| The phase separation and extraction ability of two different kinds of aqueous two-phasesystems (ATPS)(monohydroxy-alcohol/salts and polyethylene glycol/salts) were analysed toextract whey protein of water buffalo milk in South China in this paper. Two systems haddifferent behaviors, in monohydroxy-alcohol/salts system, by single factor experiments we foundthat the optimum monohydroxy-alcohol and salt were ethanol and sodium carbonate. Extractionconditions were optimized using response surface method, the optimum mass fraction of sodiumcarbonate, the mass fraction of ethanol and pH condition were15.80%,8.73%and12.13,respectively, and the recovery of whey protein was87.89%. In this system α-lactalbumin andβ-lactoglobulin were extracted to the top phase (ethanol phase). After extraction, ultravioletabsorption spectrum and fluorescence emission spectrum of whey protein from water buffalowere analysed, the experiment results proved that extraction had some affects on the structure ofwhey protein. Extraction conditions were optimized in polyethylene glycol/salts systems, such asthe kinds of PEG, the mass fraction of PEG and phosphate, the results showed that the optimumconditions were: PEG1500, PEG mass fraction:12.4%, phosphate mass fraction:18.8%, the ratioof partition coefficient of α-La and β-Lg was221.9. In this system, α-La was extracted tothe top phase (PEG phase), andβ-Lg was extracted to the bottom phase (phosphate phase), theywere basically separated showed by SDS-PAGE.Ion-exchange chromatography and gel chromatography were used to separate whey proteincompletely. Whey protein was obtained by isolating casein from defatted milk usinghydrochloric acid. Other proteins such as some caseins were precipitated by60%saturatedammonium sulfate, and supernatant was then precipitated by90%saturated ammonium sulfate toisolate whey protein, SDS-PAGE showed that by ammonium sulfate precipitation theprecipitated proteins were: α-La, β-Lg and a little bovine serum albumin (BSA). Adjust theconcentration of pure whey protein to15mg/mL using distilled water and10mL of protein wasfractionated using diethylaminoethyl (DEAE)-Sepharose CL-6B chromatography in whichβ-Lgwas separated from α-La and BSA. The latter two proteins that co-eluted in anion-exchangechromatography were then separated from each other by Sephadex G-75gel filtration. ThroughSDS-PAGE and RP-HPLC to identify the purity of the purified proteins, and the results showedthat purification effect was good. The purity of α-La andβ-Lg were both very high and RP-HPLC showed that in different elution conditions, different genetic variants of β-Lg wascollected.Due to the high value of whey protein, some cheap proteins are mixed in whey protein toreduce the production cost. SDS-PAGE and RP-HPLC two methods were using in this paper todetect whey protein which was mixed with some soy protein and gelatin. Two methods canseparate different kinds of protein completely. SDS-PAGE was more intuitive because differentkinds of protein had different bands, we can distinguish with naked eye. SDS-PAGE can be usedas a preliminary qualitative analysis. On the contrary, RP-HPLC had a short separation time,different proteins were distinguished by retention time of different absorption peak, it is hard tojudge if there is no obvious different absorption peak. According to retention time, peak heightand peak area, further quantitative analysis can be calculated. The minimum detection limit ofwhey protein mixed with soy protein and gelatin were3.8%and6.96%(m/m) by SDS-PAGEmethod, while by RP-HPLC the minimum detection limit were3.8%and4.97%(m/m),respectively.LTQ-Orbitrap mass spectrometer coupled with liquid chromatography techniques wereapplied in this paper to investigate the amino acid sequence of whey protein fractions of waterbuffalo in South China. The amino acids sequences of whey protein were acquired byexperiment and matched to the database, and this method was used to analysis bovine milk andgoat milk. The amino acids sequences of whey protein from different resource of milk werecompared with each other, results showed that the substitution of amino acids of α-La fromwater buffalo and bovine were smaller than that of goat’s milk and substitution of amino acids ofβ-Lg from water buffalo and bovine were bigger than that of goat’s milk. This means stabilityand homology of α-La’s amino acids of water buffalo and bovine were higher than goat’s whilesituation of β-Lg was opposite.During the preparation and response surface optimization, the hydrolysis condition oftrypsin was selected to optimize 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 were43℃of temperature,3.4h of time and2.5%of enzyme substrate ration. And inhibition of this antimicrobial peptides to Escherichia coli was81.34%.All these results provide the knowledge background for the purification of whey protein ofwater buffalo and production of dairy products. The results also provide some certain methodsfor rapid detection of whey protein. |
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