Rapeseed Protein Hydrolysates Research Articles

Identification of dipeptidyl peptidase IV inhibitory peptides from rapeseed proteins

This paper aims to investigate the dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from rapeseed proteins. Results showed that rapeseed protein hydrolysates by simulated gastrointestinal digestion had DPP-IV inhibitory activities. Twenty rapeseed-derived peptides with potential DPP-IV inhibitory activity were selected by an in silico approach and 12 of them were determined with DPP-IV inhibitory activities. Finally, 4 peptides IPK, LPR, VPHL and IPNQT were successfully identified from the rapeseed protein hydrolysate by LC-MS/MS and IPK showed the highest DPP-IV inhibitory activity with IC50 of 44.40 ± 2.18 μmol/L or 46.28 ± 3.62 μmol/L with Gly-Pro-pNA or Gly-Pro-AMC as substrate, respectively. IPK behaved as a competitive inhibitor and was predicted to form attractive charge, hydrophobic and hydrogen bond interactions with the residues located in the S1 and S2 pocket of DPP-IV. This result indicates that rapeseed proteins are prominent sources for DPP-IV inhibitory peptides and have the potential for the management of type 2 diabetes.

Rapeseed Protein-Derived Antioxidant Peptide RAP Ameliorates Nonalcoholic Steatohepatitis and Related Metabolic Disorders in Mice.

Rapeseed protein hydrolysates have recently shown in vitro antioxidant and anti-inflammatory activities. However, scant data exist about their in vivo activities. Here, we report that the peptide DHNNPQIR (hereinafter referred to as RAP-8), a bioactive peptide originated from rapeseed protein, exhibits excellent in vivo efficacy in mouse models of nonalcoholic steatohepatitis (NASH) and hepatic fibrosis. We demonstrated that RAP-8 significantly reduced hepatic steatosis and improved insulin resistance and lipid metabolism. Furthermore, RAP-8 showed markedly reduced hepatic inflammation, fibrosis, liver injury, and metabolic deterioration. In particular, RAP-8 directly suppressed fibrosis-associated gene expression, including α-smooth muscle actin (α-Sma) and collagen type I (Col-1α) in the liver of mice in vivo. In addtion, RAP-8 significantly decreased macrophage infiltration and reduced pro-inflammatory cytokines secretion. Finally, we found that RAP-8 administration significantly decreased oxidative stress-induced apoptosis in liver injury induced by CCl4. Therefore, our results suggest that RAP-8 could be available for treatment of NASH and NASH-related metabolic disorders as a potential therapeutic candidate.

Rapeseed Protein-Derived Antioxidant Peptide Rap Ameliorates Nonalcoholic Steatohepatitis and Related Metabolic Disorders in Mice

Rapeseed protein hydrolysates have recently shown in vitro antioxidant and anti-inflammatory activities. However, scant data exist about their in vivo activities. Here we report that the peptide DHNNPQIR (hereinafter referred to as RAP-8), a natural peptide derived from rapeseed protein, has excellent in vivo efficacy in mouse models of nonalcoholic steatohepatitis (NASH) and liver fibrosis. We demonstrated that RAP-8 significantly reduced hepatic steatosis and improved insulin resistance and lipid metabolism. Furthermore, RAP-8 showed markedly reduced hepatic inflammation, fibrosis, liver injury, and metabolic deterioration. In particular, RAP-8 directly suppressed expression of fibrosis-related genes α-smooth muscle actin (α-Sma) and collagen type I (Col-1α) in the liver of mice in vivo. In addtion, RAP-8 significantly decreased macrophage infiltration and reduced secretion of proinflammatory cytokines. Finally, we found that RAP-8 administration significantly decreased oxidative stress-induced apoptosis in liver injury induced by CCl4. Therefore, our results suggest that RAP-8 could be an attractive therapeutic candidate for treatment of NASH and NASH-related metabolic deterioration. Funding Statement: The Program for Guangdong Introducing Innovative and Enterpreneurial Teams (2016ZT06Y337). X.J. supported by the Thousand Young Talents Program. Declaration of Interests: All authors participated in this study declare no potential, perceived, or real conflict of interest and have agreed to the content of this manuscript. Ethics Approval Statement: The experimental procedure used in this study met the guidelines of the Animal Care and Use Committee of the Sun Yat-sen University.

