Intestinal delivery of encapsulated bacteriocin peptides in cross-linked alginate microcapsules.

Oral delivery of larger bioactive peptides (>20 amino acids) to the small intestine remains a challenge due to their sensitivity to proteolytic degradation and chemical denaturation during gastrointestinal transit. In this study, we investigated the capacity of crosslinked alginate microcapsules (CLAMs) formed by spray drying to protect Plantaricin EF (PlnEF) (C-EF) in gastric conditions and to dissolve and release PlnEF in the small intestine. PlnEF is an unmodified, two-peptide (PlnE: 33 amino acids; PlnF: 34 amino acids) bacteriocin produced by Lactiplantibacillus plantarum with antimicrobial and gut barrier protective properties. After 2 h incubation in simulated gastric fluid (SGF) (pH 1.5), 43.39 % ± 8.27 % intact PlnEF was liberated from the CLAMs encapsulates, as determined by an antimicrobial activity assay. Transfer of the undissolved fraction to simulated intestinal fluid (SIF) (pH 7) for another 2 h incubation resulted in an additional release of 16.13 % ± 4.33 %. No active PlnEF was found during SGF or sequential SIF incubations when pepsin (2,000 U/ml) was added to the SGF. To test PlnEF release in C-EF contained in a food matrix, C-EF was mixed in peanut butter (PB) (0.15 g C-EF in 1.5 g PB). A total of 12.52 % ± 9.09 % active PlnEF was detected after incubation of PB + C-EF in SGF without pepsin, whereas no activity was found when pepsin was included. Transfer of the remaining PB + C-EF fractions to SIF yielded the recovery of 46.67 % ± 13.09 % and 39.42 % ± 11.53 % active PlnEF in the SIF following exposure to SGF and to SGF with pepsin, respectively. Upon accounting for the undissolved fraction after SIF incubation, PlnEF was fully protected in the CLAMs-PB mixture and there was not a significant reduction in active PlnEF when pepsin was present. These results show that CLAMs alone do not guard PlnEF bacteriocin peptides from gastric conditions, however, mixing them in PB protected against proteolysis and improved intestinal release.

Investigating the impact of common migration substances found in milk packaging on proteases: A multispectral and molecular docking approach.

The effects of common migration substances in milk packaging on digestive protease were studied. We choose the common migrants found in eight types of multi-layer composite milk packaging. Enzyme activity experiments revealed that pepsin activity decreased by approximately 18 % at 500 µg/mL of stearic acid and stearamide treatment, while trypsin activity decreased by approximately 18 % only by stearic acid treatment (500 µg/mL). Subsequently, fluorescence spectroscopy, circular dichroism spectroscopy, and molecular docking technology were employed to investigate the inhibition mechanism of protease activity by migrating substances in three systems: stearic acid-trypsin, stearic acid-pepsin, and stearamide-pepsin. Results showed that the inhibitory effect of stearic acid on trypsin is a reversible mixed inhibition, whereas the inhibitory effects of stearic acid and stearamide on pepsin are non-competitive. In all three systems, ΔH < 0, ΔS < 0, and ΔG < 0, indicating the binding process between the migrant and the protease is a spontaneous exothermic process primarily driven by hydrogen bonding and van der Waals forces. In addition, their binding constants are all around 104 L/moL, indicating that there are moderate binding affinities exist between migrants and proteases. The binding process results in the quenching of the protease's endogenous fluorescence and induces alterations in the enzyme's secondary structure. Synchronized fluorescence spectroscopy showed that stearic acid enhanced the hydrophobicity near the Tyr residue of trypsin. The molecular docking results indicated that the binding affinity of stearic acid-trypsin, stearic acid-pepsin, and stearamide-pepsin was -22.51 kJ/mol, -12.35 kJ/mol, -19.28 kJ/mol respectively, which consistent with the trend in the enzyme activity results. This study can provide references for the selection of milk packaging materials and the use of processing additives, ensuring food health and safety.

Diagnostic value associated with the combination of saliva pepsin and microorganisms in functional heartburn and gastroesophageal reflux disease.

Heartburn is a common symptom shared by both gastroesophageal reflux disease (GERD) and functional heartburn (FHB), which can make it challenging to differentiate between the two conditions. However, examining oral manifestations of GERD can be a cost-effective and readily available method to aid in this differentiation process. It may serve as a valuable tool in distinguishing GERD from FHB.

