Antioxidant and nitric oxide inhibitory activities of tilapia (Oreochromis niloticus) protein hydrolysate: effect of ultrasonic pretreatment and ultrasonic‐assisted enzymatic hydrolysis

Ultrasound was incorporated to processing of fish protein hydrolysate to facilitate homogenate pretreatment and enzymatic hydrolysis of tilapia (Oreochromis niloticus) muscle protein. Their effects on Flavourzyme hydrolysis and biological activities of the tilapia hydrolysate were examined. The ultrasound‐assisted hydrolysis caused reduction in degree of hydrolysis ranging from 23% to 35% relative to that of the conventional process. The 70 W ultrasound‐assisted hydrolysis process increased DPPH radical‐scavenging activity and reducing power of tilapia hydrolysate prepared from the non‐pretreatment homogenate by 33% and 45%, respectively. All hydrolysates have no cytotoxicity on RAW264.7 cell lines at the maximum concentration of 20 mg protein mL^?1. The 70 W ultrasound pretreatment at 30 and 45 min combined with conventional hydrolysis is the suitable condition for producing tilapia hydrolysate with nitric oxide inhibitory and antioxidative activities on RAW264.7 cell lines, respectively. As a result, ultrasound could be applied to enzymatic protein hydrolysis either as pretreatment or during the hydrolysis.     

扫频超声处理对玉米醇溶蛋白酶解特性的影响

为了探索扫频超声技术促进蛋白质酶解反应的效果,提高酶解产物的血管紧张素转换酶(ACE)相对抑制活性,利用扫频超声处理玉米醇溶蛋白,进行单因素考察来寻找最佳扫频超声工作模式、超声预处理参数和最佳酶解条件。在最佳的超声预处理及最佳的酶解条件下,ACE抑制率为48.48%、水解度为11.20%,酶解产物的IC50值为3.77mg/mL,比常规酶解(无超声处理)降低了31.20%,产品得率为66.15%,比常规酶解提高了14.39%。这表明扫频超声预处理能够有效地促进玉米醇溶蛋白的酶解反应,提高产物的ACE相对抑制活性。      

Ace Inhibition and Enzymatic Resistance In Vitro of a Casein Hydrolysate Subjected to Plastein Reaction in the Presence of Extrinsic Proline and Ethanol- or Methanol-Water Fractionation

Casein was hydrolyzed by alcalase to a degree of hydrolysis of 10.9% to obtain a hydrolysate having ACE-inhibition in vitro with an IC₅₀ value of 52.6 μg/mL. The prepared hydrolysate was modified by alcalase-catalyzed plastein reaction with extrinsic proline added at 0.4 mol/mol free amino groups (on the basis of the hydrolysate), and fractionated by ethanol- or methanol-water solvents in proportions of 3:7, 5:5, or 7:3 (v/v), respectively. With the decrease of free amino groups of the modified hydrolysate as the response, the optimized plastein reaction conditions were alcalase addition of 3.1 kU/g peptides, substrate concentration of 50% (w/v), and reaction temperature of 25°C. Four modified hydrolysates prepared with different reaction times exhibited higher ACE-inhibitory activities than the original hydrolysate. The evaluation results showed that solvent fractionation of the modified hydrolysate with the maximum activity (IC₅₀ = 13.0 μg/mL) yielded the separated soluble fraction's higher activity but the precipitate fraction's lower one. Further enzymatic digestion of the modified hydrolysate with the maximum activity and its two fractionated products by four proteases in vitro caused damage to the activities, but the residual activities of the final digests were higher than that of the original hydrolysate, indicating that the plastein reaction could confer casein hydrolysate protease resistance.     

