| Heat treatment is the necessary food producing process,may cause the physical and chemical changes in food components,thus influence the quality and function of food.As the main component in food,various biological macromolecules(such as protein,sugar and lipid)show migration,degradation and combination during the heating process.Eventually,heating process not only show the new compounds in the chemical reaction,but also show the new structure in the physical reaction.The research of food and Traditional Chinese Medicine(TCM)in heating process mostly focuses on the chemical changes of components,such as the stability of active ingredients,the establishment of detection methods and the generation mechanism.There is a widespread phenomenon that food and TCM have nanostructures after heat treatment.However,there are few research to discuss the problem such as "how to self-assemble" and "what kind of biological effects exist in the nanostructures during heating process".There are research has been shown that biomacromolecules could self-assemble to nano-particles during food and TCM heating process,it could be obtained new biological activity such as oxidation resistance after formation of nano-particles,or could be loaded with biological and active composition.Through the preliminary work,this project proposed that "Nanostructures of biomacromolecules formed by self-assembly during food heating process have an important contribution to the biological function of food".Based on this scientific hypothesis,taking the "apricot kernel"(Let food be the medicine)as the research object,it investigated the self-assembly of the apricot kernel protein fraction during heating process.The characterization and application of the protein nanoparticles were also studied.In this study,the 11S globulin was sperated and purified from apricot kernel by chromatograph.The 11S globulin includes 6 monomers(A-pro)and each of them was composed of an acid and a basic subunit which were connected through disulfide bond.Study on the process of heating,we found that the protein unfolded,rearranged and self-assembed which would influence the properties of food.Then we studied the properties and application of self-assembly apricot kernel 11S globulin,and the main results were as follows:(1)The disulfide bond of protein monomer(Apro)would rupture during heating,in order to keep the stable structure,the disulfide bond would rearrange to form the intermediate product,and this process mainly occurred between the acidic subunit AS-1 and the AS-2(42-44 kDa),the molecular weight of the newly emerging protein component was observed to be about 88 kDa during the heating process.With the formation of disulfide bond,the formation of nanostructures was also observed,indicating that the self-assembly of protein was closely related to the disulfide bond.Through the study of self-assembly behavior,it was found that temperature condition for Apro self-assembly must be higher than the transition midpoint temperature of 76 ?,a-helix decreased quickly,the secondary structure was desdroyed,hydrophobic segments were exposed to surface because of the heat treatment.At the same time,Apro relied on disulfide bonds and hydrogen bonds to maintain a specific spatial structure.Nano-particles were formed by hydrophobic interaction with hydrophobic groups on the protein surface,at the same time,disulfide bond and hydrogen bond were warped inside.Therefore,Apro self-assembly was depended on the disulfide bond and hydrogen bond in the heat treatment.(2)The essence of protein self-assembly behavior is the molecular interactions.By means of regulation of the chemical factors including pH,temperature,the self-assembly behavior,the morphology of nano-particle and other characteristic were affected.The results showed that the acidic environment led to the decrease of the solubility of apricot kernel protein,and the basic environment was conducive to the self-assembly;Low concentration of sodium promote the self-assembly behavior,and high concentration of calcium salt leaded to precipitation;The lower protein concentration,the slower rate of self-assembly behavior;the higher temperature,the greater particle size of nano-particles.(3)According to the regulatory mechanism of chemical factors on self-assembly behavior,the optimal conditions of Apro nano-particles formation were showed:pH 9.0,heating temperature was 95 °C,heating time was 10 min,protein concentration was 1 mg/mL,and named Apro-NPs,the diameter of Apro-NPs was about 90 nm with uniform and unique distribution.The effect of temperature to Apro-NPs was showed the molecular tended to form polymerization during heating process,and it would tend to form nano-particles during cooling process.In addition,the nano-particles were stable at low temperature,which is conducive to preservation.(4)The nano-particles formed by food protein are more safe than synthetic nano-materials,there are advantage for preparation of nano-carriers.The results of cell safety evaluation showed that Apro-NPs increased the survival rate of normal cells and obtained higher safety than the protein.Encapsulation test showed that Apro-NPs loaded with anti-cancer drugs amygdalin and paclitaxel successfully.Due to the different loading methods,the loading efficiency of nano-particles with amygdalin and paclitaxel were different.Amygdalin was bind to the surface of the particles,and paclitaxel was embedded inside the particles.Therefore,the loading efficiency of paclitaxel was higher than amygdalin,and the efficiency of sustained-release was also better than amygdalin.In addition,the drug increased the inhibitory effect on the proliferation of cancer cells after loaded by Apro-NPs(maximum efficiency was 43%),which laid the foundation for research on targeted delivery of protein carrier. |
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