Antiproliferative activity of peptides prepared from enzymatic hydrolysates of tuna dark muscle on human breast cancer cell line MCF-7

To produce and identify antiproliferative peptides, two commercial enzymes, papain (PA) and Protease XXIII (PR) were used to hydrolyse tuna dark muscle byproduct, and the protein hydrolysates were purified, before being evaluated for antiproliferative activities against human breast cancer cell line MCF-7. The results showed that the peptide fractions with the molecular weight ranging from 390 to 1400 Da possessed the greatest antiproliferative activity. The amino acid sequences of the two antiproliferative peptides isolated from PA and PR hydrolysates were Leu-Pro-His-Val-Leu-Thr-Pro-Glu-Ala-Gly-Ala-Thr (1206 Da) and Pro-Thr-Ala-Glu-Gly-Gly-Val-Tyr-Met-Val-Thr (1124 Da), whilst they show the dose-dependent inhibition effect of the MCF-7 cells with IC₅₀ values of 8.1 and 8.8 μM, respectively. We thus conclude that antiproliferative hydrolysates from tuna dark muscle byproduct may be useful ingredients in food and nutraceutical applications.     

Purification and characterisation of antioxidative peptides from enzymatic hydrolysates of venison protein

To prepare antioxidative peptides from venison protein hydrolysates (APVPH), six proteases were employed for enzymatic hydrolysis, and the antioxidative activities of the hydrolysates were investigated using a free radical scavenging assay. Among the hydrolysates, papain hydrolysates which had the highest free radical scavenging activity were further separated into four groups and purified using consecutive chromatographic methods. Finally, two antioxidative peptides were obtained, and their sequences identified as Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly (APVPH I) and Asp-Leu-Ser-Asp-Gly-Glu-Gln-Gly-Val-Leu (APVPH II). The free radical scavenging activity of APVPH I was higher than that of APVPH II, and the IC₅₀ values of the hydroxyl, DPPH, superoxide, and peroxyl radical scavenging activities were 44, 77, 217, and 85 μg/ml, respectively. These results indicate that enzymatic hydrolysates of venison protein possess potent antioxidative activity.     

Purification and characterisation of antioxidative peptides from unfractionated rice bran protein hydrolysates

Crude rice bran protein (CRBP) was prepared by alkaline extraction and then treated with 0.6 m HCl to remove phytic acid. The phytate‐free rice bran protein (PFRBP) was hydrolysed with proteases M, N, S, P and pepsin under optimal conditions. Hydrolysates obtained from various hydrolysis periods were subjected to analysis for their degree of hydrolysis (DH) and functional properties. The hydrolysates were fractionated by reversed‐phase column chromatography on Kaseigel ODS resin (120–140 μm) using a stepwise gradient of aqueous ethanol, and their activities were measured. The 40% ethanol fraction of protease P 4 h‐hydrolysate was separated by successive reversed‐phase high‐performance liquid chromatography and the amino acid sequences of isolated antioxidative peptides were determined by a protein sequencer and matrix‐assisted laser desorption ionisation‐time of flight mass spectrometry. Crude rice bran protein had higher antioxidative activity than PFRBP, due to the presence of phytic acid. Phytate contents of rice bran, CRBP and PFRBP were 2.5%, 1.42% and 0%, respectively. The activity of PFRBP increased upon protease digestion. Protease M hydrolysates showed the highest DH, but the lowest antioxidative activity. Hydrolysates with DH below 10% had higher antioxidative activity than those above 20%. This result indicates that the antioxidative activity of the hydrolysates is inherent to their characteristics amino acid sequences of peptides depending on the protease specificities.     

