Grass carp peptides hydrolysed by the combination of Alcalase and Neutrase: Angiotensin‐I converting enzyme (ACE) inhibitory activity,antioxidant activities and physicochemical profiles

In this study, grass carp peptides were prepared by enzymatic hydrolysis of grass carp protein using the combination of Alcalase and Neutrase, and angiotensin‐I converting enzyme (ACE) inhibitory activity in vitro, antihypertensive activity in vivo, antioxidant activities, and physicochemical properties of peptides achieved from grass carp protein were characterised after ultrafiltration and desalted processes using mixed ion exchange resins. The purified peptides exhibited strong ACE inhibitory activity (IC₅₀ = 105 μg mL^?1), antihypertensive activity with the maximal drop for systolic blood pressure (SBP) of 43 mmHg at a dosage of 100 mg per kg body weight in spontaneously hypertensive rat (SHR), and antioxidant activities indicated by thiobarbituric acid‐reactive substance values in a liposome‐oxidising system, radical‐scavenging activity and chelation of metal ions (Fe²⁺). The molecular weight of peptides was <1000 Da. Compared to grass carp protein, the peptides separated from enzymatic hydrolysates possessed similar amino acid compositions, but contained higher concentrations of essential amino acids. Moreover, the peptides exhibited excellent solubility at a wide range of pH values from 2 to 10, and lower apparent viscosity than the protein. The peptides separated from enzymatic hydrolysates might be used as a promising ingredient in antihypertensive functional foods and nutraceuticals.     

花生蛋白水解产物ACE抑制活性的研究

采用胰蛋白酶、碱性蛋白酶、中性蛋白酶水解花生蛋白,研究了水解过程中水解度的变化,并对水解产物的ACE抑制活性进行了探讨。得出三种酶对花生蛋白的水解作用:碱性蛋白酶>胰蛋白酶>中性蛋白酶。碱性蛋白酶水解产物ACE抑制活性明显高于胰蛋白酶和中性蛋白酶,水解产物的ACE抑制活性高达89.73%,中性蛋白酶水解产物ACE抑制率仅为27.24%。     

花生分离蛋白碱性蛋白酶Alcalase水解物具有血管紧张素转化酶抑制活性

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Alcalase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。     

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.     

Recovery of protein hydrolysates from brewer’s spent grain using enzyme and ultrasonication

The aim of this study was to develop a green ultrasound-assisted enzymatic process to separate protein from brewer’s spent grain (BSG) to produce protein hydrolysates and determine the physicochemical properties of produced protein hydrolysates. When the enzyme (Alcalase) loading increased from 1 to 20 μL g⁻¹ BSG, the protein separation efficiency increased from 34.0% to 61.6%. The application of ultrasound pretreatment further increased protein separation efficiency to 69.8%. More promisingly, the ultrasound pretreatment was able to reduce enzyme loading by 73% and decrease enzyme incubation time by 56%. The produced protein hydrolysates had molecular weights lower than 15 kDa and high protein solubilities at the pH of 1.0–11.0. The ultrasound pretreatment improved the protein solubility to above 90%. Glutamic acid and proline were the most abundant amino acids in produced protein hydrolysates. This study demonstrated that enzymatic hydrolysis along with ultrasound pretreatment is an effective way to separate protein from BSG.     

Enzymatic hydrolysis of hard‐to‐cook bean (Phaseolus vulgaris L.) protein concentrates and its effects on biological and functional properties

Inadequate postharvest handling and storage under high temperature and relative humidity conditions produce the hard‐to‐cook (HTC) defect in beans. However, these can be raw material to produce hydrolysates with functional activities. Angiotensin I‐converting enzyme (ACE) inhibitory and antioxidant capacities were determined for extensively hydrolysed proteins of HTC bean produced with sequential systems Alcalase‐Flavourzyme (AF) and pepsin–pancreatin (Pep‐Pan) at 90 min ACE inhibition expressed as IC₅₀ values were 4.5 and 6.5 mg protein per mL with AF and Pep‐Pan, respectively. Antioxidant activity as Trolox equivalent antioxidant capacity (TEAC) was 8.1 mm  mg^?1 sample with AF and 6.4 mm  mg^?1 sample with Pep‐Pan. The peptides released from the protein during hydrolysis were responsible for the observed ACE inhibition and antioxidant activities. Nitrogen solubility, emulsifying capacity, emulsion stability, foaming capacity and foam stability were measured for limited hydrolysis produced with Flavourzyme and pancreatin at 15 min. The hydrolysates exhibited better functional properties than the protein concentrate.     

