RESUMEN
The main objective of this study was to assess the ability of the NEar Real Digestive Tract (NERDT), a computer-controlled biomimetic in vitro digestion system that considers the biomechanics of the stomach, to reproduce physiologically relevant features of skimmed milk gastric digestion. A second objective was to evaluate the influence of pepsin on the gastric coagulation and emptying of milk proteins from experiments performed with and without pepsin. A mass balance model over the stomach, assuming a perfectly stirred reactor behaviour, has been developed. The results show that the NERDT can adequately reproduce the targeted kinetics of gastric acidification and emptying, with a sieving effect that naturally leads to a delayed emptying of caseins. Milk coagulated earlier and more chyme was emptied towards the end of the experiments in the presence of pepsin than without, hence illustrating the key influence of pepsin on the gastric coagulation of caseins and subsequent hydrolysis and emptying of dairy particles. Overall, this study shows that the NERDT can be adequately controlled to achieve desired gastric digestion conditions, and appears to be a very useful tool to further improve the knowledge of the gastric digestion behaviour of complex foods such as milk.
Asunto(s)
Biomimética , Digestión , Leche , Pepsina A , Digestión/fisiología , Animales , Pepsina A/metabolismo , Humanos , Leche/química , Biomimética/métodos , Modelos Biológicos , Concentración de Iones de Hidrógeno , Vaciamiento Gástrico/fisiología , Caseínas/metabolismo , Estómago/fisiología , Cinética , Proteínas de la Leche/metabolismoRESUMEN
The gelation of milk proteins can be achieved by various means, enabling the development of diverse products. In this study, heat-set milk protein gels (15 % protein) of diverse textures were made by pH modulation and two gels were selected for dynamic in vitro gastric digestion: a spoonable soft gel (SG, pH 6.55' G' of â¼100 Pa) and a sliceable firm gel (FG, pH 5.65; G' of â¼7000 Pa). The two gels displayed markedly different structural changes and digestion kinetics during gastric digestion. The SG underwent substantial structural compaction during the first 120 min of gastric digestion into a denser and firmer gastric chyme (26.3 % crude protein, G* of â¼8500 Pa) than the chyme of the FG (15.7 % crude protein, G* of â¼3000 Pa). These contrasting intragastric structural changes of the gels reversed their original textural differences, which led to slower digestion and gastric emptying of proteins from the SG compared with the FG. The different intragastric pH profiles during the digestion of the two gels likely played a key role by modulating the proteolytic activity and specificity (to κ-casein) of pepsin. Preferential early cleavage of κ-casein in SG stimulated coagulation and compaction of solid chyme, whereas rapid hydrolysis of αS- and ß-caseins in the FG weakened coagulation. This study provided new insights into controlling the structural development of dairy-based foods during gastric digestion and modulating digestion kinetics.
Asunto(s)
Digestión , Geles , Calor , Proteínas de la Leche , Geles/química , Digestión/fisiología , Concentración de Iones de Hidrógeno , Cinética , Proteínas de la Leche/química , Proteínas de la Leche/metabolismo , Vaciamiento Gástrico , Caseínas/química , Caseínas/metabolismo , Pepsina A/metabolismo , Animales , Manipulación de Alimentos/métodos , ProteolisisRESUMEN
Utilizing different chymosin and pepsin ratios in cheesemaking may represent a potential strategy to shape the sensory profile of hard cheeses. This study investigated the impact of rennet with varying chymosin and pepsin ratios on the chemical profile and sensory attributes of Grana Padano PDO cheese at different ripening times (10 to 20 months). The research involved the analysis of hard cheese manufactured with distinct calf chymosin percentages (99 %, 95 %, and 83 %), exploiting sensory analyses and untargeted metabolomics to identify marker compounds correlating with specific sensory traits. The results demonstrated that varying the rennet composition significantly affected sensory profile; in particular, the rennet made by 83 % chymosin and 17 % pepsin generated a more complex sensory profile starting from 12 months. AMOPLS and ASCA analysis on untargeted metabolomics signatures revealed that ripening time was the only significant factor when compared with rennet type and the interaction ripening x rennet. Finally, at more advanced ripening times, 3-methylbutanoic acid and homoethone were significantly up-accumulated in cheese samples manufactured with higher pepsin percentages, likely explaining sensory outcomes. This study provides valuable insights into using rennet to tailor the sensory qualities of hard cheeses, underscoring the importance of enzyme selection in cheese manufacturing to drive innovation in the dairy industry.
