由碱性蛋白酶制备的乳清蛋白水解物抗氧化活性的研究

通过碱性蛋白酶水解乳清蛋白以研究乳清蛋白水解物的抗氧化活性。通过测定水解物对卵磷脂脂质氧化体系的氧化抑制作用、亚铁还原能力、DPPH自由基清除能力,研究乳清多肽抗氧化活性的大小。结果表明,乳清多肽的抗氧化能力与底物浓度、加酶量、水解时间、pH值、温度等有关,水解的最佳工艺条件是底物质量浓度50g／L,pH值8．5,加酶量（E／S）2％,温度65℃,水解时间为5h。研究表明,碱性蛋白酶制备的乳清蛋白水解物在卵磷脂脂肪氧化体系中可以降低硫代巴比妥酸值（TBARs）,且具有较好的还原能力和清除自由基的能力,因而具有较好的抗氧化活性。     

Antioxidant activities of squid protein hydrolysates prepared with papain using response surface methodology

Squid protein hydrolysates (SPH) were prepared from the Indian squid Loligo duvauceli using papain. Response surface methodology (RSM) was used for optimization of hydrolysis conditions, including temperature, time, and the enzyme-substrate ratio using DPPH radical scavenging activity as a response. The amino acid composition of SPH was compared with raw squid muscle. In vitro antioxidant activities were evaluated based on reducing power, metal chelation, ABTS, hydroxyl radical, and superoxide anion radical scavenging assays. SPH exhibited good ABTS radical scavenging activities of 96.50±0.90%, superoxide anion radical scavenging activities of 96.4±0.89%, reducing powers of 0.71±0.02, moderate hydroxyl radical scavenging activities of 64.03±2.11%, and metal chelating activities of 52.04±1.02%. In vivo antioxidant activities determined using a sardine minced model system showed 42% reduction in formation of secondary oxidative products as thiobarbituric acid reactive substances (TBARS), almost equivalent to reduction by ascorbic acid of 41.42% at 400 ppm.     

乳清分离蛋白酶解物的分离条件

采用试管法研究了SP Sephadex C-25对乳源抗氧化肽的分离条件,确定SP Scphadex C-25色谱分离条件为:上样量3mg/mL IE;起始缓冲液:20mmol/L的醋酸缓冲液(pH4.0);NaCI浓度为Imol/L.     

Solubility,functional properties,ACE‐I inhibitory and DPPH scavenging activities of Alcalase hydrolysed soy protein hydrolysates

Soy proteins are less soluble at acidic pH value, which impedes their utilisation in acidic beverages. Soy protein isolate (SPI) was hydrolysed using varying Alcalase concentrations (0.0001–2.0 U g^?1 protein) at different pHs (3.0–4.0). Degree of hydrolysis (DH) of soy protein hydrolysates (SPH) at pH 3.0, 3.5 and 4.0 were 5.0–10.7%, 2.3–6.1% and 0–5.4%, respectively, while solubilities ranged from 70.7 to 74.9%, 18.8 to 51.2% and 7.1 to 40.4%, respectively. The highest solubility (74.9%) was observed at pH 3.0 with 1.5 U Alcalase per g protein (DH = 9.2%). Emulsifying activities of SPHs at pH 3.0 and 4.0 ranged from 0.49 to 0.63 AU and 0.19 to 0.24 AU, respectively, while the emulsifying stabilities were 12.2–14.7 min and 18.7–56.0 min, respectively. The foaming capacity at pH 3.0 and 4.0 was 44.9–46.3 mL and 31.2–41.3 mL, respectively, whereas the foaming stability was 25.5–35.2 min and 12.8–15.1 min, respectively. However, hydrolysates had an insignificant effect on ACE‐I inhibitory and DPPH scavenging activities in comparison with SPI.     