Dynamic changes in the aggregation-depolymerization behavior of Ovomucin-Complex and its binding to urease during in vitro simulated gastric digestion.

Ovomucin-Complex extracted from egg white is expected to have a barrier function similar to gastric mucin. In this study, the dynamic changes in structure, rheological properties and binding ability of Ovomucin-Complex during in vitro simulated gastric digestion were investigated. The results from HPLC and CLSM showed that extremely acidic pH (pH = 2.0) promoted Ovomucin-Complex to form aggregation. Acid-induced aggregation may hinder its binding to pepsin, thus rendering Ovomucin-Complex resistant to pepsin. Consequently, most of the polymer structure and weak gel properties of Ovomucin-Complex retained after simulated gastric digestion as verified by HPLC, CLSM and rheological measurement, although there was a small breakdown of the glycosidic bond as confirmed by the increased content of reducing sugar. The significantly reduced hydrophobic interactions of Ovomucin-Complex were observed under extremely acidic conditions and simulated gastric digestion compared with the native. Noticeably, the undigested Ovomucin-Complex after simulated gastric digestion showed a higher affinity (KD = 5.0 ± 3.2 nm) for urease - the key surface antigen of Helicobacter pylori. The interaction mechanism between Ovomucin-Complex and urease during gastric digestion deserves further studies. This finding provides a new insight to develop an artificial physical mucus barrier to reduce Helicobacter pylori infection.

Use of Betaine HCl with Pepsin in Esophageal Cancer Patient: A Case Report.

The principal mechanisms surrounding gastrointestinal (GI) side effects due to chemotherapy are unclear, whereas the information regarding symptom management of patients with esophageal cancer post-esophagectomy is lacking. Esophagectomy patients are left with significant anatomical changes to the GI tract, including the cutting of the vagus nerve, which regulates gastric secretions, gastric acid pH, and motility. A 76-year-old male patient self-referred himself to the clinical dietitian for nutritional management of chronic nausea, fatigue, weight loss, and dumping syndrome 9 months post-esophagectomy, which was not responsive to medications. A physical functional nutritional assessment with evaluation of diet history and elimination suggested gastric hypochlorhydria. Gastric acid is needed for the active absorption of iron, zinc, B complex vitamins, especially B12, and digestion of consumed proteins. A digestive supplement, betaine hydrochloric acid with pepsin (BHClP), was introduced, and the patient ingested 1 capsule containing 500 mg betaine hydrochloride and 23.5 mg pepsin prior to protein-containing meals and reported a substantial decrease in GI symptoms while eating a regular diet with no limitations. He gained necessary weight and energy for daily activities. After a few months, the patient discontinued BHClP, and GI symptoms and dumping syndrome returned, leading to a loss of 7.5% of his body weight. The patient reinitiated the supplement and GI symptoms dissipated, and weight was restored. BHClP provided metabolic therapeutic benefit to optimize the patient's oral intake, preventing further complications and malnutrition. The success with BHClP for this patient case suggests that more research is needed to fully realize the mechanisms and clinical usage.

Pepsin immobilization on activated carbon and functionalized with glutaraldehyde and genipin for the synthesis of antioxidant peptides of goat casein.

In this article, the synthesis of antioxidant peptides in the enzymatic hydrolysis of caprine casein was analyzed at three different time points (60 min, 90 min, and 120 min) using immobilized pepsin on activated and modified carbon (AC, ACF, ACG 50, ACG 100). The immobilization assays revealed a reduction in the biocatalysts' activity compared to the free enzyme. Among the modified ones, ACG 50 exhibited greater activity and better efficiency for reuse cycles, with superior values after 60 min and 90 min. Peptide synthesis was observed under all studied conditions. Analyses (DPPH, ß-carotene/linoleic acid, FRAP) confirmed the antioxidant potential of the peptides generated by the immobilized enzyme. However, the immobilized enzyme in ACG 50 and ACG 100, combined with longer hydrolysis times, allowed the formation of peptides with an antioxidant capacity greater than or equivalent to those generated by the free enzyme, despite reduced enzymatic activity.

Jellyfish protein hydrolysates: Multifunctional bioactivities unveiled in the battle against diabetes, inflammation, and bacterial pathogenesis.

