Preparation of sesame peptide and evaluation of antibacterial activity on typical pathogens

The antibacterial activity of sesame peptides on common pathogen was evaluated. The preparation of sesame protein hydrolysate was carried out in an enzymatic membrane reactor followed by fractionation through several ultrafiltration stages to obtain desired peptide fractions and evaluation of their antibacterial activity using two pathogens namely, Pseudomonas aeruginosa and Bacillus subtilis. Potential sesame peptides were characterized by mass spectroscopy (TOF-MS) followed by determination of their corresponding amino acid compositions. Sesame peptide fraction of molecular mass less than 1 kDa exhibited significant inhibition against growth of P. aeruginosa as compared to B. subtilis. Thus, these findings confirm the bacteriostatic effect of sesame peptide on growth of pathogens which can be exploited in bacteriostat composition.     

Selective separation and characterisation of dual ACE and DPP-IV inhibitory peptides from rainbow trout (Oncorhynchus mykiss) protein hydrolysates

Microwave pretreatment and hydrolysis were applied to rainbow trout (Oncorhynchus mykiss) by-products to produce bioactive peptides with dual in vitro angiotensin-I converting enzyme (ACE) and dipeptidyl-peptidase IV (DPP-IV) inhibitory activities. Peptides were fractionated using the single step electrodialysis with ultrafiltration membrane (EDUF). Concentration of cationic peptides (CP) increased in the recovery solution, reaching 125 μg mL⁻¹ after a 4-h treatment with migration rate of 15.68 ± 2.98 g m⁻² h. CP fractions displayed ACE and DPP-IV I inhibitory properties, with IC₅₀ values of 0.0036 mg mL⁻¹ and 1.23 mg mL⁻¹ respectively. The bioactivity was attributed to the low molecular weight peptides (300–500 Da) recovered. CP exhibited non-competitive inhibition patterns for ACE and DPP-IV, which were dose dependent. These results showed that bioactive peptides can successfully be separated from complex hydrolysate mixtures by EDUF. The fractionated peptides can serve as potential functional food ingredients or nutraceuticals for the management of hypertension and diabetes.     

Recovery of valuable peptides from marine protein hydrolysate by electrodialysis with ultrafiltration membrane: impact of ionic strength

Electrodialysis with ultrafiltration membrane (EDUF) has been used to selectively separate and concentrate peptides from protein hydrolysates. Therefore, comprehension and optimization of electrodialytic parameters involved in electrodialysis with ultrafiltration membrane (EDUF) are crucial for its industrial application. The aim of the present work was to study the effect of KCl concentrations (1, 3 and 5 g/L) in recovery compartment on local electric field (LEF), total charge transport, energy consumption, peptide migration rate and selectivity during EDUF of snow crab by product hydrolysate (SCBH). The highest peptide migration rate (13.76 ± 3.64 g/m².h) with the lowest relative energy consumption (132.85 ± 15.67 W.h/g) was recovered for the first time to our best knowledge in EDUF process whatsoever the KCl conditions. Furthermore, constant LEF and linear peptide migration rate were obtained for all the conditions tested by regulating the conductivities to their initial values during the EDUF process. More than 85% of total peptides in KCl fractions had molecular sizes that ranged from 300 to 600 Da. In addition, KCl fractions were enriched with cationic peptides containing arginine and lysine and/or free arginine (Arg) and lysine (Lys). Furthermore, the relative abundance of Arg increased by 48%, 99% and 114% and that of Lys by 87%, 150% and 180% respectively in 1, 3 and 5 g/L KCl concentrations in comparison to SCBH. An increase in ionic strength amplified the negative charge density of UF membrane. This increase in negative charge density would induce (1) an increase in effective pore size due to the electrostatic repulsion between ions and (2) a thinner hydration layer due to salting out effect at pore walls.     