超声预处理时间对金枪鱼皮酶解过程ACE抑制肽释放的影响

以水解度、ACE抑制率为指标,并通过电泳、HPLC、红外光谱(FTIR)、热稳定性分析(DSC)研究不同超声预处理时间对金枪鱼皮酶解过程ACE抑制肽释放的影响。结果表明,酶解5 min后,超声预处理组酶解液的水解度与未处理组呈现显著性差异(p<0.05),并且ACE抑制率均高于未处理组,说明超声预处理可改变金枪鱼皮胶原酶解模式,加快短时酶解过程中ACE抑制肽的快速释放。电泳和HPLC分析表明,随着酶解过程的进行,10 kDa以下小分子组分含量呈现往复增减变化,超声预处理会加速该过程进行,从而提高其释放效率。FTIR和DSC分析可知,超声处理会破坏鱼皮胶原蛋白内部氢键的平衡,使其三螺旋结构松散,更多酶切位点暴露,说明超声处理能促进短时酶解过程ACE抑制肽的快速释放。     

超声波-离子液体耦合预处理制备蚕蛹蛋白质ACE抑制肽的工艺优化

为提高蚕蛹蛋白质酶解产物的ACE抑制活性,利用超声波-离子液体耦合法对蚕蛹蛋白质进行预处理。以酶解产物ACE抑制活性为指标,采用单因素结合响应面分析法,研究超声波-离子液体耦合预处理蚕蛹蛋白质的工艺条件,并通过SDS-聚丙烯酰胺凝胶电泳,研究预处理前后蚕蛹蛋白质及其酶解产物相对分子质量的变化。结果表明,各因素对酶解产物ACE抑制活性的影响程度由大到小依次为:液料比、超声波功率、预处理时间。确定最佳预处理工艺条件为:液料比27.2 mL/g,处理时间31.9 min,超声波功率406.8 W。在此优化条件下,蚕蛹蛋白质酶解产物的ACE抑制率为75.7%(IC50=0.071 mg/mL)。与未处理、超声波预处理的相比,超声波-离子液体耦合预处理在制备蚕蛹蛋白质ACE抑制肽上具有明显优势。超声波-离子液体耦合预处理后,蚕蛹蛋白质的分子质量无明显变化,但其酶解产物的分子质量(1.43 ku)变小。     

Effect of pretreatment on enzymatic hydrolysis of bovine collagen and formation of ACE-inhibitory peptides

Bovine collagen was pre-treated (boiled or high pressure (HP)-treated) and then hydrolysed by 6 proteases. The degree of hydrolysis (DH) and the angiotensin-converting enzyme (ACE)-inhibitory activity of hydrolysates were measured. All enzymes used were able to partly degrade collagen and release ACE-inhibitory peptides. The highest ACE-inhibitory activity was obtained with Alcalase. Pretreatment significantly influenced the DH and ACE-inhibition. For most enzymes, boiling for 5 min resulted in a significantly higher DH and ACE-inhibitory activity. With Alcalase and collagenase, hydrolysis and release of ACE-inhibitory peptides occurred without any pretreatment, but HP-treatment significantly improved the DH and ACE-inhibitory activity. HP did not markedly affect the hydrolysis with the other enzymes. The major peptides obtained with Alcalase were identified; all were released from the triple helix structure of collagen. Many of these peptides had C-terminal sequences similar to known ACE-inhibitory peptides. The present results suggest that collagen-rich food materials are good substrates for the release of potent ACE-inhibitory peptides, when proper pre-treatment and enzymatic treatment is applied.     

Investigation of the peptides with calcium chelating capacity in hydrolysate derived from spent hen meat

Calcium peptide chelates are developed as efficient supplements for preventing calcium deficiency. Spent hen meat (SHM) contains a high percentage of proteins but is generally wasted due to the disadvantages such as hard texture. We chose the underutilized SHM to produce peptides to bind calcium by proteolysis and aimed to investigate chelation between calcium and peptides in hydrolysate for a sustainable purpose. The optimized proteolysis conditions calculated from the result of response surface methodology for two-step hydrolysis were 0.30% (w_enzyme/w_meat) for papain with a hydrolysis time of 3.5 h and 0.18% (w_enzyme/w_meat) for flavourzyme with a hydrolysis time of 2.8 h. The enzymatic hydrolysate (EH) showed a binding capacity of 63.8 ± 1.8 mg calcium/g protein. Ethanol separation for EH improved the capacity up to a higher value of 68.6 ± 0.6 mg calcium/g protein with a high association constant of 420 M⁻¹ (25°C) indicating high stability. The separated fraction with a higher amount of Glu, Asp, Lys, and Arg had higher calcium-binding capacity, which was related to the number of ─COOH and ─NH₂ groups in peptide side chains according to the result from amino acid analysis and Fourier transform infrared spectroscopy. Two-step enzymatic hydrolysis and ethanol separation were an efficient combination to produce peptide mixtures derived from SHM with high calcium-binding capacity. The high percentage of hydrophilic amino acids in the separated fraction was concluded to increase calcium-binding capacity. This work provides foundations for increasing spent hen utilization and developing calcium peptide chelates based on underutilized meat.     

