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.     

Peptide–peptide and protein–peptide interactions in mixtures of whey protein isolate and whey protein isolate hydrolysates

The extent of aggregation in whey protein isolate (WPI) hydrolysates induced by Bacillus licheniformis protease was quantified as a function of degree of hydrolysis (DH), temperature and ionic strength. The capacity of the hydrolysates to aggregate added intact protein was also studied. The amount of aggregated material and the size of the aggregated peptides were measured by nitrogen content and size exclusion chromatography, respectively. Aggregation increased with DH up to the practical end point of hydrolysis (DH 6.8%). The aggregates formed under the various conditions studied consisted of peptides with masses ranging from 1.4 to 7.5 kDa. The hydrolysates were also able to aggregate added WPI. The additional amount of aggregated material increased with increasing DH. Peptides involved in peptide–peptide interactions were also involved in protein–peptide interactions. It is hypothesized that hydrophobic interactions dominated peptide–peptide interactions, while protein–peptide interactions depended on the balance between hydrophobic attraction and electrostatic repulsion.     

Whey protein isolate hydrolysates obtained with free and immobilized Alcalase: Characterization and detection of residual allergens

Protein antigenicity can be reduced by enzymatic hydrolysis, which can be performed either by free or immobilized enzyme. The immobilized enzyme is removed from the reaction medium and reused, while the free enzyme must be inactivated to stop the reaction, generally by heating. Here we have shown that hydrolysates produced with free or immobilized Alcalase on glyoxyl-agarose bead presented different physicochemical properties (hydrophilicity profile, molecular mass distribution, surface hydrophobicity) and different levels of residual milk allergens (α-lactalbumin and β-lactoglobulin). Although, under the studied conditions, the hydrolysis with immobilized enzyme did not reduce the residual allergen levels as efficiently as the free enzyme, the results suggest potential applications of immobilized Alcalase for production of hypoallergenic hydrolysates.     

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.     

Antioxidant activity,functional properties and bioaccessibility of whey protein hydrolysates

The main objective of this study was to produce the functionally improved whey protein hydrolysate with high bioaccessible antioxidant activity. The hydrolysate with highest antioxidant activity, mainly composed of hydrophilic antioxidant peptides and suitable for preventing oxidation in polar food matrices, was produced by tryptic hydrolysis conducted at 37 °C and an enzyme/substrate (E/S) ratio of 0.5%. The hydrolysate exerted significantly improved antioxidant activity (80.0%), digestibility (95.9%) and bioaccessibility (124.6%) compared to the native whey proteins (32.1%, 87.1% and 106.3%, respectively) as widely used food supplement. The high bioaccessibility indicates the preservation of hydrolysate bioactivity during the process of gastrointestinal digestion, as a particular challenge in its application.     

Dual function peptides from pepsin hydrolysates of whey protein isolate

The aim of this study was to investigate the effect of pepsin hydrolysates of whey protein isolate (WPI) on vascular relaxation and emulsifying capacity. WPI was subjected to pepsin hydrolysis for 5 h. The chromatographic profiles of the samples showed the formation of a wide variety of peptides. Addition of WPI hydrolysates in phenylephrine-contracted rat aortic rings induced a similar concentration-dependent relaxation in both endothelium-intact and endothelium-denuded preparations. In endothelium-denuded vessels the maximum relaxation induced by WPI fractions increased along the time, reaching over 70% after 3 h-hydrolysis on. In addition, the vascular relaxation was not associated with an inhibition of the angiotensin-converting enzyme or activation of K⁺ channels. Hydrolysed fractions were further evaluated for the emulsifying capacity (EC) and all tested fractions were able to keep an EC over 60%. These results reinforce the potential of WPI pepsin-hydrolysates as an option in the search for dual function peptides from whey proteins.     

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.     

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.     

Pilot-scale production of hydrolysates with altered bio-functionalities based on thermally-denatured whey protein isolate

Whey protein isolate (WPI) solutions (100 g L⁻¹ protein) were subjected to a heat-treatment of 80 °C for 10 min. Unheated and heat-treated WPI solutions were hydrolysed with Corolase^® PP at pilot-scale to either 5 or 10% degree of hydrolysis (DH). Hydrolysates were subsequently processed via cascade membrane fractionation using 0.14 μm, and 30, 10, 5 and 1 kDa cut-off membranes. The compositional and molecular mass distribution profiles of the substrate hydrolysates and membrane processed fractions were determined. Whole and fractionated hydrolysates were assayed for both angiotensin-I-converting enzyme (ACE) inhibitory activity and ferrous chelating capabilities. A strong positive correlation (P < 0.01) was established between the average molecular mass of the test samples and the concentration needed to chelate 50% of the iron (CC₅₀) in solution. The lowest ACE inhibition concentration (IC₅₀ = 0.23 g L⁻¹ protein) was determined for the 1 kDa permeate of the heat-treated 10% DH hydrolysate.     

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.     

In vitro cytomodulatory and immunomodulatory effects of bovine colostrum whey protein hydrolysates

Bovine colostrum possesses biological compounds involved in the development of the newborn. Among them, the proteins have drawn attention as a source of bioactive peptides. This study shows the in vitro cytotoxic and immunostimulatory potential of two colostrum whey protein (CWP) hydrolysates obtained by in vitro digestion with pepsin and pancreatin. MTT cell viability, apoptosis induction, polymorphonuclear proliferation and phagocytic activity assays were performed. Treatment with the hydrolysates induced a significant decrease in the viability of MDA-MB-231 cell lines due to apoptosis and also a significant increase in the proliferation of blood mononuclear cells. It could also be observed that for the RM-1 and PC-3 prostate cancer cell lines and for the two times of exposure (24 and 48 h), the hydrolysate H1 is significantly more cytotoxic than CWP. These results showed the potential of bovine CWP and its hydrolysates for the treatment of chronic diseases such as cancer.     

High pressure can reduce the antigenicity of bovine whey protein hydrolysates

The combined effect of high pressure (HP) and enzymatic treatments on the proteolysis and antigenicity of hydrolysates from bovine whey proteins (WP) was studied. Four food grade protease preparations (Alcalase, Neutrase, Corolase 7089 and Corolase PN-L) were used. Hydrolysis was performed at 40 °C for Corolase PN-L and 50 °C for the other enzymes, for 15 min, after or during HP treatment. The degree of hydrolysis and RP-HPLC peptide profile were evaluated. An indirect ELISA test using polyclonal rat anti β-lactoglobulin antibodies was used to determine the residual antigenicity. The results showed that HP treatment enhanced the hydrolysis of bovine WP. For most enzymes, the best results were obtained at a pressure of 300 MPa. For two enzymes, Corolase PN-L and Neutrase, differences in peptide profiles were obtained due to the pressure applied during the enzymatic hydrolysis. Based on these differences, the residual antigenicity of these preparations were determined. An important reduction was found in the antigenicity of the hydrolysates obtained with Corolase PN-L and Neutrase in combination with HP treatment (300 MPa), prior to or during enzymatic hydrolysis, respectively. These results suggest that HP can enhance the WP hydrolysis, and, depending on the choice of enzymes, reduce the residual antigenicity of the hydrolysates. The reduced antigenicity of hydrolysates obtained by the combined treatments could have a relevant application in the development of hypoallergenic infant formulae.     

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.     

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.     

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。     

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.