Effect And Mechanism Of Ultrasound On Enzymatic Preparation Of ACE-inhibitory Peptides From Wheat Germ Protein

Wheat germ protein(WGP) is a byproduct of the flour milling industry.The precious WGP source has poor utility for human applications due to its bad color and taste.The objective of this study was to prepare ACE-inhibitory peptides from WGP by ultrasound-assisted enzymatic hydrolysis.The effects of ultrasound on protease activity and its mechanism were studied.The impacts of ultrasonic treatment during enzymatic hydrolysis on kinetics of the hydrolysis of WGP were investigated.The ultrasonic pretreatment technology for WGP was optimized.The impacts of ultrasonic pretreatment on structure of WGP were studied by the methods of ultraviolet spectrometry,fluorescence spectrometry,infrared spectrometry,circular dichroism and scanning electron microscope(SEM),etc.Moreover,the relation between structure of ultrasound-pretreated WGP and ACE-inhibitory activity of its hydrolysates was analyzed.Based on previous publications,the structural relationship of ACE-inhibitory peptide was studied.The result showed that the most favorable N-terminus residues were Arg,Tyr,Gly,Val,Ala,Ile and Leu,and the most favorable C-terminus residues were Tyr,Pro,Trp,Phe and Leu.Compared with the characteristic of N-terminal amino acid for ACE-inhibitory activity,that of C-terminal amino acid was more important.Amino acids at C-terminal were mainly hydrophobic amino acids and played an important role in ACE-inhibitory peptide activity.The water-soluble albumin and salt-soluble globulin were the main protein fractions in WGP.Alcalase hydrolysates of albumin,globulin,gliadin and glutelin isolated from WGP had antihypertensive activity in vitro.Albumin and globulin give the greatest contribution to ACE-inhibitory activity of WGP hydrolysate.According to ACE-inhibitory activity of hydrolysates,Alcalase was chosen from six proteases,which were used to prepare antihypertensive peptides from WGP,and its condition for enzymatic hydrolysis was also optimized.The optimum technological conditions for ultrasonic pretreatment of WGP were decided by response surface methodology based on single factor analysis.The results showed that the optimum pretreatment conditions were ultrasonic power 1900 W, ultrasonic time 19.5 min and initial temperature of pretreatment 40℃for probe-type ultrasound,and were feed concentration 1.0g/100mL,ultrasonic time 140 min and sweep cycle 170 ms for sweep plate-type ultrasound.The ultrasonic pretreatment for WGP had little effect on conversion rate of hydrolysates,but caused remarkable increase in ACE-inhibitory activity of hydrolysates.Analysis of free sulfhydryl and disulfide bond contents,ultraviolet spectra, fluorescence spectra,hydrophobicity,infrared spectra,and circular dichroism revealed that ultrasonic pretreatment could cause a change on molecular structure of WGP. Analysis of SEM showed that particle size of ultrasound-pretreated WGP was smaller and more homogeneous compared to the control.Moreover,it was found that hydrolyzed WGP after ultrasonic pretreatment had more holes than that without pretreatment.The results suggested that ultrasound might loosen the tissue of WGP aggregate,and help the enzyme to attack the interior of WGP aggregate easily which resulted in the release of hydrophobic amino acids from WGP aggregate.Amino acid composition revealed that hydrolysates of ultrasound-pretreated WGP had a higher relative concentration of hydrophobic amino acid,with particularly noticeable increase in Pro content,as compared to the control.These changes play an important role in improvement of ACE-inhibitory activity of hydrolysates.Analysis results of thermodynamic parameters for Alcalase showed that the values of E_a,ΔH,ΔS andΔG could be decreased after ultrasonic treatment.In addition,the analysis results of ultraviolet spectra,fluorescence spectra,and circular dichroism showed that ultrasonic treatment led to slight change on molecular structure of Alcalase,which was related to increased activity of Alcalase.WGP hydrolysis under ultrasonic irradiation caused an increase in the value of k_A, and a decrease in the value of K_M,compared with that obtained without ultrasonic treatment.The ultrasound during enzymatic treatment for WGP by plate-type ultrasound at 24±2 and 24 kHz increased the conversion rate and ACE-inhibitory activity of hydrolysates.The ultrasound during enzymatic treatment for WGP by probe-type ultrasound had less effect on ACE-inhibitory activity of hydrolysates, while caused an increase in conversion rate of hydrolysates.The performance tests showed that antihypertensive peptides obtained from WGP had good acidic,alkaline,and thermal stability and ability of anti-intestinal digestion, and had clear decreases in systolic blood pressure(SBP) after being administed to spontaneously hypertensive rats(SHR).