This study investigates the multifunctional bioactivities of pepsin-hydrolyzed jellyfish by-products (Rhopilema hispidum and Lobonema smithii), focusing on their anti-α-glucosidase activity, anti-inflammatory effects, anti-bacterial properties, and ability to inhibit biofilm formation of Staphylococcus aureus. Our findings revealed that jellyfish protein hydrolysates, particularly from Rhopilema hispidum, exhibit significant anti-α-glucosidase activity, surpassing the well-known α-glucosidase inhibitor Acarbose. Furthermore, we demonstrated the anti-inflammatory capabilities of these hydrolysates in suppressing lipopolysaccharide (LPS)-induced nitric oxide production in murine macrophage cells. This effect was dose-dependent and non-cytotoxic, highlighting the hydrolysate potential in treating inflammation-related conditions. Regarding anti-bacterial activity, pepsin-hydrolyzed jellyfish selectively exhibited a potent effect against S. aureus, including Methicillin-susceptible and Methicillin-resistant strains. This activity was evident at minimum inhibitory concentrations (MIC) of 25 µg/mL for S. aureus ATCC10832, while a modest effect was observed against other Gram-positive strains. The hydrolysates effectively delayed bacterial growth dose-dependently, suggesting their use as alternative agents against bacterial infections. Most notably, pepsin-hydrolyzed jellyfish showed significant anti-biofilm activity against S. aureus. The umbrella section hydrolysate of Rhopilema hispidum was particularly effective, reducing biofilm formation through downregulating the icaA gene, crucial for biofilm development. Furthermore, the hydrolysates modulated the expression of the agrA gene, a key regulator in the pathogenesis of S. aureus. In conclusion, pepsin-hydrolyzed jellyfish protein hydrolysates exhibit promising multifunctional bioactivities, including anti-diabetic, anti-inflammatory, antibacterial, and anti-biofilm properties. These findings suggest their potential application in pharmaceutical and nutraceutical fields, particularly in managing diabetic risks, inflammation, bacterial infections, and combating the biofilm-associated pathogenicity of S. aureus.

Combined techniques for revealing the mechanism beneath the inhibition effects of pectin on gluten digestibility using static in vitro gastro-duodenal protocols.

In the current study, how pectin retards the digestibility of wheat gluten was investigated using a static in vitro gastric-duodenal model. The degree of protein hydrolysis was estimated using the o-phthaldialdehyde method, while the in vitro digestograms were mathematically fitted using a single first-order kinetics model. Peptides' profile, free amino acids compositions, gluten-pectin interactions and their effects on enzymatic activities of proteolytic enzymes as well as on the gluten secondary structures under digestive conditions were studied using combined techniques. Results showed that pectin could retard gluten digestibility through 1). preferential absorption to insoluble gluten aggregates by electrostatic interactions; 2). increasing the helix and reducing the ß-sheet content of the solubilized gluten protein fractions in terms of their secondary molecular structures; 3). reducing pepsin activity by forming negatively charged pectin-gluten mixtures which then interacted with the positively charged pepsin molecules. The deeper insight into gluten-pectin interactions and their influences on gluten digestibility under gastrointestinal conditions provides important clues for developing effective forms of dietary fiber to improve the nutritional benefits of plant protein in individuals.

In-Membrane Enrichment and Peptic Digestion to Facilitate Analysis of Monoclonal Antibody Glycosylation.

The number of therapeutic monoclonal antibodies (mAbs) is growing rapidly due to their widespread use for treating various diseases and health conditions. Assessing the glycosylation profile of mAbs during production is essential to ensuring their safety and efficacy. This research aims to rapidly isolate and digest mAbs for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of glycans and monitoring of glycosylation patterns, potentially during manufacturing. Immobilization of an Fc region-specific ligand, oFc20, in a porous membrane enables the enrichment of mAbs from cell culture supernatant and efficient elution with an acidic solution. Subsequent digestion of the mAb eluate occurred in a pepsin-modified membrane within 5 min. The procedure does not require alkylation and desalting, greatly shortening the sample preparation time. Subsequent LC-MS/MS analysis identified 11 major mAb N-glycan proteoforms and assessed the relative peak areas of the glycosylated peptides. This approach is suitable for the glycosylation profiling of various human IgG mAbs, including biosimilars and different IgG subclasses. The total time required for this workflow is less than 2 h, whereas the conventional enzymatic release and labeling of glycans can take much longer. Thus, the integrated membranes are suitable for facilitating the analysis of mAb glycosylation patterns.