A novel angiotensin I converting enzyme inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in spontaneously hypertensive rats

Tuna frame protein was hydrolysed using Alcalase, Neutrase, pepsin, papain, α-chymotrypsin and trypsin. Peptic hydrolysate exhibited the highest ACE I inhibitory activity among them and was fractionated into three ranges of molecular weight (below 1, 1–5 and 5–10 kDa) using an ultrafiltration membrane bioreactor system. The 1–5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. During consecutive purification, a potent ACE inhibitory peptide from tuna frame protein (PTFP), which was composed of 21 amino acids, Gly-Asp-Leu-Gly-Lys-Thr-Thr-Thr-Val-Ser-Asn-Trp-Ser-Pro-Pro-Lys-Try-Lys-Asp-Thr-Pro (MW: 2,482 Da, IC₅₀: 11.28 μm), was isolated. Lineweaver–Burk plots suggest that PTFP plays as a non-competitive inhibitor against ACE. Furthermore, antihypertensive effect in spontaneously hypertensive rats (SHR) also revealed that oral administration of PTFP can decrease systolic blood pressure significantly (P < 0.01). These results suggest that the PTFP would be a beneficial ingredient for nutraceuticals and pharmaceuticals against hypertension and its related diseases.     

Value-added utilization of yak milk casein for the production of angiotensin-I-converting enzyme inhibitory peptides

Yak (Bos grunniens) milk casein derived from Qula, a kind of acid curd cheese from northwestern China, was hydrolysed with alcalase. The hydrolysates collected at different hydrolysis times (0 min, 60 min, 120 min, 180 min, 240 min, 300 min, 360 min) were assayed for the inhibitory activity of angiotensin-I-converting enzyme (ACE), and the one obtained at 240 min hydrolysis showed the highest ACE inhibitory activity. The active hydrolysate was further consecutively separated by ultrafiltration with 10 kDa and then with 6 kDa molecular weight cut-off membranes into different parts, and the 6 kDa permeate showed the highest ACE-inhibiting activity. This active fraction was further purified to yield two novel ACE-inhibiting peptides, whose amino acid sequences were Pro–Pro–Glu–Ile–Asn (PPEIN)(κ-CN; f156–160) and Pro–Leu–Pro–Leu–Leu (PLPLL) (β-CN; f136–140), respectively. The molecular weight and IC₅₀ value of the peptides were 550 Da and 566.4 Da, and 0.29 ± 0.01 mg/ml and 0.25 ± 0.01 mg/ml, respectively.     

Enhancing the anti-adipogenic activity of soy protein by limited hydrolysis with Flavourzyme and ultrafiltration

Limited hydrolysis of soy protein isolate (SPI) with Flavourzyme for 2 h to obtain the hydrolysate (FH2h) revealed much higher suppression of glycerol-3-phosphate dehydrogenase (GPDH) activity and relative lipid accumulation (RLA) than intact SPI in 3T3-L1 preadipocytes during differentiation. Lower GPDH activity or RLA indicates higher anti-adipogenic activity. The GPDH significantly decreased from 673 to 477 U/mg protein (p < 0.05). Sequentially fractionating FH2h with 30–1 kDa (kilo-daltons) molecular weight cut-off (MWCO) membranes to obtain the 1 kDa permeate resulted in further reduction of 59% GPDH activity. When comparing the high-performance size-exclusion chromatography (HPSEC) profiles, the most active peptide fraction for the anti-adipogenic activity was primarily composed of small peptides with molecular weight less than 1300 Da. According to the Western immunoblot analysis, 1 kDa permeate inhibits adipogenesis by affecting the expression of peroxisome proliferators-activated receptor γ (PPARγ) and the CCAAT/enhancer binding protein α (C/EBPα) during 3T3-L1 cells differentiation.     

