Effects Of Sweeping Frequency Ultrasonic Pretreatment On ?-Lactoglobulin Enzymatic Sensitivity And Aggregation Behavior

Ultrasound treatment on proteins can improve their enzymatic properites by changing their structure.However,if the power too strong or the treatment time too long will make the molecules approach each other to form large aggregations,which is not conducive for the enzymatic hydrolysis reaction.Therefore,it is necessary to study the regular relation of ultrasound conditions on enzymatic properties,structure and aggregation behavior of proteins.The enzymatic hydrolysis characteristics of?-lactoglobulin??-LG?,such as the hydrolysis degree?DH?,extraction rate of peptide,antioxidant activity were studied.Changes of the structure,such as sulfhydryl?-SH?,disulfide bond?S-S?content,surface hydrophobicity?H₀?,fluorescence spectrum,circular dichroism?CD?,surface morphology and the aggregation kinetics and thermodynamic stability were also studied.Those studies were done to explore the relationship between the enzymatic hydrolysis characteristics and aggregation behavior of?-LG under sweeping frequency ultrasound?SFU?and the main conclusions were as follows:?1?The DH,extraction rate of peptides and antioxidant were taken as indicators for enzymatic hydrolysis of?-LG under the effect of single-frequency and dual frequency SFU pretreatments.40 kHz and 68-40 kHz SFU improved the DH and the antioxidant activity of enzymatic hydrolysates.SFU pretreatment increased the contents of tyrosine?Tyr?,proline?Phe?and histidine?His?of the hydrolysate of?-lactoglobulin.After SFU treatments,the H₀ of enzymatic hydrolysates increased which indicated that more hydrophobic substances were released and polypeptides with molecular weight 200²000 Da increased significantly which improved the antioxidant activity of?-LG hydrolysates.However,the DH decreased after treated by SFU for a long time?60⁹0 min?and the extraction rate of peptides decreased significantly after treating for 60 min,due to the protein forming large aggregations.?2?Effects of sonication time on particle size,microstructure and molecular structure were studied.With 40 kHz and 68-40 kHz sonication,the protein size decreased first and then increased.After 20 and 30 min of SFU treatment,the particle size of?-LG decreased significantly and the fluorescence intensity increased,indicating that the tryptophan residues in the protein were exposed.After 20 min of SFU treatment,the content of-SH increased significantly,which indicating that the molecular structure of protein became loose.The H₀ of?-LG increased significantly after 30 min of SFU treatment,indicating that the protein molecules were stretched.The results of CD showed that the content of?-helix decreased after 20⁶0 min of SFU traetment.After long-time SFU?60,90 min?treatment,the partical size of?-LG increased and the fluorescence intensity decreased,which indicated that the exposed tryptophan residues aggregated into the protein when the ultrasound time was too long.After SFU for 60 and 90 min,the content of-SH of?-LG decreased while the content of disulfide bond?S-S?increased significantly,and the H₀ of protein decreased indicating that proteins were close to each other under hydrophobic interaction,which made the exposed hydrophobic groups protected by the protein molecule and was a sign of protein aggregation.Hydrophobicity and disulfide bond cross-linking of proteins formed protein aggregations.The surface roughness of proteins increased and cracks appeared on the surface of?-LG which indicated that SFU increased the surface area of proteins and increased contact opportunities between proteins and enzymes.?3?Particle size distribution of protein was measured by dynamic light scattering,the peak intensity of the particle size distribution and treatment time was fitted by linear regression and the slope was the aggregation reaction rate?k?.It was shown that40 kHz and 68-40 kHz SFU treatments increased the aggregation reaction rate?k?.The apparent activation energy??H?of the aggregation reaction decreased by58.45%and 54.99%respectively under 40 kHz and 68-40 kHz SFU treatments.