Research On The Protein Structure Differences And Antibacterial Peptides Preparation Of Milk Casein From Different Species

Casein, which was the main component of southern buffalo milk, was selected to be theobject of this study. Kinds of spectroscopy, such as Far-UV CD, fluorescence spectrum, NanoZSand so on were used in the research on the structure differences of casein. During this study, thestructure differences between casein from buffalo and other species, casein by differentsterilizations (pasteurization and UHT), and different components (α-、β-、κ-casein) werecompared, and the method and conditions of preparing antibacterial peptides from casein byenzymatic hydrolysis was studied. The above research on the structure difference of buffalocasein provide a theoretical reference for the dairy processing of buffalo milk, and lay thefoundation for future process improvements, the development of new milk products andmilk-derived protein biological activity products, such as the immune active peptides,antihypertensive peptides, anti-thrombotic peptides and other bioactive peptides.In the research on the structure differences of milk-derived casein and the differencesbetween pasteurized and UHT casein, Far-UV CD, fluorescence spectrum, SEM and NanoZSwere used respectively to study the secondary structure, the hydrophobic property and theposition of tryptophan and the structure of micelle and the size distribution of casein. During theresearch on the structure differences between α-, β-and κ-casein from bovine, in addition to theresearch of the secondary structure by Far-UV CD and the hydrophobic property and the positionof tryptophan by fluorescence spectrum, LTQ-Orbitrap coupled to liquid chromatographytechniques were applied to study the amino acid sequence. During the research on thepreparation of antibacterial peptides, the degree of hydrolysis and the antibacterial activity ofhydrolysis products from buffalo casein was compared. The hydrolysis condition by trypsin wasoptimized by single factor experiments, whose hydrolysis products own the highest antibacterialactivity. Based on the center composite experiments of Box-Behnken response surface, theprevious results were optimized again by Minitab14data statistical analysis software. Theexperimental results in this article are as follows:1. In the research on the structure differences between milk-derived casein, the secondarystructure, the hydrophobic property, the structure of micelle and the size distribution of water buffalo casein and goat casein were analyzed. The research on the secondary structure of buffaloand goat casein by Far-UV CD suggest the secondary structure of goat casein was more orderly,which contained a large number of α-helix and a small amount of random coil. The results offluorescence spectrum suggested Trp of water buffalo and goat casein located in the hydrophobicenvironment. The results of SEM suggested the casein particles of water buffalo casein wereinterconnected into small micelles, and linked into a more dense network-like structure. Thedegree of aggregation of goat casein was much lower than water buffalo casein and few of caseinparticle interconnected into short micelle, but can’t linked into network-like structure. The dataof NanoZS showed the average diameter of water buffalo casein particles was182.3nm, whichwas smaller than goat casein of173.8nm.2. During the research on the structure differences between pasteurized and UHT bovinecasein, the secondary structure, the hydrophobic property, the structure of micelle and the sizedistribution were analyzed. The structure differences between pasteurized and UHT bovinecasein were mainly in the following aspects. First, the stability of UHT Casein decreased andspatial structure was more irregular. Second, the results of fluorescence spectrum suggestedsome Trp on the surface of casein structure moved into the hydrophobic environment of theinside. Third, the results of SEM suggested the cover composed of κ-casein was destroyed,which caused the spherical structure of the casein particles and micelle structure were destroyedfurther. Fourth, the diameter of the UHT bovine casein particles was smaller than the pasteurizedbovine casein. This illustrated the structure of αs1-, αs2-and β-casein was destroyed, whichcaused the hydrophobic interaction in the process of aggregation become weaker.3. In the research on the structure differences between α-, β-and κ-casein from bovinemilk, the differences of the secondary structure, the hydrophobic property and the amino acidsequence was analyzed. Hydrolysis peptides by trypsin was separated and purified by C18reverse phase column chromatography and identified by LTQ-Orbitrap coupled to liquidchromatography techniques. Amino acids sequences of α-, β-and κ-casein were acquired andmatched with the amino acids sequences from the database, results show that the number andsequence of amino acid were significant different. The research on the secondary structure byFar-UV CD showed α-casein had the highest content of ordered structure and the lowest contentof disordered structure, which was opposite to β-casein. The results of fluorescence spectrum suggested the most tryptophan residues located in the surface of α-casein, but the tryptophanresidues positioned in the internal structure of β-casein.4. During the preparation and response surface optimization, the hydrolysis condition oftrypsin was selected to optimized by single factor experiments and response surface experimentsafter comparing the antibacterial activities. Single factor experiments suggest the besttemperature, time and enzyme substrate ratio were41℃、3h、2%, respectively. Center compositeexperiments of Box-Behnken response surface suggest the best hydrolysis conditions were41℃of temperature,3h of time,2%of enzyme substrate ratio. Inhibition of this antimicrobialpeptides to Escherichia coli was81.34%.