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.     

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.     

Purification and identification of antihypertensive peptides from seaweed pipefish (Syngnathus schlegeli) muscle protein hydrolysate

Angiotensin-I-converting enzyme (ACE-I) inhibitory peptides were purified from the seaweed pipefish muscle protein using papain, alcalase, neutrase, pronase, pepsin and trypsin. Among them, the alcalase hydrolysate exhibited the highest ACE-I inhibitory activity. The alcalase hydrolysate was separated into four fractions (Fr1, Fr2, Fr3, and Fr4) by fast protein liquid chromatography (FPLC) on a Hiprep 16/10 DEAE FF anion exchange column. Among four fractions, Fr3 has shown the highest ACE-I inhibitory activity and it was further purified into three fractions (Fr3-I, Fr3-II, and Fr3-III) using reverse-phase high performance liquid chromatography (RP-HPLC) on a Primesphere 10 C18 (20 × 250 mm) column. The Fr3-II has exhibited the highest ACE-I inhibition (IC₅₀, 0.62 mg/ml) than the Fr3-III (IC₅₀, 1.44 mg/ml). The amino acid sequences of the obtained peptides from Fr3-II and Fr3-III were identified as Thr-Phe-Pro-His-Gly-Pro (MW, 744 Da) and His-Trp-Thr-Thr-Gln-Arg (MW, 917 Da) respectively. Furthermore, cell viability assay showed that no cytotoxicity of alcalase hydrolysate on human lung fibroblasts cell line (MRC-5). These results suggest that peptides derived from seaweed pipefish can be developed as antihypertensive ingredients in functional foods.     

Purification and identification of antioxidative peptides from loach (Misgurnus anguillicaudatus) protein hydrolysate by consecutive chromatography and electrospray ionization-mass spectrometry

Loach protein was hydrolyzed by papain to obtain antioxidative peptides. The results showed that the loach protein hydrolysate (LPH) could scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) (IC₅₀ = 17.0 ± 0.54 mg/mL) and hydroxyl radicals (IC₅₀ = 2.64 ± 0.29 mg/mL). It could chelate cupric ion and inhibit the lipid peroxidation in a linoleic acid emulsion system. The hydrolysate was isolated and purified by ultrafiltration and consecutive chromatographic methods including ion-exchange chromatography, gel filtration chromatography and a two-step reverse high-performance liquid chromatography (RP-HPLC). The purified antioxidant peptide was identified as Pro-Ser-Tyr-Val (464.2 Da) using RP-HPLC connected on-line to an electrospray ionization (ESI) mass spectrometer. The purified peptide showed a 9.14-fold higher scavenging activity for hydroxyl radical compared with the crude LPH. Therefore, it is possible to produce natural antioxidative peptides from loach protein by enzymatic hydrolysis and purification.     

大米蛋白抗氧化肽的活性以及组成鉴定研究

采用碱性蛋白酶酶解大米蛋白制备大米蛋白抗氧化肽。研究表明大米蛋白酶解产物具有螯合金属铁离子、清除H2O2、羟自由基、DPPH自由基、抑制亚油酸自然氧化的作用。通过应用高解析离子淌度质谱（HD-MS）分析鉴定米蛋白抗氧化肽的序列主要为Met-Pro-Pro-Ser-Ser-Pro-His（376.68u）,Leu-Ala-Gly-Pro-Lys-Phe-Ala-Leu（408.75u）和Met-Pro-Arg-His-Asp-Pro-Gln（440.70u）。      

超滤法浓缩富集牡蛎蛋白抗氧化活性肽

为研究牡蛎蛋白酶解产物中发挥抗氧化活性的肽片段,采用超滤的方法对其进行初步分离富集,测定了各级组分的体外抗氧化能力和氨基酸组成。结果表明:<4000u超滤液对羟自由基、DPPH自由基和超氧自由基清除活性的IC50值分别为17.29、2.82、7.02mg/mL,对肝脂质过氧化抑制作用的IC50值为14.40mg/mL,均低于原液和其他超滤组分;<4000u超滤液的还原力均高于同浓度的原液和其他超滤组分。经氨基酸分析,超滤后组氨酸、脯氨酸、甲硫氨酸、半胱氨酸、酪氨酸、色氨酸及苯丙氨酸的含量得到了富集。<4000u的超滤液经凝胶层析后,活性部分主要集中在550～3890u。      