基于超声预处理的脱脂玉米胚芽ACE抑制肽的制备

为了研究超声辅助酶解制备血管紧张素转化酶(ACE)抑制肽的较优工艺,通过三种超声设备对脱脂玉米胚芽预处理,碱性蛋白酶酶解,酶解液体外模拟胃肠消化,以消化液ACE抑制率和酶解过程中玉米胚芽水解度(DH)为指标对超声预处理和酶解的参数进行单因素逐级优化。实验结果表明,最佳超声工作模式为20~40 kHz聚能式逆流双频交替超声模式;超声工作参数为功率密度120 W/L,超声预处理时间15 min,初始温度30℃,物料浓度5%;酶解条件为加酶量3000 U/g,酶解时间30 min,pH9.0,酶解温度50℃。在此条件下,酶解液的IC₅₀为4.166 mg/mL,比对照组降低了5.08%;胃肠消化液的IC₅₀为3.986 mg/mL,比对照降低了4.44%。制备的酶解产物,经模拟胃肠消化后具有较强的ACE抑制活性。优化获得的制备脱脂玉米胚芽ACE抑制肽的工艺是可行的。     

Physicochemical and bitterness properties of enzymatic pea protein hydrolysates

ABSTRACT:  The effects of different proteolytic treatments on the physiochemical and bitterness properties of pea protein hydrolysates were investigated. A commercial pea protein isolate was digested using each of 5 different proteases to produce protein hydrolysates with varying properties. After 4 h of enzyme digestion, samples were clarified by centrifugation followed by desalting of the supernatant with a 1000 Da membrane; the retentates were then freeze-dried. Alcalase and Flavourzyme™ produced protein hydrolysates with significantly higher ( P < 0.05) degree of hydrolysis when compared to the other proteases. Flavourzyme, papain, and alcalase produced hydrolysates that contained the highest levels of aromatic amino acids, while trypsin hydrolysate had the highest levels of lysine and arginine. Papain hydrolysate contained high molecular weight peptides (10 to 178 kDa) while hydrolysates from the other 4 proteases contained predominantly low molecular weight peptides (≤ 23 kDa). DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging activity of the Flavourzyme hydrolysate was significantly ( P < 0.05) the highest while alcalase and trypsin hydrolysates were the lowest. Inhibition of angiotensin converting enzyme (ACE) activity was significantly higher ( P < 0.05) for papain hydrolysate while Flavourzyme hydrolysate had the least inhibitory activity. Sensory analysis showed that the alcalase hydrolysate was the most bitter while papain and α-chymotrypsin hydrolysates were the least. Among the 5 enzymes used in this study, papain and α-chymotrypsin appear to be the most desirable for producing high quality pea protein hydrolysates because of the low bitterness scores combined with a high level of angiotensin converting enzyme inhibition and moderate free radical scavenging activity.     

High‐pressure microfluidisation pretreatment disaggregate peanut protein isolates to prepare antihypertensive peptide fractions

High‐pressure microfluidisation (HPM) pretreatment was applied to increase in vitro antihypertensive activity of peanut peptide fractions (PPF). The morphology of protein in aqueous dispersion revealed that peanut protein isolate (PPI) disaggregated at relatively low pressure (≤120 MPa) and re‐aggregated at relatively high pressures (150–210 MPa). The treated pressure of 120 MPa could lead to the most disaggregation of PPI. Small peptides contents, trichloroacetic acid‐nitrogen soluble index (TCA‐NSI) and degree of hydrolysis (DH) of peanut protein hydrolysates (PPH) all reached the highest at 120 MPa. Consequently, it possessed the highest angiotensin converting enzyme (ACE) and renin inhibitory activity. The highest surface hydrophobicity occurred at 120 MPa pretreatment samples. Thirty‐nine oligopeptides at 120 MPa pretreatment were identified by ultra‐performance liquid chromatography‐quadrupole time‐of‐flight (UPLC‐Q‐TOF) mass spectrometer combined with Progenesis QI for Proteomics software compared with 29 and 35 at control and 210 MPa, respectively. This meant that disaggregation of PPI at 120 MPa resulted in the release of new hydrophobic peptide.     

