Interaction Mechanism Between Lactoferrin And Casein In Bovine Milk

Lactoferrin mainly exists in bovine whey protein. LF has many biologicalfunctions, such as it can resist parasite, resist antitumor, regulate immune function andso on. Because of the low content, and it also interacts with casein micelles which isnegatively charged colloid to form large colloidal complex(LF-CM) in bovine milk, itresults in the lower content of lactoferrin in whey and low yield of lactoferrin ofseparation. In this study, skim milk was choosed as raw material and determinated LFcontent by enzyme linked immunosorbent assays(ELISA). Regulating the systemfactors to determine the best optimal factors that can promote LF to release fromLF-CM. Through the laser diffraction technique inspected particle size changes ofLF-CM under different condition. Moreover, to research the thermodynamic parametersbetween LF and different caseins (a-casein(ACN), β-casein(BCN), κ-casein(KCN) byisothermal titration calorimetry(ITC).This present study used fresh milk to get skim milk under the condition of4℃,6000r/min, centrifugal15min. Further optimized LF concentration by ELISA method.When the LF concentration was diluted to90.80ug/L got the most accurate result8.91mg/mL that was98.13%of the theoretical value. The LF was detected in caseinprecipitation, and at the same time got LF from casein precipitation solution bySDS-PAGE, all these indicates the LF interacted with CM in the milk.Through directdrying method, the residual LF in casein precipitate was far less than that of caseinprecipitation that combination with CM. Meanwhile it further verified CM interactionswith LF. Also it indicated the ELISA method can only detect the free LF content.By adjusting the pH、ionic strength and temperature of skim milk, determined LFcontent by ELISA, got the optimal parameters that promote LF to release from LF-CM:300mmol/L NaCl, pH6.4, temperature35℃. The highest release quantity oflactoferrin was48.18mg/L. Milk particle size distribution under different processingconditions showed that the particle size of casein solutions became small under pH6.4、temperature35℃,300mmol/L NaCl. That further verified LF will release fromLF-CM under this condition and made the CM particle size became small. When pHwas4.6, the soluble casein in solution became little, and the casein particle size becamesmaller, and it was1.262μm. When4.6<pH <5.6, the soluble casein moleculesincreased, so the particle size increased with the increased pH, when pH5.6it was3.170μm. When pH>6.6, the electrostatic repulsion between the micelle became big that made the micelle stability decreased. Casein monomolecular gradually disintegratesand particle size became smaller. When pH among6.6~8.6, the small particles volumefraction increased and large particles volume fraction decreases. The presents of largeparticles in casein solution increase and the small particles volume percentage decreasedwith increased adding NaCl, the size of the large and small particle size is3.170μm and21.70μm respectively.To study interaction between LF and caseins(ACN、BCN、KCN) by ITC knowedLF could interact with three kinds of caseins monomers under the condition of25℃spontaneously. According to the different affinity, LF has two ACN binding sites; threeBCN binding sites; three KCN binding sites.By studying the influence factors of the interaction beween casein micelles andlactoferrin and the thermodynamic parameters, such as binding point、combinedenthalpy、 combined entropy and so on. It will further expound the interactionmechanism between LF and casein micelles, and it will lay the foundation forextraction and separation of LF in scientific research and industrial production.