Purification and identification of antioxidant peptides from corn gluten meal

Corn gluten meal was hydrolyzed by alkaline protease and Flavourzyme to obtain the antioxidant peptides. The antioxidant activities of the hydrolysates or peptides were evaluated by free radical scavenging capacity (1,1-diphenyl-2-picrylhydrazyl/2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt/hydroxyl radical/superoxide radical anion), metal ion (Fe²⁺/Cu²⁺) chelating activity and lipid peroxidation inhibitory capacity. The hydrolysates were separated by ultrafiltration, and those with molecular weight <10 kDa exhibited highest antioxidant activity in all relevant assays. The hydrolysates were subsequently purified by gel filtration chromatography, and fraction F3 showed the highest antioxidant activity. Three peptides were identified from fraction F3 using LC–ESI–Q–TOF MS/MS as Leu-Pro-Phe (375.46 Da), Leu-Leu-Pro-Phe (488.64 Da) and Phe-Leu-Pro-Phe (522.64 Da). These peptides exhibited good free radical scavenging activity and lipid peroxidation inhibitory effect. Thus, corn gluten meal may be used as a potential source of antioxidant peptides for food and nutraceutical applications.     

Purification and characterisation of a novel antioxidant peptide from porcine haemoglobin hydrolysate

Porcine haemoglobin, which is normally discarded as a by‐product of meat industry, was hydrolysed using pepsin, AS1398 neutrase, trypsin, flavorzyme, papain and alcalase respectively. The peptic hydrolysate exhibited the highest antioxidant activities than those of other hydrolysates, which was separated using ultrafiltration membranes, and consecutively using chromatographic methods including ion‐exchange chromatography on SP Sephadex C‐25 column, gel filtration chromatography on Sephadex G‐25 column and reversed‐phase high performance liquid chromatography. Finally, a novel antioxidant peptide from porcine haemoglobin (APPH) was purified, and its sequence was identified to be ARRLGHDFNPDVQAA (1666 Da) using mass spectrometry. APPH exhibited significant higher lipid peroxidation inhibitory ability than that of α‐tocopherol as positive control (P < 0.05), and efficiently quenched hydroxyl radical (IC₅₀ = 26.9 μm ). APPH agrees with the 115–129 residues of the β‐chain from porcine haemoglobin. These results indicate that APPH would be a beneficial ingredient for functional food and pharmaceuticals.     

Purification and identification of novel antioxidant peptides from enzymatic hydrolysates of sardinelle (Sardinellaaurita) by-products proteins

In order to utilise sardinelle (Sardinellaaurita) protein by-products, which is normally discarded as industrial waste in the process of fish manufacturing, heads and viscera proteins were hydrolysed by different proteases to obtain antioxidative peptides. All hydrolysates showed different degrees of hydrolysis and varying degrees of antioxidant activities. Hydrolysate generated with crude enzyme extract from sardine (Sardinapilchardus) displayed high antioxidant activity, and the higher DPPH radical-scavenging activity (87 ± 2.1% at 2 mg/ml) was obtained with a degree of hydrolysis of 6%. This hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P₁–P₈). Fraction P₄, which exhibited the highest DPPH scavenging activity, was then fractionated by reversed-phase high performance liquid chromatography (RP-HPLC). Seven antioxidant peptides were isolated. The molecular masses and amino acids sequences of the purified peptides were determined using ESI-MS and ESI-MS/MS, respectively. Their structures were identified as Leu-His-Tyr, Leu-Ala-Arg-Leu, Gly-Gly-Glu, Gly-Ala-His, Gly-Ala-Trp-Ala, Pro-His-Tyr-Leu and Gly-Ala-Leu-Ala-Ala-His. The first peptide displayed the highest DPPH radical-scavenging activity (63 ± 1.57%; at 150 μg/ml) among these peptides.     

核桃蛋白ACE抑制肽分离纯化研究

核桃ACE抑制肽经超滤后,其ACE抑制率由76.58%增至78.43%,再经DA201-C大孔吸附树脂脱盐纯化后,脱盐率达到98.70%,ACE抑制率升高至80.73%;采用Sephadex G-15凝胶分离后,核桃ACE抑制肽被分为三个组分,其中活性最大的G2组分,其ACE抑制率达83.10%,且IC50为1.308 mg/mL,G2组分主要分布在500 Da¹80 Da,系为2180 Da,系为2⁴个氨基酸残基组成小肽。     

Purification and identification of an ACE-inhibitory peptide from walnut protein hydrolysate

Angiotensin I-converting enzyme (ACE) is a dipeptidyl carboxypeptidase. It plays an important physiological role in regulating blood pressure in human bodies. ACE-inhibitory peptides inhibit the activity of ACE, thereby decreasing the tension of blood vessels and the blood volume, thus lowering blood pressure. ACE-inhibitory peptides derived from food proteins due to their safety properties and beneficial effects on human health have attracted more and more attentions on their ACE-inhibitory activity. In the present study, a novel ACE-inhibitory peptide, P-1a1, was homogeneously purified from walnut protein hydrolysate by ultrafiltration, consecutive column chromatography and high performance liquid chromatography. The purified peptide was characterized by Edman degradation, matrix-assisted laser desorption ionization time-of-flight mass spectrophotometer and a liquid-phase peptide sequencer. The amino acid sequence of P-1a1 was determined to be LPGRPPIKPWPL. The potent ACE-inhibitory peptide showed a high ACE-inhibitory activity with the IC₅₀ value of 128.98 μg/mL (95.2 μmol/L). The purified peptide could be used in functional food products as a bioactive component with good ACE-inhibitory activity.     