超声预处理辅助酶解玉米胚芽ACE抑制肽的研究

旨在研究不同工作模式的超声预处理对玉米胚芽蛋白酶解制备血管紧张素转换酶(Angiotensin-I Converting Enzyme,ACE)抑制肽的影响。以蛋白转化率和高活性肽占比为指标,利用聚能逆流双频、发散三频和对振双频的超声设备,对玉米胚芽粕进行预处理,得到最优的超声预处理模式;采用单因素逐级优化法来确定最佳超声预处理参数;在最优超声处理条件下,优化酶解反应条件。结果表明分子量在300~1000 Da的多肽ACE抑制活性最高,IC₅₀值为0.78 mg/mL;最优的超声模式为20/40 kHz交替双频,最佳超声预处理参数为功率密度100 W/L、底物浓度为8%、超声时间20 min、超声温度30 ℃,酶解条件为加酶量2000 U/g蛋白、酶解时间2.5 h。在最优条件下,蛋白转化率为85.00%,相比于未超声组的73.01%提高了16.42%;高活性肽占比为29.63%,相比于未超声组的26.00%提高了13.96%。因此,逆流双频超声波辅助酶解法能有效提高蛋白转化率和产物ACE抑制活性,有利于ACE抑制肽的制备。     

Effects of sweeping frequency ultrasound treatment on enzymatic preparations of ACE-inhibitory peptides from zein

In this study, the effect of sweeping frequency ultrasound (SFU) treatment on the degree of hydrolysis of zein and the angiotensin I-converting enzyme (ACE)-inhibitory activity of its hydrolysates were investigated. The mechanism through which ultrasonic pretreatment releases peptides with ACE-inhibitory activity was also studied by fluorescence, circular dichroism (CD), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Compared to the control, SFU and fixed frequency ultrasound (FFU) increased the degree of zein hydrolysis by approximately 11.5 %. Sweeping frequency ultrasound pretreatment increased ACE-inhibitory activity of zein hydrolysates by 12.3–116.7 % over the control. At 40 ± 2 kHz, SFU-treated zein hydrolysates had 42.9 % ACE-inhibitory activity, representing an increase of 116.7 % over the control. The fluorescence intensity of SFU- and FFU-treated zein was weaker than in untreated zein, indicating that more Phe, Trp, and Tyr residues were exposed outside the polypeptide chains. CD spectra show that SFU treatment resulted in increase in the α-helix content by 3.4 %, and β-sheet, β-turns, and random coils content by 24.4 %. Analyses of microstructure by SEM and AFM revealed that ultrasonic pretreatment ruptured the fine meshwork structure of zein resulting in the appearance of several micro-holes. We conclude that the SFU pretreatment for 40 ± 2 kHz can remarkably raise the degree of zein hydrolysis and ACE-inhibitory activity of the hydrolysates by altering the second structure of zein and rupturing the smooth surface of protein.     

超声波对麦胚蛋白性质及其酶解物ACE抑制活性的影响

为了提高麦胚分离蛋白酶解产物的降血压活性,采用超声波处理麦胚分离蛋白。研究了超声波功率对麦胚分离蛋白的溶解度、表面疏水性、荧光光谱、巯基含量变化、水解度和酶解产物对血管紧张素转换酶(ACE)相对抑制活性的影响。结果表明:经超声波处理后,小麦胚芽球蛋白的荧光光谱和巯基含量均发生了显著的变化。麦胚分离蛋白的溶解度和表面疏水残基含量随着超声波功率的增加而提高,但当超声功率达到800 W后,增幅趋于平缓。经超声波前处理后,ACE抑制活性明显提高,而麦胚分离蛋白的酶解产物水解度没有明显变化,因此ACE抑制活性的提高是由于蛋白结构的变化造成的。在超声功率800 W时,酶解产物的ACE相对抑制活性提高了41.09%。     