Obtaining of Brassica carinata protein hydrolysates enriched in bioactive peptides using immobilized digestive proteases

Brassica carinata protein hydrolysates were obtained by sequential hydrolysis with immobilized trypsin, chymotrypsin, and carboxypeptidase A on glioxyl-agarose supports. The final protein hydrolysate with a 36% degree of hydrolysis was made up of peptides smaller than 15 kDa. Three peptide fractions were obtained after gel filtration chromatography and antioxidative, hypocholesterolemic, and inhibitory of angiotensin converting enzyme activities were assayed in comparison with the starting materials (protein isolate and hydrolysate). Total protein hydrolysate achieved the best results in the reduction of micellar cholesterol and fraction II, composed by peptides between 1800 and 1400 Da, showed the best antioxidant and inhibitory of angiotensin converting enzyme activities. These results show that B. carinata seed proteins may represent an useful source of bioactive peptides after hydrolysis with digestive proteases such as trypsin, chymotrypsin, and carboxypeptidase A.     

Limited hydrolysis of β‐casein by cell wall proteinase and its effect on hydrolysates's conformational and structural properties

Optimisation of enzymatic hydrolysis of β‐casein with cell envelope proteinase (CEP) from Lactobacillus acidophilus JQ‐1 to produce the angiotensin‐I‐converting enzyme (ACE) inhibitory peptides using response surface methodology (RSM). Under optimal conditions (enzyme‐to‐substrate ([E]/[S]) ratio (w/w) of 0.132 and pH of 8.00 at 38.8 °C), the ACE inhibitory activity of hydrolysates was 72.06% and the total peptides was 11.75 mg mL^?1. Scanning electron microscopy (SEM) micrographs indicated that the tightness of the β‐casein surface structure was gradually weakened and small holes appeared after enzymatic treatment, while Fourier transform infrared spectroscopy (FTIR) spectra indicated remarkable changes in the chemical composition and macromolecular conformation of β‐casein after enzymatic hydrolysis. Differential scanning calorimetry (DSC) analysis indicated that the corresponding hydrolysates had higher thermal stability. The enzymatic hydrolysis also led to an increase in the free sulfhydryl content of β‐casein hydrolysates compared with raw β‐casein, which led to the increase in the antioxidant activity of β‐casein hydrolysates.     

Angiotensin I‐converting enzyme inhibitory activity of protein hydrolysates prepared from three freshwater carps (Catla catla,Labeo rohita and Cirrhinus mrigala) using Flavorzyme

Fish protein hydrolysates from three freshwater carps, Catla catla, Labeo rohita and Cirrhinus mrigala with different degree of hydrolysis (DH) (5%, 10%, 15% and 20%), were prepared using Flavorzyme enzyme and designated as HCF, HRF and HMF, respectively. The angiotensin I‐converting enzyme (ACE) inhibitory activity of hydrolysates was found to vary from 43 ± 2% to 71 ± 3%. Based on ACE inhibitory activity, HRF with DH‐15% was taken up for further study. The mode of ACE activity inhibition by HRF‐DH 15% was mixed type as revealed by Lineweaver–Burk plot. Sequential digestion of HRF‐DH 15% using pepsin and pancreatin decreased the ACE inhibitory activity from 76% to 63%. Partial purification of HRF‐DH 15% by size exclusion chromatography gave three different fractions designated as F‐1, F‐2 and F‐3 with the molecular mass in the range of 6456–407 Da. Fraction 2 had significantly higher ACE inhibitory activity than the other fractions.     

Effect of ultrasonic pretreatment on the antioxidant properties of porcine liver protein hydrolysates

In this study, the effect of ultrasonic pretreatment on the antioxidant activity of porcine liver protein hydrolysates (PLPHs) was investigated. The results showed that the degree of hydrolysis (DH) and peptide contents of the PLPHs increased as the time of ultrasonication increased. The hydrolysate pretreated with ultrasonication for 60 s exhibited the highest DH and peptide contents. The hydrolysate pretreated with ultrasonication for 45 s exhibited the highest ferrous ion chelating ability and reducing power. The hydrolysate pretreated with ultrasonication for 30 s exhibited the highest 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging activity and the higher inhibitory activity in the linoleic acid autoxidation system. The molecular weight of peptides in the hydrolysates was less than 6.2 kDa. The results clearly demonstrated that ultrasonic pretreatment enhances the antioxidant activities of the PLPHs in a short period of time (15–30 s).     

