Identification, rapid screening, docking mechanism and in vitro digestion stability of novel DPP-4 inhibitory peptides from wheat gluten with ginger protease.

To better understand the hypoglycemic potential of wheat gluten (WG), we screened dipeptidyl peptidase IV (DPP-4) inhibitory active peptides from WG hydrolysates. WG hydrolysates prepared by ginger protease were found to have the highest DPP-4 inhibitory activity among the five enzymatic hydrolysates, from which a 1-3 kDa fraction was isolated by ultrafiltration. Further characterization of the fraction with nano-HPLC-MS/MS revealed 1133 peptides. Among them, peptides with P'2 (the second position of the N-terminal) and P2 (the second position of the C-terminal) as proline residues (Pro) accounted for 12.44% and 43.69%, respectively. The peptides including Pro-Pro-Phe-Ser (PPFS), Ala-Pro-Phe-Gly-Leu (APFGL), and Pro-Pro-Phe-Trp (PPFW) exhibited the most potent DPP-4 inhibitory activity with IC50 values of 56.63, 79.45, and 199.82 µM, respectively. The high inhibitory activity of PPFS, APFGL, and PPFW could be mainly attributed to their interaction with the S2 pocket (Glu205 and Glu206) and the catalytic triad (Ser630 and His740) of DPP-4, which adopted competitive, mixed, and mixed inhibitory modes, respectively. After comparative analysis of PPFS, PPFW, and PPF, Ser was found to be more conducive to enhancing the DPP-4 inhibitory activity. Interestingly, peptides with P2 as Pro also exhibited good DPP-4 inhibitory activity. Meanwhile, DPP-4 inhibitory peptides from WG showed excellent stability, suggesting a potential application in type 2 diabetes (T2DM) therapy or in the food industry as functional components.

Phenolic compounds in walnut pellicle improve walnut (Juglans regia L.) protein solubility under pH-shifting condition.

This study focused on the interaction of walnut protein with phenolic extracts of walnut pellicle (PEWP) under alkaline condition, leading to enhancement of protein solubility under neutral condition. First, the change of PEWP under alkaline condition was determined by RP-HPLC and mass spectrometry, and the results showed that most ellagitannins in PEWP could be retained under alkaline condition within 3 h. Interaction between PEWP and walnut protein under pH-shifting condition resulted in the remarkable increase of protein solubility (above 90%) at neutral pH. The results from SDS-PAGE and SEC showed that the improved solubility lied in the formation of large and soluble protein aggregates due to the covalent interaction among walnut protein and polyphenols. A significant change in tertiary structure of protein-phenolic complex was witnessed by fluorescence spectrum and near-UV circular dichroism. Meanwhile, walnut protein-polyphenol interaction led to a slight increase in ß-turn while a slight decrease in ß-sheet. Combined with amino acid composition, it could be illustrated that the covalent bonding for walnut protein with polyphenol mainly occurred at Lysine residues.

Comparison of aroma active compounds in cold- and hot-pressed walnut oil by comprehensive two-dimensional gas chromatography-olfactory-mass spectrometry and headspace-gas chromatography-ion mobility spectrometry.

Aroma composition of cold-pressed walnut oil (CWO) and hot-pressed walnut oil (HWO) was analyzed by comprehensive two-dimensional gas chromatography-olfactory-mass spectrometry (GC × GC-O-MS) and headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS). A total of 83 and 197 compounds were identified in the CWO and HWO, respectively; among these, 76 and 123 compounds were sniffed exclusively by GC × GC-O-MS, respectively. A total of 36 volatile compounds were detected by HS-GC-IMS, of which 10 in CWO and 32 in HWO. Based on of flavor dilution (FD) factors, odor-activity values (OAVs), and recombination and omission experiments, 1-octen-3-ol, cyclohexanol, and benzaldehyde were found to be the key aroma-active compounds in CWO, while 3-methylbutanal, (E,E)-2,4-nonadienal, nonanal, 1-octen-3-ol, 3-pentanol, 1-octanol, and furfural were the key aroma-active compounds in HWO. Moreover, Maillard reaction and lipid oxidation were found to play an important role in flavor formation in HWO. This study provides a guide to improve the quality of walnut oil based on aroma characteristics.

