Cunshan Zhou

Pretreatment of defatted wheat germ proteins (by-products of flour mill industry) using ultrasonic horn and bath reactors: Effect on structure and preparation of ACE-inhibitory peptides

The ultrasonic horn and bath reactors were compared based on production of angiotensin-converting-enzyme (ACE) inhibitory peptides from defatted wheat germ proteins (DWGP). The DWGP was sonicated before hydrolysis by Alcalase. Degree of hydrolysis, ACE-inhibitory activity, surface hydrophobicity, fluorescence intensity, free sulfhydryl (SH), and disulfide bond (SS) were determined. The highest ACE-inhibitory activity of DWGP hydrolysate was obtained at power intensity of 191.1 W/cm(2) for 10 min in the ultrasonic horn reactor. The fixed frequency of 33 kHz and the sweep frequency of 40±2 kHz resulted in the maximum ACE-inhibitory activity. The combined irradiation of dual fixed frequency (24/68 kHz) produced significant increase in ACE-inhibitory activity compared with single frequency (33 kHz). The ultrasonic probe resulted in significant higher ACE-inhibitory activity compared with ultrasonic bath operating at single or dual fixed and sweep frequencies. The changes in conformation of the DWGP due to sonication were confirmed by the changes in fluorescence intensity, surface hydrophobicity, SHf and SS contents and they were found in conformity with the ACE-inhibitory activity in case of the ultrasonic horn reactor but not in bath reactor.

Effects of multi-frequency power ultrasound on the enzymolysis and structural characteristics of corn gluten meal

The aim of this study was to investigate the effect of multi-frequency power ultrasound (sweeping frequency and pulsed ultrasound (SFPU) and sequential dual frequency ultrasound (SDFU)) on the enzymolysis of corn gluten meal (CGM) and on the structures of the major protein fractions (zein, glutelin) of CGM. The results showed that multi-frequency power ultrasound pretreatments improved significantly (P<0.05) the degree of hydrolysis and conversion rate of CGM. The changes in UV-Vis spectra, fluorescence emission spectra, surface hydrophobicity (H0), and the content of SH and SS groups indicated unfolding of zein and glutelin by ultrasound. The circular dichroism analysis showed that both pretreatments decreased α-helix and increased β-sheet of glutelin. The SFPU pretreatment had little impact on the secondary structure of zein, while the SDFU increased the α-helix and decreased the β-sheet remarkably. Scanning electron microscope indicated that both pretreatments destroyed the microstructures of glutelin and CGM, reduced the particle size of zein despite that the SDFU induced aggregation was observed. In conclusion, multi-frequency power ultrasound pretreatment is an efficient method in protein proteolysis due to its sonochemistry effect on the molecular conformation as well as on the microstructure of protein.

Effects of ultrasound and ultrasound assisted alkaline pretreatments on the enzymolysis and structural characteristics of rice protein.

The objectives of this study were to investigate the effects of multi-frequency energy-gathered ultrasound (MFEGU) and MFEGU assisted alkaline pretreatments on the enzymolysis and the mechanism of two pretreatments accelerating the rice protein (RP) proteolysis process. The results showed that MFEGU and MFEGU assisted alkaline pretreatments improved significantly (P<0.05) the degree of hydrolysis (DH) and the protein elution amount of RP. Furthermore under the same DH conditions, ultrasound and ultrasound assisted alkaline pretreatments were more save the enzymolysis time than the unpretreatment. The changes in UV-vis spectra, fluorescence emission spectra indicated unfolding and destruction of RP by MFEGU and MFEGU assisted alkaline pretreatments. The circular dichroism analysis showed that both pretreatments decreased α-helix but increased β-sheet and random coil of RP. Amino acid composition revealed that MFEGU and MFEGU assisted alkaline pretreatments could increase the protein elution amount and the ratio of hydrophobic amino acids. Atomic force microscopy (AFM) indicated that both pretreatments destroyed the microstructures and reduced the particle size of RP. Therefore, MFEGU and MFEGU assisted alkaline pretreatments are beneficial to improving the degree of hydrolysis due to its sonochemistry effect on the molecular conformation as well as on the microstructure of protein.

Effects of multi-frequency power ultrasound on the enzymolysis of corn gluten meal: Kinetics and thermodynamics study.