Antioxidant and biochemical properties of protein hydrolysates prepared from Silver carp (Hypophthalmichthys molitrix)

The antioxidant and biochemical properties of enzymatically hydrolyzed silver carp (Hypophthalmichthys molitrix) protein were studied. The molecular weight of the main peaks of the hydrolysates by both Alcalase and Flavourzyme was lower than 5000 Da. The hydrolysates treated by Alcalase for ?1.5 h (hydrolysis time) showed that the relative proportion of <1000 Da fraction was more than 60%. For the biochemical properties, hydrolysis by both enzymes increased protein solubility to above 75% over a wide pH range; and when the hydrolysis time was prolonged (>3 h), the colour of the hydrolysates turned yellowish. The protein hydrolysates exhibited significant hydroxyl radical-scavenging activity and inhibited linoleic acid peroxidation. For Alcalase treatment, the hydroxyl radical-scavenging activity and the inhibition of linoleic acid peroxidation of hydrolysates appeared to reach a maximum level for 1.5, 2.0 h of hydrolysis, respectively; and their antioxidant activity was close to that of α-tocopherol in a linoleic acid emulsion system, and carnosine in the 2-deoxyribose oxidation system. The hydrolysate with lower molecular weight distribution possessed stronger Fe²⁺ chelation ability at a sample concentration of 5.0 mg/mL. The results suggested that the antioxidant activity of silver carp protein hydrolysates were related to its degree of hydrolysis (DH), hydrolysis time and molecular weight.     

Purification and characterization of antioxidative peptides derived from rice bran protein hydrolysates

Rice bran protein fraction (RBPF)—albumin, globulin, glutelin and prolamin were hydrolyzed with proteases M, N, P, S and pepsin under their optimal conditions for 24 h. Hydrolysates of various hydrolysis periods were collected and subjected to peptide mapping and the antioxidative activity measured by the 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) method. Protease M hydrolysates showed high degree of hydrolysis (DH), but low antioxidative activity. On the contrary, pepsin hydrolysates showed low DH with high activity. Albumin and globulin hydrolysates had higher DH values, but lower values for glutelin and prolamin. The globulin hydrolysate (Opep2) from 2 h-pepsin hydrolysis was separated by using three consecutive purification steps with RP-HPLC. Nineteen antioxidative peptides were isolated and their amino acid sequences were determined by a gas-phase protein sequencer and MALDI-TOF mass spectrometry. These peptides were composed of 6–30 amino acid residues with molecular masses ranging from 670–3,611 Da. Tyr-Leu-Ala-Gly-Met-Asn had the highest antioxidative activity among them.     

Compositions,functional properties and antioxidative activity of protein hydrolysates prepared from round scad (Decapterus maruadsi)

Composition, functional properties and antioxidative activity of a protein hydrolysate prepared from defatted round scad (Decapterus maruadsi) mince, using Flavourzyme, with a degree of hydrolysis (DH) of 60%, were determined. The protein hydrolysate had a high protein content (48.0%) and a high ash content (24.56%). It was brownish yellow in colour (L^∗ = 58.00, a^∗ = 8.38, b^∗ = 28.32). The protein hydrolysate contained a high amount of essential amino acids (48.04%) and had arginine and lysine as the dominant amino acids. Na⁺ was the predominant mineral in the hydrolysate. The protein hydrolysate had an excellent solubility (99%) and possessed interfacial properties, which were governed by their concentrations. The emulsifying activity index of the protein hydrolysate decreased with increasing concentration (p < 0.05). Conversely, the foaming abilities increased as the hydrolysate concentrations increased (p < 0.05). During storage at 25 °C and 4 °C for 6 weeks, the antioxidative activities and the solubility of round scad protein hydrolysate slightly decreased (p < 0.05). Yellowness (b^∗-value) of the protein hydrolysate became more intense as the storage time increased but the rate of increase was more pronounced at 25 °C than at 4 °C.     

