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Effects of single factors on DH and yield. a Effect of hydrolysis time on DH and yield. b Effect of temperature on DH and yield. c Effect of E/S ratio on DH and yield
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The purpose of this study was to separate and purify antioxidant peptides from the scorpion (Buthus martensii Karsch) protein hydrolysates (SPHs). Scorpion protein (SP) was first hydrolyzed by trypsin, papain, and alcalase, respectively. Results from hydrolysis tests revealed that peptides hydrolyzed with papain showed the highest degree of hydroly...
Citations
... The ABTS radical scavenging activity of HBMP hydrolysate was determined according to previous reports [28] with slight modifications. 19.20 mg of ABTS powder dissolved in 5 mL of distilled water (7 mmol/L) and 3.31 mg of potassium persulfate powder dissolved in 5 mL of distilled water (2.45 mmol/L) and mixed, then reacted for 16 h at room temperature in the dark and diluted with absolute ethanol to an absorbance value of 0.70 ± 0.02 at 734 nm. ...
... HBMP-AH had shown a higher scavenging effect on ABTS free radicals (Fig. 14B) and a significant dose-dependent relationship within the range of 0 ~ 0.5 mg/mL. The IC 50 value of HBMP-AH on ABTS free radical was 0.044 mg/ mL, it was much stronger than native HBMP, it may be related to the lower Mw of the HBMP-AH as well as the amino acid composition and peptide sequence [26,28]. ...
This study aimed to investigate the structural features and antioxidant activity in vitro of horse bone marrow protein (HBMP) hydrolysate based on the activity-originated approach. Antioxidant peptides were isolated from the HBMP with different enzymes. Then, the structural features of the obtained peptide were characterized by extensive chemical, spectroscopic, and chromatographic methods. Antioxidative properties were evaluated through the determination of free radicals scavenging activity. The results showed that alcalase was determined to be the most effective for the generation of antioxidant peptides, and the highest degree of hydrolysis (39.89%) and free radical scavenging activity (55.73%) were obtained under the condition of enzyme concentration 0.9%, pH 9.5, temperature 50℃, solid-liquid ratio 1:16 g/mL through optimized by response surface methodology. HBMP and its hydrolysates were rich in beneficial amino acids. Fourier transforms infrared spectroscopy (FT-IR) and Circular dichroism (CD) revealed key functional structures of obtained peptides whereas Scanning electron microscope-energy dispersive X-ray (SEM-EDX) exhibited its characteristic morphology and elemental composition. The molecular weight distribution of the HBMP alcalase hydrolysate was 2189.014-11561.368 Da determined by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis. The antioxidant activity test showed that enzymatic hydrolysis can significantly enhance the activity of the HBMP (P < 0.05). The results indicated that HBMP exhibited significant antioxidant activity in vitro, supporting the potential application of HBMP as antioxidants in food processing, pharmaceuticals, and other industries.
... The DPPH radical scavenging activity of TSP was determined according to the previous method of Wali, A. et al., with minor modifications [25]. Glutathione (GSH) and the sample were prepared with the test solution with concentration gradients of 2, 4, 6, 8 and 10 mg/mL, respectively. ...
Antioxidants, which can activate the body’s antioxidant defence system and reduce oxidative stress damage, are important for maintaining free radical homeostasis between oxidative damage and antioxidant defence. Six antioxidant peptides (P1–P6) were isolated and identified from the enzymatic hydrolysate of tilapia skin by ultrafiltration, reversed-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography–tandem mass spectrometry (LC–MS/MS). Moreover, the scavenging mechanism of the identified peptides against DPPH (2,2-Diphenyl-1-picrylhydrazyl) and ABTS (2-azido-bis (3-ethylbenzothiazoline-6-sulfonic acid) was studied by molecular docking. It was found that Pro, Ala and Tyr were the characteristic amino acids for scavenging free radicals, and hydrogen bonding and hydrophobic interactions were the main interactions between the free radicals and antioxidant peptides. Among them, the peptide KAPDPGPGPM exhibited the highest DPPH free radical scavenging activity (IC50 = 2.56 ± 0.15 mg/mL), in which the hydrogen bond between the free radical DDPH and Thr-6 was identified as the main interaction, and the hydrophobic interactions between the free radical DDPH and Ala, Gly and Pro were also identified. The peptide GGYDEY presented the highest scavenging activity against ABTS (IC50 = 9.14 ± 0.08 mg/mL). The key structures for the interaction of this peptide with the free radical ABTS were identified as Gly-1 and Glu-5 (hydrogen bond sites), and the amino acids Tyr and Asp provided hydrophobic interactions. Furthermore, it was determined that the screened peptides are suitable for applications as antioxidants in the food industry, exhibit good water solubility and stability, are likely nonallergenic and are nontoxic. In summary, the results of this study provide a theoretical structural basis for examining the mechanism of action of antioxidant peptides and the application of enzymatic hydrolysates from tilapia skin.
