Affinity purification and characterisation of chelating peptides from chickpea protein hydrolysates

A chickpea protein hydrolysate produced with pepsin and pancreatin was used for the affinity purification of chickpea chelating peptides. Three chelating peptide fractions were obtained after affinity chromatography with immobilised copper. These peptide fractions showed a higher chelating activity and histidine contents than the original protein hydrolysate. Chelating activity was positively correlated with the histidine content of the purified fractions. Different subfractions were also obtained after gel filtration chromatography from the affinity purified peptide fractions. Some of these subfractions showed a higher chelating activity and histidine contents than the original fractions. These results suggest that a combination of high His contents, around 20–30%, and small peptide size provide the best chelating activities. Thus sequential purification with affinity and gel filtration chromatography is a useful procedure for the purification of chickpea peptides with high chelating activity. These results show that a range of chelating peptides are generated during digestion of the chickpea proteins that, after metal chelation, may prevent the generation of reactive oxygen species (ROS) and favour metal absorption.     

Production of copper-chelating peptides after hydrolysis of sunflower proteins with pepsin and pancreatin

Sunflower protein hydrolysates obtained with pepsin and pancreatin were used for purification of copper-chelating peptides by affinity chromatography with copper immobilized on solid supports. The chelating activity of purified peptides was indirectly measured by the inhibition of β-carotene oxidation in the presence of copper. The protein hydrolysate obtained after 180 min incubation with pepsin plus 60 min with pancreatin was the most inhibitory of β-carotene oxidation. Purified chelating peptides were 2.5 times more antioxidant than the parent protein hydrolysate. Chelating peptides were enriched in certain amino acids, such as histidine and arginine, with respect to the original hydrolysate. This work shows that chelating peptides may be generated during digestion of sunflower proteins and have a protective role, due to their antioxidative activity, and favour mineral bioavailability.     

Combined effects of high-pressure and enzymatic treatments on the hydrolysis of chickpea protein isolates and antioxidant activity of the hydrolysates

A chickpea protein isolate (CPI) was pretreated before hydrolysis under a pressure that varied between 100 and 600MPa. The hydrolysis rate increased significantly with pressure above 300MPa. At 40min, the DH of the control was 15.3%, while the DH of the CPI treated at 300MPa was 18.5%, which reached 23.74% post treatment at 400MPa. The pretreatment of CPI above 300MPa enhanced the superoxide anion capturing rate of enzymatic hydrolysis. Pretreatment at 400MPa significantly reduced the hydrolysis time with the release of antioxidant peptides. While hydrolysis by Alcalase during treatment at high pressure (100-300MPa) significantly increased the degree of hydrolysis (DH), its maximum value peaked after hydrolysis at 200MPa for 30min. In addition, hydrolysates obtained at high pressure (100-300MPa) had a higher superoxide anion capturing rate. High-pressure treatment at 200MPa for 20min resulted in products with high antioxidative activity. The molecular-weight (MW) determination of the enzymatic hydrolysates indicated that hydrolysis at high pressure could significantly increase the amount of low-molecular-weight peptides.     

菜籽源锌螯合肽的制备、纯化和结构解析

采用碱性蛋白酶水解菜籽蛋白制备水解物,运用固定金属亲和层析(IMAC-Zn2+)和葡聚糖凝胶G25(Sephadex G25)层析分离纯化;利用反相高效液相色谱(RP-HPLC)和电喷雾电离质谱(ESI-MS)鉴定序列;采用EDTA络合滴定法(ECT)、双硫腙比色法(DCM)、原子吸收光谱(AAS)和电感耦合等离子体原子发射光谱法(ICP-AES)检测锌螯合率。试验表明:菜籽蛋白的4 h水解物锌螯合潜力最佳;分离该水解物获得3个肽组分(I,Ⅱ和Ⅲ),其中Ⅱ和Ⅲ具有锌螯合能力;进一步分离后分别获得3个(Ⅱ-1,Ⅱ-2,Ⅱ-3)和2个肽组分(Ⅲ-1和Ⅲ-2),组分Ⅱ-3的锌螯合率最高,高于同浓度的谷胱甘肽(GSH)(P0.05);鉴定Ⅱ-3序列获得4条肽,即Ala-Arg(AR),Asn-Ser-Met(NSM),Gly-Lys-Arg(GKR)和Glu-Pro-SerHis(EPSH)。其中NSM的锌螯合率最高,来源于菜籽蛋白NADH-enoyl-ACP reductase Chain A序列中的296-298氨基酸残基。因此,菜籽蛋白可以作为微量元素螯合肽的优良来源。     

