蛋白酶对大豆蛋白和花生蛋白溶解性的影响

为了改善醇法大豆浓缩蛋白和花生粕的溶解性,利用Alcalase碱性蛋白酶和Validase FPconcentrate蛋白酶对它们进行限制性水解。结果表明,利用Alcalase碱性蛋白酶水解醇法大豆浓缩蛋白的最佳水解条件为:温度50℃、pH 8.0、水解时间3 h,底物浓度为10%,酶浓度为0.5%(占底物),在此水解条件下醇法大豆浓缩蛋白的氮溶解性指数(NSI)由4.37%提高到60.34%,水解度为6.37%。利用Validase FP concentrate蛋白酶对花生粕进行水解时的最佳水解条件为:温度50℃、pH 7.0、水解时间4 h,底物浓度10%,酶浓度1%(占底物),在此水解条件下花生粕的氮溶解性指数(NSI)由42.40%提高到87.54%,水解度为12.90%。     

双酶法制备大豆降胆固醇活性肽的研究

通过比较4种酶对大豆分离蛋白的水解效果,并通过单因素及L9(34)正交试验优化其水解工艺条件,研究其最佳水解工具酶及最佳酶解参数。结果表明:木瓜蛋白酶和植物蛋白酶联合应用可作为大豆分离蛋白的水解工具酶;其最佳酶解参数为:酶解温度55℃、初始pH7.0、底物浓度12%、酶添加量8%、植物蛋白酶与木瓜蛋白酶的质量之比为1∶2,水解度可达14.20%;用双蛋白酶水解大豆分离蛋白,水解度为14.71%的产物降胆固醇活性最高,对胆固醇胶束溶解度的抑制率为61.67%。     

双酶解法提取大豆蛋白的工艺研究

以豆粕粉为原料,加入高效蛋白酶和胰蛋白酶进行水解研究,对反应温度、底物浓度、反应时间、pH值、酶的用量及酶加入间隔的时间等工艺参数进行优化,并分析了大豆多肽的含量及蛋白质的水解度与各影响因素之间的关系.经过优化得到的反应条件为温度45℃,pH 7.5,液固比7: 1,加酶量为高效蛋白酶和胰蛋白酶各0.2 g,反应时间8 h,加酶间隔时间3 h.在此条件下,酶解后的蛋白质含量达85%、水解度为17.72%.     

微波辅助酶解花生粕同步提取多糖和抗氧化肽的工艺研究

本文以花生饼粕为原料通过微波辅助碱性蛋白酶(Alcalase)水解技术同步提取花生多糖和抗氧化肽,研究了提取反应时间、底物浓度、加酶量及pH值等因素对提取花生多糖和抗氧化肽抗氧化活性的影响。在单因素实验基础上,进行响应面分析实验。结果表明,花生多糖和抗氧化肽的最佳同步提取条件为:底物浓度12%,加酶量0.8mL,反应液pH值8.0,温度50℃。     

Alcalase2．4L碱性内切蛋白酶改性大豆蛋白的工艺研究

以大豆分离蛋白为研究对象,采用碱性内切蛋白酶Alcalase 2.4L作为水解大豆分离蛋白的酶制剂,探讨碱性内切蛋白酶Alcalase 2.4L对大豆分离蛋白的影响.本实验以水解度和蛋白质分散性指数为评价指标,确定了Alcalase 2.4L酶解大豆分离蛋白的最佳酶解反应条件:加酶量0.004 8AU/g、pH值为7.4、底物浓度11%、反应温度65℃、酶解时间50min.该条件下大豆分离蛋白的分散性得到显著改善,蛋白质分散指数PDI值达到92.8%.     