Growing of fungi on the stored low denatured defatted soybean meals and the hydrolysis of proteins and isoflavone glycosides by fungal enzymes.

Recently, more and more attention has been paid to the effects of fungal contamination and fungal enzymes secreted in raw grain on product quality. As the starting material of protein and active components, the quality of low denatured defatted soybean meals (LDSM) directly determines the qualities of subsequent products. In previous studies, we have revealed that infection with Aspergillus ochraceus protease causes significant hydrolysis of proteins. In this study, growing of fungi on the stored low denatured defatted soybean meals (LDSM) was analyzed by high-throughput sequencing and real-time PCR, which revealed that the abundance of Aspergillus increased significantly after storage. Twenty fungal proteases and 9 fungal glucosidases were found in stored LDSM and zymography showed that the proteases were of serine-type with some cysteine and aspartic activities. Proteolysis of the soybean storage proteins mainly occurred after the hydration of LDSM and the average molecular weight of soy proteins decreased from 57.9 kDa to 30.7 kDa after 60 min's of hydrolysis. Two-dimensional electrophoresis (2-DE) analysis found the polypeptide fragments from soybean 7S and 11S proteins with molecular weight around 10-25 kDa in the hydrated LDSM. Glycosylated isoflavones were hydrolyzed in both dry and hydrated stored LDSM which resulted in significant (p < 0.05) increase in the contents of isoflavone aglycones. This study suggested that fungi contamination be a new factor affecting the properties of LDSM derived soy protein products.

Characterization of proteases from Irpex lacteus grown on minimally denatured soybean meal.

BACKGROUND: Acid and thermal stabilities are important properties for the preparation of acidic protein beverage. It is an important method for enzymatic modification to improve the functional properties of protein. Irpex lacteus protease showed a selective hydrolysis to soy proteins. The purpose of this study was to investigate the mechanism of enzymatic hydrolysis and its effects on acid and thermal stabilities of soy proteins. RESULTS: The I. lacteus protease selectively hydrolyzed the α and α' subunits of the native soybean ß-conglycinin (7S globulin) to produce products that presented as the 55 kDa band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequences of 55 kDa polypeptides were analyzed in gel multi-enzyme digestion followed by liquid chromatography-mass spectrometry. By matching the multi-enzyme digestion peptides with the published polypeptide chain sequences of the α and α' subunits, it was confirmed that the 55 kDa polypeptides were formed by eliminating amino acid residues on both sides of the N- and C-terminals. From the published protein structure database (https://www.uniprot.org/), it is known that the cleaved peptide bonds were in extension regions. Non-selective enzyme hydrolysis of both ß-conglycinin (7S globulin) and glycinin (11S globulin), with corresponding drastic increases in the degree of hydrolysis, was observed when the substrates were preheated to the denaturation degree of 40% and above. However, 55 kDa hydrolyzed products and B polypeptides showed some extent of resistance to the proteolysis by I. lacteus protease even if denaturation degree was 100%. Both selective and non-selective hydrolysis of soy proteins by I. lacteus protease improved the acid and heat stabilities under the same hydrolysis conditions (enzyme/substrate ratio, time, and temperature). CONCLUSION: Enzymatic hydrolysis of soybean proteins by the I. lacteus protease can effectively improve the acid and thermal stabilities of proteins. This discovery is significant to avoid aggregation during processing in the beverage industry. In the near future, the protease has potential application value for modification of other proteins. © 2022 Society of Chemical Industry.

Raw walnut kernel: A natural source for dietary proteases and bioactive proteins.