The effects of multi-frequency power ultrasound (MPU) pretreatment on the kinetics and thermodynamics of corn gluten meal (CGM) were investigated in this research. The apparent constant (KM), apparent break-down rate constant (kA), reaction rate constants (k), energy of activation (Ea), enthalpy of activation (ΔH), entropy of activation (ΔS) and Gibbs free energy of activation (ΔG) were determined by means of the Michaelis-Menten equation, first-order kinetics model, Arrhenius equation and transition state theory, respectively. The results showed that MPU pretreatment can accelerate the enzymolysis of CGM under different enzymolysis conditions, viz. substrate concentration, enzyme concentration, pH, and temperature. Kinetics analysis revealed that MPU pretreatment decreased the KM value by 26.1% and increased the kA value by 7.3%, indicating ultrasound pretreatment increased the affinity between enzyme and substrate. In addition, the values of k for ultrasound pretreatment were increased by 84.8%, 41.9%, 28.9%, and 18.8% at the temperature of 293, 303, 313 and 323 K, respectively. For the thermodynamic parameters, ultrasound decreased Ea, ΔH and ΔS by 23.0%, 24.3% and 25.3%, respectively, but ultrasound had little change in ΔG value in the temperature range of 293-323 K. In conclusion, MPU pretreatment could remarkably enhance the enzymolysis of CGM, and this method can be applied to protein proteolysis industry to produce peptides.

Effects and mechanism of dual-frequency power ultrasound on the molecular weight distribution of corn gluten meal hydrolysates.

The impact of dual-frequency power ultrasound (DPU) on the molecular weight distribution (MWD) of corn gluten meal (CGM) hydrolysates and its mechanism were investigated in the present study. The mechanism was studied from aspects of structural and nano-mechanical characteristics of the major protein fractions of CGM, viz. zein and glutelin. The results of molecular weight distribution indicated that DPU pretreatment of CGM was beneficial to the preparation of peptides with molecular weights of 200-1000Da. Moreover, FTIR spectral analysis and atomic force microscopy characterization showed that the DPU pretreatment changed the contents of secondary structure of proteins, decreased the particle height and surface roughness of glutelin, reduced the Youngs modulus and stiffness of zein while increased its adhesion force. In conclusion, DPU pretreatment of proteins before proteolysis is an efficient alternative method to produce short-chain peptides because of its positive effects originating from acoustic cavitation on the molecular conformation, nano-structures and nano-mechanical properties of proteins as well.

Antioxidant peptides from corn gluten meal: Orthogonal design evaluation

Protamex catalyzed corn gluten meal (CGM) hydrolysis peptides (CHP) were prepared. Orthogonal design L16 (4(5)) was used to optimize processing variables of CGM concentration, CGM heat pretreatment (121 °C) time, and enzymolysis pH, temperature, and time. Degree of hydrolysis (DH), undigested residue ratio, molecular weight (MW) distribution and DPPH radical inhibition were selected as analysis indicators. Optimum variables were CGM concentration of 18%, heat pretreatment time of 40 min, and enzymolysis pH, temperature and time of 7.5, 55 °C and 24h, respectively. Verification test showed that CHP IC50 for scavenging hydroxyl radical was the best and then followed by reducing power. Oligopeptides improved after hydrolysis at the expense of di- and tripeptides, suggesting formation of soluble aggregates from low MW peptides. The increase in the DH, oligopeptides, Alanyl-Tyrosine, and antioxidant free amino acids coincided with the improvement in the antioxidant activity of CHP.

Study of ultrasonic cavitation during extraction of the peanut oil at varying frequencies

The ultrasonic extraction of oils is a typical physical processing technology. The extraction process was monitored from the standpoint of the oil quality and efficiency of oil extraction. In this study, the ultrasonic cavitation fields were measured by polyvinylidene fluoride (PVDF) sensor. Waveform of ultrasonic cavitation fields was gained and analyzed. The extraction yield and oxidation properties were compared. The relationship between the fields and cavitation oxidation was established. Numerical calculation of oscillation cycle was done for the cavitation bubbles. Results showed that the resonance frequency, fr, of the oil extraction was 40kHz. At fr, the voltage amplitude was the highest; the time was the shortest as reaching the amplitude of the waveform. Accordingly, the cavitation effect worked most rapidly, resulting in the strongest cavitation intensity. The extraction yield and oxidation properties were closely related to the cavitation effect. It controlled the cavitation oxidation effectively from the viewpoint of chemical and physical aspects.