Protex7L中性蛋白酶改性大豆分离蛋白的工艺研究

利用中性蛋白酶Protex 7L对大豆分离蛋白进行改性研究,以水解度和蛋白质分散指数为评价指标,确定了中性蛋白酶Protex 7L改性大豆分离蛋白的最佳酶解反应条件:加酶量13.5AU/g大豆分离蛋白(SPI)、反应温度55℃、底物浓度为10%、pH值为7.0,酶解时间为1h.在上述条件下大豆分离蛋白的分散性得到显著改善,蛋白质分散指数(PDI值)达到91.8%.     

酶法制备大豆多肽的研究

本文研究了以Alcalase碱性蛋白酶和风味蛋白酶水解大豆蛋白制备大豆肽的工艺。确定了酶水解的最佳温度、底物浓度、酶与底物浓度比,得到了Alcalase碱性蛋白酶和风味蛋白酶水解大豆分离蛋白的最佳水解工艺。     

花生蛋白碱性蛋白酶水解物对血管紧张素转化酶抑制作用的初步研究

分别以碱性蛋白酶Alcalase和中性蛋白酶Neutrase对花生分离蛋白进行水解．制备花生分离蛋白水解物．并测定不同水解时间所得产物对血管紧张素转化酶(ACE)的抑制活性。未水解的花生分离蛋白没有ACE抑制活性．用中性蛋白酶Neutrase水解所得的水解物显示弱ACE抑制活性。然而，碱性蛋白酶Alcalasc水解物具有很强的ACE抑制活性．水解0．5h时水解物活性最高，其半抑制浓度为(IC50)0．56mg／mL。本研究表明，当用碱性蛋白酶Alcalase水解时，花生分离蛋白是生产ACE抑制肽的良好蛋白质来源，花生分离蛋白碱性蛋白酶Alcalase水解物可作为具有降压功能的功能食品添料。     

饲用大豆小肽生产工艺的研究

采用酶解法制备饲用大豆小肽,对反应温度、底物浓度、反应时间、pH值、酶用量等生产工艺参数及饲用大豆小肽的脱色、分离等精制工艺流程进行了系统的研究.结果表明:最适生产工艺参数为温度45～48℃,pH8.0,底物浓度6%～8%,酶用量2%(W/V),反应时间4 h.此条件下水解度高达80%左右,低聚肽(分子量≤500D)含量达到80%以上,以2～4个氨基酸链为主.此生产工艺具有成本低,工序简单,小肽含量高,质量好等优点.     

响应面法优化限制性酶解花生浓缩蛋白制备工艺研究

研究超声波辅助蛋白酶制备限制性酶解花生浓缩蛋白,为扩大花生浓缩蛋白的应用提供理论基础。以限制性酶解花生浓缩蛋白的溶解度和持水性为考察指标,研究了底物浓度、pH值、加酶量、超声波频率、超声波功率、温度和时间对溶解度和持水性的影响,确定最佳工艺条件为:底物浓度8.1%、pH值9.0、加酶量12.5μL/g、超声波频率45kHz、超声波功率150w、温度51℃和时间21min。此工艺下溶解度和持水性理论值分别为77.63%和9.77mL/g。验证试验得到的平均溶解度和持水性分别为78.89±0.53%和9.58±0.26mL/g,与理论值分别相差1.62%和1.94%,说明模型与实际情况拟合较好,验证了预测模型的正确性。限制性酶解花生浓缩蛋白具有较好的功能特性和清除自由基、还原力、金属离子螯合力、抑制脂质过氧化等4大类抗氧化活性。     

Angiotensin I Converting Enzyme Inhibitory Peptides Derived from Phycobiliproteins of Dulse Palmaria palmata