Asunto(s)
Queso , Quimosina , Manipulación de Alimentos , Metabolómica , Pepsina A , Gusto , Queso/análisis , Quimosina/metabolismo , Pepsina A/metabolismo , Metabolómica/métodos , Manipulación de Alimentos/métodos , Animales , Bovinos , HumanosRESUMEN
AIM: Pepsin is an enzyme that helps digest protein secreted only from the gastric chief cell in an inactive state. Pepsin is a good marker for acidic gastroesophageal reflux (GER). Its presence in sputum or saliva is considered pathologic. In GER, cough is stimulated by broncho-esophageal neurogenic reflex and aspiration of gastric contents into the airways. GER is the most common cause of cough. Gastric acid reflux is also thought to play a role in Interstitial Lung Disease (ILD) etiology. In many studies, pepsin and bile acid levels in bronchial lavage were high in patients with interstitial lung disease and chronic cough. In our study, we aimed to evaluate pepsin levels in bronchial lavage in patients with ILD and chronic cough and to investigate the relationship between symptoms and reflux treatment. METHODS: Between January 2021 and February 2022, 212 patients who underwent bronchoscopy in our tertiary clinic were evaluated. These patients were divided into three groups: 52 patients with interstitial lung disease, 81 patients with chronic cough, and 79 patients who underwent bronchoscopy with a pre-diagnosis of lung cancer as the control group. Bronchial lavage obtained by bronchoscopy was analyzed for pepsin levels. RESULTS: Shortness of breath and cough were the most common symptoms in all three groups. Pepsin levels were 16.71 ± 8.6 ng/ml in the chronic cough group, 15.6 ± 8.9 ng/ml in the ILD group, and 10.58 ± 5.4 ng/ml in the lung cancer (control) group. Pepsin levels in the ILD and chronic cough group were statistically significantly higher than in the lung cancer group (p:0.00). There was no statistical difference between the ILD group and the chronic cough group regarding pepsin levels. It was found that pepsin levels were lower in the three groups who received anti-reflux treatment. There was no difference in pepsin levels between ILD subgroups. CONCLUSION: Pepsin levels in bronchial lavage were higher in the ILD and chronic cough groups. This suggests that reflux may be involved in the etiology of chronic cough and ILD. Low pepsin values in patients receiving anti-reflux therapy have shown that occult reflux may occur. In our study, the high level of pepsin in bronchial lavage, especially in the chronic cough and ILD group, may be instructive in the etiology and treatment planning of the disease.