Walnut kernels are health-promoting nuts, which are mainly attributed to polyunsaturated fatty acids, phenolics, and phytosterols. However, the information concerning benefits of walnut proteins are limited. In this study, endopeptidases, aminopeptidases, carboxypeptidases, superoxide dismutases, catalases, and phospholipases with respective relative abundance of 2.730, 1.728, 0.477, 3.148, 0.743, and 0.173‰ were identified by liquid chromatography tandem mass spectrometry. These endogenous proteases exhibited activity in a broad pH range of 2-6.5, and optimal at pH 4.5 and 50 °C. Aspartic endopeptidases were predominant endopeptidases, followed by cysteine ones. There were two types of aspartic endopeptidases, one (not inhibited by pepstatin A) exerted activity at pH 2-3 and the other (inhibited by pepstatin A) optimal at pH 4.5. Carboxypeptidases were optimal at pH 4.5, and aminopeptidases exerted activity at pH near 6.5. These endogenous proteases assisted the digestion of walnut proteins, and soaking, especially peeling, greatly improved the in vitro digestibility.

Endopeptidases, exopeptidases, and glutamate decarboxylase in soybean water extract and their in vitro activity.

The proteolytic activity of some soybean endogenous proteases have been clarified in the previous studies, but the information concerning the roles of these proteases and some other unknown ones during soybean processing are scarce. Herein, 16 endopeptidases, 13 exopeptidases, 24 inhibitors (two serpin-ZX and one subtilisin inhibitor firstly identified), and one glutamate decarboxylase were identified in the soybean water extract by the liquid chromatography tandem mass spectrometry analysis. Amongst the identified endopeptidases, just the aspartic endopeptidases (optimal at pH 2.5-3 and 35-45 °C) showed the detectable proteolytic activity by the tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis and protease inhibitor assay analyses, whereas serine, cysteine, and metallo- endopeptidases (except P34 probable thiol protease) did not. Free amino acid analysis showed that the exopeptidases and glutamate decarboxylase were optimal at pH 6 and 45 °C, and by 6 h incubation, the free amino acids and γ-aminobutyric acid almost doubled.

Separation, identification and molecular binding mechanism of dipeptidyl peptidase IV inhibitory peptides derived from walnut (Juglans regia L.) protein.

Walnut protein was hydrolyzed with different proteases to evaluate the hydrolytic efficiency and dipeptidyl peptidase IV (DPP-IV) inhibitory activity in vitro. All of walnut protein hydrolysates (WPHs) exhibited DPP-IV inhibitory activity and Alcalase-derived hydrolysate (WPH-Alc) with better DPP-IV inhibitory activity of 33.90% (at 0.50 mg/mL) was subsequently separated by ultrafiltration and cation exchange chromatography on a SP Sephadex C-25 column. The results showed that fractions with lower molecular weight and higher basic amino acid residues possessed stronger DPP-IV inhibitory activity. Comparably, the obtained fraction B with the yield of 19.80% had the highest DPP-IV inhibitory activity of 76.19% at 0.25 mg/mL. Moreover, nine novel DPP-IV inhibitory peptides were identified using MALDI-TOF/TOF-MS. Molecular docking revealed the peptides could interact with DPP-IV through hydrogen bonds, salt bridges, hydrophobic interactions, π-cation bonds and π-π bonds. The walnut DPP-IV inhibitory peptides showed better stability with heating treatment, pH treatment, or in vitro gastrointestinal digestion.

Characterization of endogenous endopeptidases and exopeptidases and application for the limited hydrolysis of peanut proteins.

Research concerning the utilization of oilseed endogenous proteases is scarce. Herein, we investigated the peanut proteases and their effects on peanut proteins. Liquid chromatography tandem mass spectrometry analysis showed that peanut contained several endopeptidases and exopeptidases. Protease inhibitor assay and analysis of cleavage sites showed that the obvious proteolytic activity at pH 2-5 and 20-60 °C was from aspartic endopeptidases (optimal at pH 3) and one legumain (pH 4). The above endopeptidases destroyed five and six IgE-binding epitopes of Ara h 1 at pH 3 and 4, respectively. Ara h 1 (>95%) and arachin (50-60%) could be hydrolyzed to generate 10-20 kDa and <4 kDa peptides at pH 3, which was enhanced by the pH 3 â†’ 4 incubation. Further, the limited hydrolysis improved the gel-forming ability and in vitro digestibility (approximately 15%) of peanut proteins. Free amino acid analysis showed that the activity of exopeptidases was low at pH 2-5.