We examined the inhibitory activity of angiotensin I converting enzyme (ACE) in protein hydrolysates from dulse, Palmaria palmata. The proteins extracted from dulse were mainly composed of phycoerythrin (PE) followed by phycocyanin (PC) and allophycocyanin (APC). The dulse proteins showed slight ACE inhibitory activity, whereas the inhibitory activity was extremely enhanced by thermolysin hydrolysis. The ACE inhibitory activity of hydrolysates was hardly affected by additional pepsin, trypsin and chymotrypsin treatments. Nine ACE inhibitory peptides (YRD, AGGEY, VYRT, VDHY, IKGHY, LKNPG, LDY, LRY, FEQDWAS) were isolated from the hydrolysates by reversed-phase high-performance liquid chromatography (HPLC), and it was demonstrated that the synthetic peptide LRY (IC₅₀: 0.044 μmol) has remarkably high ACE inhibitory activity. Then, we investigated the structural properties of dulse phycobiliproteins to discuss the origin of dulse ACE inhibitory peptides. Each dulse phycobiliprotein possesses α-subunit (Mw: 17,477–17,638) and β-subunit (Mw: 17,455–18,407). The sequences of YRD, AGGEY, VYRT, VDHY, LKNPG and LDY were detected in the primary structure of PE α-subunit, and the LDY also exists in the APC α- and β-subunits. In addition, the LRY sequence was found in the β-subunits of PE, PC and APC. From these results, it was suggested that the dulse ACE inhibitory peptides were derived from phycobiliproteins, especially PE. To make sure the deduction, we carried out additional experiment by using recombinant PE. We expressed the recombinant α- and β-subunits of PE (rPEα and rPEβ, respectively), and then prepared their peptides by thermolysin hydrolysis. As a result, these peptides showed high ACE inhibitory activities (rPEα: 94.4%; rPEβ: 87.0%). Therefore, we concluded that the original proteins of dulse ACE inhibitory peptides were phycobiliproteins.     

Characterization of ACE Inhibitory Peptides Prepared from Pyropia pseudolinearis Protein

More than 7000 red algae species have been classified. Although most of them are underused, they are a protein-rich marine resource. The hydrolysates of red algal proteins are good candidates for the inhibition of the angiotensin-I-converting enzyme (ACE). The ACE is one of the key factors for cardiovascular disease, and the inhibition of ACE activity is related to the prevention of high blood pressure. To better understand the relationship between the hydrolysates of red algal proteins and the inhibition of ACE activity, we attempted to identify novel ACE inhibitory peptides from Pyropia pseudolinearis. We prepared water soluble proteins (WSP) containing phycoerythrin, phycocyanin, allophycocyanin, and ribulose 1,5-bisphosphate carboxylase/oxygenase. In vitro analysis showed that the thermolysin hydrolysate of the WSP had high ACE inhibitory activity compared to that of WSP. We then identified 42 peptides in the hydrolysate by high-performance liquid chromatography and mass spectrometry. Among 42 peptides, 23 peptides were found in chloroplast proteins. We then synthesized the uncharacterized peptides ARY, YLR, and LRM and measured the ACE inhibitory activity. LRM showed a low IC₅₀ value (0.15 μmol) compared to ARY and YLR (1.3 and 5.8 μmol). In silico analysis revealed that the LRM sequence was conserved in cpcA from Bangiales and Florideophyceae, indicating that the novel ACE inhibitory peptide LRM was highly conserved in red algae.     

醋制对黑豆蛋白含量及多肽ACE抑制活性的影响

为考察醋制对黑豆蛋白含量及多肽ACE抑制活性的影响,为对黑豆醋浸过程中蛋白组分及含量变化进行分析,并对相应ACE抑制肽活性进行测定。黑豆经醋浸不同时间后,采用顺序抽提法提取清蛋白、球蛋白、醇溶蛋白和谷蛋白,并采用凯氏定氮法及SDS-PAGE电泳进行定量和定性分析;各蛋白组分经胃蛋白酶水解,经超滤(截留分子量3 k D)后收集滤液,获得多肽组分,RP-HPLC法进行ACE抑制活性评价。结果表明:经醋浸14 d后,黑豆总蛋白含量变化幅度小于±0.5%;清蛋白含量由55.13%(0 d)降低至9.61%(14 d);球蛋白由7.04%(0 d)增加到20.34%(14 d);醇溶蛋白由0.81%(0 d)增加到1.72(14 d);谷蛋白由21.11%(0 d)增加到59.45%(14 d),含量最高。醋浸处理降低了清蛋白源多肽的ACE抑制活性,但提高了球蛋白、醇溶蛋白及谷蛋白多肽的ACE抑制活性,其中,谷蛋白多肽抑制率由18.18%(0 d)增加至37.58%(14 d),抑制活性升高。醋浸可改变黑豆各蛋白组分的含量和对应多肽的ACE抑制活性,其中,谷蛋白含量及其多肽ACE抑制活性均有增加。研究结果表明可进一步采用分离纯化技术从谷蛋白多肽中获得高活性降压肽。     