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Tos , Reflujo Gastroesofágico , Enfermedades Pulmonares Intersticiales , Pepsina A , Humanos , Tos/metabolismo , Tos/etiología , Pepsina A/análisis , Pepsina A/metabolismo , Enfermedades Pulmonares Intersticiales/metabolismo , Enfermedades Pulmonares Intersticiales/complicaciones , Enfermedad Crónica , Masculino , Femenino , Persona de Mediana Edad , Reflujo Gastroesofágico/complicaciones , Reflujo Gastroesofágico/metabolismo , Anciano , Líquido del Lavado Bronquioalveolar/química , Lavado Broncoalveolar/métodos , Broncoscopía/métodos , Biomarcadores/metabolismo , Biomarcadores/análisis , Tos CrónicaRESUMEN
Immunoglobulin G (IgG) exhibits potent antiviral, antibacterial, and immunological activities. The digestion process and bioavailability of IgG are often a concern. Dietary hydrocolloids are crucial for regulating healthy digestion and the bioavailability of protein as functional components. Understanding the effects of dietary hydrocolloids on the digestive kinetics of IgG is requisite. Herein, the pepsin and trypsin digestion of IgG was investigated using ordered porous layer interferometry (OPLI). The real-time variation in the interference spectral shift reflected by OPLI can be converted into changes in the optical thickness (OT) to obtain a degradation kinetics curve. The impact of dietary hydrocolloids, including alginic acid sodium salt (ALG), polydextrose (PD), and konjac glucomannan (KG), on IgG degradation was evaluated using OPLI. The results demonstrated that ALG significantly inhibited the degradation of IgG by pepsin under acidic conditions, whereas the addition of PD increased the Michaelis-Menten constant for IgG degradation by trypsin. Notably, this dependence is not based on the hydrocolloid viscosity, but relies more on the electrical properties. The study enhances our understanding of how hydrocolloids affect IgG digestion and could provide valuable insights into preserving IgG activity and facilitating the development of oral drugs or health products related to IgG.
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Coloides , Inmunoglobulina G , Pepsina A , Proteolisis , Tripsina , Inmunoglobulina G/química , Tripsina/química , Tripsina/metabolismo , Coloides/química , Pepsina A/metabolismo , Pepsina A/química , Cinética , Humanos , AnimalesRESUMEN
Despite advancements in therapeutic monoclonal antibodies (mAbs) and cell line engineering, separating host cell proteins (HCPs) from mAbs during downstream purification remains challenging. Therefore, in this study, we developed a novel multimodal chromatography (MMC) resin to enhance HCP removal during mAb polishing processes. We evaluated the impact of both ligand structure and pore size of the MMC resin by purifying a post-protein A chromatography solution in flow-through mode. We observed that the efficiency of HCP clearance depended on the hydrophobic moiety structure of the ligand and predicted the mAb purification capability of MMC through linear salt-gradient elution experiments involving a mixture of transferrin, bovine serum albumin (BSA), and pepsin. Our findings revealed that the prototype immobilized 1,12-dodecanediamine via the formyl group exhibited the best performance attributed to its long alkyl chain. Furthermore, an investigation of effects of base bead pore size on HCP capacity using cellulose base beads of five different pore sizes showed that larger pore resin base beads had the highest HCP removal capacity. Specifically, MMC resins with a pore diameter exceeding 440 nm reduced the HCP level by three orders of magnitude under high mAb loading conditions (> 1000 mg/mL-resin). The MMC resin developed in this study, along with the insights gained into ligand structure and pore size, not only enhances mAb polishing efficiency but also contributes to improving downstream processes in mAb biopharmaceutical production.
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Anticuerpos Monoclonales , Cricetulus , Albúmina Sérica Bovina , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Animales , Células CHO , Ligandos , Albúmina Sérica Bovina/química , Porosidad , Cromatografía de Afinidad/métodos , Proteína Estafilocócica A/química , Transferrina/química , Transferrina/aislamiento & purificación , Pepsina A/química , Pepsina A/metabolismo , Proteínas/aislamiento & purificación , Proteínas/química , Resinas Sintéticas/química , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Pepsin as an aspartic acid protease member and one of the three foremost proteolytic enzymes in the digestive system is essential to be detected. An electrochemically polymerized tyrosine film on carbon paste electrode (pTyr/CPE) has been synthesized by electro-polymerization donating an affordable electrochemical sensor to sense salivary pepsin as a diagnostic technique for gastroesophageal reflux disease (GRD) due to saliva collection is non-invasive and relatively comfortable. The pTyr/CPE was applied for Voltammetric sensing of pepsin and its quantification in phosphate buffer solution of pH 2.0 (PBS). Scanning electron microscopy (SEM) was conducted to learn the surface morphology. Cyclic voltammetry (CV), differential pulse voltammetry (DPVs), chronoamperometry (CA), and electrochemical impedance spectroscopy (EIS) were developed to realize the electrocatalytic activity of the sensor. The pTyr/CPE proceeded as a sensitive detector to pepsin with two linear ranges from 1 to 20 & 20 to 100 ng/mL donating two limits of detection as 0.5 & 0.09 ng/mL, respectively, and high selectivity toward pepsin, as well as stability and fast response of 1.5 s. Consequently, it is guessed that the pTyr/CPE sensor could be supportive for the initial diagnosis of GRD through the detection of pepsin in saliva. Finally, we quantified the pepsin levels in saliva samples of LPR patients (n = 2), showing that the results were agreeable with those from the electrochemical sensor.
Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas , Pepsina A , Saliva , Pepsina A/análisis , Saliva/química , Humanos , Técnicas Electroquímicas/métodos , Técnicas Biosensibles/métodos , Electrodos , Tirosina/análisis , Espectroscopía Dieléctrica , Reflujo Gastroesofágico/diagnóstico , Límite de DetecciónRESUMEN
The pathophysiology of laryngopharyngeal reflux (LPR) and its impact on the vocal fold is not well understood, but may involve acid damage to vocal fold barrier functions. Two different components encompass vocal fold barrier function: the mucus barrier and tight junctions. Mucus retained on epithelial microprojections protects the inside of the vocal fold by neutralizing acidic damage. Tight junctions control permeability between cells. Here we developed an in vitro experimental system to evaluate acidic injury and repair of vocal fold barrier functions. We first established an in vitro model of rat vocal fold epithelium that could survive at least one week after barrier function maturation. The model enabled repeated evaluation of the course of vocal fold repair processes. Then, an injury experiment was conducted in which vocal fold cells were exposed to a 5-min treatment with acidic pepsin that injured tight junctions and cell surface microprojections. Both of them healed within one day of injury. Comparing vocal fold cells treated with acid alone with cells treated with acidic pepsin showed that acidic pepsin had a stronger effect on intercellular permeability than acid alone, whereas pepsin had little effect on microprojections. This result suggests that the proteolytic action of pepsin has a larger effect on protein-based tight junctions than on phospholipids in microprojections. This experimental system could contribute to a better understanding of vocal fold repair processes after chemical or physical injuries, as well as voice problems due to LPR pathogenesis.
Asunto(s)
Pepsina A , Uniones Estrechas , Pliegues Vocales , Animales , Pepsina A/metabolismo , Pepsina A/farmacología , Pliegues Vocales/efectos de los fármacos , Pliegues Vocales/patología , Pliegues Vocales/metabolismo , Pliegues Vocales/lesiones , Ratas , Uniones Estrechas/metabolismo , Uniones Estrechas/efectos de los fármacos , Ratas Sprague-Dawley , Masculino , Reflujo Laringofaríngeo/metabolismo , Reflujo Laringofaríngeo/tratamiento farmacológico , Reflujo Laringofaríngeo/patología , Concentración de Iones de HidrógenoRESUMEN
D-amino acids can affect the action of digestive enzymes, hence the protein digestion. In this work the behaviour of the main stomach and gut digestive enzymes (pepsin, trypsin, and chymotrypsin) in the presence of D-amino acids in the protein chain was monitored over time using a model peptide, Ac-LDAQSAPLRVYVE-NH2 (belonging to ß-lactoglobulin, position 48-60), where L-amino acids were systematically substituted by D-amino acids. The results showed several changes in the behaviour of digestive enzymes, not only when the D-amino acids are inserted at the specific cleavage sites (after Val-57), but in some cases also when in distant positions. The effect seemed more pronounced in the case of pepsin rather than the gut enzymes, possibly indicating a better resilience of the upper gut phase of digestion to racemization. These results demonstrated that racemization could impair nutritional value by slowing down digestibility and has different effects according to the enzyme/amino acids involved.