Angiotensin I-converting enzyme inhibitor derived from soy protein hydrolysate and produced by using membrane reactor

Five different proteolytic enzymes, including Alcalase, Flavourzyme, trypsin, chymotrypsin and pepsin were employed to hydrolyze isolated soy protein (ISP) to produce the hydrolysates, respectively. The result indicated that hydrolysis of ISP for 0.5–6 h with Alcalase produced the highest ACE inhibitory activity. Therefore, Alcalase was selected for further study on optimization of hydrolysis conditions. The optimum conditions for Alcalase to hydrolyze ISP to produce the lowest IC₅₀ value were: E/S = 0.01, hydrolysis temperature = 50 °C, pH 9.0 and hydrolysis time = 6 h. Under these conditions, the IC₅₀ value of ISP was significantly reduced from 66.4 to 0.67 mg protein/ml. The lower IC₅₀ value represented the higher the ACE inhibitory activity. Moreover, several membranes with molecular weight cut-offs (MWCFs) of 1000–30,000Da were used to filter the hydrolysate. The 10 kDa permeate obtained from the treatment of the hydrolysate by 10,000 Da MWCF membrane could further reduce its IC₅₀ value from 0.668 to 0.078 mg protein/ml with a peptide recovery of 67.5%. An operation stability study showed that the membrane reactor system could maintain a steady production of ISP hydrolysate for over 8 h. The in vitro effect of gastrointestinal protease on ACE inhibitory activity of 10 kDa permeate was also investigated. The results suggested that gastrointestinal proteases have very little effect on the ACE inhibitory activity of 10 kDa permeate.     

Casein hydrolysis by Bifidobacterium longum KACC91563 and antioxidant activities of peptides derived therefrom

Milk protein is a well-known precursor protein for the generation of bioactive peptides using lactic acid bacteria. This study investigated the antioxidant activity of bovine casein hydrolysate after fermentation with Bifidobacterium longum KACC91563 using the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay and total phenolic content (TPC). The antioxidant activities of the 24-h and 48-h hydrolysates were higher than that of the 4-h hydrolysate (2,045.5 and 1,629.3 μM gallic acid equivalents, respectively, vs. 40.3 μM) in the ABTS assay. In contrast, TPC values showed activities of 43.2 and 52.4 μM gallic acid equivalents for the 4-h and 24-h hydrolysates, respectively. Three fractions (≥10 kDa, ≥3 but <10 kDa, and <3 kDa) were separated from the 24-h hydrolysate by ultrafiltration. Among these fractions, the <3 kDa fraction exhibited the highest antioxidant activity (936.7 μM) compared with the other fractions (42.1 and 34.2 μM for >10 kDa and 3–10 kDa fractions, respectively). Through liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, 2 peptides, VLSLSQSKVLPVPQK and VLSLSQSKVLPVPQKAVPYPQRDMPIQA, containing the fragment VLPVPQ that has antioxidant properties, were identified in the <3 kDa fraction after 24 h of hydrolysis. The present study demonstrates the possibility of antioxidant peptide production from bovine casein using Bifidobacterium longum.     

Purification and characterisation of antibacterial peptide-containing compound derived from palm kernel cake

Palm kernel cake (PKC), the most useful by-product resulted from palm kernel oil production. In this study, PKC-derived protein product was found suitable for use as an antimicrobial agent with potent antibacterial activity, particularly against Bacillus species, after enzymatic hydrolysis with alcalase. The hydrolysate was further purified by gel filtration chromatography. The purified fraction was found to have 14.63 ± 0.70% (w/w) protein, a molecular mass of 2.4 kDa and low hemolytic activity (<50% hemolysis of human erythrocytes at concentration of 1000 μg/ml). The presence of lysine and the major component lauric acid derivative, as indicated by electrospray ionisation-mass spectrometry (ESI-MS) direct infusion and nuclear magnetic resonance (NMR) spectroscopy, may have contributed to the antibacterial effect of purified PKC fraction. This study suggests that the antibacterial PKC compound may be not a pure peptide but instead a peptide-containing compound high in lauric acid derivative.     

