Study On The Interaction Mechanism Of Tetracycline Hydrochloride With Three Kinds Of Milk Protein And ?-galactosidase

Tetracycline hydrochloride(TCH),as an important class of tetracycline antibiotics,has been widely used in livestock and poultry bacterial infection treatment.Apart from this,it has been used as feed additives to enhance growth of food-producing animals or protect animals from disease,usage increased year by year.In recent years,food safety issues have been reported frequently,in order to study the effect of tetracycline residues on the quality of milk,we conducted a series of studies.In this study,fluorescence spectroscopy,UV-Vis absorption spectroscopy,fluorescence excitation-emission matrix spectroscopy,circular dichroism and molecular docking techniques were used to explore the interaction between tetracycline hydrochloride(TCH)and ?-casein(?-CN),?-lactalbumin(?-LA),lactoferrin(LF),?-galactosidase(?-Gal).Meanwhile,the effect of TCH on milk protein function was further analyzed on the basis of studying the influence of TCH on the structure of milk protein,which was important to milk industry and food safety.This paper consists of three parts,as follows:In the first chapter,the causes and the harm of excessive antibiotic residues,the structure and function of TCH,three kinds of milk protein(?-CN,?-LA,LF)and ?-Gal were summarized.Then this chapter introduced the commonly used methods in the study of the interaction between TCH and proteins.Finally,concluded the current research situation about the interaction between TCH and proteins.In the second chapter,UV-Vis absorption spectroscopy,fluorescence spectroscopy,fluorescence excitation-emission matrix spectroscopy,circular dichroism and molecular docking techniques were used to study the interaction between TCH and three proteins(?-CN,?-LA and LF)under simulated milk pH(6.6)and normal temperature(300 K)conditions.Research(fluorescence spectra and time-resolved fluorescence experiment)results showed that TCH induced the intrinsic fluorescence quenching of these three proteins,and the quenching types were static quenching;thermodynamic parameters and molecular docking results represented electrostatic forces played major roles in the interaction between TCH and ?-CN,LF,while Van der Waals forces and hydrogen bonds were the main force for ?-LA-TCH complex.The binding distances of TCH and ?-CN,?-LA and LF were 3.70 nm,3.19 nm and 2.11 nm,respectively.Synchronous fluorescence spectra,fluorescence excitation-emission matrix spectra and CD spectra results indicated TCH could interact with ?-CN,?-LA and LF and partially destroyed the secondary structures of proteins.In addition,it was found that the presence of TCH could result in the change of the function of milk protein by measuring the surface hydrophobicity of protein and the change of sulfhydryl and disulfide bond.In the chapter three,multi-spectral method and molecular docking techniques were used to study the interaction between TCH and ?-Gal under simulated milk pH(6.6)and normal temperature(300 K)conditions.The results of the study showed that the TCH inhibited ?-Gal activity reversibly in a competitive manner.Thermodynamic parameters revealed that electrostatic forces played a major role in the interaction between ?-Gal and TCH.In addition,the effect of TCH on the enzyme activity was further analyzed;the interaction mechanism and the inhibition type between TCH and ?-Gal were judged;the effect of different temperature and pH on the stability of the enzyme was also discussed.