Mechanism for improving the in vitro digestive properties of coconut milk by modifying the structure and properties of coconut proteins with monosodium glutamate.

In this paper, the effect of monosodium glutamate (MSG) on coconut protein (CP) solubility, surface hydrophobicity, emulsification activity, ultraviolet spectroscopy and fluorescence spectroscopy was investigated. Meanwhile, the changes in the in vitro digestive properties of coconut milk were also further analyzed. MSG treatment altered the solubility and surface hydrophobicity of CP, thereby improving protein digestibility. Molecular docking showed that CP bound to pepsin and trypsin mainly through hydrogen bonds and salt bridges. And MSG increased the cleavable sites of pepsin and trypsin on CP, thus further improving the protein digestibility. In addition, MSG increased the Na+ concentration in coconut milk, promoted flocculation and aggregation between coconut milk droplets, which prevented the binding of lipase and oil droplets and inhibited lipid digestion. These findings may provide new ideas and insights to improve the digestive properties of plant-based milk.

Analysis of Protein Digestion and Absorption Using a Ussing Chamber to Simulate the Environment in the Digestive Tract.

The Ussing chamber is a tool for analyzing drug absorption. We investigated whether the Ussing chamber can be used to analyze the process from digestion to absorption of protein in the gastrointestinal tract. Mixtures containing infant formula, whole cow's milk, processed soy milk, enteral nutrition, or human breast milk, were placed in the apical membrane side equipped with Caco-2 cells. After the addition of first pepsin then pancreatin, samples from the apical and basal membranes were collected. Infant formula showed the highest digestibility and absorption rate. This may be attributed to the presence of whey protein, which is rapidly digested and absorbed. The digestion and absorption of human breast milk showed different results in each donor, suggesting that digestion and absorption may vary among individuals. We concluded that the Ussing chamber can continuously analyze the process from digestion to absorption of proteins in the gastrointestinal tract.

Pepsin-mediated inflammation in laryngopharyngeal reflux via the ROS/NLRP3/IL-1ß signaling pathway.

BACKGROUND: Laryngopharyngeal reflux (LPR) is one of the most common disorders in otorhinolaryngology, affecting up to 10% of outpatients visiting otolaryngology departments. In addition, 50% of hoarseness cases are related to LPR. Pepsin reflux-induced aseptic inflammation is a major trigger of LPR; however, the underlying mechanisms are unclear. The nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome has become an important bridge between stimulation and sterile inflammation and is activated by intracellular reactive oxygen species (ROS) in response to danger signals, leading to an inflammatory cascade. In this study, we aimed to determine whether pepsin causes LPR-associated inflammatory injury via mediating inflammasome activation and explore the potential mechanism. METHODS: We evaluated NLRP3 inflammasome expression and ROS in the laryngeal mucosa using immunofluorescence and immunohistochemistry. Laryngeal epithelial cells were exposed to pepsin and analyzed using flow cytometry, western blotting, and real-time quantitative PCR to determine ROS, NLRP3, and pro-inflammatorycytokine levels. RESULTS: Pepsin expression was positively correlated with ROS as well as caspase-1 and IL-1ß levels in laryngeal tissues. Intracellular ROS levels were elevated by increased pepsin concentrations, which were attenuated by apocynin (APO)-a ROS inhibitor-in vitro. Furthermore, pepsin significantly induced the mRNA and protein expression of thioredoxin-interacting protein, NLRP3, caspase-1, and IL-1ß in a dose-dependent manner. APO and the NLRP3 inhibitor, MCC950, inhibited NLRP3 inflammasome formation and suppressed laryngeal epithelial cell damage. CONCLUSION: Our findings verified that pepsin could regulate the NLRP3/IL-1ß signaling pathway through ROS activation and further induce inflammatory injury in LPR. Targeting the ROS/NLRP3 inflammasome signaling pathway may help treat patients with LPR disease.

Structural analyzes suggest that MiSSP13 and MiSSP16.5 may act as proteases inhibitors during ectomycorrhiza establishment in Laccaria bicolor.