Digestibility, toxicity and metabolic effects of rapeseed and sunflower protein hydrolysates in mice

The digestibility (in vitro), toxicity and metabolic effects of rapeseed (RPH) and sunflower (SPH) protein hydrolysates have been evaluated in a murine animal model. The enzyme Alcalase® was employed to obtain a mild enzymatic hydrolysis of rapeseed and sunflower defatted seed meals (DSM) protein isolates. Both hydrolysates showed higher in vitro digestibility than the respective DSM, presumably as a consequence of the hydrolysis process that they had undergone. In vivo, RPH and SPH were well tolerated. Body and organ weights, biochemical blood parameters from treated male mice were comparable to controls. Food intake was regular in RPH and SPH animals, suggesting a good palatability of the hydrolysates. Not relevant perturbations of the principal hepatic and renal drug metabolism enzymes were observed in RPH or SPH mice. In conclusion, protein hydrolysates from sunflower and rapeseed DSM did not determine relevant toxicological effects; therefore, they could be considered as alternative protein sources and/or food ingredients.

Impact of Power Ultrasound on Antihypertensive Activity, Functional Properties, and Thermal Stability of Rapeseed Protein Hydrolysates

The effects of power ultrasound pretreatments on the degree of hydrolysis (DH), angiotensin-I-converting enzyme (ACE) inhibitory activity, amino acid composition, surface hydrophobicity, protein solubility, and thermal stability of ACE inhibition of rapeseed protein hydrolysates were evaluated. Ultrasonic pretreatments before enzymolysis in terms of power and exposure time increased the DH and ACE inhibitory activities over the control (without sonication). In this study, maximum DH 22.07% and ACE inhibitory activity 72.13% were achieved at 600 W and 12 min pretreatment. Compared to the hydrolysates obtained without sonication, the amino acid profile of ultrasound pretreated hydrolysates showed significant changes particularly in the proline content and hydrophobic amino acids with an increased rate of 2.47% and 6.31%, respectively. Ultrasound pretreatment (600 watts, 12 min) improved functional properties of protein hydrolysates over control by enhancing surface hydrophobicity and solubility index with an increased rate of 130.76% and 34.22%. Moreover, the stability test showed that the ACE inhibitory activity remains stable against heat treatments. However, extensive heat, prolonged heating time, and alkaline conditions were not in the favor of stability test, while under mild heat and acidic conditions their ACE inhibitory activities were not significantly different from unheated samples.

Study on Antioxidant Activity and Amino Acid Analysis of Rapeseed Protein Hydrolysates

Alkaline extraction followed by acid precipitation were employed to extract rapeseed protein and, Alcalase 2.4 L was used to obtain rapeseed protein hydrolysates. Three groups of rapeseed protein hydrolysates were obtained by purifying with membrane ultrafiltration and a Sephacryl S-100HR gel column. The antioxidant activities were then determined. Group 3 had the best antioxidant activities according to the oxygen radical absorbance capacity, peroxyl radical-scavenging capacity, and cellular antioxidant activity assays, with the following antioxidant values: an oxygen radical absorbance capacity value of 1610 ± 113 µmol TE/(g sample), a peroxyl radical-scavenging capacity value of 622 ± 30 mg VC/(100 g sample), and a cellular antioxidant activity value of 25 ± 2 µmol QE/(g sample) and corresponding EC50 value of 58 ± 3 µg/mL. Six peaks of group 3 were collected and well separated by reversed phase–high-performance liquid chromatography. Peak 5 were identified to exhibit a higher antioxidant activity, the amino acid sequence of which was found to be Trp-Ile (Leu)-Tyr, as determined by liquid chromatography–mass spectrometry.

Antihypertensive and free radical scavenging properties of enzymatic rapeseed protein hydrolysates

In this study, rapeseed protein isolate (RPI) was digested with various proteases to produce rapeseed protein hydrolysates (RPHs), which were then separated into different peptide fractions (<1, 1–3, 3–5, and 5–10kDa) by membrane ultrafiltration. Membrane fractionation showed that peptides with sizes <3kDa had significantly (p<0.05) reduced surface hydrophobicity when compared to the RPHs and peptide fractions with sizes >3kDa. In contrast, the <3kDa peptides showed significantly (p<0.05) higher oxygen radical scavenging ability when compared to the >3kDa peptides and RPHs. In vitro inhibition of angiotensin I-converting enzyme (ACE) was significantly (p<0.05) higher for the Thermolysin, Proteinase K and Alcalase RPHs when compared to the pepsin+pancreatin (PP) and Flavourzyme RPHs. The Alcalase RPH had significantly (p<0.05) higher renin inhibition among the RPHs, while with the exception of Thermolysin, the 5–10kDa peptide fraction had the least renin-inhibitory ability when compared to the <5kDa peptide fractions. Oral administration (100mg/kg body weight) of the RPHs and RPI to spontaneously hypertensive rats (SHR) showed the Alcalase RPH to be the most effective in blood pressure (BP) reduction (∼24mmHg) while Proteinase K RPH was the least effective (∼5mmHg) after 8h. However, the PP RPH had the most prolonged effect with BP reduction of ∼20mmHg after 24h of oral administration. We conclude that the strong BP-lowering ability of Alcalase and PP RPHs could be due to high resistance of the peptides to structural degradation coupled with high absorption rate within the gastrointestinal tract.

