| Pine nut meal is a by-product of processing pine nut oil.The food-grade pine nut meal is usually used to process protein powder or hydrolyze to produce functional peptides.The non-food-grade pine nut meal is generally used as feed.The high value utilization of non-food grade pine nut meal is an urgent problem to be solved.Gold nanoparticles have excellent catalytic function and can effectively catalyze the degradation of pollutants?phenols and azo compounds?in sewage,which can eventually be transferred to humans through the food chain.With the improvement of environmental protection awareness,the preparation of gold nanoparticles by non-toxic natural substances such as proteins has become a new trend.Therefore,it is possible to use non-food-grade pine nut meal to prepare gold nanoparticles with the function of good catalytic degradation of pollutants in the environment,so as to expand the use range of non-food-grade pine nut meal and improve its economic value.In this paper,water-soluble protein extract from pine nut meal was used to prepare gold nanoparticles with good catalytic capacity and its formation mechanism was described at the molecular level.The protein extract of pine nut meal with little protein damage and high ABTS radicals scavenging ability was obtained by homogenate extraction.Through the purification technology,exclusion chromatography technology and on-line ABTS radical scavenging ability detection technology,the components with high activity and antioxidant molecular weight were screened out,and gold nanoparticles were prepared by the one-pot method.By adjusting the parameters of gold ion concentration,protein concentration,reaction temperature and reaction time,the gold nanoparticles with small particle size was prepared.The gold nanoparticles were characterized by means of spectroscopy,transmission electron microscopy,X-diffraction and exclusion chromatography,and their antioxidant capacity and catalytic degradation of p-nitrophenol and Congo red were evaluated.The mechanism of preparing gold nanoparticles by pine nuts 7S globulin was studied with analysis techniques,such as ultraviolet-visible spectrum,fluorescence spectrum,circular dichroism spectrum,and transmission electron microscopy,Stern-volmer equation,chemical equilibrium equation,Gipps free energy equation,and Foerster's dipole-dipole non-radiation energy transfer theory.The molecular docking technology was used to study the reaction mechanism at the molecular level.The main research contents and conclusions are as follows:?1?Study on extraction technology of water-soluble protein from pine nutCompared with ultrasonic extraction,microwave extraction and hot water extraction methods,the high-speed homogenization extraction method is more conducive to retaining the antioxidant power and reducing the destruction of protein secondary structure,and its extract is more suitable for preparing gold nanoparticles.Compared with the other three common extraction methods,the high-speed homogenization method has the highest water-soluble protein extraction rate?11.68±0.14%?,the highest ABTS radical scavenging rate?85.29±0.97%?,the shortest extraction time?less than 6min?,the lowest energy consumption for extraction,the hignter proportion of protein ordered structure?80.05%?,and the lower price of extraction equipment.The optimal process parameters for extracting water-soluble glycoproteins from pine nut meal by high-speed homogenization extraction method were:liquid-to-material ratio of 43.23 mL·g-1,extraction time of 170 s,extraction temperature of 35?,extraction speed of 20000 r·min-1 for two continuously extractions.?2?Isolation of gold nanoparticles from water-soluble protein of pine nut mealAnion exchange chromatography was used to preliminary purify the water-soluble protein extract from pine nut meal.The exclusion chromatography and online ABTS radical scavenging detection technology were used to analyze each fraction to quickly find proteins with antioxidant activity and the higher molecular weight.The fraction 6 with antioxidant activity and higher molecular weight protein.The fraction 6 is mainly composed of pine nut 7S globulin trimer?203 ku?and pine nut 2S albumin?7.2 ku?,and the ABTS radical clearance rate of the fraction 6 was?91.56±2.33?%.?3?Preparation,characterization and catalytic function determination of pine nuts protein complexes with gold nanoparticlesGold-nanoparticle protein complex was prepared by one-pot method using the 6th fraction of pine nut meal water-soluble protein and chloroauric acid as raw materials.The best parameters for preparing small-sized gold nanoparticles were:gold ion concentration of 0.76 mmol·L-1,protein concentration of 0.71mg·mL-1,reaction temperature of 43? and reaction time of 47 min.Under these conditions,the hydrodynamic diameter of the gold nanoparticles is 25.28±1.26 nm.The prepared gold nanoparticles have typical plasmon resonance absorption peaks at 540 nm.Most of the gold nanoparticles are well dispersed,round,average particle size of 7 nm,and have a typical crystalline structure.The prepared gold nanoparticles have no anti-oxidation ability,but the gold nanoparticle protein complex has good catalytic ability.Its catalytic ability is better than that of gold nanoparticles prepared by some chemical methods,and it can quickly complete the catalytic degradation process of p-nitrophenol.The catalytic reaction rate can reach 7.9 ×10-3 s-1.As the number of cycles increases,the catalytic performance gradually decreases.The catalyst can be used at least 5?6 times while ensuring the conversion rate.The prepared gold nanoparticle pine nut 7S globulin complex can quickly complete the catalytic degradation process of Congo red,and the catalytic reaction rate can reach 1.7×10-3 s-1.?4?Establishment of detection method for specific protein components connected to gold nanoparticlesThe dual-wavelength molecular exclusion chromatography established in this paper can quickly and accurately analyze the specific protein components connected to gold nanoparticles.The minimum can detect gold nanoparticles prepared by 0.02 mmol·L-1 gold ions,which is 20 times that of traditional spectrometry.This method analyzes the interaction between gold nanoparticles and pine 7S globulin trimer to form a strong complex.?5?Study on formation mechanism of gold nanoparticles 7S globulin complexThe interaction between gold nanoparticles and pine nut 7S globulin is a spontaneous process of spontaneous heat generation with increased entropy and reduced Gibbs free energy.The interaction of gold nanoparticles with 7S globulin affected the tertiary structure of 7S globulin of pine nuts.The preparation mechanism of the gold nanoparticle pine nut 7S globulin complex were:gold ions reversibly affect the secondary structure of pine nut 7S globulin,and the reducing substances in the system reduce gold ions to gold atoms.The key amino acid of pine nuts 7S globulin,histidine at position 120,adsorbs one gold nanoparticle by electrostatic force.This gold atoms act as nucleus adsorption free gold ions in the system.These gold ions will be reduced to gold atoms by reducing agents and gradually form larger gold nanoparticles.With the increase of gold nanoparticles,some gold nanoparticles adsorb to each other to form larger gold nanoparticles.Due to a certain electrostatic repulsion force between the 7S globulin proteins,the proteins adsorbed by gold nanoparticles will interact with each other.Therefore,these pine nuts 7S globulin will gradually form a spherical shell around the newly formed larger gold nanoparticles.This protein shell helps form spherical gold nanoparticles while controlling their particle size.To sum up,this paper successfully prepared gold nanoparticle pine nut 7S globulin complex with good catalytic activity by using water-soluble protein extract from pine nut meal and chloroauric acid.The formation mechanism of gold nanoparticle pine nut 7S globulin complex was explained at the molecular level,which provided a new idea and theoretical basis for the high value utilization of non-food pine nut meal. |
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