The effects of pretreatments on antioxidative activities of protein hydrolysate from the muscle of brownstripe red snapper (Lutjanus vitta)

Different pretreatments of mince from brownstripe red snapper (Lutjanus vitta) including 1) washing; 2) membrane separation; 3) washing followed by membrane separation and 4) membrane separation followed by washing were conducted prior to hydrolysis. Among the resulting minces, that subjected to membrane separation with subsequent washing (MS/W) contained the lowest remaining myoglobin content, phospholipid content, heme iron and non-heme iron contents (p < 0.05) and showed the lowest TBARS values throughout 9 days of storage at 4 °C in the presence and absence of 0.15 mol L⁻¹ cupric acetate (p < 0.05). When hydrolysates from 1) mince, 2) MS/W and 3) protein isolate from MS/W (PI) with different degree of hydrolysis (DH) (20, 30 and 40%) were prepared using proteases from pyloric caeca of brownstripe red snapper, antioxidative activities determined by DPPH, ABTS radical scavenging activities, ferric reducing antioxidant power and metal chelating activity varied with hydrolysates and DH. Antioxidative activities increased with increasing DH up to 40% (p < 0.05). At all DH tested, hydrolysate prepared from MS/W exhibited the highest antioxidative activities determined by all assays, compared to those from mince and PI (p < 0.05). Hydrolysate from MS/W with 40% DH had the molecular weight lower than 6.5 kDa as determined by SDS-PAGE. In liposome oxidation system, the addition of hydrolysate from MS/W resulted in the lower TBARS, compared with the control throughout the incubation period of 48 h at room temperature (25-28 °C). Therefore, fish mince with membrane separation followed by washing was the most appropriate source for production of hydrolysate possessing antioxidative activity with the lowered amount of lipids and pro-oxidants.     

Purification and characterisation of an antioxidative peptide from enzymatic hydrolysates of duck processing by-products

Duck processing by-products were hydrolysed using eight proteases to produce an antioxidative peptide. Of the various hydrolysates produced, the pepsin extract exhibited the highest hydroxyl radical scavenging activity. The derived peptide was purified using high-performance liquid chromatography (HPLC) to identify any potent radical scavenging activity. The sequence of the antioxidative peptide obtained was identified as Asp-Val-Cys-Gly-Arg-Asp-Val-Asn-Gly-Tyr, with a molecular weight of 1096 Da. The IC₅₀ value of purified peptide for hydroxyl radical scavenging activity was 75 μg/ml as the measurement by an electron spin resonance (ESR) spectrometer. In addition, the purified peptide exhibited a protective effect on H₂O₂-induced DNA damage. These results indicate that the purified peptide possesses a potent antioxidative activity and protective effect of DNA against H₂O₂-induced oxidative damage.     

Protein hydrolysis by protease isolated from tuna spleen by membrane filtration: A comparative study with commercial proteases

Membrane filtration is considered to be a low-cost and large scale method for recovery and purification of enzymes. The trypsin-like serine protease (TSP) was recovered and purified by microfiltration and ultrafiltration from the extract of the spleen of the yellowfin tuna (Thunnus albacores). The partially purified TSP has the activity of 96.5 U/mL and purity of 74.2 U/mg protein based on casein digesting unit. It has the molecular weight of 24 kDa proved by SDS-PAGE. The potential application of TSP in the protein hydrolysis was investigated in comparison to the use of two commercial proteases, i.e. Alcalase^® 0.6 L and Delvo-Pro under their optimal hydrolysis conditions. The effects of enzymes, substrates and enzyme to substrate ratio on the degree of hydrolysis (DH) were studied. Alcalase showed the highest DH for both casein and soybean isolate. TSP showed higher DH than Delvo-Pro when casein was employed as the substrate. However, TSP showed the lowest DH when soybean protein was used as the substrate. The present study proved that TSP recovered and purified from the tuna canning waste by membrane filtration can be used for protein hydrolysis as well as other commercial proteases.     