Fractionation and identification of a novel hypocholesterolemic peptide derived from soy protein Alcalase hydrolysates

A novel hypocholesterolemic peptide was fractionated by gradient ethanol elution from a macroporous adsorption resin (MAR DA201-C), and then separated on Sephadex G-15 and RP-HPLC from a soy protein hydrolysate (SAPH DH 18%). Identification of the hypocholesterolemic peptide structure was accomplished with HPLC–MS. The peptide with the highest hypocholesterolemic activity was found in 75% ethanol fraction among the four fractions from gradient ethanol elution with MAR DA201-C. The calculated average hydrophobicity by amino acid composition of each ethanol eluted fraction suggested that the peptides could be separated in terms of hydrophobicity with MAR DA201-C. Four peaks were obtained with further fractionation on Sephadex G-15, the highest cholesterol micellar solubility inhibition rate, 81.3%, was obtained in Peak 2, corresponding to the molecular weight fraction of 300–800 Da. Fifteen main peaks were obtained with RP-HPLC fractionation, the highest cholesterol micellar solubility inhibition rate (94.3%) was in Peak 7. The amino acid sequence of this peptide was identified as WGAPSL with LC–MS and amino acid composition analysis.     

Antioxidant and free radical-scavenging activities of smooth hound (Mustelus mustelus) muscle protein hydrolysates obtained by gastrointestinal proteases

We have investigated the antioxidative activity of five hydrolysates from smooth hound (Mustelus mustelus) meat obtained by various gastrointestinal proteases: crude enzyme extract, low molecular weight (LMW) alkaline protease and trypsin-like protease from M. mustelus intestine, pepsin from M. mustelus stomach, and bovine trypsin.     

Antioxidative activity of whey protein hydrolysates in a liposomal system

Whey protein isolate (WPI) with or without preheating (90 degrees C for 5 min) was hydrolyzed for 0.5 to 6 h using four pure enzymes (pepsin, papain, trypsin, and chymotrypsin) and three commercial crude proteases. After determining the degree of hydrolysis, the hydrolysates were incubated (37 degrees C, 1 h) with a liposome oxidizing system (50 mM FeCl3/0.1 mM ascorbate, pH 7.0). Lipid oxidation was measured by determining the concentrations of TBA-reactive substances (TBARS). The degree of hydrolysis of WPI ranged from 4 to 37% depending on the enzymes used and whether the substrate was heated or not. WPI hydrolysates prepared by pure enzyme treatments did not prevent TBARS formation in the oxidative model system, but WPI hydrolyzed by the commercial crude enzymes, especially protease F, exhibited antioxidant activity. The antioxidative potential of hydrolyzed WPI was not affected by the degree of hydrolysis, and it was improved by preheat treatment in only some samples.     

Anticoagulant activities of goby muscle protein hydrolysates

The anticoagulant activities of protein hydrolysates prepared from goby muscle by treatment with various bacterial alkaline proteases were investigated. All proteases exhibited varying degrees of hydrolysis (DH) and all goby protein hydrolysates (GPHs) caused a significant prolongation of both the thrombin time (TT) and the activated partial thromboplastin time (APTT). The hydrolysate generated by the crude protease from Bacillus licheniformis NH1 displayed the highest anticoagulant activity, and the higher TT (about 32 s) at a concentration of 5 mg/mL was obtained with hydrolysate having a DH of 8.86%. This hydrolysate was then fractionated by size exclusion chromatography on a Sephadex G-25 column into five major fractions (F1–F5). Fraction F2, which exhibited the highest anticoagulant activity, was then fractionated by reversed-phase high-performance liquid chromatography. The molecular masses and amino acid sequences of four peptides in peptide sub-fraction F2–6, which exhibited the highest anticoagulant activity, were determined using ESI-MS and ESI-MS/MS, respectively. The structures of these peptides were identified as Leu-Cys-Arg, His-Cys-Phe, Cys-Leu-Cys-Arg and Leu-Cys-Arg-Arg.     

超高压与Alcalase协同作用制备牛乳清蛋白抗氧化肽

为探讨超高压与碱性蛋白酶Alcalase协同作用下乳清蛋白抗氧化肽的制备,以牛乳清分离蛋白(WPI)为原料,采用Alcalase分别对100～600MPa的超高压处理中和超高压处理后的WPI进行水解,并采用邻苯三酚自氧化法对其水解产物的超氧阴离子自由基清除能力进行测定。结果表明,超高压与Alcalase协同作用显著地促进了WPI的水解,其水解产物的抗氧化活性也显著提高;分子量小于3ku的组分具有最强的超氧阴离子自由基清除能力,其半抑制浓度IC50值最小,为411.62μg/mL。因此,超高压与Alcalase协同作用于乳清蛋白可用于开发新型天然抗氧化剂。     