Identification of the Major ACE-Inhibitory Peptides Produced by Enzymatic Hydrolysis of a Protein Concentrate from Cuttlefish Wastewater

The aim of this work was the purification and identification of the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate recovered from a cuttlefish industrial manufacturing effluent. This process consisted on the ultrafiltration of cuttlefish softening wastewater, with a 10 kDa cut-off membrane, followed by the hydrolysis with alcalase of the retained fraction. Alcalase produced ACE inhibitors reaching the highest activity (IC₅₀ = 76.8 ± 15.2 μg mL⁻¹) after 8 h of proteolysis. Sequential ultrafiltration of the 8 h hydrolysate with molecular weight cut-off (MWCO) membranes of 10 and 1 kDa resulted in the increased activity of each permeate, with a final IC₅₀ value of 58.4 ± 4.6 μg mL⁻¹. Permeate containing peptides lower than 1 kDa was separated by reversed-phase high performance liquid chromatography (RP-HPLC). Four fractions (A–D) with potent ACE inhibitory activity were isolated and their main peptides identified using high performance liquid chromatography coupled to an electrospray ion trap Fourier transform ion cyclotron resonance-mass spectrometer (HPLC-ESI-IT-FTICR) followed by comparison with databases and de novo sequencing. The amino acid sequences of the identified peptides contained at least one hydrophobic and/or a proline together with positively charged residues in at least one of the three C-terminal positions. The IC₅₀ values of the fractions ranged from 1.92 to 8.83 μg mL⁻¹, however this study fails to identify which of these peptides are ultimately responsible for the potent antihypertensive activity of these fractions.     

Anti-hypertensive substances in fermented soybean,natto

Natto is a traditional Japenese fermented food made by fermenting boiled soy beans withBacillus natto. Its contents of inhibitors against the angiotensin converting enzyme (ACE, EC3.4.15.1) were investigated. Relatively strong inhibitory activity (IC₅₀:0.4 mg/ml, 11.8 inhibition units/g natto) was detected in natto extracts and the inhibitory activity observed in the viscous fraction was more potent than in the bean extract. Two groups of inhibitors in the viscous material, high and low molecular weight inhibitors, were resolved by dialysis test. The inhibitor of high molecular weight was a protein with low IC₅₀ value (0.12 mg/ml). The two types of low molecular weight inhibitors were detected in ethanol extracts (IC₅₀: 0.53 mg/ml and 0.95 mg/ml) and they were found to be stable over a wide range of pH and temperature up to 100°C. They were different in the mode of ACE inhibition. One is competitive, and the other noncompetitive against the hydrolysis of Bz-Gly-His-Leu by ACE.     