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Aminoácidos , Quimotripsina , Digestión , Pepsina A , Péptidos , Tripsina , Aminoácidos/química , Aminoácidos/metabolismo , Tripsina/química , Tripsina/metabolismo , Péptidos/química , Péptidos/metabolismo , Quimotripsina/química , Quimotripsina/metabolismo , Pepsina A/química , Pepsina A/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Lactoglobulinas/química , Lactoglobulinas/metabolismo , Modelos BiológicosRESUMEN
A sensitive and specific bioanalytical method was required to measure the exposure of a LAGA-mutated surrogate mouse IgG2a monoclonal antibody in mouse plasma, but the lack of highly specific reagents for the LAGA mutant hindered the development of a ligand-binding assay. Equally problematic is that no sensitive unique tryptic peptides suitable for quantitative mass spectrometric analysis could be identified in the mIgG2a complementarity-determining regions. To overcome these challenges, a trypsin alternative pepsin, an aspartic protease, was systematically investigated for its use in digesting the mutated mIgG2a antibody to allow generation of signature peptides for the bioanalytical quantification purpose. After a series of evaluations, a rapid one-hour pepsin digestion protocol was established for the mutated Fc backbone. Consequently, a new pepsin digestion-based liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was successfully developed to support the mouse pharmacokinetic (PK) sample analysis. In brief, robust and reproducible C-terminal cleavage of both leucine and phenylalanine near the double mutation site of the mutated mIgG2a was accomplished at pH ≤2 and 37°C. Combined with a commercially available rat anti-mIgG2a heavy-chain antibody, the established immunoaffinity LC/MS/MS assay achieved a limit of quantitation of 20 ng/mL in the dynamic range of interest with satisfactory assay precision and accuracy. The successful implementation of this novel approach in discovery PK studies eliminates the need for tedious and costly generation of specific immunocapturing reagents for the LAGA mutants. The approach should be widely applicable for developing popular LAGA mutant-based biological therapeutics.
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Inmunoglobulina G , Pepsina A , Espectrometría de Masas en Tándem , Animales , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Espectrometría de Masas en Tándem/métodos , Ratones , Cromatografía Liquida/métodos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/genética , Ratas , Mutación , Cromatografía de Afinidad/métodosRESUMEN
Biological enzyme-driven degradation of environmental pollutants has attracted widespread attention because it is ecofriendly and highly efficient. Immobilized enzyme technology has emerged as a promising technique in enzymology that addresses the limitations associated with free enzymes. Traditional solid-loaded enzyme substrates are often affected by blockages and restricted substrate accessibility. In this study, we synthesized an efficient heterogeneous pepsin catalyst, named PEP@M-MIL100(Fe), by covalently combining carboxylated ferrite structural expanded metal-organic frameworks with pepsin. This catalyst demonstrated excellent environmental adaptability and remarkable catalytic degradation capabilities. Notably, it rapidly degraded the persistent microplastic pollutant diisononyl phthalate (DINP) within just 150 min, with a removal efficiency of up to 95.88%. Impressively, even after 10 consecutive uses, the catalyst maintained its high performance. We proposed an innovative steady-state heterogeneous enzyme-catalyzed degradation mechanism, i.e., diffusion (D)-absorption (A)-binding (B)-reaction (R)-degradation (D)-link mechanism, which emphasizes the influence of substrate diffusion rates in this process. This work presents the first successful application of pepsin to DINP degradation and offers a sustainable and effective approach for addressing contemporary pollution challenges.