Antioxidant activity of peptides isolated from alfalfa leaf protein hydrolysate

Alfalfa leaf protein, a potential source of high quality protein for human consumption, was hydrolyzed with protease. Alfalfa leaf protein hydrolysate was fractionated by ultrafiltration and the obtained peptides were purified by dynamical adsorption. The antioxidant activity of alfalfa leaf peptides (ALPs) was investigated and compared with that of a native antioxidant, reduced glutathione (GSH), which was used as a reference. The reducing power of ALPs was 0.69 at 2.00 mg/mL. ALPs at 1.60 mg/mL and 0.90 mg/mL exhibited 79.71% and 67.00% of scavenging activities on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and superoxide radical, respectively. In addition, ALPs showed 65.15% chelating effect on ferrous ion at 0.50 mg/mL. The molecular weight of the peptides was determined and 67.86% of the total amount was below 1000 Da. Combined with the result of the amino acid profiles, ALPs was believed to have high nutritive value in addition to antioxidant activity.     

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.     

玉米胚芽蛋白水解物的抗氧化活性研究

以玉米胚芽蛋白水解物为底物,通过对其羟基自由基清除能力、超氧阴离子自由基清除能力、二苯基苦味酸基苯肼(DPPH)自由基清除能力、Cu2+螯合能力、在亚油酸体系中的抗氧化性、油脂过氧化值(POV)等指标的测定,研究玉米胚芽蛋白水解物的抗氧化活性.结果表明,玉米胚芽蛋白水解物对羟基自由基、超氧阴离子自由基、DPPH自由基具有一定的清除能力,且浓度为6mg/mL时,清除效果最好;对Cu2+具有螯合能力,并且螯合能力随着玉米胚芽蛋白水解物浓度的增加而呈上升趋势;对亚油酸氧化具有抑制作用,作用效果与剂量成正相关,同时对油脂的POV值具有抑制作用,浓度为0.1％时抑制作用最强.     

Influence of preparation methods on physicochemical and gelation properties of chickpea protein isolates

Two protein isolates were prepared from defatted chickpea seed flour by applying alkaline extraction followed by isoelectric precipitation or ultrafiltration (TpI and TUF, respectively), while another one (TF) was obtained by a combination of protein extraction at a mildly acidic environment and ultrafiltration processes. The isolates differed in composition, with the TpI and TF containing mainly the chickpea globulins and the albumins, respectively, whereas the TUF isolate consisted of both types of proteins with the globulin fraction dominating over the albumins. The differences in protein composition between the isolates as well as the impact of extraction conditions were reflected in their protein solubility, surface hydrophobicity, sulfhydryl group content, thermal properties and the onset of gelation during heating. On the other hand, the protein isolate gelling behavior depended mainly on the method applied for their preparation rather than the protein composition, with the isolates obtained by ultrafiltration exhibiting lower gelling concentrations and gel networks of higher elasticity at protein contents below 12% (w/v).     