蚕蛹蛋白水解前处理和水解产物鲜味增效的研究

研究超声波、亚硫酸钠、酸等前处理对蛋白酶解效率的影响,并探讨谷氨酸钠和氯化钠对酶解产物的滋味的影响。实验表明,低浓度的亚硫酸钠处理能改善对蚕蛹蛋白的水解效果,而酸和超声波处理能显著提高蚕蛹蛋白回收率和水解度,其中超声处理效果最佳,使蚕蛹蛋白回收率和水解度分别提高5%和3%（p<0.05）。前处理都能提高酶解产物中小肽的比例,分子量小于1000 u的小肽占75%以上,1000~3000 u的组分占20%左右;其中超声前处理,分子量小于1000 u的小肽从占比66.44%提高到77.58%。通过单纯形重心设计,探讨在酶解液中氯化钠和谷氨酸钠的相互作用对滋味的影响。氯化钠和谷氨酸钠的浓度对超声前处理的酶解产物的鲜味有显著性的影响（p<0.05）,当味精和氯化钠的浓度分别是0.37%和0.85%时,鲜味增效达到最佳值,感官评分为7.3分。     

Impact of ultrasound on egg white proteins as a pretreatment for functional hydrolysates production

The objective of this study was to investigate the effects of different ultrasound pretreatment on enzymatic hydrolysis of egg white proteins (EWPs) by Alcalase as well as evaluating some functional and antioxidant properties of hydrolysates obtained by various proteases treatment and ultrasound technology. The effects of chosen ultrasound pretreatment parameters including frequency of ultrasonic waves (35 and 40 kHz), temperature (25 and 55 °C), time of pretreatment (15–60 min) and pH of egg white solution (7.00–10.00) were examined. It appeared that controlled ultrasound treatment can improved the hydrolysis process compared with untreated samples, but optimization of the power and length of sonication was important. The optimal ultrasound pretreatment at calorimetric power of 21.3 W and frequency of 40 kHz for 15 min at 25 °C and with naturally basic egg white (pH 9.25) resulted in increased initial rate and equilibrium degree of Alcalase hydrolysis by about 139.8 and 13.86 % compared with the control, respectively. EWP hydrolysates with ≈27.0 % degree of hydrolysis obtained with heat pretreatment and ultrasound pretreatments under optimal conditions were further separated by sequential ultrafiltration into 4 hydrolysate fractions (<1, 1–10, 10–30 and >30 kDa) which were investigated for protein content, peptide yield and antioxidant activity. The hydrolysis after heat pretreatment generated more peptides <1 kDa (19.04 ± 1.02 %) than ultrasound pretreatment did (11.90 ± 0.53 %), whereas the proportion of peptides <10 kDa were higher in the second case (28.80 ± 0.07 vs. 20.46 ± 0.39 %). The fraction obtained by the ultrasound pretreatment containing peptides with a molecular weight between 1 and 10 kDa demonstrated the strongest ABTS radical scavenging efficacy among the fractions (97.54 ± 0.30) with IC₅₀ value of 4.31 mg/mL. Compared with single-enzyme processes, the two-stage enzymatic processes did not significantly improve both antioxidant and functional hydrolysates’ properties.     

Angiotensin I-converting enzyme inhibitory activity in milk fermented by wild-type and peptidase-deletion derivatives of Lactobacillus helveticus CNRZ32

Development of molecular methods has enabled detailed studies of the proteolytic system of Lactobacillus helveticus, which has a central role in the release of bioactive peptides during the fermentation of milk. The impact of general aminopeptidase (PepN) and X-prolyl dipeptidyl aminopeptidase (PepX) activity of L. helveticus on the level of angiotensin I-converting enzyme (ACE) inhibitory activity in fermented milk was elucidated by taking advantage of peptidase-negative derivatives of L. helveticus CNRZ32. According to the results, increased level of ACE-inhibitory activity was attained in milk fermented by the peptidase-deficient mutants. The ACE-inhibitory activity determined with the PepN-deficient strain decreased towards the end of fermentation, suggesting that PepN gives rise to a provisional accumulation of ACE-inhibitory peptides before their hydrolysis to shorter peptides or free amino acids. The ACE-inhibitory activity determined with the PepX-deficient strain increased throughout the fermentation, indicating specific blocking of the further hydrolysis of ACE-inhibitory peptides.     