... Our research results agreed with the literature, according to which low-molecular-mass products from protein hydrolysis were more active antioxidants than their high-molecular-mass precursors [12,13]. The amino-acid composition of peptides is known to play an important role in their antioxidant properties [14,15]. All 17 amino acids except Trp (which decomposed under the acid-hydrolysis conditions) were identified in all three fractions from centrifugal membrane ultrafiltration in the present study. ...
Casein was removed from defatted bovine colostrum by precipitation at pH 4.3. Serum proteins were hydrolyzed by trypsin, alkalase, and papain. The degree of hydrolysis (DH) of colostrum serum proteins was determined from the reaction with o-phthalaldehyde (OPA). The obtained fractions were placed in the following order: alkalase > trypsin > papain. The peptides obtained by alkalase treatment were fractionated by membrane ultrafiltration to produce three fractions: PBC-a >10 kDa, PBC-b 3–10 kDa, and PBC-c <3 kDa. Fraction PBC-c showed the highest antioxidant activity with EC50 = 132.92 ± 0.06 μg/mL for ABTS•+ cation-radical and 405.96 ± 0.11 μg/mL for OH•– hydroxyl radical.
... The presence of molecules with antioxidant effects in scorpions has been reported in the literature (Wali et al., 2020). The Stigmurin (FFSLIPSLVGGLISAFK-NH2), cationic peptide of T. stigmurus, showed hydroxyl radical scavenging above 70% at 10 μM (Daniele- . ...
... A peptide fraction isolated from the venom of Buthus occitanus has been demonstrated to exhibit the antioxidant and free radical scavenger effects (Bekheet et al., 2013). Antioxidant peptides from B. martensii Karsch were separated and purified, and showed the highest ABTS + -scavenging activity and the highest DPPH-scavenging activity, but the OH-scavenging activities of these peptides were not significant (Wali et al., 2020). ...
... The substrate oxidation process consists of three stages (initiation, propagation, and termination). Antioxidants can act at any of these steps, and the more steps a compound intervenes at, the better the antioxidant it is (Wali et al., 2020). Several in vitro antioxidant tests are available to assess the antioxidant activity of biomolecules (Gulcin, 2020). ...
Anionic peptides of scorpions are molecules rich in aspartic and/or glutamic acid residues and correspond to a class of peptides without disulfide bonds that are still little explored. TanP is a linear anionic peptide (50 amino acid residues and net charge −20) present in the venom gland of the scorpion, Tityus stigmurus , with chelating properties for Cu ²⁺ ion and immunomodulatory properties. The therapeutic application of chelating molecules is related to cases of acute or chronic intoxication by metals, neurodegenerative diseases, hematological diseases, healing of skin wounds, cardiovascular diseases, and cancer. In this approach, the chelating activity of TanP was evaluated in relation to new metal ions (Fe ²⁺ and Zn ²⁺ ) of biological importance, as well as its antioxidant, hemostatic, immunomodulatory, and healing potential, aiming to expand the biological and biotechnological potential of this peptide. TanP (25 µM) was able to form stable complexes with Fe ²⁺ in a ratio of 1:5 (TanP: Fe ²⁺ ). Theoretical results suggest that TanP can work as a sensor to identify and quantify Fe ²⁺ ions. The fluorescence intensity of TanP (1.12 µM) decreased significantly after the addition of Fe ²⁺ , obtaining the highest ratio 1: 7.4 (TanP: Fe ²⁺ ) that led to the lowest fluorescence intensity. For Zn ²⁺ , no relevant spectral change was noted. TanP (50 µM) showed a maximum of 3% of hemolytic activity, demonstrating biocompatibility, as well as exhibiting a 1,1-diphenyl-2-picrylhydrazyl radical–scavenging activity of above 70% at all the concentrations tested (1–25 μM), and 89.7% iron-chelating activity at 25 μM and 96% hydroxyl radical–scavenging activity at 73.6 μM. In addition, TanP (12.5 and 25 µM) revealed an anticoagulant effect, prolonging the clotting time in prothrombin time and activated partial thromboplastin time assays, with no fibrinogenolytic activity. TanP (12.5 and 25 µM) induced the release of TNF-α by murine macrophages, in the absence of lipopolysaccharides, with a concentration-dependent increase and also stimulated the migration of 3T3 cells in the in vitro healing assay. Thus, TanP revealed a multifunctional potential, being useful as a prototype for the development of new therapeutic and biotechnological agents.