•The signaling process during mycorrhiza establishment involves intense molecular communication between symbionts. It has been suggested that a group of protein effectors, the so-called MiSSPs, plays a broader function in the symbiosis metabolism, however, many of these remain uncharacterized structurally and functionally. •Herein we used three-dimensional protein structure modeling methods, ligand analysis, and molecular docking to structurally characterize and describe two protein effectors, MiSSP13 and MiSSP16.5, with enhanced expression during the mycorrhizal process in Laccaria bicolor. •MiSSP13 and MiSSP16.5 show structural homology with the cysteine and aspartate protease inhibitor, cocaprin (CCP1). Through structural analysis, it was observed that MiSSP13 and MiSSP16.5 have an active site similar to that observed in CCP1. The protein-protein docking data showed that MiSSP13 and MiSSP16.5 interact with the papain and pepsin proteases at sites that are near to where CCP1 interacts with these same targets, suggesting a function as inhibitor of cysteine and aspartate proteases. The interaction of MiSSP13 with papain and MiSSP16.5 with pepsin was stronger than the interaction of CCP1 with these proteases, suggesting that the MiSSPs had a greater activity in inhibiting these classes of proteases. Based on the data supplied, a model is proposed for the function of MiSSPs 13 and 16.5 during the symbiosis establishment. Our findings, while derived from in silico analyses, enable us formulate intriguing hypothesis on the function of MiSSPs in ectomycorrhization, which will require experimental validation.

Oral infectivity through carnivorism in murine model of Trypanosoma cruzi infection.

Introduction: Oral transmission of T. cruzi is probably the most frequent transmission mechanism in wild animals. This observation led to the hypothesis that consuming raw or undercooked meat from animals infected with T. cruzi may be responsible for transmitting the infection. Therefore, the general objective of this study was to investigate host-pathogen interactions between the parasite and gastric mucosa and the role of meat consumption from infected animals in the oral transmission of T. cruzi. Methods: Cell infectivity assays were performed on AGS cells in the presence or absence of mucin, and the roles of pepsin and acidic pH were determined. Moreover, groups of five female Balb/c mice were fed with muscle tissue obtained from mice in the acute phase of infection by the clone H510 C8C3hvir of T. cruzi, and the infection of the fed mice was monitored by a parasitemia curve. Similarly, we assessed the infective capacity of T. cruzi trypomastigotes and amastigotes by infecting groups of five mice Balb/c females, which were infected orally using a nasogastric probe, and the infection was monitored by a parasitemia curve. Finally, different trypomastigote and amastigote inoculums were used to determine their infective capacities. Adhesion assays of T. cruzi proteins to AGS stomach cells were performed, and the adhered proteins were detected by western blotting using monoclonal or polyclonal antibodies and by LC-MS/MS and bioinformatics analysis. Results: Trypomastigote migration in the presence of mucin was reduced by approximately 30%, whereas in the presence of mucin and pepsin at pH 3.5, only a small proportion of parasites were able to migrate (∼6%). Similarly, the ability of TCTs to infect AGS cells in the presence of mucin is reduced by approximately 20%. In all cases, 60-100% of the animals were fed meat from mice infected in the acute phase or infected with trypomastigotes or amastigotes developed high parasitemia, and 80% died around day 40 post-infection. The adhesion assay showed that cruzipain is a molecule of trypomastigotes and amastigotes that binds to AGS cells. LC-MS/MS and bioinformatics analysis, also confirmed that transialidase, cysteine proteinases, and gp63 may be involved in TCTs attachment or invasion of human stomach cells because they can potentially interact with different proteins in the human stomach mucosa. In addition, several human gastric mucins have cysteine protease cleavage sites. Discussion: Then, under our experimental conditions, consuming meat from infected animals in the acute phase allows the T. cruzi infection. Similarly, trypomastigotes and amastigotes could infect mice when administered orally, whereas cysteinyl proteinases and trans-sialidase appear to be relevant molecules in this infective process.

[Analysis of clinical characteristics of children with adenoid hypertrophy and pharyngolaryngeal reflux].

