| Hypertension is a major risk factor for the development of end-stage renal disease,cardiovascular diseases,and hemorrhagic stroke among human beings.Therefore,prevention and treatment of high blood pressure at domestic and abroad is a daunting task.Over the past few years,a growing number of consumers are interested in functional foods,beyond the basic functions of providing nutrition,claiming to promote health and disease prevention.In this respect,the most important thing is that there are treatments to preserve not only the nutritional quality but also some biologically active ingredients.Numerous methods,especially acid,alkali,fermentation,and enzymatic hydrolysis,can be used to hydrolysis proteins and generate potential bioactive peptides that can prevent several diseases most likely blood pressure lowering bioactive peptides.It is known that enzymatic treatment of various proteases is the most commonly used technique for producing hydrolysed protein bioactive peptides/hydrolysates with no side effects and to upgrade functional and nutritional features of food proteins.Rapeseed protein is low cost and abundant by-product from theoil industry,considered to be a potential resource of bioactive peptides such as angiotensin-I-converting enzyme(ACE)inhibitory peptides are greatly welcomed by patients for the benefits of easy absorption,being safer and stable.In chapter 3,the main purpose of the study was to screen effective proteolytic enzymes for producing hydrolysates from rapeseed protein and to optimize hydrolysis conditions using response surface methodology(RSM)design to prepare hydrolysates with maximum ACE inhibitor activity.RSM design was successfully applied to the hydrolysis conditions on the basis of single factor experiments which further derived a statistical model for experimental validation.The molecular weight distribution of rapeseed protein hydrolysates with different degree of hydrolysis was also investigated.The results of enzyme screening test showed that the rapeseed protein hydrolyzed with alcalase was highest in terms of DH(15.35%)and the ACE inhibitory activity(55.07%)the five enzymes were screened followed by neutrase and chymotrypsin(13.81%,50.43%,and 13.52%,46.45%)after 2 hr of hydrolysis time.Aiding RSM design the highest ACE inhibitory activity 56.3%was achieved under optimum hydrolysis conditions at the hydrolysis time,pH,hydrolysis temperature,and enzyme dosage were at 90.11 min,8.88,50? and 3580.36 Ug-1,respectively.The mathematical model demonstrated a good fit with experimental results.Furthermore,the molecular weight distribution of rapeseed protein hydrolysates showed remarkable changes,most notably the<500 Da fractions of the rapeseed protein hydrolysates.Enzymatic hydrolysis and response surface methodology found good techniques in order to achieve hydrolysates with maximum ACE inhibitory activity.In this study,a positive relationship(r=0.76(p<0.05)was obtained between the DH and ACE inhibitory activity.It can be concluded that DH to some extent is important to release ACE inhibitory peptides during the hydrolysis process of rapeseed protein.Although the use of ultrasound in food processing is well known,some new and interesting applications for improving the technical performance and biological activity of food have emerged in the past few years.In chapter 4,the effects of multi-frequency ultrasound pretreatment with various frequency modes on rapeseed protein enzymolysis,ACE inhibitory activity,and its mechanism were studied.Following processing parameters,including substrate concentration,enzyme dosage,power density,pretreatment time and initial temperature on the degree of hydrolysis(DH)and ACE inhibitory activities of the hydrolysates,were evaluated using single-factorial experiments.The structural characteristics were analyzed using circular dichroism spectra(CD)and scanning electron microscopy(SEM).The results showed that during the screening of multi-frequency ultrasonic pretreatment,combined fixed frequency 24/28 kHz significantly(p<0.05)improved the DH and ACE inhibitory activity over the control.The experimental verification test results revealed that the processing factors that were studied had a significant effect on the DH and ACE inhibitory activity,which increased by 19.12%and 35.08%over the control,respectively.Moreover,the results of theeffect of combined fixed frequency ultrasonic pretreatment on enzymolysis time revealed that less time was needed to reach the maximum ACE inhibitory activity.In the present study,the highest ACE inhibitory activity(78.97%)was recorded as 78.97%at 75 min.CD spectral analysis showed that Combined fixed frequency(CFF)ultrasonic pretreatment significantly decreased in the a-helix content by 1.25%,while the ?