Surface topography, nano-mechanics and secondary structure of wheat gluten pretreated by alternate dual-frequency ultrasound and the correlation to enzymolysis.

The effects of alternate dual-frequency ultrasound (ADFU) pretreatment on the degree of hydrolysis (DH) of wheat gluten (WG) and angiotensin I-converting enzyme (ACE) inhibitory activity were investigated in this research. The surface topography, nano-mechanics and secondary structure of WG were also determined using atomic force microscope (AFM) and circular dichroism (CD). The correlations of ACE inhibitory activity and DH with surface topography, nano-mechanics and secondary structure of WG were determined using Pearsons correlation analysis. The results showed that with an increase in either pretreatment duration or power, the ACE inhibitory activity of the hydrolysate also increases, reaching maximum at 10 min and 150 W/L, respectively, and then decreases thereafter. Similarly, AFM analysis showed that as the pretreatment duration or power increases, the surface roughness also increase and again a decrease occurs thereafter. As the pretreatment duration or power increased, the Youngs modulus and adhesion of WG also increased and then declined. Youngs modulus and adhesions average values were compared with ACE inhibitory activity reversely. The result of the CD spectra analysis exhibited losses in the relative percentage of α-helix of WG. Pearsons correlation analysis showed that the average values of Youngs modulus and the relative percentage of α-helix correlated with ACE inhibitory activity of the hydrolysates linearly and significantly (P<0.05); the relative percentage of β-sheet correlated linearly with DH of WG significantly (P<0.05). In conclusion, ADFU pretreatment is an efficient method in proteolysis due to its physical and chemical effect on the Youngs modulus, α-helix and β-sheet of WG.

Effects of multi-frequency ultrasound pretreatment under low power density on the enzymolysis and the structure characterization of defatted wheat germ protein

The effects of ultrasonic frequency mode, power density, pretreatment time and other parameters under low power density on the degree of hydrolysis (DH) of defatted wheat germ protein (DWGP) and angiotensin-I-converting enzyme (ACE) inhibitory activity of DWGP hydrolysate were studied in this research. Ultraviolet-visible (UV-Vis) spectra, free sulfhydryl (SH), disulfide bond (SS), surface hydrophobicity and hydrophobic protein content of ultrasound-pretreated protein and hydrophobic amino acid (HAA) content of alcalase-hydrolysate of DWGP were measured under optimized ultrasonic condition. The ultrasonic frequency mode with dual-fixed frequency combination of 28/40kHz showed higher ACE inhibitory activity of DWGP hydrolysate compared with that of other ultrasound frequency modes and all the ultrasonic frequency combinations involving in 28kHz showed higher ACE inhibitory activity. Under the dual-fixed frequency ultrasound mode of 28/40kHz, ultrasonic power density of 60W/L, pretreatment time of 70min, temperature of 60°C and substrate concentration of 60g/L, the ACE inhibitory activity of DWGP hydrolysate was the highest with its value of 74.75% (increased by 62.30% compared to control). However, all the ultrasonic pretreatment did not increase the DH of DWGP significantly (p>0.05). The changes in UV-Vis spectra, SH and SS groups, surface hydrophobicity and hydrophobic protein content indicated that the structure of DWGP unfolded after ultrasound pretreatment. The HAA content of hydrolysate from the pretreated DWGP increased significantly (p<0.05). The results proved that ultrasound pretreatment loosed the protein structure and exposed more HAA residues of protein to be attacked easily by alcalase. This resulted in the increase in the HAA content which related to the ACE inhibitory activity.

Effect of ultrasonic treatment on the morphology of casein particles.

In this study, the effect of ultrasonic treatment duration on the morphology of self-assembled casein particles was investigated by atomic force microscopy (AFM), low vacuum scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In the case of AFM images, the particle analysis which was carried out by the SPIP program showed that the self-assembled casein particles after being ultrasonically treated for 2 min got smaller in size compared to the casein particles that have not been exposed to any ultrasonic treatment. Surprisingly, however, increasing the ultrasonic time exposure of the particles resulted in an opposite effect where larger particles or aggregates seemed to be present. We show that by comparing the results obtained by AFM, SEM and TEM, the information extracted from the AFM images and analyzed by SPIP program give more detailed insights into particle sizes and morphology at the molecular level compared to SEM and TEM images, respectively.