大豆肽的超滤分离及其清除自由基活性研究

采用不同截留相对分子质量的超滤膜进行超滤分离大豆分离蛋白酶解液,研究超滤过程中操作压力、料液质量浓度、超滤时间对超滤膜通量的影响,确定最佳超滤条件,并测定各超滤级分清除·OH与DPPH·的能力。结果表明:在操作压力0.2 MPa、料液质量浓度50 g/L、通过相对分子质量为10、3、1 k D超滤膜的操作时间80 min、通过相对分子质量为5 k D的超滤膜的操作时间100min的超滤条件下分离大豆肽,超滤效果较好;相对分子质量小于3 k D的小分子大豆肽具有较高的自由基清除率,且对于·OH与DPPH·的清除率具有较高的一致性。     

Antioxidant properties of papain hydrolysates of wheat gluten in different oxidation systems

Enzymatic hydrolysis was used for preparing hydrolysates from wheat gluten which is by-product during production of wheat starch. The enzyme used for the hydrolysis was papain. The hydrolysate was separated based on the molecular weight of the peptides by membrane ultrafiltration (UF) with a molecular weight cut-off of 5 kDa into permeate (P) and retentate (5-K) fractions. The antioxidative activities of the hydrolysate and its UF fractions were investigated by using the TBA method and scavenging effect of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The three fractions showed strong antioxidative activities in the linoleic acid oxidation system, and exhibited DPPH radical scavenging activity. The antioxidative activity of the P fraction was almost the same as that of vitamin E at pH 7.0. The molecular weight distribution of the P fraction was concentrated in 4.2 kDa (86.5%) after gel permeation chromatography fractionation using an HPLC system.The P and 5-K fractions had higher surface hydrophobicities (H₀) at pH7.0 compared with the hydrolysate. The resulting UF fractions were superior to the hydrolysate in terms of antioxidative activities.     

Isolation and identification of two novel umami and umami-enhancing peptides from peanut hydrolysate by consecutive chromatography and MALDI-TOF/TOF MS

Peanut hydrolysate produced by crude protease extract from Aspergillus oryzae HN 3.042 was found to elicit intense umami and umami-enhancing effect. Taste profiles, amino acid and organic acid composition of peanut hydrolysate and its separation fractions by ultrafiltration were evaluated. The results revealed that peanut hydrolysate was mainly low molecular weight compounds. Fractions of 1-3 kDa and below 1 kDa prominently contributed to the umami taste and umami-enhancing effect of the peanut hydrolysate. The two fractions were further purified, using gel filtration chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC), in combination with sensory evaluation, to obtain a umami peptide and umami-enhancing peptide. The active peptides were identified as Ser-Ser-Arg-Asn-Glu-Gln-Ser-Arg (SSRNEQSR, 963.9 Da) and Glu-Gly-Ser-Glu-Ala-Pro-Asp-Gly-Ser-Ser-Arg (EGSEAPDGSSR, 1091.1 Da), by MALDI-TOF/TOF MS, respectively.     

Angiotensin-I-converting enzyme (ACE)-inhibitory and antihypertensive properties of squid skin gelatin hydrolysates

Gelatin extracted from squid (Dosidicus eschrichitii Steenstrup) skin was hydrolysed with pepsin to prepare Angiotensin-I-Converting Enzyme (ACE) inhibitory peptide. The ACE-inhibitory activity was measured by spectrophotometric assay. The hydrolysate was fractionated into three ranges of molecular weight (6 kDa < HSSG-I < 10 kDa, 2 kDa < HSSG-II < 6 kDa, HSSG-III < 2 kDa) using an ultrafiltration unit. The HSSG-III showed the most potent ACE inhibitory activity in vitro with IC₅₀ of 0.33 mg/ml. Renovascular hypertensive rats (RHR) model was made with two-kidney one clip assay, and antihypertensive effects were studied in RHR treated with HSSG-III for 30 days by oral administration. Arterial blood pressure were measured respectively. The HSSG-III remarkably reduced the arterial blood pressure of RHR. These results suggested that hydrolysate of squid skin gelatin obtained by treatment with pepsin was a good source of peptides with ACE-inhibitory activity and had an antihypertensive effect by oral administration.     