Insights into the antibacterial activity of cottonseed protein-derived peptide against Escherichia coli.

In the study, antibacterial peptides were separated and identified from cottonseed protein hydrolysates and the interactions between antibacterial peptides and Escherichia coli were further investigated. Firstly, by using a combined strategy of Amberlite CG-50 ion exchange chromatography and reversed-phase high-performance liquid chromatography, three peptides with antibacterial activity were purified and identified, including HHRRFSLY, KFMPT, and RRLFSDY. Interestingly, HHRRFSLY and RRLFSDY exhibited higher inhibition activity with the IC50 value of 0.26 mg mL-1 and 0.58 mg mL-1 (p < 0.05), respectively. Flow cytometry results showed that the incubation of antibacterial peptides with E. coli could cause damage to the integrity of the E. coli cell membrane. Transmission electron microscopy and scanning electron microscopy results revealed the damage caused to the bacterial cell surface and the leakage of cytoplasmic content by the antibacterial peptides. Molecular docking studies indicated that HHRRFSLY, KFMPT, and RRLFSDY have a good binding affinity to the active sites of the surface protein (OmpF) mainly through a hydrogen bond and salt bridge. The results here showed that the antibacterial peptides derived from cottonseed protein could be used as a good choice for functional foods or related drugs, and also shed light on further studies of antibacterial mechanism.

Propofol inhibits parthanatos via ROS-ER-calcium-mitochondria signal pathway in vivo and vitro.

Parthanatos is a new form of programmed cell death. It has been recognized to be critical in cerebral ischemia-reperfusion injury, and reactive oxygen species (ROS) can induce parthanatos. Recent studies found that propofol, a widely used intravenous anesthetic agent, has an inhibitory effect on ROS and has neuroprotective in many neurological diseases. However, the functional roles and mechanisms of propofol in parthanatos remain unclear. Here, we discovered that the ROS-ER-calcium-mitochondria signal pathway mediated parthanatos and the significance of propofol in parthanatos. Next, we found that ROS overproduction would cause endoplasmic reticulum (ER) calcium release, leading to mitochondria depolarization with the loss of mitochondrial membrane potential. Mitochondria depolarization caused mitochondria to release more ROS, which, in turn, contributed to parthanatos. Also, we found that propofol inhibited parthanatos through impeding ROS overproduction, calcium release from ER, and mitochondrial depolarization in parthanatos. Importantly, our results indicated that propofol protected cerebral ischemia-reperfusion via parthanatos suppression, amelioration of mitochondria, and ER swelling. Our findings provide new insights into the mechanisms of how ER and mitochondria contribute to parthanatos. Furthermore, our studies elucidated that propofol has a vital role in parthanatos prevention in vivo and in vitro, and propofol can be a promising therapeutic approach for nerve injury patients.

Selective Extraction and Antioxidant Properties of Thiol-Containing Peptides in Soy Glycinine Hydrolysates.

A highly selective procedure to extract thiol-containing peptides (TCPs) from complicated soy glycinin hydrolysates (SGHs) was described. This procedure included the reduction of disulfide bonds by 1,4-dithiothreitol (DTT) and enrichment of TCPs through Thiopropyl-Sephrose 6B covalent chromatography. TCPs were confirmed using a strategy based on mass shift after differential alkylation of sulfhydryl groups with iodoacetamide and N-ethylmaleimide by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS). The antioxidant activities of TCPs were evaluated using chemical assays. DTT reduction increased the concentration of sulfhydryl groups from 1.8 µmol/g to 113.8 µmol/g. The efficiency of the extraction was improved by optimizing the loading of sample, extraction and desorption time and the content of desorption reagent. Both of the adsorption and desorption process were found to fit a pseudo-second order model. MALDI-TOF-MS showed that 36 of the 45 extracted peptides were TCPs. The EC50 of TCPs for DPPH, hydroxyl radical, and superoxide anion radical was 0.1, 1.49 and 0.084 mg/mL, respectively. The reducing power of TCPs (0.2 mg/mL) was of 0.375. These results suggest that the combination of DTT reduction and Thiopropyl-Sepharose 6B covalent chromatograph was a successful pathway to extract TCPs from SGHs and the TCPs could be used as potential antioxidants.