Characterization of ACE Inhibitory Peptides from Mactra veneriformis Hydrolysate by Nano-Liquid Chromatography Electrospray Ionization Mass Spectrometry (Nano-LC-ESI-MS) and Molecular Docking

Food-derived bioactive compounds are gaining increasing significance in life sciences. In the present study, we identified angiotensin I-converting enzyme (ACE)-inhibitory peptides from Mactra veneriformis hydrolysate using a nano-LC-MS/MS method. Mactra veneriformis hydrolysate was first separated into four fractions (F1–F4) based on molecular weight by ultrafiltration. The fraction with molecular weight lower than 1 kDa (F1) showed the highest ACE inhibitory activity. F1 was then analyzed by a high throughput nano-LC-MS/MS method and sequences of peptides in F1 were calculated accordingly. The 27 peptides identified as above were chemically synthesized and tested for ACE-inhibitory activity. The hexapeptide VVCVPW showed the highest potency with an IC₅₀ value of 4.07 μM. We then investigated the interaction mechanism between the six most potent peptides and ACE by molecular docking. Our docking results suggested that the ACE inhibitory peptides bind to ACE via interactions with His383, His387, and Glu411 residues. Particularly, similar to the thiol group of captopril, the cysteine thiol group of the most potent peptide VVCVPW may play a key role in the binding of this peptide to the ACE active site.     

Preparation and bioactivity evaluation of low salt peptide from sunf lower seed meal

Sunflower seed meal peptide as one sort of bioactive peptide has intensively application prospects. However, preparation of low salt peptide from sunflower seed meal with high efficiency remains a challenge. In this study, single and compound proteases were optimized to hydrolyze protein. Results showed that hydrolysis at pH 7.0 by proteases resulted in ash content in the range of 5.66%-7.37% and small peptides. Among all hydrolysis processes, sequential hydrolysis of Alcalase with Flavourzyme and Alcalase with Protamex showed higher nitrogen recovery ratio (67.66% and 66.49%, respectively). Furthermore, biological activities of peptides were investigated by testing their ABTS (2,2-azinobis (3-ethylben-zothiazoline-6-sulfonic acid) diammonium salt) radical scavenging activity, DPPH (2,2-diphenyl-1-picrylhydrazil) radical scavenging activity and angiotensin converting enzyme (ACE) inhibitory activity. Peptide hydrolyzed by Alcalase with Papain presented the highest antioxidant activity, followed by Alcalase with Protamex, with ABTS scavenging rate as 63.01% and 31.75%, and DPPH scavenging rate as 56.04% and 28.06%, respectively. Synchronously, peptide hydrolyzed by Alcalase with Protamex and Alcalase with Alcalase had the highest ACE inhibitory activity (56.74%, 56.76%). In conclusion, hydrolysis by proteases Alcalase with Protamex at pH 7.0 was the most effective method for the preparation of low salt peptide from sunflower seed meal, which could be an alternative for anti-oxidants and anti-vasoconstrictor.     

Characterization of Protein Hydrolysates from Fish Discards and By-Products from the North-West Spain Fishing Fleet as Potential Sources of Bioactive Peptides

Fish discards and by-products can be transformed into high value-added products such as fish protein hydrolysates (FPH) containing bioactive peptides. Protein hydrolysates were prepared from different parts (whole fish, skin and head) of several discarded species of the North-West Spain fishing fleet using Alcalase. All hydrolysates had moisture and ash contents lower than 10% and 15%, respectively. The fat content of FPH varied between 1.5% and 9.4% and had high protein content (69.8–76.6%). The amino acids profiles of FPH are quite similar and the most abundant amino acids were glutamic and aspartic acids. All FPH exhibited antioxidant activity and those obtained from Atlantic horse mackerel heads presented the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, reducing power and Cu²⁺ chelating activity. On the other hand, hydrolysates from gurnard heads showed the highest ABTS radical scavenging activity and Fe²⁺ chelating activity. In what concerns the α-amylase inhibitory activity, the IC₅₀ values recorded for FPH ranged between 5.70 and 84.37 mg/mL for blue whiting heads and whole Atlantic horse mackerel, respectively. α-Glucosidase inhibitory activity of FPH was relatively low but all FPH had high Angiotensin Converting Enzyme (ACE) inhibitory activity. Considering the biological activities, these FPH are potential natural additives for functional foods or nutraceuticals.