Identification of dipeptidyl peptidase-IV inhibitory peptides from mare whey protein hydrolysates

Inhibition of dipeptidyl peptidase-IV (DPP-IV) activity is a promising strategy for treatment of type 2 diabetes. In the current study, DPP-IV inhibitory peptides were identi?ed from mare whey protein hydrolysates obtained by papain. The results showed that all the mare whey protein hydrolysates obtained at various hydrolysis durations possessed more potent DPP-IV inhibitory activity compared with intact whey protein. The 4-h hydrolysates showed the greatest DPP-IV inhibitory activity with half-maximal inhibitory concentration of 0.18 mg/mL. The 2 novel peptides from 4-h hydrolysate fractions separated by successive chromatographic steps were characterized by liquid chromatography–electrospray ionization tandem mass spectrometry. The novel peptides Asn-Leu-Glu-Ile-Ile-Leu-Arg and Thr-Gln-Met-Val-Asp-Glu-Glu-Ile-Met-Glu-Lys-Phe-Arg, which corresponded to β-lactoglobulin 1 f(71–77) and β-lactoglobulin 1 f(143–155), demonstrated DPP-IV inhibitory activity with half-maximal inhibitory concentrations of 86.34 and 69.84 μM, respectively. The DPP-IV inhibitory activity of the 2 peptides was retained or even improved after simulated gastrointestinal digestion in vitro. Our findings indicate that mare whey protein-derived peptides may possess potential as functional food ingredients in the management of type 2 diabetes.     

Antioxidant properties of whey protein hydrolysates as measured by three methods

Four microbial proteases (Alcalase, Flavourzyme, Neutrase and Protamex) were used for the preparation of whey protein hydrolysates. The aim of this research was to find out whether these hydrolysates can be used as a source of whey derived antioxidants. Hydrolyzed samples, including their unhydrolyzed protein solutions were tested by the ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) decolorization assay, by the total radical-trapping potential method and by the assay of liposomes peroxidation (fluorescence photometry). Antioxidant properties were enhanced by hydrolysis in most of cases. Alcalase hydrolysates were found as the most effective antioxidants as determined by ABTS assay (~50% of antioxidant activity at 0.1 mg ml⁻¹ of hydrolysate in reaction) and fluorescence photometry. Liposomes were oxidized ~50% less (1.1 μM of α-tocopherol equivalent) with Alcalase hydrolysates additive (at 5.85 mg ml⁻¹ of hydrolysate in reaction). Hydrolysates did not inhibit the oxidation of liposomes at concentrations below 1.0 mg ml⁻¹ in reaction. On the contrary, results of total trapping potential method did not agree with findings observed in other tests. In this assay, Neutrase hydrolysates showed the best antioxidant properties. Pro-oxidant properties were observed in solutions containing (prior to the enzyme Protamex addition only) intact whey protein as determined by the measurement of the liposome peroxidation. The ABTS assay was optimized for the evaluation of the antioxidant activity in whey protein hydrolysates. The reaction time should be prolonged to avoid underestimation of the antioxidant activity.     

Improving antioxidant activities of whey protein hydrolysates obtained by thermal preheat treatment of pepsin,trypsin, alcalase and flavourzyme

Antioxidant activity of whey protein concentrate (WPC) hydrolysates was evaluated. Hydrolysates were obtained by pepsin, trypsin, alcalase and flavourzyme enzymatic reaction and preheat treatment of 95 °C for 5 or 10 min. The degree of hydrolysis (DH) was determined by 2,4,6‐trinitrobenzene sulphonic acid method, and antioxidant properties were determined by three spectrophotometric methods: ferricyanide method, ferric reducing/antioxidant power assay and diphenyl‐picryl hydrazinyl radical‐scavenging activity. For all the enzymes, briefly preheat treatment (95 °C/5 min) increased DH of WPC. Alcalase hydrolysates showed the highest antioxidant activity by three methods. The changes in antioxidant activity was coincidental with the changes in DH (R² = 0.988). Hydrolysates analysed by polyacrylamide gel electrophoresis and high performance liquid chromatography indicated that the α‐La was hydrolysed completely by pepsin, trypsin and alcalase and was resistant to flavourzyme to some extent; β‐lactoglobulin was only completely hydrolysed by trypsin and alcalase. Results indicated that antioxidant activity of hydrolysates was greatly related to the exposure of amino acid residues.     