2种蛋白酶的大豆蛋白水解物对血管紧张素转换酶的抑制作用

报道了 2种工业化酶ProteaseA和OrientaseB的大豆蛋白水解物的高活性ACEI肽的筛选工作 ,并且对所得到的活性较高的水解物进行了感官评价。这 2种酶的水解物的ACE抑制活性均随着水解时间的增加呈指数归律增加。其中OrientaseB水解大豆蛋白 1 0h ,其产物的ACE抑制活性最高达到 95 %,ProteaseA的最高也可达到 91 33%。ProteaseA水解 4～ 1 0h的产物以及Ori entaseB水解 6～ 1 0h的产物 ,是由 2～ 5个氨基酸组成的寡肽 ,ACE的抑制活性高 ,且苦味轻微或没有。这些肽因其天然、安全、作用温和而有望作为良好的降压成分进入人们的日常膳食。     

Separation of angiotensin I‐converting enzyme inhibitory peptides from bovine connective tissue and their stability towards temperature,pH and digestive enzymes

Bovine collagen was isolated from connective tissue, a by‐product in the meat processing industry and characterised by SDS‐PAGE. Alcalase and papain were employed to generate collagen hydrolysates with different degree of hydrolysis (DH). In vitro angiotensin I‐converting enzyme (ACE) inhibitory activities were evaluated and the two most potent hydrolysates from each enzyme were separated by two‐step purification. Both alcalase‐catalysed and papain‐catalysed hydrolysates exhibited strong ACE inhibitory capacities with IC₅₀ values of 0.17 and 0.35 mg mL^?1, respectively. Purification by ion‐exchange chromatography and gel filtration chromatography revealed higher ACE inhibitory activities in one fraction from each enzyme with IC₅₀ values of 3.95 and 7.29 μg mL^?1. These peptide fractions were characterised as 6‐12 amino acid residues by MALDI‐TOF/MS. The peptides retained their activity (>90%) after exposure to processing temperature and pH and in vitro simulated gastrointestinal digestion. The present results demonstrated that collagen peptides can be utilised for developing high value‐added ingredients, for example ACE inhibitory peptides.     

Bioactive properties of enzymatic hydrolysates from abdominal skin gelatin of yellowfin tuna (Thunnus albacares)

Gelatin (90.6 ± 0.1%) was optimally prepared by response surface methodology from yellowfin tuna (Thunnus albacares, YT) abdominal skin. To investigate bioactive properties of enzymatic hydrolysates from the abdominal skin gelatin (ASG), ASG was hydrolysed with alcalase, protamex, neutrase and flavourzyme as affected by hydrolysis time. Antioxidant, nitrite scavenging and angiotensin‐I converting enzyme (ACE) inhibitory activities of the hydrolysates were determined. Antioxidant activities of the hydrolysates were found through linoleic acid peroxidation inhibitory effects. Alcalase‐derived hydrolysates (AHs) were more effective than others in metal ions chelating, superoxide anion scavenging and hydroxyl radical scavenging activities (P < 0.05). AHs showed significantly stronger nitrite scavenging activities (44.4–60.7%) than others (P < 0.05). Fraction A from AH showed strong ACE inhibitory activity (IC₅₀ of 0.75 mg mL^?1). These results suggest that YT ASG and its enzymatic hydrolysates could be functional food and/or pharmaceutical ingredients with potent antioxidant, anticarcinogenic and antihypertensive benefits.     

Isolation,purification and characterisation of angiotensin I‐converting enzyme–inhibitory peptides derived from catfish (Clarias batrachus) muscle protein thermolysin hydrolysates

The angiotensin I‐converting enzyme (ACE)‐inhibitory activities of catfish (Clarias batrachus) muscle protein hydrolysates were investigated. Thermolytic digests of C. batrachus sarcoplasmic and myofibrillar proteins exhibited inhibitory activity towards ACE and were purified with the aim of ultrafiltration, gel filtration and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). The amino acid sequences of hydrolysates with the highest ACE‐inhibitory activities were determined using electrospray quadrupole time‐of‐flight tandem mass spectrometry (ESI‐TOFQ MS/MS). The sequences of GPPP (IC₅₀ = 0.86 μm ) and IEKPP (IC₅₀ = 1.2 μm ) corresponding to the fragments 986–989 and 441–445 of myosin‐I heavy chain were identified for the sarcoplasmic and myofibrillar protein hydrolysates, respectively. Peptide GPPP exhibited a mixed‐type inhibition whereas peptide IEKPP could only bind to the active sites of ACE. The results demonstrate that hydrolysates of C. batrachus muscle proteins obtained by thermolysin may contain bioactive peptides.     