Mig6 reduces inflammatory mediators production by regulating the activation of EGFR in LPS-induced endotoxemia.

Epithelial growth factor receptor (EGFR), a tyrosine kinase receptor, plays a critical role in lipopolysaccharide (LPS)-induced endotoxemia. Meanwhile, EGFR signaling is regulated by multiple feedback regulators, including mitogen-inducible gene 6 protein (Mig6). However, as an EGFR regulator, the role of Mig6 in endotoxemia is still remained unknown. Here, we reported for the first time that LPS treatment increased the expression of Mig6 and this effect could be inhibited by EGFR inhibitor, PD168393 or erlotinib. Furthermore, knocking down of Mig6 expression led to increased EGFR activation and inflammatory mediators (TNF-α, il-1ß) production in response to LPS treatment. On the other hand, the increased EGFR activation and TNF-α or il-1ß production in LPS treatment could be inhibited by Mig6 overexpression. Besides, in LPS-induced endotoxemia, ERK1/2 and p-38 activation required Mig6. All these results indicated that Mig6 regulates the production of inflammatory mediators (TNF-α, il-1ß) through inhibiting the over activation of EGFR, which in turn inhibit MAPKs signaling (ERK1/2, p-38). These finding suggested that Mig6 may be a novel potential target for controlling the over inflammatory response in endotoxemia.

A two-chain aspartic protease present in seeds with high affinity for peanut oil bodies.

Peanut seeds are rich in oil, which exists as oil bodies (OBs). By extraction, peanut crude OBs are obtained and can be used as a food ingredient. In a previous study, it was found that the crude OBs contained an unknown protease, which hydrolyzed the oleosins. This would disrupt the integrity of OBs, and therefore, affect their physical and oxidative stability. In this study, the protein composition of crude OBs and some properties of the unknown protease were examined. The results showed that the protease was a two-chain (32 and 9kDa) aspartic protease, which showed high affinity for OBs. The optimal pH and temperature for oleosin hydrolysis by the protease were pH 4.0 and 60°C. Interestingly, the aspartic protease not only hydrolyzed OB intrinsic proteins (oleosin, caleosin, and steroleosin), but also extrinsic proteins (especially Ara h 1 allergen and 26-30kDa arachin).

Soybean P34 Probable Thiol Protease Probably Has Proteolytic Activity on Oleosins.

P34 probable thiol protease (P34) and Gly m Bd 30K (30K) show high relationship with the protease of 24 kDa oleosin of soybean oil bodies. In this study, 9 day germinated soybean was used to separate bioprocessed P34 (P32) from bioprocessed 30K (28K). Interestingly, P32 existed as dimer, whereas 28K existed as monomer; a P32-rich sample had proteolytic activity and high cleavage site specificity (Lys-Thr of 24 kDa oleosin), whereas a 28K-rich sample showed low proteolytic activity; the P32-rich sample contained one thiol protease. After mixing with purified oil bodies, all P32 dimers were dissociated and bound to 24 kDa oleosins to form P32-24 kDa oleosin complexes. By incubation, 24 kDa oleosin was preferentially hydrolyzed, and two hydrolyzed products (HPs; 17 and 7 kDa) were confirmed. After most of 24 kDa oleosin was hydrolyzed, some P32 existed as dimer, and the other as P32-17 kDa HP. It was suggested that P32 was the protease.

Heat-induced inactivation mechanism of soybean Bowman-Birk inhibitors.

Due to the complications of the soymilk system, the heat-induced Bowman-Birk inhibitor (BBI) inactivation mechanism is not well known. In this study, two BBI samples with low and high purities were prepared from soymilk. It was confirmed that three groups (A, C, and D) of BBI, which are contained in soybean seeds, were transferred into soymilk during processing. On heating, it was found that 1) the two subdomains of BBI were not equally heat stable, 2) the conformation of BBI gradually changed, 3) some amino acid residues (namely, cystine, serine and lysine) in BBI were degraded, 4) BBI did not tend to form intermolecular cross-links with another BBI, but did slightly with non-BBI proteins. Based on some previous studies, the conformational change of BBI was attributed to ß-elimination reactions on the amino acid residues of BBI and the subsequent intramolecular reactions induced by the products yielded by the ß-elimination reactions.