Zinc incorporation capacity of whey protein nanoparticles prepared with desolvation with ethanol

Whey protein isolate (WPI) nanoparticles were prepared using ethanol desolvation, and their capacity to incorporate ZnCl(2) was analysed. Desolvation was carried out at pH 9 and the volume of added ethanol was 0-3 times the volume of protein solution. The desolvated solutions were dispersed in acidified water (pH 3) immediately after desolvation. The size of the WPI nanoparticles increased with the volume ratio of ethanol:water used, as well as with the amount of ZnCl(2). The nanoparticles showed high incorporation efficiencies, and remained stable after 30 days of storage at 22 °C. The amount of zinc incorporated in the WPI particle suspensions was within the range of daily zinc requirements for healthy adults.     

Optimisation, by response surface methodology, of degree of hydrolysis and antioxidant and ACE-inhibitory activities of whey protein hydrolysates obtained with cardoon extract

The hydrolysis of bovine whey protein concentrate (WPC), α-lactalbumin (α-La) and caseinomacropeptide (CMP), by aqueous extracts of Cynara cardunculus, was optimized using response surface methodology. Degree of hydrolysis (DH), angiotensin-converting enzyme (ACE)-inhibitory activity and antioxidant activity were used as objective functions, and hydrolysis time and enzyme/substrate ratio as manipulated parameters. The model was statistically appropriate to describe ACE-inhibitory activity of hydrolysates from WPC and α-La, but not from CMP. Maximum DH was 18% and 9%, for WPC and α-La, respectively. 50% ACE-inhibition was produced by 105.4 (total fraction) and 25.6 μg mL⁻¹ (<3 kDa fraction) for WPC, and 47.6 (total fraction) and 22.5 μg mL⁻¹ (<3 kDa fraction) for α-La. Major peptides of fractions exhibiting ACE-inhibition were sequenced. The antioxidant activities of WPC and α-La were 0.96 ± 0.08 and 1.12 ± 0.13 μmol trolox equivalent per mg hydrolysed protein, respectively.     

Antibody binding and functional properties of whey protein hydrolysates obtained under high pressure

This paper examines the potential of high hydrostatic pressure to produce whey protein hydrolysates that combine low immunoglobulin (Ig)G- and IgE-binding with acceptable functional properties, with the aim to produce milk-based ingredients with reduced potential allergenicity that could be used in hypoallergenic foods. Treatment with pepsin and chymotrypsin under high pressure produced, in minutes, hydrolysates in which α-lactalbumin and β-lactoglobulin were totally proteolysed, giving rise to large and hydrophobic peptides. Such hydrolysates presented reduced antigenicity and human IgE-binding properties. The hydrolysates obtained with pepsin at 400 MPa showed improved heat stability, particularly at a pH, close to the isoelectric point of the whey proteins, and their emulsion activity indexes at pH 7.0 were superior to those of the untreated whey proteins. These results suggest that the peptides present retained low antigenicity together with sufficient capacity to form emulsions.     

Antioxidant activity of bigeye tuna (Thunnus obesus) head protein hydrolysate prepared with Alcalase

The antioxidant activities of tuna head protein hydrolysate (THPH) prepared with Alcalase were evaluated. THPH showed evident radical scavenging activity in a dose‐dependent manner with the IC₅₀ values for 1,1‐diphenyl‐2‐pycrylhydrazyl (DPPH), superoxide and hydroxyl radicals being 1.34, 1.20 and 2.84 mg mL^?1, respectively, and its reducing power was 0.948 at 12.5 mg mL^?1. THPH showed good inhibitory activity in soybean oil peroxidation after accelerated oxidation at 60 °C, and the oils with 0.01%, 0.05% and 0.1% THPH had significantly (P < 0.05) lower peroxide values than the control, after storage at 60 °C. Moreover, the inhibited oxidation effect of 0.1% THPH was similar to that of 0.01% butylated hydroxytoluene (BHT). The molecular weight distribution of THPH revealed that 70.5% of the total amount was peptides with molecular weight lower than 5000 Da, composing mostly of low molecular weight peptides located at 1020–2585 Da (30.78%) and 241–1020 Da (37.15%).     