-sheet content increased significantly by 25.94%,and there was a significant reduction in random coil content of 20.40%over the control.Furthermore,the findings molecular weight distribution showed that molecular weight of the?500 Da fraction of the CFF hydrolysate was significantly increased by 42.28%over the hydrolysate without the pretreatment.The molecular conformation of rapeseed protein indicated that sonication had destroyed the microstructure and increased surface area.In conclusion pretreatment with combined fixed frequency is an effective way because of its sonochemical effect on themolecular structure and improved the ACE inhibitory activity of rapeseed protein hydrolysates by altering thesecondary structure and the molecular weight distribution(MW).Ultrasound is a promising non-thermal and physical processing technology.Recently,ultrasound technology is appealing much interest in the field of food science and food bioprocessing.In chapter 5 our focus was to evaluate the effect of sequential dual-frequency ultrasound(SDFU)pretreatments on rapeseed protein enzymolysis,using alcalase as a model enzyme.Hydrolysed protein concentrations,enzymolysis kinetics,and thermodynamic parameters were investigated.Additionally,the structural changes induced by the SDFU were also studied with fluorescence emission and Fourier Transform infrared(FTIR)spectra.The structural analysis results illustrate that the hydrolysed rapeseed protein concentration following SDFU pretreatments was higher compared to that of the control for up to 75 min of enzymolysis at various substrate concentrations of 5-25 gL-1;both control and SDFU pretreatment groups showed first-order reaction kinetics.Compared to the control,the Michaelis-Menten constant(KM)value decreased remarkably by 17.61%,while an increase in the binding frequency between enzyme and substrate(KA)by 10.47%was observed.The thermodynamic parameters,enthalpy;entropy,and activation energy were reduced in the SDFU pretreatment group compared to the control by 31.78%18.0%and 29.56%,respectively.SDFU pretreatment showed little effect on Gibbs free energy at the various temperatures studied.Results obtained from the fluorescence emission and FTIR spectra provided evidence about the changes induced by sonochemical effects of the SDFU pretreatments,thus affecting the enzymolysis rate.Hydrolysates from plant protein sources with high biological activities can be used as substitutes for the complete protein and elemental formulation in functional food development.In chapter6 the focus was on the exploration of rapeseed protein pretreatment by high-energy power ultrasound before initiation of 2 h enzymatic hydrolysis with alcalase on the ACE inhibitory,functional properties and thermal stability of protein hydrolysates.The ultrasonic power ranged from 200 to 1200 Watt while the exposure time of pretreatment was kept in the range of 3 to 18 min.The effects of ultrasound pretreatments on the DH,inhibitory effect of ACE,amino acid composition,surface hydrophobicity,protein solubility were evaluated.Additionally,in this study,the thermal stability of ACE inhibitory activity in the resulting rapeseed protein hydrolysates obtained from ultrasound pretreatment followed by enzymatic hydrolysis was also examined.Ultrasonic pretreatments before enzymolysis in terms of power and sonication exposure time impacted significantly thus,increased in DH and ACE inhibitory activity over the control(without sonication).However,the maximum DH 22.07%and ACE inhibitory activity 72.13%was achieved at 600 W and 12 min of pretreatment.Rapeseed hydrolysate that had been subjected to ultrasonic pretreatment showed anotable increase in proline content by 2.47%while therelatively higher concentration of hydrophobic amino acids with an increased rate of 6.31%was observed when compared to the control.Effect of different sonication powers on the surface hydrophobicity,the results showed the surface hydrophobicity from 200 to 600 W significantly improved.However,the maximum impact on surface hydrophobicity was observed in the 600 W followed by the 800 W pretreated samples and their rate of increase compared to the control was recorded as 130.76%and 122.83%respectively.The solubility index after enzymolysis with and without sonication remarkably increased over native protein by 34.22%,and 63.81%respectively,while the increased rate between ultrasound pretreated and without pretreated hydrolysates was 22.05%.Additionally,the stability test showed that the ACE inhibitory activity remains stable against heat treatments.However,extensive heat,prolonged heating time and alkaline conditions were not in the favor of stability test,while under mild heat and acidic conditions their ACE inhibitory activities were not significantly different from unheated samples. |
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