... Next, sericin was subjected to enzymatic hydrolysis by the reported method [5,6]. The obtained sericin hydrolysate was passed through an ultrafiltration membrane of pore size 3 kDa (FS010K10C, Millipore, Guangzhou, China). ...
... The enzymatic hydrolysis was performed at 37°C for 8 h. When the reaction time expired, the enzyme was inactivated by refluxing the mixture for 10 min [6]. ...
... Data collection and processing used Analyst 1.6.2 software [6,12]. ...
Sericin was obtained from Bombyx mori cocoons in 28% yield with 92.06% purity. Its hydrolysate was produced by an enzymatic method. A fraction with molecular mass < 3 kDa was collected using ultrafiltration. The contents of 19 peptides with molecular masses in the range 600–3000 Da were determined using MALDI TOF/TOF. Their amino-acid sequences were found. The antioxidant activity of sericin hydrolysate (IC50) was 0.07 mg/mL according to absorption of ABTS radicals and 1.09 mg/mL according to absorption of OH radicals. These were 4.5 and 1.2 times greater, respectively, than that of sericin.
... As is shown in Table 7, 3 kDa permeate part has the highest hydroxyl and DPPH radical scavenging activity, followed by the 10 kDa retentate part. Accordingly, based on the results of the present study, it was likely to be caused by the differences between Mw of protein from different separation parts from BBM, and this was in accordance with the previous reports (Liu et al. 2018a, b;Wali et al. 2019b). ...
Salt extraction and Ultrafiltration membrane (UFM) separation technology of bovine bone marrow protein (BBMP) were optimized, and the structures of proteins and peptides, as well as biological activities were evaluated. The maximum protein content and yield obtained were 57.40 mg/mL, 56.80% respectively by salt extraction under the following conditions: concentration 0.5 mol/L, extraction of 2 times per 3 h; temperature, 45 °C. Maximum membrane flux and protein content reached to 25.29 L/(m 2 h), 76.9 mg/mL using UFMs method optimized by the response surface methodology (RSM) at 10 kDa membrane, operation pressure 0.20 MPa; concentration rate 5, and temperature 40 °C. Among them, the 3 kDa permeate part has the highest radical scavenging capacity against DPPH and hydroxyl free radicals (41 ± 0.20%, 53 ± 0.35%, respectively). These isolated parts were characterized by Fourier transform infrared spectrometry (FT-IR), scanning electron microscopy (SEM), atomic force microscope (AFM), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Liquid chromatography-Mass spectrometer-Mass spectrometer (LC-MS/MS). The key functional groups, molecular weight, and mass data accordance with the UFM separation results. Therefore, the separation of protein and peptides with different molecular weights can be achieved by UFM. This study can provide technical support for the full utilization and scientific application of BBM resources, as well as the technical and theoretical foundation for the development of peptides for potential health foods or drugs supplementary materials.