酪蛋白水解物的类蛋白反应修饰及其产物ACE抑制活性特征

采用碱性蛋白酶水解酪蛋白,制备水解度为10.9%、IC50值为52.6μg/mL的酪蛋白水解物,并利用响应面法优化碱性蛋白酶催化的类蛋白反应修饰条件。修饰反应时间固定为6h时,适宜的条件为酶添加量3.1kU/g pro、底物质量浓度50g/100mL、反应温度25℃。制备9个修饰程度不同的修饰产物,结果显示：修饰产物ACE抑制活性均提高,并且活性最高的修饰产物的IC50降低至14.9μg/mL。该修饰产物离心分级后,上清液部分和沉淀部分的ACE抑制活性分别低于和高于修饰产物,表明沉淀部分是提高ACE抑制活性的主要原因;Tricine-SDS-PAGE电泳分析表明,修饰产物及沉淀部分有较大分子质量的肽分子生成;该修饰产物和上清液部分、沉淀部分的进一步酶水解处理则显示,酶水解会导致它们的ACE抑制活性降低,但是仍然高于最初的酪蛋白水解物。     

Activity and bioavailability of food protein-derived angiotensin-I-converting enzyme–inhibitory peptides

Angiotensin-I-converting enzyme (ACE) inhibitory peptides are able to inhibit the activity of ACE, which is the key enzymatic factor mediating systemic hypertension. ACE-inhibitory peptides can be obtained from edible proteins and have the function of antihypertension. The amino acid sequences and the secondary structures of ACE-inhibitory peptides determine the inhibitory activities and stability. The resistance of ACE-inhibitory peptides to digestive enzymes and peptidase affect their antihypertensive bioactivity in vivo. In this paper, the mechanism of ACE-inhibition, sources of the inhibitory peptides, structure–activity relationships, stability during digestion, absorption and transportation of ACE-inhibitory peptides, and consumption of ACE-inhibitory peptides are reviewed, which provide guidance to the development of new functional foods and production of antihypertensive nutraceuticals and pharmaceuticals.     

ENZYMATIC HYDROLYSIS OF DEFATTED WHEAT GERM BY PROTEASES AND THE EFFECT ON THE FUNCTIONAL PROPERTIES OF RESULTING PROTEIN HYDROLYSATES

Endogenous enzymes of wheat germ were inactivated by heating for 15 min at 105C in an oven. The germ was defatted using supercritical carbon dioxide, smashed and solubilized by enzymatic hydrolysis. The enzyme hydrolysate was continuously stirred under conditions of optimum hydrolysis and centrifuged. Defatted wheat germ protein (DWGP) was obtained when the supernatant was spray‐dried. The enzymes were used to enhance the solubilization of protein so as to increase the yield of DWGP. Alcalase and flavourzyme® solubilized 85 and 80% of the protein, respectively, while papain, neutrase and protamex solubilized 73, 66 and 61%, respectively. The functional properties of DWGP solubilized using alcalase were as follows: nitrogen solubility, 74% at pH 6; emulsifying activity, capacity and stability were 64, 62 and 57%, respectively. Water retention of DWGP solubilized using alcalase was 232% at pH 7 and temperature of 70C. DWGP is a potential source of functional protein for possible food applications.     

Modification of Soybean Protein Hydrolysates by Alcalase-Catalyzed Plastein Reaction and the ACE-Inhibitory Activity of the Modified Product In Vitro