An advance for removing antinutritional protease inhibitors: Soybean whey purification of Bowman-Birk chymotrypsin inhibitor by combination of two oppositely charged polysaccharides.

Two successive and selective coacervations induced by chitosan (Ch) and carrageenan (CG) were applied to remove antinutritional protease inhibitors and purify Bowman-Birk protease inhibitor (BBI) from soybean whey. At the first coacervation induced by Ch (66.7, 200, and 510kDa), only Kunitz trypsin inhibitor (KTI) and BBI complexed with Ch were extracted, while ß-amylase and soybean agglutinin remained in supernatant. The binding constants for the interaction increased on the order Ch-66.7

Differential role of intravenous anesthetics in colorectal cancer progression: implications for clinical application.

Anesthetics are unavoidable to colorectal cancer (CRC) patients who underwent surgical treatment. Thus, the molecular mechanisms underlying the role of the intravenous anesthetics in CRC metastasis are still unclear. In this study, the effects of intravenous anesthetics, such as propofol, etomidate and dexmedetomidine, on cell migration were determined. The migration of CRC cells was inhibited by propofol in vitro, but not in vivo. Etomidate, however, promoted the migration of CRC cells both in vitro and in vivo. Epithelial-mesenchymal transition (EMT) mediated the promotive effect of propofol and etomidate on the migration of CRC cells through PI3K/AKT signaling pathway. Dexmedetomidine alone or in combination with propofol or etomidate had minor effect on the migration of CRC cells. These findings indicate that propofol inhibites CRC cell migration in vitro. Etomidate playes a role for prompting CRC metastasis progression by activating (PI3K)/AKT signaling and inducing EMT. It provides an important hint for the clinical application of these anesthetics.

Effects of pH on protein components of extracted oil bodies from diverse plant seeds and endogenous protease-induced oleosin hydrolysis.

Plant seeds are used to extract oil bodies for diverse applications, but oil bodies extracted at different pH values exhibit different properties. Jicama, sunflower, peanut, castor bean, rapeseed, and sesame were selected to examine the effects of pH (6.5-11.0) on the protein components of oil bodies and the oleosin hydrolysis in pH 6.5-extracted oil bodies. In addition to oleosins, many extrinsic proteins (globulins, 2S albumin, and enzymes) were present in pH 6.5-extracted oil bodies. Globulins were mostly removed at pH 8.0, whereas 2S albumins were removed at pH 11.0. At pH 11.0, highly purified oil bodies were obtained from jicama, sunflower, peanut, and sesame, whereas lipase remained in the castor bean oil bodies and many enzymes in the rapeseed oil bodies. Endogenous protease-induced hydrolysis of oleosins occurred in all selected plant seeds. Oleosins with larger sizes were hydrolysed more quickly than oleosins with smaller sizes in each plant seed.

Behaviors of particle size and bound proteins of oil bodies in soymilk processing.

In unheated soymilk, extrinsic proteins are bound to intact oil bodies coated by one monolayer of phospholipids and oil body intrinsic oleosins. In this study, effects of heating (70-100°C; 0-30min) on particle size and bound proteins of oil bodies were examined in suspension (oil bodies from unheated soymilk into deionized water) and soymilk. Mass ratio of extrinsic proteins/oleosins of oil bodies in unheated suspension and soymilk was respectively 1.1 and 2.5. By heating, extrinsic proteins released from oil bodies with different rates, and Z-average size of oil bodies increased in the beginning; afterwards, residual extrinsic proteins (extrinsic proteins/oleosins: 0.31 in suspension and 0.74 in soymilk; 100°C, 4min) and Z-average size were affected little. The amount of residual proteins at 80-100°C was negatively correlated with Z-average size of oil bodies in suspension (R(2)=0.996) and soymilk (R(2)=0.890).