Characterization of antioxidant activity and volatile compounds of Maillard reaction products derived from different peptide fractions of peanut hydrolysate

Five peptide fractions isolated from peanut hydrolysate were purified by ultrafiltration and gel filtration chromatography. Their reaction degrees with glucose or lecithin in Maillard reaction were compared. Glucose consumption, oxygen radical absorbance capacity (ORAC) values and volatile compounds composition of peanut hydrolysate (PH) and its peptide fractions after thermal treatment and Maillard reaction were evaluated. Results revealed that the peptide fraction of 1-3 kDa showed the highest antioxidant activity. The peptides with smaller molecular weight showed a higher reaction degree to increase antioxidant activity and to produce more volatile compounds through Maillard reaction. Analysis of molecular weight distribution showed that peptide degradation and cross-linking simultaneously occurred during the Maillard reaction. In addition, lecithin addition to peptide-glucose system caused a significant increase of antioxidant activity possibly due to more nitrogen-containing compounds formed.     

Low molecular weight flaxseed protein-derived arginine-containing peptides reduced blood pressure of spontaneously hypertensive rats faster than amino acid form of arginine and native flaxseed protein

Flaxseed protein isolate (FPI) contains high amount of arginine, which plays important physiological roles especially as nitric oxide precursor in the vascular endothelium. Arginine-rich peptides can be generated from FPI and used as a source of nitric oxide, which can produce in vivo vasodilatory effects during hypertension. Enzymatic hydrolysis of FPI with trypsin and pronase resulted in a hydrolysate that was fractionated using electrodialysis-ultrafiltration (EDUF). EDUF experiment resulted in migration of peptides to the anionic and cationic recovery compartments. Compared to FPI with 11% arginine, about one-third of the cationic fraction was composed of arginine. Thirteen potential peptide sequences were identified to be present in the cationic compartment of which 12 contained at least one arginine residue. None of the peptides identified from the anionic compartment contained arginine. Oral administration of the cationic peptides (200 mg/kg body wt.) to spontaneously hypertensive rats resulted in a more rapid decrease in systolic blood pressure when compared to similar amounts of FPI or the amino acid form of arginine. It was concluded that the rapid effect of the arginine-rich peptide product suggests faster rate of peptide absorption than amino acids and this may be exploited to provide fast relief from hypertension.     

Enhancing the lipolysis-stimulating activity of soy protein using limited hydrolysis with Flavourzyme and ultrafiltration

The aim of this study was to explore the lipolysis-stimulating activity of soy protein isolate (SPI) hydrolysate using 3T3-L1 adipocytes. Intracellular triglyceride residue (TR) was employed as a marker for lipolysis in cells. The lower TR represents the better lipolysis-stimulating activity. SPI was hydrolysed with Flavourzyme for 2 h to obtain the hydrolysate FH2h, which showed lipolysis-stimulating activity in adipocytes at 400–1600 ppm levels. The sequential fractionation of FH2h with 30–0.3 kDa molecular weight cut-off (MWCO) membranes in order to obtain a 1 kDa retentate resulted in further enhancement of its lipolysis-stimulating activity in the cells. The TR decreased significantly from 2.73 to 2.30 μmole/mg protein at the 400 ppm level (p < 0.05). Based on the western immunoblot and immunostaining analysis, the 1 kDa retentate promotes lipolysis by increasing phosphorylation and translocation of the hormone-sensitive lipase in 3T3-L1 adipocytes.     

Preparation and antioxidant activity of enzymatic hydrolysates from purple sea urchin (Strongylocentrotus nudus) gonad

Purple sea urchin (Strongylocentrotus nudus) gonad was treated separately with neutral protease, papain, pepsin and trypsin. The resultant hydrolysates were fractionated using a series of ultrafiltration membranes (molecular weight cut-offs of 10, 5, 3 and 1 kDa). Five fractions were prepared from each hydrolysate and the corresponding molecular weight ranges were below 10 kDa, 5-10 kDa, 3-5 kDa, 1-3 kDa and below 1 kDa. The peptide fractions were evaluated for antioxidant activity by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and reducing power assay. Results indicated that all peptide fractions possessed DPPH radical scavenging capacity and reducing power in a dose-dependent manner. For all four hydrolysates, the below 1 kDa fractions exhibited the highest DPPH radical scavenging capacity. The below 1 kDa fractions prepared with neutral protease, papain and pepsin, and the 1-3 kDa fraction prepared with trypsin showed the highest reducing capacity among corresponding hydrolysates.     