Soybean protein hydrolysates were prepared by hydrolyzing soybean protein isolates with a protease alcalase to a degree of hydrolysis of 16.6%, and then modified by alcalase-catalyzed plastein reaction to reveal the impact of plastein reaction on the ACE-inhibitory activity of the modified product in vitro. The suitable conditions of plastein reaction of soybean protein hydrolysates were selected based on the results of response surface methodology with the decreased amount of the free amino groups of the modified product as response. When reaction temperature was fixed at 30°C, the selected conditions were as follows: concentration of soybean protein hydrolysates of 45% (w/w), addition level of alcalase of 275 U/g peptides, and reaction time of 3 to 4 h. Soybean protein hydrolysates and eight modified products were evaluated for their ACE-inhibitory activities in vitro. The assay results highlighted that plastein reaction improved the ACE-inhibitory activity of the modified product. The IC₅₀ of the modified products ranged from 0.64 to 1.11 mg/mL, while that of soybean protein hydrolysates was 1.45 mg/mL. The decreased amount of the free amino groups of the modified product showed influence on the ACE-inhibitory activity in vitro. Analysis results from size exclusion chromatography confirmed that some plasteins with higher molecular weights were formed in the modified product. Our results showed that alcalase-catalyzed plastein reaction could be applied as a potential approach to enhance the ACE-inhibitory activity of soybean protein hydrolysates in vitro.     

Ultrasound Pretreatment as an Useful Tool to Enhance Egg White Protein Hydrolysis: Kinetics,Reaction Model,and Thermodinamics

The impact of ultrasound waves generated by probe‐type sonicator and ultrasound cleaning bath on egg white protein susceptibility to hydrolysis by alcalase compared to both thermal pretreatment and conventional enzymatic hydrolysis was quantitatively investigated. A series of hydrolytic reactions was carried out in a stirred tank reactor at different substrate concentrations, enzyme concentrations, and temperatures using untreated, and pretreated egg white proteins (EWPs). The kinetic model based on substrate inhibition and second‐order enzyme deactivation successfully predicts the experimental behavior providing an effective tool for comparison and optimization. The ultrasound pretreatments appear to greatly improve the enzymatic hydrolysis of EWPs under different conditions when compare to other methods. The apparent reaction rate constants for proteolysis (k₂) are 0.009, 0.011, 0.053, and 0.045 min^?1 for untreated EWPs, and those pretreated with heat, probe‐type sonicator, and ultrasound cleaning bath technologies, respectively. The ultrasound pretreatment also decreases hydrolysis activation (E_a) and enzyme deactivation (E_d) energy, enthalpy (ΔH), and entropy (ΔS) of activation and for the probe‐type sonication this decrease is 61.7%, 61.6%, 63.6%, and 32.2%, respectively, but ultrasound has little change in Gibbs free energy value in the temperature range of 318 to 338 K. The content of sulfhydryl groups and ζ potential show a significant increase (P < 0.05) for both applied ultrasound pretreatments and the reduction of particle size distribution are achieved, providing some evidence that the ultrasound causes EWP structural changes affecting the proteolysis rate.     

Angiotensin I-converting enzyme (ACE) inhibitory peptide derived from Tenebrio molitor (L.) larva protein hydrolysate

Short peptides from different sources have been proven to be very efficient inhibitors of angiotensin I-converting enzyme (ACE), an enzyme with a major role in the regulation of blood pressure. In order to obtain ACE-inhibitory peptides from Tenebrio molitor (L.) larva, enzymatic hydrolysis was performed using Alcalase. The hydrolysate (DH 20 %) with the highest ACE inhibition was fractionated into four peaks according to their relative molecular weight by gel filtration on Sephadex G-15. The IC₅₀ value of peak 2 (with the relative molecular weight 180–500 Da) was 0.23 mg/mL, while it was 0.39 mg/mL of hydrolysate before fractionation determined by reversed-phase high performance liquid chromatography (RP-HPLC). Antihypertensive activity of peak 2 was proven by single oral administration to spontaneously hypertensive rats (SHR). Multiple dose oral administration (100, 200, and 400 mg/kg body weight) to SHR led to a significant decrease in blood pressure for peak 2. The reduction of systolic blood pressure even reached 27 mm Hg at 4 h post-administration at a dose of 400 mg/kg body weight. Additionally, further separation and purification of the ACE-inhibitory peptides from peak 2 were performed using RP-HPLC on C₁₈ column. Then a novel peptide Tyr–Ala–Asn was identified by tandem mass spectrometry with an IC₅₀ value of 0.017 mg/mL. The result of research indicates that Tenebrio molitor (L.) larva protein may be a suitable candidate to prepare ACE-inhibitory peptides which could be used for food industry and nutraceuticals.