... Furthermore, the DH and BCAA contents were higher in the target fraction when the hydroxyl-radical-scavenging activity was higher in our study. Some studies have confirmed that hydrolysates with high DH contents exhibit greater antioxidant activity [31], and the crude protein and amino acid contents increased with increasing DH values [32]. Finally, we obtained 13 peptides from OP-III. ...
Oyster (Crassostrea talienwhanensis) protein was hydrolyzed by trypsin to produce peptides with different response values, and response surface methodology (RSM) was applied to optimize the hydrolysis conditions. The highest degree of hydrolysis (DH) of the oyster peptide (OP) was obtained at an enzyme concentration of 1593.2 U/g, a pH of 8.2, a hydrolysis temperature of 40.1 °C, a hydrolysis time of 6.0 h, and a water/material ratio of 8.2. The greatest hydroxyl-radical-scavenging activity of OP was obtained at an enzyme concentration of 1546.3 U/g, a pH of 9.0, a hydrolysis temperature of 50.2 °C, a hydrolysis time of 5.1 h, and a water/material ratio of 5.6. The largest branched-chain amino acid (BCAA) content of OP was obtained at an enzyme concentration of 1323.8 U/g, a pH of 8.3, a hydrolysis temperature of 41.7 °C, a hydrolysis time of 6.7 h, and a water/material ratio of 4.8. The three experimental values were significantly in agreement with the predicted values within the 95% confidence interval. Furthermore, ultrafiltration and chromatographic methods were used to purify the OP, and 13 peptides that were rich in Lys, Arg, His, and Thr were identified by LC-MS/MS. The results of this study offer different optimum hydrolysis conditions to produce target peptides from oyster protein by using RSM, and this study provide a theoretical basis for the high-value utilization of oyster protein.
This study evaluated the structural characteristics, processing properties, and antioxidant properties of hydrolysates prepared from donkey milk (DM) whey protein using different proteases (Alcalase, Neutrase, papain, and Flavourzyme). The results showed that enzymatic hydrolysis significantly increased hydrolysate solubility and reduced average particle size compared to those of DM whey protein. Neutrase and Flavourzyme hydrolysates exhibited higher degrees of hydrolysis (DH), along with elevated emulsification properties and surface hydrophobicity. The choice of protease influenced secondary and tertiary protein structures and amino acid composition. Enzymatic hydrolysis led to decreased molecular weight of DM whey proteins. Moreover, all hydrolysates exhibited higher fluorescence intensity at λmax compared to DM whey protein, implying distinct properties due to the varied impacts of the four proteases on DM whey protein structure. The preparation of hydrolysates from DM whey proteins using proteases contributes to the development of integrated-value DM products.
Turtle shell as a food residue of Pelodiscus sinensis (a type of edible aquatic animal) is widely used in Traditional Chinese Medicine for hepatic fibrosis therapy. Previous studies have demonstrated that the peptides (<6 kDa) derived from turtle shells are considered effective components. The protein of turtle shells has important potential as a source of bioactive peptides which may play a role as ingredients in functional foods. In the present study, the protein of turtle shell was hydrolyzed using a two-enzyme combination. It was found that the hydrolysates obtained by a combination of pepsin and trypsin showed the highest anti-liver fibrosis activity relative to other combinations in a cell viability assay. The hydrolysates were separated and purified by ultra-filtration (<6 kDa), gel filtration chromatography (GFC) and high-performance liquid chromatography (HPLC). Subsequently, the sequences of purified peptides were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). Molecular docking was used to analyze the interaction of these peptides with the transforming growth factor-β1 (TGF-β1) receptor. Two (GPPGVPGPGPL, TSLPVPAPV) of these novel peptides displayed lower binding energies to the TGF-β1 receptor (-8.18 kcal mol-1, -8 kcal mol-1). Finally, the above two peptides were synthesized chemically and their in vitro anti-liver fibrosis activity was verified by MTT assay. Among them, GPPGVPGPGPL showed a better in vitro anti-liver fibrosis activity (IC50: 80.13 μM). We established a method to obtain anti-liver fibrosis peptides from turtle shells by using bioactivity-guided isolation with molecular docking. Turtle shell protein is an excellent source of anti-liver fibrosis peptides which can offer therapeutic and commercial benefits as an ingredient in functional foods.