Bioavailability of selenium in the defatted dark muscle of tuna

The content and bioavailability of selenium (Se) in the dark muscle of tuna (DMT) were studied. Fluorometric analysis showed that DMT contained 2.0-4.7 µg g^-1 Se. A large part of Se of the DMT was recovered in the dry powder of the defatted fraction prepared by successive treatment with acetone and n-hexane/ethanol. On trypsin digestion of the defatted DMT, release of Se paralleled that of nitrogen and about 70% of Se was released within 4 h. Male weanling ddY mice were fed a Torula yeast-based Se-deficient diet (basal diet) for 3 weeks, and then fed the basal diet or a diet supplemented with a 0.05-0.25 µg g^-1 Se as either sodium selenite or the defatted DMT for a further 1 week. Se contents and GSHPx activities in the liver increased gradually with increases in the amount of supplemented Se. No differences were observed between selenite and defatted DMT in the effect on Se content. At low Se levels, the effect of Se in the defatted DMT on the liver GSHPx activities was inferior to that of selenite, but at a high Se level, Se in the defatted DMT showed a greater effect. The bioavailability of Se in the defatted DMT, as assessed by slope ratio analysis using selenite as the reference Se, was 87% for the liver Se content and 168% for the GSHPx activity. The results indicate that the defatted DMT contains high levels of Se in a nutritionally available form.     

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.     

鹅血红蛋白抗氧化活性肽的制备及工艺研究

以新鲜鹅血为原料提取血红蛋白,利用碱性蛋白酶、中性蛋白酶及风味蛋白酶来酶解鹅血红蛋白,采用甲醛滴定法测定鹅血红蛋白水解液的水解度,通过研究水解液对DPPH自由基清除能力来检测水解产物的抗氧化活性,并分析了酶解温度、时间、pH及酶与底物比对鹅血红蛋白水解产物的影响.结果表明,采用中性蛋白酶获得的酶解产物抗氧化活性最高.在中性蛋白酶的作用下,通过单因素及正交实验,优化出产物抗氧化活性较高的酶解工艺条件:温度50℃,酶解时间8h,pH7.0,酶与底物比8000U/g,在最佳酶解条件下,产物对DPPH自由基的清除率为89.6％.     

水解蛋白来源的抗氧化肽研究进展

采用水解技术制备抗氧化肽可大大拓宽其来源。本文介绍了影响水解技术制备抗氧化肽的因素，综述了国内外在这一领域的研究进展，并展望了此种方法制备抗氧化肽产业化的可能性。     

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.     

Purification and characterization of antioxidative peptide derived from muscle of conger eel (Conger myriaster)

The tryptic hydrolysate of conger eel (Conger myriaster) muscle protein was fractionated according to the molecular weights using ultrafiltration (UF) membrane system. The lowest molecular weight fraction (<1 kDa) with higher antioxidative properties was purified using consecutive chromatographic techniques and designated conger eel antioxidative peptide (CEAP). Sequence determination revealed that it contained nine amino acids in its sequence (LGLNGDDVN) and the molecular mass was identified to be 928 Da. CEAP performed better than the natural antioxidant, α-tocopherol for the prevention of lipid peroxidation in vitro. Additionally, it scavenged hydroxyl radicals and carbon-centered radicals at IC₅₀ values of 74.1 μM and 78.5 μM, respectively. Therefore these results suggested that the peptides derived from conger eel protein hydrolysates are responsible for higher antioxidative properties and molecular weight as well as presence of hydrophobic amino acids highly contributed for their antioxidation.     

Purification and identification of immunomodulating peptides from enzymatic hydrolysates of Alaska pollock frame

To prepare immunomodulating peptides from Alaska pollock frame (APF) hydrolysates, trypsin was employed for enzymatic hydrolysis, and the immunomodulating activities of the hydrolysates were studied using splenic lymphocytes proliferation assay. The highest activity of the hydrolysates was reached when DH ranged from 15% to 18%. The peptide fractions which exhibited the highest activity were further purified using ion exchange chromatography, gel filtration chromatography, and RP-HPLC. The peptides were identified using nano-LC-ESI mass spectrometry. Finally, three immunomodulating peptides were obtained, and the amino acid sequences were Asn-Gly-Met-Thr-Tyr, Asn-Gly-Leu-Ala-Pro, and Trp-Thr, respectively. The lymphocyte proliferation rates were 35.92%, 32.96%, and 31.35% in the presence of 20 μg/ml purified peptides, respectively. Therefore, the results demonstrated that the APF proteins hydrolysates prepared by trypsin could serve as a source of peptides with immunomodulating activity. It provided a scientific basis for the preparation of immunomodulating peptides.     