Purification and characterisation of antioxidant and nitric oxide inhibitory peptides from Tilapia (Oreochromis niloticus) protein hydrolysate

Nitric oxide (NO)‐inhibitory and antioxidative activities of tilapia hydrolysates were prepared using ultrasound pretreatment at 70 W for 30 and 45 min, respectively, followed by Flavourzyme hydrolysis for 1 h. Both hydrolysates were fractionated using size exclusion chromatography on Sephadex G‐25 column and purified by RP‐HPLC. The amino acid sequence of the most potent and purified fractions was determined using LC/MS/MS. The antioxidant peptide (KAFAVIDQDKSGFIEEDELKLFLQNFSAGARAGDSDGDGKIGVDEFAALVK, MW: 6334.49 KDa) and NO‐inhibitory peptide (AFAVIDQDKSGFIEEDELKLFLQNFSAGARAGDSDGDGKIGVDEFAALVK, MW: 6309.49 Da) produced no cytotoxicity in RAW264.7 macrophage cell lines at the concentration of 100 mg mL^?1. The purified peptides at the concentration 100 μg mL^?1 possessed antioxidative and NO‐inhibitory activities 83.0 ± 1.1% and 40.9 ± 0.2%, respectively, which were about 100 times those of their counterpart crude hydrolysates.     

Influence of degree of hydrolysis on functional properties and angiotensin I‐converting enzyme‐inhibitory activity of protein hydrolysates from cuttlefish (Sepia officinalis) by‐products

BACKGROUND: In Tunisia the cuttlefish‐processing industry generates large amounts of solid wastes. These wastes, which may represent 35% of the original material and constitute an important source of proteins, are discarded without any attempt at recovery. This paper describes some functional properties and the angiotensin I‐converting enzyme (ACE)‐inhibitory activity of protein hydrolysates prepared by hydrolysis of cuttlefish (Sepia officinalis) by‐products with crude enzyme extract from Bacillus licheniformis NH1. RESULTS: Cuttlefish by‐product protein hydrolysates (CPHs) with different degrees of hydrolysis (DH 5, 10 and 13.5%) were prepared. All CPHs contained 750–790 g kg^?1 proteins. Solubility, emulsifying capacity and water‐holding capacity increased while fat absorption and foaming capacity decreased with increasing DH. All hydrolysates showed greater fat absorption than the water‐soluble fraction from undigested cuttlefish by‐product proteins and casein. CPHs were also analysed for their ACE‐inhibitory activity. CPH3 (DH 13.5%) displayed the highest ACE inhibition (79%), with an IC₅₀ value of 1 mg mL^?1. CONCLUSION: Hydrolysis of cuttlefish by‐product proteins with alkaline proteases from B. licheniformis resulted in a product with excellent solubility over a wide pH range and high ACE‐inhibitory activity. This study suggests that CPHs could be utilised to develop functional foods for prevention of hypertension. Copyright © 2010 Society of Chemical Industry     

Potential of innovative pre-treatment technologies for the revalorisation of residual materials from the chicken industry through enzymatic hydrolysis

The effect of microwave, ultrasound and high-pressure pre-treatment prior to enzymatic hydrolysis of mechanically deboned chicken residuals was evaluated. Increased protein extraction yield was observed for high-pressure pre-treatment at ≥400 MPa. Enhanced antioxidant activity was found in hydrolysates pre-treated with high-pressure (200 MPa) and ultrasound (900 W). Hydrolysates with higher protein solubility over pH were found after pre-treatments at 200 and 600 MPa, and 1 and 10 min microwave at 40 °C, with the most efficient option being the shortest microwave treatment. Furthermore, pre-treatments with ultrasound at 600 W, microwave for 1 min at 40 °C, and high-pressure at 200 MPa seem to have the potential to induce formation of peptides with higher lipid associating properties.Industrial relevanceThe increase in meat consumption resulting from the rise in human population, has led to increased demand for relevant nutritional (protein) sources together with the need for proper utilisation of residual materials and reduction of food waste footprint. Thus, assessment and validation of novel technologies that allow unexploited materials, earlier regarded as waste, to be transformed into high-value products is of great interest for the industry within the current global framework. Enhanced extraction yield and bio- and techno-functionality will lead to high-value markets, while minimising environmental footprint. This article focuses on the potential of microwave, ultrasound and high-pressure technologies as pre-treatment strategies prior to enzymatic hydrolysis of mechanically deboned chicken residuals towards production of high-value peptides. The results show that the applied technologies are able to enhance the properties of the resulting peptides in terms of overall protein extraction and bio- and techno-functional properties (such as protein solubility over pH, antioxidant activity). Microwave pre-treatment is shown as a rapid method for increased protein solubility over pH; ultrasound pre-treatment increases the antioxidant activity of the peptides in a power-dependent manner; high-pressure pre-treatment has the potential to increase protein solubility, antioxidant properties and protein extraction, depending on the applied pressure. To achieve certain bio- or techno-functional peptide properties, choosing specific technologies and operational conditions is needed. Thus, relevant industry sectors will benefit from these results in light of their strategy towards valorisation of residual materials similar to mechanically deboned chicken, through enzymatic hydrolysis.