Characterization of protein isolates from different Indian chickpea (Cicer arietinum L.) cultivars

Protein isolates prepared by alkaline solubilization followed by isoelectric precipitation and freeze drying from desi (PBG-1, PDG-4, PDG-3, GL-769, and GPF-2) and kabuli (L-550) chickpea cultivars were evaluated for functional (water and oil absorption capacities, least gelation concentration, foaming capacity and stability) and thermal properties. Significant difference (P ? 0.05) in properties of kabuli and desi chickpea protein isolates was observed. Kabuli chickpea protein isolate showed significantly (P ? 0.05) higher ash (1.14%), protein (94.4%), L^∗, ΔE value, oil absorption capacity (OAC) and lower water absorption capacity (WAC) than their corresponding desi chickpea protein isolates. The solubility-pH profile of different protein isolates showed minimum solubility in the pH between 4.0 and 5.0 and two regions of maximum solubility at pH 2.5 and 7.0. Foaming capacity of all protein isolates increased with the increase in concentration. Kabuli chickpea protein isolate showed the highest foam stability (94.7%) after 120 min of storage. The thermal properties of protein isolates from different chickpea cultivars were studied by differential scanning calorimetry (DSC). Protein isolates from both the chickpea types differed significantly (P ? 0.05) in peak denaturation temperature (T_d) and heat of transition (ΔH). Kabuli type protein isolate exhibited lower T_d and ΔH value as compared to those from desi types. The interrelationships between characteristics of protein isolates showed a significant (P ? 0.05) negative correlation of T_d with protein content and OAC. It was also observed that cultivars with high fat content had high ΔH and lower WAC.     

Antibacterial peptides from barbel muscle protein hydrolysates: Activity against some pathogenic bacteria

Peptides obtained by enzymatic hydrolysis of fish proteins exhibit not only nutritional but also biological properties of dietary uses, or even therapeutic potential. The objective of the present study was to isolate and characterize peptides from the protein hydrolysates of barbel muscle with antibacterial activity against Gram-positive (Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis, Micrococcus luteus and Bacillus cereus) and Gram-negative (Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter sp.) bacteria. Barbel muscle protein hydrolysates (BMPHs), obtained by treatment with Alcalase^® (DH = 6.6%), was fractionated by size exclusion chromatography on a Sephadex G-25 and purified by reversed-phase high performance liquid chromatography (RP-HPLC). The molecular masses and amino acid sequences of these peptides were determined using ESI–MS and ESI–MS/MS, respectively. Eleven peptides in F_II-1, F_II-2, F_II-3 and F_II-4 sub-fractions separated by RP-HPLC were identified. The most active peptide fraction (F_II-3) contained three peptides: Ala–Ala–Ala–Leu; Ala–Ala–Gly–Gly–Val and Ala–Ala–Val–Lys–Met.     

鹰嘴豆肽、大豆肽功能性质的研究

研究对比了鹰嘴豆肽和大豆肽的水解度、抗氧化性、吸油性、吸湿及保湿性等功能特性的差异,以及蛋白酶种类对肽产物功能性质的影响。结果表明,①酶Ⅱ(Protease from Bacillus sp.)制备的蛋白肽抗氧化性和吸油能力最好,且在低湿度下的吸湿性和高湿度下的保湿性也最好;酶Ⅲ(Papain from papaya latex)制备的蛋白肽水解度最高;酶Ⅰ(Protease fromAspergillus melleus)制备的蛋白肽在不同湿度条件下都有较好的吸湿性能。②大豆肽的水解度和抗氧化能力比鹰嘴豆肽好。③Desi肽吸油能力最强,在抗氧化性上仅次于大豆肽,高低湿度环境下都有很好的保湿能力,其中Desi肽Ⅱ这些特征最为明显。④Kabuli肽在不同湿度条件下的吸湿和保湿能力都较好。     

Emulsifying properties of chickpea protein isolates: Influence of pH and NaCl

Structure and emulsifying properties of chickpea protein isolates (CPI) as a function of protein concentration, oil volume, pH and ionic strength were studied. The optimum protein concentration 2 g l⁻¹ used to determine the emulsifying properties was obtained. Emulsifying activity index (EAI) increased from 244 to 376 m² g⁻¹ with pH from 3.0 to 11.0 except the protein isoelectric point (pI 5.0), where the EAI was 20 m² g⁻¹ and emulsion droplet size was the largest. At lower ionic strengths (0.0–0.1 M NaCl, pH 7.0), EAI decreased from 253 to 72.4 m² g⁻¹; however, it increased from 72.4 to 231.4 m² g⁻¹ at higher ionic strengths (0.1–1.0 M NaCl). A positive relation between EAI and surface hydrophobicity (S₀) of CPI at various ionic strengths was obtained, while EAI was independent of S₀ under different pH values. α-Helix was the major configuration of CPI at the pI or lower ionic strength.     