蒸煮对金枪鱼肉及其蛋白质热变性的影响

试验测定了金枪鱼肉在蒸气加热过程中的传热曲线以及重量、水分含量和蛋白质组分随中心温度的变化情况.结果显示:中心温度随着加热时间的延长而不断上升,加热初期的升温速度较后期快;蒸煮损失随着中心温度的升高而不断增大,80 ℃时的失重率和失水率分别为19.86%和14.43%;蛋白质组分在加热过程中发生了明显变化,随着中心温度的上升,水溶性蛋白和盐溶性蛋白的含量急剧减少,碱溶性蛋白的含量不断增加,此外,碱不溶性蛋白在60 ℃时开始迅速减少,非蛋白氮的含量随着中心温度的上升也有少量增加.     

Production and functional characterisation of antioxidative hydrolysates from corn protein via enzymatic hydrolysis and ultrafiltration

Corn protein was hydrolysed by three microbial proteases and further separated by sequential ultra-filtration to 12 hydrolysate fractions which were investigated for free radical scavenging capacity and chelating activity. The oxygen radical absorbance capacity (ORAC) of the hydrolysates varied significantly between 65.6 and 191.4μmoles Trolox equivalents (TE)/g dried weight with a small peptide fraction (NP-F3) produced by neutral protease (NP) possessing the highest antioxidant activity. The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH()) scavenging activities of the hydrolysate fractions also varied significantly between 18.4 and 38.7μmoles TE/g. Two fractions (AP-F2 and AP-F3) produced by alkaline protease (AP) showed the strongest activity. However, no significant difference was detected on the chelating activity of the fractions. NP-F3, AP-F2, and AP-F3 were incorporated into ground beef to determine their effects on lipid oxidation during 15-day storage period. NP-F3 was the only fraction that inhibited lipid oxidation at both 250 and 500μg/g levels by as much as 52.9%.     

水酶法从花生中提取油与水解蛋白的研究

采用水酶法从花生中同时提取油与水解蛋白质。研究蛋白酶Alcalase用量和酶解时间对花生油与水解蛋白得率的影响。结果表明：在60℃时，1．5％(E／S)的酶作用5h总游离油得率及水解蛋白得率即可达91.3％和83.2％。工艺过程中所得乳状液经-24℃冻结随后在35℃解冻，再经3500r／min离心20min后，乳状液中油回收率可达92％。用光学显微镜观察冷冻温度对乳状液中油滴聚集状态的影响，发现随冷冻温度下降，乳状液中的油滴聚集加剧，粒径增大，乳状液稳定性下降。     

Antioxidative properties of peptides prepared from tuna cooking juice hydrolysates with orientase (Bacillus subtilis)

The antioxidative activity of isolated peptides from of tuna cooking juice hydrolysates by orientase was determined. The results showed that, among the hydrolysates obtained under different hydrolysis time, the highest antioxidative activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capacity were presented at 60 min. Then, the protein hydrolysate was subjected to a Sephadex G-25 gel filtration chromatography, and five major fractions were obtained. Peptide fractions ranging from 400 to 1500 Da showed the highest antioxidative activity among all fractions. The peptide fractions were further isolated using the two-step high performance liquid chromatography (HPLC-1 and HPLC-2), and six and three fractions were obtained, respectively. The final three antioxidative peptides comprised 4–10 amino acids sequences were observed, and the structures of the peptides were Pro-Val-Ser-His-Asp-His-Ala-Pro-Glu-Tyr (1305 Da), Pro-Ser-Asp-His-Asp-His-Glu (938 Da), and Val-His-Asp-Tyr (584 Da), respectively. We thus conclude that antioxidative hydrolysates from tuna cooking juice may be useful ingredients in food and nutraceutical applications.