Antioxidant activities of enzymatic rapeseed protein hydrolysates and the membrane ultrafiltration fractions

In this study, rapeseed protein isolate was hydrolyzed with various proteases to obtain hydrolysates that were separated by membrane ultrafiltration into four molecular size fractions (<1, 1–3, 3–5, and 5–10kDa). Alcalase hydrolysis significantly (p<0.05) produced the highest yield of protein hydrolysate while Flavourzyme produced the least. The <1kDa fraction was the most abundant after the membrane ultrafiltration of the protein hydrolysates, which indicates that the proteases were efficient at reducing the native rapeseed proteins into low molecular weight peptides. Antioxidant properties of the resulting hydrolysates and membrane fractions were characterized and results showed the Pepsin+Pancreatin (P+P) protein hydrolysate had significantly highest (p<0.05) scavenging activity against DPPH radical among the unfractionated enzymatic hydrolysates. But the P+P hydrolysate was not as effective as other hydrolysates during long-term inhibition of linoleic acid oxidation. For most of the samples, fractionation into the <1kDa peptides significantly (p<0.05) improved DPPH and superoxide scavenging properties when compared to the unfractionated protein hydrolysates. Only the <1kDa fraction showed ferric reducing antioxidant power and the effect was dose-dependent. Overall, Alcalase and Proteinase K seem to be more efficient proteases to release antioxidant peptides from rapeseed proteins when compared to P+P, Flavourzyme and Thermolysin.

Purification and Characterization of a Radical Scavenging Peptide from Rapeseed Protein Hydrolysates

AbstractWe previously reported that crude rapeseed peptides (CRPs) and peptide fractions (RP25 and RP55) prepared from aqueous enzymatic extraction of rapeseed exhibited marked antioxidant activities, among which RP55 showed the most potent effects. In the present study, RP55 was further purified using consecutive chromatographic methods for identification of antioxidant peptides. The α,α‐diphenyl‐β‐picrylhydrazyl (DPPH) radical scavenging effects of peptides were measured at each purification step to evaluate the antioxidant activities. RP55 was first fractionated by anion‐exchange chromatography, and three fractions (E1, E2, and E3) were obtained. All of them showed significantly lower scavenging activities compared to RP55, which was very probably due to the remarkable loss of tannin during the separation. Next, the active fraction E2 with higher protein content was sequentially purified by gel filtration chromatography (GFC) and reversed‐phase high performance liquid chromatography (RP‐HPLC). The purified peptide, of which the median effective dose (ED50) value for DPPH radical scavenging was 0.063 mg/mL, was identified to be Pro‐Ala‐Gly‐Pro‐Phe (487 Da) using electrospray ionization (ESI) mass spectrometry. The unique amino acid composition and sequence in the peptide might play an important role in expression of its antioxidant activity.

Antioxidant Activities of Rapeseed Protein Hydrolysates

Rapeseed protein hydrolysates (RPH) were obtained by enzymatic hydrolysis of rapeseed protein using Alcalase 2.4 L FG. The degree of hydrolysis (DH) of RPH was about 25% using pH-stat method. The antioxidant activities of RPH were investigated by employing several in vitro assay, including the 1,1-diphenyl-2-picrylhydrazyl (DPPH)/superoxide/hydroxyl radical scavenging assays, and reducing power assay. RPH showed scavenging activity against free radicals such as DPPH, superoxide, and hydroxyl radicals. The radical scavenging effect was in a dose-dependent manner, and the EC50 values for DPPH, superoxide, and hydroxyl radicals were found to be 0.71, 1.05, and 4.92 mg/mL, respectively. In addition, the RPH also exhibited notable reducing power, which was 0.51 at 2.00 mg/mL. The data obtained by in vitro systems obviously established the antioxidant potency of RPH. Combined with the results of the amino acid profiles, RPH were believed to have high nutritive value in addition to antioxidant activities.