Isolation and characterization of three antioxidant pentapeptides from protein hydrolysate of monkfish (Lophius litulon) muscle

In the current study, an efficient method had been developed to acquire the antioxidant hydrolysate of monkfish muscle protein (MPH) using trypsin by an orthogonal (L₉(3)⁴) test. Under the optimum conditions of enzymolysis time 4 h, enzyme-to-substrate ratio (E/S) 2%, enzymolysis temperature 40 °C and pH 8.0, the DH (Degree of hydrolysis) and hydroxyl radical scavenging activity of MPH reached 19.83 ± 0.82% and 58.05 ± 3.01%, respectively. By using ultrafiltration, gel filtration chromatography and reversed phase high performance liquid chromatography (RP-HPLC), three antioxidant pentapeptides were isolated from MPH, and their amino acid sequences were identified as Glu-Trp-Pro-Ala-Gln (MPH-P1), Phe-Leu-His-Arg-Pro (MPH-P2), and Leu-Met-Gly-Gln-Trp (MPH-P3) with molecular weights of 629.68 Da, 668.80 Da, and 633.77 Da, respectively. MPH-P1, MPH-P2, and MPH-P3 exhibited good scavenging activities on hydroxyl radical (EC₅₀ 0.269, 0.114 and 0.040 mg/ml), DPPH radical (EC₅₀ 2.408, 3.751, and 1.399 mg/ml), and superoxide anion radical (EC₅₀ 0.624, 0.101, and 0.042 mg/ml) in a dose-dependent manner. MPH-P3 was also effective against lipid peroxidation in the model system. The antioxidant activities of MPH-P1, MPH-P2, and MPH-P3 were due to their small sizes and the presence of antioxidant and hydrophobic amino acid residues within their sequences. The results of this study suggested that the protein hydrolysate and/or its isolated peptides might be effectively used as food additives for retarding lipid peroxidation occurring in foodstuffs.     

Characterisation of antioxidant peptides from enzymatic hydrolysate of golden melon seeds protein

The purpose of present research was to study novel antioxidant peptides from Golden melon seeds. Alkaline protease was used to hydrolyse the Golden melon seeds protein to obtain the hydrolysed peptides. These antioxidant peptides were purified and identified by ultrafiltration, gel filtration chromatography and RP-HPLC-ESI-MS/MS. Results showed that the peptide fraction (GMSHp3) with molecular weight (MW) <3 kDa obtained by ultrafiltration had the highest antioxidant capacity. This fraction was further purified via gel filtration chromatography into six sub-fractions, among which GMSHp3-3 exhibited the highest hydroxyl radical scavenging effect. Fraction GMSHp3-3 was further purified via RP-HPLC-ESI-MS/MS and sequenced as six potential antioxidant peptides with amino acids sequences of RMSFPVMCRN, LMRVLAQLG, ALAPLVALPAA, LVGKPAPD, LPAAHKA and AHAAGYGG, among which LMRVLAQLG, LPAAHKA and AHAAGYGG possessed effective ferric reducing power. These results indicated that novel antioxidant peptides from golden melon seeds protein hydrolysates might be potential antioxidant source of functional foods or nutraceutical supplements.     

Calcium-binding peptides derived from tilapia (Oreochromis niloticus) protein hydrolysate

Calcium-binding peptide was derived from protein hydrolysates. In this study, tilapia protein at a concentration of 2 % (w/v) was hydrolyzed using various proteases including Alcalase 2.4 L, Flavourzyme 1,000L, Protease GN, and papain at 50 °C, pH 8 for 6 h. It was found that the degree of hydrolysis increased with the time of the incubation in all cases. The highest calcium-binding capacity of the hydrolysate was 65 mg/g protein at 27.7 % degree of hydrolysis by Alcalase 2.4 L. The molecular weight of the calcium-binding peptides characterized by gel-filtration chromatography on a Sephadex G-25 was 1.2 kDa. The calcium-binding motif of the hydrolyzed peptides identified by the automated Edman degradation was a short peptide (Trp-Glu-Trp-Leu-His-Tyr-Trp). The results of this study suggested that tilapia protein is a good source for calcium-binding peptides.