Production and fractionation of tuna by-product protein hydrolysate by ultrafiltration and nanofiltration: Impact on interesting peptides fractions and nutritional properties

The production of fish protein hydrolysates (FPH) is a promising route to add value to fish by-products due to their potential application as a source of interest peptide fractions. Using Alcalase for hydrolysis of tuna dark muscle by-product, the influence of enzyme/substrate ratio and hydrolysis time on the rate of interest peptide fractions (1–4 kDa) was studied. The rate of this fraction obtained under optimized conditions (temperature 55 °C, pH 8.5, enzyme/substrate ratio of 1% and 60 min of hydrolysis reaction) was 26%. The performance of the combined process associating ultrafiltration and nanofiltration membranes was evaluated for fractionation of produced hydrolysate in order to isolate fractions enriched in peptides of specific molecular weight (MW). The interest peptide fraction (1–4 kDa) was isolated and the positive effect of diafiltration on peptide purification process was underlined. The peptide fractions produced have a high nutritional quality which can be used in human nutrition and they have a potential for applications in aquaculture diets.     

Studies on purification and the molecular mechanism of a novel ACE inhibitory peptide from whey protein hydrolysate

This study sought to purify and identify a novel angiotensin I-converting enzyme (ACE) inhibitory peptide from whey protein hydrolysed by trypsin. The peptide’s amino acid sequence, as well as the molecular mechanism of the interactions between the peptide and the ACE, were also studied. Using ultrafiltration, the hydrolysate was separated into three fractions. The fraction with molecular weight of <6 kDa had the greatest ACE inhibitory activity and was further separated by size exclusion chromatography on Sephadex G-25 and G-10 columns. Reverse-phase high performance liquid chromatography (RP-HPLC) was used to separate the most active fraction. The amino acid sequence of the peptide with the greatest ACE inhibitory characteristics was confirmed as Leu–Leu (LL). The molecular mechanisms, position, type, and energy of the LL/ACE interaction were investigated by using flexible molecule docking technology.     

Characteristic and antioxidant activity of retorted gelatin hydrolysates from cobia (Rachycentron canadum) skin

Alkali-pretreated cobia (Rachycentron canadum) skin was extracted in a retort (121 °C) for 30 min to obtain a retorted skin gelatin hydrolysate (RSGH). The molecular mass distributions and antioxidant activities of cobia RSGH and enzyme-treated RSGHs (ET-RSGHs) derived from bromelain, papain, pancreatin, and trypsin digestion were then characterized. The molecular mass distribution of the RSGH ranged mainly between 20,000 and 700 Da and those of ET-RSGHs ranged between 6500 and 700 Da. The DPPH (α,α-diphenyl-β-picrylhydrazyl) radical scavenging effects (%) of 10 mg/ml of RSGH and 10 mg/ml of the four ET-RSGHs were 55% and 51–61%, respectively. The lipid peroxidation inhibition (%) of RSGH and ET-RSGHs (10 mg/ml) were 58% and 60–71% on the fifth day in a linoleic acid model system, respectively. The 3Kd-ET-RSGHs, obtained by using a series of centrifugal ultrafiltration filters (molecular weight cut-offs of 10, 5, and 3 kDa done sequentially with decreasing pore size), exhibited dramatically improved antioxidant activity, with most of the molecular mass ranging below 700 Da. Compared to 10 mg/ml of the RSGH, 10 mg/ml of 3Kd-ET-RSGHs exhibited 45–65% more scavenging of DPPH radical and 24–38% more inhibition of lipid peroxidation. The peptides with molecular masses below 700 Da in the ET-RSGHs or 3Kd-ET-RSGHs significantly affect the antioxidant properties. These peptides are composed of a small number of amino acids or free amino acids and have the potential to be added as antioxidants in foods.