Study On The Interaction Of The Main Component Of Spices Piperine With Myoglobin And Hemoglobin And Evaluation Of Its Effect On Beef Preservation

The color of meat products is an important reason to influence consumers’purchase.The study of pigment protein changes in meat products is an important link to regulate the color changes during processing and storage of meat products,which has great scientific and industrial development potential.Myoglobin（Mb）and hemoglobin（Hb）are the two most important pigment proteins in meat products that affect the color of meat.Piperine（PIP）is an alkaloid compound found mainly in black and white pepper.It has received increasing attention because of its potential health benefits,the most prominent of which is antioxidant activity.This work investigates the interaction mechanism between Mb/Hb and PIP at the molecular level,and the results provide a theoretical basis for the molecular mechanism of changes in the structural properties of the protein.The antioxidant capacity of PIP in meat products was further understood,and the findings are useful to guide the potential applications of PIP in related meat products.The results of the study include the following three parts.1.Study on the interaction mechanism between Mb/Hb and PIP.This section analyzes the interaction mechanism between Mb/Hb and PIP.Computer simulations were used to predict the binding location,force type,binding energy,and dynamic trajectory of PIP within the protein.The quenching mechanism between Mb/Hb and PIP was investigated by fluorescence spectroscopy,and the quenching mechanism was further verified by time-resolved fluorescence spectroscopy.The effect of PIP on protein secondary structure was determined by circular dichroism（CD）spectroscopy.Isothermal Titration Calorimetry（ITC）and thermodynamic calculations were performed to analyze various thermodynamic parameters during the combination of Mb/Hb and PIP.The results are as follows:（1）Molecular docking results based on YASARA software showed that PIP binds to the flexible region of Mb/Hb with binding energies of-6.37 k J·mol^-1 and-8.32 k J·mol^-1,respectively.Molecular dynamics simulations showed that the Mb-PIP and Hb-PIP complexes were more stable than the proteins alone.（2）PIP is a static quenching mechanism for both Mb and Hb,and the extent of PIP quenching for Mb is greater than that for Hb.The quenching mechanism was further demonstrated by time-resolved fluorescence spectroscopy.The binding of PIP to proteins alters the microenvironment of protein amino acid residues.（3）CD spectroscopy and synchronous fluorescence suggest that PIP changes the secondary structure conformation of Mb/Hb.（4）ITC and thermodynamic results showed that the interaction between Mb and Hb and PIP was spontaneous,with K_a>10⁵ indicating a moderate affinity binding between PIP and the protein.2.In vitro antioxidant activity study of Mb-PIP/Hb-PIP complex.In this section,changes in the in vitro antioxidant properties of PIP before and after binding Mb and Hb were assessed using DPPH and ABTS radical scavenging capacity assays as well as ferrous reducing antioxidant power assay based on the interaction mechanism.The antioxidant activity of PIP was further characterized by the release of free iron from the protein and the color change of the protein.The results are as follows:（1）The radical scavenging capacity of DPPH and ABTS of the complexes bound to PIP was significantly higher than that of PIP at the same concentration.（2）The FRAP analysis showed that the Mb/Hb-PIP complex system had higher antioxidant properties than PIP alone compared to different concentrations of PIP alone.（3）The release of free iron from the protein increased significantly with the increase of PIP concentration.The color analysis showed that the oxidative discoloration of the Mb/Hb-PIP complex system was down.The best color appeared at the concentration of PIP in 80 mg/L.3.Evaluation of the fresh beef preservation effect of PIP at 4°CThis section determines the effects of PIP,black pepper and white pepper on fresh beef during storage at 4°C through sensory evaluation,color change,texture,TBARS,and Met Mb%.（1）The sensory quality of the beef was scored and observed,and the results showed that the black pepper,white pepper and PIP groups deteriorated more slowly,and that PIP had the best preservation effect.The results of measuring a^*,b^*and L^*values showed that PIP had the best color protection.（2）The results of texture measurements showed that PIP was able to better maintain the height,hardness,elasticity,chewiness,cohesiveness and recovery of beef.（3）The results of TBARS values showed that TBARS values increased to 7.49-fold,6.00-fold,5.96-fold and 5.93-fold in the blank control,black pepper,white pepper and PIP groups,respectively.PIP was effective in inhibiting lipid oxidation in meat.（4）The results of Met Mb%measurement showed that Met Mb%increased 4.77-fold,4.05-fold,4.10-fold and 3.58-fold in the blank control,black pepper,white pepper and PIP groups,respectively.PIP significantly inhibited the increase in Met Mb%.