Downstream Processes for Aqueous Enzymatic Extraction of Rapeseed Oil and Protein Hydrolysates

AbstractDownstream processes following aqueous enzymatic extraction (AEE) of rapeseed oil and protein hydrolysates were developed to enhance the oil and protein yields as well as to purify the protein hydrolysates. The wet precipitate (meal residue) from the AEE was washed with twofold water at 60 °C, pH 11 for 1 h. Emulsions from the AEE and the washing step were pooled and submitted to a stepwise demulsification procedure consisting of storage‐centrifugation and freezing–thawing followed by centrifugation. Aqueous phases were pooled and adsorbed onto macroporous adsorption resins (MAR) to remove salts and sugars. Following extensive rinsing with deionized water (pH 4), desorption was achieved by washing with 85% ethanol (v/v) to obtain crude rapeseed peptides (CRPs). In a separate experiment, stepwise desorption was carried out with 25, 55, and 85% ethanol to separate the bitter peptides from the other peptides. Using a combination of the AEE process, washing and demulsification steps, the yields of the total free oil and protein hydrolysates were 88–90% and 94–97%, respectively. The protein recovery was 66.7% and the protein content was enriched from 47.04 to 73.51% in the CRPs. No glucosinolates and phytic acid were detected in the CRPs. From the stepwise desorption, a non‐bitter fraction RP25 (containing 64–66% of total desorbed protein) had a bland color and significantly higher protein content (81.04%) and hence was the more desirable product.

Prediction of short peptides composition by RP-HPLC coupled to ESI mass spectrometry

The combination of reversed-phase chromatography and electrospray mass spectrometry was used to predict the amino acid composition of low molar weight peptides present in a complex mixture of rapeseed protein hydrolysates. First, amino acid retention times on a hydrophobic stationary phase were evaluated to determine hydrophobicity coefficients for each amino acid and then global hydrophobicity coefficients were established for standard peptides. In this way, a correlation between peptide hydrophobicity coefficients and retention time was established. Then, a C language program was developed to calculate all potential amino acid combinations from the mass of a peptide (molar mass < 1000 Da) and determine theoretical hydrophobicity coefficients corresponding to these combinations. Comparison between the theoretical retention time determined by the model and the experimental retention time resulted in the establishment of a sorted potential composition table. This methodology has been applied to two different rapeseed protein hydrolysates peptides that have been purified and sequenced.

Optimization of the Aqueous Enzymatic Extraction of Rapeseed Oil and Protein Hydrolysates

AbstractAn aqueous enzymatic extraction method was developed to obtain free oil and protein hydrolysates from dehulled rapeseeds. The rapeseed slurry was treated by the chosen combination of pectinase, cellulase, and β‐glucanase (4:1:1, v/v/v) at concentration of 2.5% (v/w) for 4 h. This was followed by sequential treatments consisting of alkaline extraction and an alkaline protease (Alcalase 2.4L) hydrolysis to both produce a protein hydrolysate product and demulsify the oil. Response surface methodology (RSM) was used to study and optimize the effects of the pH of the alkaline extraction (9.0, 10.0 and 11.0), the concentration of the Alcalase 2.4L (0.5, 1.0 and 1.5%, v/w), and the duration of the hydrolysis (60, 120, and 180 min). Increasing the concentration of Alcalase 2.4L and the duration of the hydrolysis time significantly increased the yields of free oil and protein hydrolysates and the degree of protein hydrolysis (DH), while the alkaline extraction pH had a significant effect only on the yield of the protein hydrolysates. Following an alkaline extraction at pH 10 for 30 min, we defined a practical optimum protocol consisting of a concentration of 1.25–1.5% Alcalase 2.4L and a hydrolysis time between 150 and 180 min. Under these conditions, the yields of free oil and protein hydrolysates were 73–76% and 80–83%, respectively. The hydrolysates consisted of approximately 96% of peptides with a MW less than 1500, of which about 81% had a MW less than 600 Da.

Utilisation of rapeseed protein isolates for production of peptides with angiotensin I-converting enzyme (ACE)-inhibitory activity

ACE activity is related to increased arterial pressure and coronary diseases. A rapeseed protein isolate was hydrolyzed with the protease Alcalase in order to investigate the possible presence of ACE inhibitory peptides in the resulting hydrolysates. Hydrolysis for 30 min yielded a hydrolysate with the highest ACE inhibitory activity. Two fractions of this hydrolysate obtained by Biogel P2 gel filtration chromatography were used for further purification of ACE inhibitory peptides. Three fractions with ACE inhibitory activity were purified by reverse-phase HPLC of Biogel P2 f ractions. This demonstrates that rapeseed protein hydrolysates represent a good source of ACE inhibitory peptides .

Partially hydrolyzed rapeseed protein isolates with improved functional properties

AbstractLimited rapeseed protein hydrolysates ranging from 3.1 to 7.7% hydrolysis were produced from isoelectric‐precipitated protein isolate. Water absorption, oil absorption, whippability, foam capacity and stability, emulsifying activity, and emulsion stability of the hydrolysates were determined. All protein hydrolysates showed better functional properties than the original protein isolate. Foam and emulsion stability decreased as the degree of hydrolysis increased. The hydrolysate with the lowest degree of hydrolysis showed the best functional properties. These improved functional properties make rapeseed protein hydrolysates a useful product to be used in foods such as breads, cakes, ice creams, meat products, desserts, and salad dressings.