Objectives: To explore the clinical characteristics of children with adenoid hypertrophy (AH) and laryngopharyngeal reflux (LPR) by detecting the expression of pepsin in adenoids as a standard for AH with LPR. Methods: A total of 190 children who were admitted for surgical treatment due to AH were included in the study. The main clinical symptoms of the patients were recorded, and the degree of adenoid hypertrophy was evaluated. Before the surgery, Reflux Symptom Index (RSI) and Reflux Finding Score (RFS) were used to evaluate the reflux symptoms. After the surgery, pepsin immunohistochemical staining was performed on the adenoid tissue, and according to the staining results, the patients were divided into study group (pepsin staining positive) and control group (pepsin staining negative). SPSS 19.0 software was used for statistical analysis. Quantitative data conforming to normal distribution between the two groups were tested by two-independent sample t test, and quantitative data with skewed distribution were tested by Mann-Whitney U test. Results: The positive rate of pepsin staining in the 190 AH patients was 78.4% (149/190). The study group had higher levels of preoperative symptoms such as erythema and/or congestion of the pharynx(2.1±0.7 vs. 1.8±0.6,t=2.23), vocal cord edema[1.0(0, 1.0) vs. 1.0(0, 1.0), Z=2.00], diffuse laryngeal edema[0(0, 1.0) vs. 0(0, 0), Z=2.48], posterior commissure hypertrophy[(1.4±0.6 vs. 1.1±0.5), t=2.63], and a higher total score on the RFS scale than the control group(6.2±2.7 vs. 5.0±2.6, t=2.47), with statistical differences (P<0.05). The sensitivity and specificity of RFS score in diagnosing AH with LPR were 24.8% and 80.5%, respectively. When RFS>5 was used as the positive threshold, the sensitivity and specificity of RFS score in diagnosing AH with LPR were 61.1% and 58.5%, respectively. There was a statistical difference in the number of positive cases of RFS score between the study group and the control group(91 vs. 17,χ2=5.04,P=0.032). Conclusions: LPR is common in AH children. Children with AH and LPR have specific performance in electronic laryngoscopy, such as erythema with edema in the pharynx, posterior commissure hypertrophy, and vocal cord edema.

Chemically Bonded Pepsin via Its Inert Center to Diazo Functionalized Silica Gel through Multipoint Attachment Mode: A Way of Restoring Biocatalytic Sustainability over "Wider pH" Range.

Proteolytic enzymes play a pivotal role in the industry. Still, because of denaturation, the extensive applicability at their level of best catalytic efficiency over a more comprehensive pH range, particularly in alkaline conditions over pH 8, has not been fully developed. On the other hand, enzyme immobilization following a suitable protocol is a long pending issue that determines the conformational stability, specificity, selectivity, enantioselectivity, and activity of the native enzymes at long-range pH. As a bridge between these two findings, in an attempt at a freezing temperature 273-278 K at an alkaline pH, the diazo-functionalized silica gel (SG) surface has been used to rapidly diazo couple pepsin through its inert center, the O-carbon of the phenolic -OH of surface-occupied Tyr residues in a multipoint mode: when all the various protein groups, viz., amino, thiol, phenol, imidazole, carboxy, etc., in the molecular sequence including those belonging to the active sites, remain intact, the inherent inbuilt interactions among themselves remain. Thereby, the macromolecule's global conformation and helicity preserve the status quo. The dimension of the SG-enzyme conjugate confirms as {Si(OSi)4 (H2O)1.03}n {-O-Si(CH3)2-O-C6H4-N═N+}4·{pepsin}·yH2O; where the values of n and y have been determined respectively as 347 and 188. The material performs the catalytic activity much better at 7-8.5 than at pH 2-3.5 and continues for up to six months without any appreciable change.

Study of an inhibitory effect of plant polyphenolic compounds against digestive enzymes using bench-working experimental evidence predicted by molecular docking and dynamics.

The substantial nutritional content and diversified biological activity of plant-based nutraceuticals are due to polyphenolic chemicals. These chemicals are important and well-studied plant secondary metabolites. Their protein interactions are extensively studied. This relationship is crucial for the logical development of functional food and for enhancing the availability and usefulness of polyphenols. This study highlights the influence of protein types and polyphenols on the interaction, where the chemical bindings predominantly consist of hydrophobic interactions and hydrogen bonds. The interaction between polyphenolic compounds (PCs) and digestive enzymes concerning their inhibitory activity has not been fully studied. Therefore, we have examined the interaction of four digestive enzymes (α-amylase, pepsin, trypsin, and α-chymotrypsin) with four PCs (curcumin, diosmin, morin, and 2',3',4'-trihydroxychalcone) through in silico and in vitro approaches. In vitro plate assays, enzyme kinetics, spectroscopic assays, molecular docking, and simulations were performed. We observed all these PCs have significant docking scores and preferable interaction with the active site of the digestive enzymes, resulting in the reduction of enzyme activity. The enzyme-substrate binding mechanism was determined using the Lineweaver Burk plot, indicating that the inhibition occurred competitively. Among four PCs diosmin and morin has the highest interaction energy over digestive enzymes with IC50 value of 1.13 ± 0.0047 and 1.086 ± 0.0131 µM. Kinetic studies show that selected PCs inhibited pepsin, trypsin, and chymotrypsin competitively and inhibited amylase in a non-competitive manner, especially by 2',3',4'-trihydroxychalcone. This study offers insights into the mechanisms by which the selected PCs inhibit the enzymes and has the potential to enhance the application of curcumin, diosmin, morin, and 2',3',4'-trihydroxychalcone as natural inhibitors of digestive enzymes.

