Effects Of Ultrasound Treatment On Structure And Functional Properties Of Whey Protein

The study belongs to the project of Chinese ministry of science and technology project-" Technology and application of whey protein"(2013BAD18B07).Whey protein is an excellent natural protein with high nutritional value and bioavailability.?-Lactoglobulin is a major component of whey protein with high proportion of amino acids and branched-chain amino acids.?-Lactoglobulin has unique conformation and physical-chemical properties,and is widely used in food industry.Ultrasound is a new technology with a strong penetrating power,good directivity and great energy.High intensity Ultrasound technology has been used to enhance food protein quality and functional properties in recent years.In this study,high intensity ultrasound was used to modifiy whey protein nanoparticles,?-lactoglobulin and its enzyme products.This dissertation includes the following aspects:(1)Effect of high intensity ultrasound on physical-chemical properties of ?-lactoglobulin and optimization of ultrasonic conditions.(2)Effect of high intensity ultrasound on structure of ?-lactoglobulin and its enzyme products.(3)Effect of high intensity ultrasound on the mechanisms of ?-lactoglobulin and its enzyme products against oxidative stress in Caco-2 cell.(4)Effect of high intensity ultrasound on the changes in immune function of macrophages induced by ?-lactoglobulin and its enzyme products.(5)Effect of high ultrasound treatment on antimicrobial property of whey protein-totarol nanoparticles.(1)The purity of separated ?-lactoglobulin from raw cow milk was 91% by HPLC.?-Lactoglobulin was treated with Ultrasound at different amplitudes,temperatures and durations.The optimized conditions of ultrasound parameter treated on ?-lactoglobulin were to be 45 oC,20 min and amplitude of 30% using Box-Behnken design.The surface hydrophobicity and free sulfhydryl group of ?-lactoglobulin were significantly increased after ultrasound treatment(p < 0.05).The maximal surface hydrophobicity and free sulfhydryl group were 5812.08 and 5.97 ?mol/g,respectively.Ultrasound treatment changed the physicochemical properties of ?-lactoglobulin including particle size(from 1.21 ± 0.05 to 1.66 ± 0.03 nm),particle size distribution(from 0.44 ± 0.23 to 0.67 ± 0.32)and absolute zeta potential(from 15.47 ± 1.60 to 27.63 ± 3.30 m V).Ultrasound treatment increased ?-helix structure(from 16.9 ± 0.16 to 29.1 ± 0.20%)and ?-sheet structure(from 38.0 ± 0.09 to 49 ± 1.17%)of ?-lactoglobulin,while decreased random coil structure(from 28.2 ± 0.63 to 2.5 ± 0.09%).CD data indicated that Ultrasound-treatment increased both ?-helix and ?-sheet contents of ?-lactoglobulin and altered Trp residues,which resulted in the secondary and tertiary structure changes.DSC datas showed that Ultrasound-treated ?-lactoglobulin contained larger aggregates than untreated.Ultrasound treatment inceased the degree of degeneration(from 0.59 ± 0.038 to 0.83 ± 0.17 J/g),reduced the denaturation temperature(from 71.08 ± 2.78 to 58.13 ± 2.29oC).Ultrasound treatment reduced the thermal stability of ?-lactoglobulin.There were no significant changes in protein electrophoretic patterns of ?-lactoglobulin.(2)High intensity ultrasound can improve enzymatic hydrolysis of ?-lactoglobulin.There were no significant changes in protein electrophoretic patterns of ?-lactoglobulin,but a significant change in protein electrophoretic patterns of enzyme products.High intensity ultrasound had a significant effect on the HPLC elution peak,more elution peaks at 13,18,35 and 37 min and decreased peaks for native ?-lactoglobulin were observed in the chromatography of ?-lactoglobulin with ultrasound and enzymatic treatments.Ultrasound treatment increased ?-helix structure(from 16.9 ± 0.16 to 35.1 ± 0.14%)and ?-sheet structure(from 33.3 ± 0.2 to 59.2 ± 0.14%)of ?-lactoglobulin and its enzyme products.Intrinsic fluorescence intensity of ultrasound and enzymatic-treated ?-lactoglobulin was increased with shift of ?max from 334 to 329 nm.The results indicated that the structure of ultrasound and enzymatic ?-lactoglobulin tended to be loose.Trp residues moved from weak hydrophobic area to strong hydrophobic area leading to an increase of fluorescence intensity.UV absorption of ultrasound and enzymatic-treated ?-lactoglobulin was decreased with shift of ?max from 288 to 285 nm after ultrasound treatment.UV spectrum showed some conformational changes to the surface region of protein molecule were occurred.The split and precipitation behavior of ?-lactoglobulin was better than that of the exposure of chromogenic group under ultrasound and enzymatic treatment,which was embedded inside the molecule due to the changes in the structure of ?-lactoglobulin.(3)Ultrasound treatment had considerable impact on the antioxidant activity of ?-lactoglobulin and its enzyme products.?-lactoglobulin and its enzyme products showed significantly higher ABTS+ radical scavenging activity,DPPH radical scavenging activity and oxygen radical absorbance capacity.It also showed significantly higher oxygen scavenging activities in Caco-2 cells models.?-Lactoglobulin and its enzyme products significantly inhibited the generation of MDA and intracellular ROS,increased the activities of antioxidant enzymes in Caco-2 cells.The increasing acoustic energy could break up the agglomerates of ?-lactoglobulin,and increase the specific surface area providing more reactive sites with free radicals.So,it could protect Caco-2 cells from H2O2-induced oxidative stress through the elevation of cellular antioxidant activities of enzymes.(4)Ultrasound-treated ?-lactoglobulin and its enzyme products had obvious influence on the immune regulation function of macrophages.It had no toxic and promotive effects on the proliferation of macrophages at concentration from 50 to 800 ?g /m L.It enhanced the expression of TNF-?,IFN-? and NO release in a dose-dependent manner.At 800 ?g /m L,the maximum of TNF-?,IFN-? and NO release were 703.00 ± 2.15 pg/m L,160.76 ± 1.22 pg/m L and 57.87 ± 0.24 ?M,respectively.Ultrasound-treated ?-lactoglobulin and its enzyme products could enhance the phagocytosis from 38.3 to 60.5%.(5)Ultrasound treatment can improve the antibacterial activities of whey proteintotarol nanoparticles against S.aureus.The particle size of whey protein-totarol nanoparticles was reduced by Ultrasound treatment(31.24 ± 5.31 to 24.20 ± 4.02 nm)and size distribution was also narrowed by the treatment(0.46 ± 0.06 to 0.47 ± 0.02).The data of viscosity and modulus indicated that the flow behaviors of whey protein-totarol nanoparticles seemed to be a Newtonian and exerted a typical viscoelastic fluid at protein content of 15%(w/v).The minimal inhibitory concentration(MIC)of whey protein-totarol nanoparticles after ultrasound treatment decreased from 4 to 2 ?g/m L compared to that without ultrasound treatment.Whey protein-totarol nanoparticles treated with Ultrasound resulted in a significant(p < 0.05)decrease in time-killing after 24 h.The agar diffusion results showed that the inhibition zones of whey protein-totarol nanoparticles were 12 and 36 mm for untreated and treated with ultrasound,respectively.The bacterial membrane damages and the microstructure changes also proved that whey protein-totarol nanoparticles treated with ultrasound had strong antibacterial activities against S.aureus.