Purification and identification of kokumi-enhancing peptides from chicken protein hydrolysate

The Maillard reaction products (MRPs) from chicken protein hydrolysate were demonstrated to have intense umami and kokumi-enhancing effects. To find the main flavour-enhancing compounds in the chicken protein hydrolysate, the fractions with different molecular weights were obtained by ultrafitration. The evaluation of taste characteristics revealed that the fractions with molecular weights ranging from 1000 to 5000 Da predominantly contributed to the umami and kokumi-enhancing effects. After further purification by using ultrafiltration, gel filtration chromatography, and ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in combination with sensory evaluation, three peptides were identified, an octapeptide (WVNEEDHL), a nonapeptide (NSLEGEFKG) and a decapeptide (KDLFDPVIQD). Sensory evaluation results showed that all three peptides could significantly enhance the meat flavour, umami taste and thickness of the chicken powder solution. The results indicate that the peptides have potential application as effective chicken flavour-enhancing ingredients in the food industry.     

Effect of digestive enzymes on the activity of camel‐milk insulin

This study investigated the effect of digestive enzymes on the activity of camel‐milk insulin. The digestion was performed using the sequential action of pepsin and pancreatin. Proteolysis degree was estimated using the O‐phthaldialdehyde method. Insulin concentration was determined using an enzyme‐linked immunosorbent assay (ELISA). Results revealed that milk proteins were partially digested by pepsin alone and the degradation was increased during the pepsin–pancreatin digestion as compared to control. Insulin lost its activity after 30 min of pepsin digestion, and it was not detected by ELISA. This study strongly suggests that insulin is not responsible for the antidiabetic action of camel's milk.     

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.     

Purification of angiotensin I-converting enzyme-inhibitory peptides from the enzymatic hydrolysate of defatted canola meal

Defatted canola meals from seeds of different processing origins were hydrolyzed by Alcalase to give hydrolysates that inhibited angiotensin converting enzyme (ACE) activity. Heat treated meals yielded protein hydrolysates with 50% ACE-inhibitory concentrations of 27.1 and 28.6 μg protein/ml compared with 35.7 and 44.3 μg protein/ml for the none-heat treated meals. Separation of the hydrolysate on a Sephadex G-15 gel permeation column (GPC) yielded a fraction with an IC₅₀ value of 2.3 μg protein/ml. Amino acid analysis showed that the GPC fraction contained 45% content of aromatic amino acids in comparison to 8.5% of the hydrolysate. Two peptides, Val-Ser-Val (IC₅₀ = 0.15 μM) and Phe-Leu (IC₅₀ = 1.33 μM) were purified, and located in the primary structure of canola napin and cruciferin native proteins. The results suggest that canola protein hydrolysate is a potential ingredient for the formulation of hypotensive functional foods.     

Influence of the extent of enzymatic hydrolysis on the functional properties of protein hydrolysate from grass carp (Ctenopharyngodon idella) skin

Protein hydrolysates from grass carp skin were obtained by enzymatic hydrolysis using Alcalase^®. Hydrolysis was performed using the pH-stat method. The hydrolysis reaction was terminated by heating the mixture to 95 °C for 15 min. At 5.02%, 10.4%, and 14.9% degree of hydrolysis (DH), the hydrolysates were analyzed for functional properties. The protein hydrolysates had desirable essential amino acid profiles. Results demonstrated that the hydrolysates had better oil holding and emulsifying capacity at low DH. The water holding capacity increased with increased levels of hydrolysis. Enzymatic modification was responsible for the changes in protein functionality. These results suggest that grass carp fish skin hydrolysates could find potential use as functional food ingredients as emulsifiers and binder agents.