不同蛋白酶水解棉籽蛋白制备抗氧化多肽的研究

利用6种蛋白酶对棉籽蛋白进行酶解,测定了各酶在水解过程中的水解度及其变化,对酶解产物的抗氧化活性进行了分析比较。研究表明,各蛋白酶在水解的前2h内,水解度迅速增加,2h之后水解曲线变得平缓。其中胃蛋白酶的水解能力最强,其4h水解产物水解度最大,为30.40%;胰蛋白酶的水解能力最差,最终水解产物的水解度为17.61%。中性蛋白酶水解产物的抗氧化活性较强,经测定其DPPH清除能力为54.95%,羟自由基清除能力为68.98%,超氧阴离子自由基清除能力为58.38%。     

Use of macroporous adsorption resin for simultaneous desalting and debittering of whey protein hydrolysates

Whey protein isolate (WPI) was hydrolysed to whey protein hydrolysates (WPH) of degree of hydrolysis equal to 15% using Protease N ‘Amano’ G (IUB 3.4.24.28) in a batch reactor at 55 °C and pH 7.0 according to the pH‐stat procedure. Ash was removed by adsorbing WPH onto macroporous adsorption resins (MAR). Following rinsing with deionised water, desorption was achieved by washing with 20%, 40% and 75% alcohol (v v^?1) to obtain the three fractions HS20, HS40 and HS75. Ash reduced from 15.71% (WPH) to 4.38% (HS20), 2.02% (HS40) and 2.38% (HS75). Similarly, the protein content was enriched from a low of 64.89% (WPH) to 94.74% (HS20), 95.32% (HS40) and 92.00% (HS75). The fractions were analysed for surface hydrophobicity (SH_o), angiotensin‐I converting enzyme (ACE) inhibition, emulsifying activity index, total amino acids composition and molecular weight distribution. Fraction HS75 was objectionably bitter, showed superior ACE inhibition (lowest IC₅₀), had the highest content of hydrophobic and essential amino acids and contained about 71% of <600 Da with no fractions exceeding 4142 Da. Desorption with alcohol weakened the hydrophobic interaction forces between the peptides and resins and hence eluted the peptides, with the bitter HS75 being extracted.     

Effect of heat and enzymatic treatment on the antihypertensive activity of whey protein hydrolysates

The influence of heat and enzymatic treatments on the hypotensive activity of hydrolysates derived from whey protein isolate was examined. The whey protein isolate (WPI) was previously denatured at 65 or 95 °C and hydrolyzed using the enzymes Alcalase, α-chymotrypsin or Proteomix. The hydrolysates thus obtained were characterized and studied with regard to their angiotensin converting enzyme (ACE) inhibitory activity and hypotensive activity in spontaneously hypertensive rats (SHR). The enzyme α-chymotrypsin was found to produce hydrolysates with the highest ACE inhibitory activity. The hydrolysate that most effectively reduced blood pressure in SHR was obtained from WPI previously denatured at 65 °C and treated with the enzyme Alcalase. The hydrolysate with the highest ACE inhibitory activity was able to reduce the arterial blood pressure of the animals only after intraperitoneal administration, suggesting an interference of gastrointestinal enzymes in the absorption of active peptides from this hydrolysate.     

Antioxidant and free radical-scavenging activities of chickpea protein hydrolysate (CPH)

Chickpea protein hydrolysate (CPH) was fractionated by gel filtration on Sephadex G-25. The antioxidant and free radical-scavenging activities of four CPH fractions (Fra.I, Fra.II, Fra.III, and Fra.IV) were measured using reducing power, inhibition of linoleic acid autoxidation, and 1,1-diphenyl-2- pycrylhydrazyl (DPPH)/superoxide/hydroxyl radical-scavenging assay. The antioxidant activity of Fra.IV (81.13%) was closer to that of α-tocopherol (83.66%) but lower than that of BHT (99.71%) in the linoleic acid oxidation system. Amino acid analyses showed that Fra.IV with the strongest antioxidant activity also had the highest total hydrophobic amino acids content (38.94% THAA) and hydrophobicity (125.62 kcal/mol amino acid residue) compared with the other three fractions. The molecular weight distribution of Fra.IV was found to vary from 200 to 3000 Da.