group illustrated that Cos group was conjugated to Cas;CD spectrum analysis demonstrated that the content ofα-helix andβ-sheet of glycosylation products increased,and the content of random coils decreased;the results of fluorescence spectrum showed that the access of Cos would have a certain shielding effect on its endogenous fluorescent groups,and the maximum fluorescence intensity would be reduced;the surface hydrophobicity index was 879,which increased by 320;the emulsifying activity was 53.29 m~2/g,which decreased by 11.88 m~2/g;the emulsion stability was 46.95%,which increased by 5.68%.(2)Cas and Cas-Cos empty nanoparticles(_n-Cas,_n-Cas-Cos respectively)were prepared by ultrasonic self-assembly method.Firstly,the average particle size(D_Z)and dispersion coefficient(PDI)were used as indicators to investigate the effect of protein concentration,ultrasonic power,and p H on D_Z and PDI,so as to determine the optimal preparation process of empty nanoparticles;secondly,the freeze-thaw stability and storage stability of nanoparticles were analyzed.The results illustrated that when the protein concentration of Cas and Cas-Cos was 4g/L,the ultrasonic power was 200w,and the p H of the solution was 5.8,the D_Z of _n-Cas and _n-Cas-Cos was the smallest,which were 169.1 nm and 125.6 nm respectively,PDI was 0.217 and 0.161,respectively;freeze-thaw stability and storage stability results showed that _n-Cas,_n-Cas-Cos had good stability.(3)The Que was encapsulated into empty nanoparticles(_n-Cas,_n-Cas-Cos),and its encapsulation efficiency(EE),load capacity(LC),D_Z,PDI and structural characteristics were characterized.The results revealed that when the mass ratio of protein to Que was 40:1,the encapsulation efficiency was 74.14%and 85.21%respectively;the load capacity was 1.85%and 2.13%respectively;D_Z was 183.2 nm and 137.6 nm respectively;PDI was 0.257 and 0.177 respectively;FTIR analysis indicated that Que had been successfully encapsulated in the hydrophobic core of empty nanoparticles,and was combined with proteins through hydrogen bonding and hydrophobic interaction;the results of X-ray diffraction(XRD)displayed that Que was dispersed as an amorphous form in the protein;Differential scanning calorimetry(DSC)and thermogravimetric analysis(TG)demonstrated that casein glycosylation enhanced its thermal stability,and _n-Cas-Cos had better protection against Que;the results of transmission electron microscopy showed that the empty nanoparticles were spherical,and the shape of the particles did not change significantly before and after loading Que.(4)The storage stability,thermal stability,in vitro anti-digestibility,bioaccessibility,anti-oxidation and inhibitory effect on human prostate cancer PC-3cells of Que nanoparticles(_n-Cas-Que and _n-Cas-Cos-Que,respectively).The storage stability results expressed that the two kinds of nanoparticles can enhance the stability of Que during storage(conditions were 4℃avoid light,25℃avoid light,25℃natural light)and the protection effect of _n-Cas-Cos-Que on Que better than _n-Cas-Que;thermal stability results indicated that _n-Cas-Cos-Que had the best protection effect on quercetin under different heating temperatures(37℃,60℃,99℃);anti-digestion results pointed out that _n-Cas-Cos-Que had a better anti-digestion effect,which was conducive to ensuring the stable structure of the target in the human digestive tract and both nanoparticles had improved the biological accessibility of Que to a certain extent(increased by 1.72～3.83 times);Antioxidant results showed that Quercetin ethanol solution(Que-ethanol)had the strongest ability to remove DPPH and ABTS.the scavenging effect of _n-Cas-Que and _n-Cas-Cos-Que on DPPH and ABTS were far better than that of quercetin aqueous solution(Que-water)Under water conditions;the inhibition test of human prostate cancer PC-3 cells illustrated that when the concentration of quercetin reached 80μg/m L,The survival rates of Que,_n-Cas-Que and _n-Cas-Cos-Que were 73.25%,56.84%,and 49.88%,respectively(p<0.05),indicated that the quercetin nanoparticle solution had better anticancer activity than the quercetin solution.In summary,_n-Cas-Cos Can encapsulate Que and improve its water solubility,stability and biological activity effectively by ultrasonic self-assembly method.In addition,the raw materials used in this study are all components in food.The raw materials from natural and green source guaranteed the safety of the composite nanomaterials.In the research and development field of food functional factors,especially for small molecule compounds represented by flavonoids,nanotechnology had shown very broad application value and promising industrial prospects. |
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