Structure, physicochemical properties, and biological activities of protein hydrolysates from Zanthoxylum seed.

BACKGROUND: Zanthoxylum seed, as a low-cost and easily accessible plant protein resource, has good potential in the food industry. But protein and its hydrolysates from Zanthoxylum seed are underutilized due to the dearth of studies on them. This study aimed to investigate the structure and physicochemical and biological activities of Zanthoxylum seed protein (ZSP) hydrolysates prepared using Protamex®, Alcalase®, Neutrase®, trypsin, or pepsin. RESULTS: Hydrolysis using each of the five enzymes diminished average particle size and molecular weight of ZSP but increased random coil content. ZSP hydrolysate prepared using pepsin had the highest degree of hydrolysis (24.07%) and the smallest molecular weight (<13 kDa) and average particle size (129.80 nm) with the highest solubility (98.9%). In contrast, ZSP hydrolysate prepared using Alcalase had the highest surface hydrophobicity and foaming capacity (88.89%), as well as the lowest foam stability (45.00%). Moreover, ZSP hydrolysate prepared using Alcalase exhibited the best hydroxyl-radical scavenging (half maximal inhibitory concentration (IC50 ) 1.94 mg mL-1 ) and ferrous-ion chelating (IC50 0.61 mg mL-1 ) activities. Additionally, ZSP hydrolysate prepared using pepsin displayed the highest angiotensin-converting enzyme inhibition activity (IC50 0.54 mg mL-1 ). CONCLUSION: These data showed that enzyme hydrolysis improved the physicochemical properties of ZSP, and enzymatic hydrolysates of ZSP exhibited significant biological activity. These results provided validation for application of ZSP enzymatic hydrolysates as antioxidants and antihypertensive agents in the food or medicinal industries. © 2023 Society of Chemical Industry.

On the effect of pepsin incubation on type I collagen from horse tendon: Fine tuning of its physico-chemical and rheological properties.

Type I collagen is commonly recognized as the gold standard biomaterial for the manufacturing of medical devices for health-care related applications. In recent years, with the final aim of developing scaffolds with optimal bioactivity, even more studies focused on the influence of processing parameters on collagen properties, since processing can strongly affect the architecture of collagen at various length scales and, consequently, scaffolds macroscopic performances. The ability to finely tune scaffold properties in order to closely mimic the tissues' hierarchical features, preserving collagen's natural conformation, is actually of great interest. In this work, the effect of the pepsin-based extraction step on the material final properties was investigated. Thus, the physico-chemical properties of fibrillar type I collagens upon being extracted under various conditions were analyzed in depth. Correlations of collagen structure at the supramolecular scale with its microstructural properties were done, confirming the possibility of tuning rheological, viscoelastic and degradation properties of fibrillar type I collagen.

Evidence for paraquat-pepsin interaction: In vitro and silico study.

The use of the herbicide paraquat (PQ) has raised concerns about potential environmental consequences due to its toxicity and persistence in the environment. Considering the affinity of dangerous compounds to biological molecules, it is necessary to know their binding properties. This article focuses on the behavior of the pepsin enzyme following its contact with paraquat poison, and the interaction between paraquat and pepsin has been investigated in laboratory conditions and simulated physiological conditions using multispectral techniques. Fluorescence experiments showed that PQ uses a static method to quench pepsin's intrinsic fluorescence. By causing structural damage to pepsin, PQ may be detrimental as it alters its conformational function based on FT-IR spectroscopy. The coupling reaction is a spontaneous process caused by hydrogen bonding and van der Waals forces according to the analysis of the thermodynamic parameters of each system at three different temperatures. The molecular structure of pepsin changes when it binds to PQ. Also, the results showed that PQ is a pepsin inhibitor that changes the function of the enzyme.