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Ésteres , Estructuras Metalorgánicas , Pepsina A , Ácidos Ftálicos , Estructuras Metalorgánicas/química , Ácidos Ftálicos/química , Pepsina A/química , Pepsina A/metabolismo , Ésteres/química , Catálisis , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Contaminantes Ambientales/químicaRESUMEN
BACKGROUND: Tracheoesophageal speech is one of the most effective method used for voice rehabilitation after laryngectomy. The main limitation is the need for periodic voice prothesis (VP) replacements. The process of developing VP usage complications is still unexplored. The aim of this study was to assess the level of cytokines (IL-1ß, IL-6, IL-8, IL-10, TNFα) and pepsin in saliva as potential factors reducing VP longevity. METHODS: Prospective double-blind randomized clinical trial was conducted (NCT04268459). Patients were randomly divided into two groups depending on VP replacement regimen (regular-every 3 months, or irregular-when complications occur). Levels of IL-1ß, IL-6, IL-8, IL-10, TNFα, and pepsin in saliva samples (fasting and after eating) of laryngectomized patients were measured using ELISA tests. RESULTS: Fifty-two patients (26 in both groups) with control group (7 patients) participated in the study. The level of IL-1ß, IL-6, IL-8, IL-10, TNFα, and pepsin did not differ according to regularity of VP replacements (p = 0.301-0.801). IL-6 levels were significantly higher when VP complications occurs (p = 0.012). CONCLUSIONS: The saliva components were not significantly different depending on the frequency of VP replacements. IL-6 plays an important role in the development of VP use complications.
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Citocinas , Laringectomía , Laringe Artificial , Pepsina A , Saliva , Humanos , Saliva/química , Saliva/metabolismo , Método Doble Ciego , Masculino , Femenino , Persona de Mediana Edad , Pepsina A/metabolismo , Anciano , Citocinas/metabolismo , Estudios ProspectivosRESUMEN
The study aimed to assess the extent to which protein aggregation, and even the modality of aggregation, can affect gastric digestion, down to the nature of the hydrolyzed peptide bonds. By controlling pH and ionic strength during heating, linear or spherical ovalbumin (OVA) aggregates were prepared, then digested with pepsin. Statistical analysis characterized the peptide bonds specifically hydrolyzed versus those not hydrolyzed for a given condition, based on a detailed description of all these bonds. Aggregation limits pepsin access to buried regions of native OVA, but some cleavage sites specific to aggregates reflect specific hydrolysis pathways due to the denaturation-aggregation process. Cleavage sites specific to linear aggregates indicate greater denaturation compared to spherical aggregates, consistent with theoretical models of heat-induced aggregation of OVA. Thus, the peptides released during the gastric phase may vary depending on the aggregation modality. Precisely tuned aggregation may therefore allow subtle control of the digestion process.
Asunto(s)
Digestión , Calor , Ovalbúmina , Pepsina A , Ovalbúmina/química , Ovalbúmina/metabolismo , Pepsina A/química , Pepsina A/metabolismo , Hidrólisis , Péptidos/química , Agregado de Proteínas , Concentración de Iones de Hidrógeno , AnimalesRESUMEN
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.
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Alginatos , Bacteriocinas , Cápsulas , Alginatos/química , Péptidos/química , Intestino Delgado/metabolismo , Lactobacillus plantarum/metabolismo , Concentración de Iones de Hidrógeno , Reactivos de Enlaces Cruzados/química , Pepsina A/metabolismoRESUMEN
Monitoring the gastric digestive function is important for the diagnosis of gastric disorders and drug development. However, there is no report on the in situ and real-time monitoring of digestive functions. Herein, we report a flexible fully organic sensor to effectively monitor protein digestion in situ in a simulated gastric environment for the first time. The sensors are made of a blend of gluten that is a protein and poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) that is a conducting polymer. During the protein digestion, the breakdown of the polypeptides increases the level of separation among the PEDOT chains, thereby increasing the resistance. The resistance variation is sensitive to various conditions, including the concentration of pepsin that is the enzyme for protein digestion, temperature, pH value, and digestive drugs. Hence, these sensors can provide real-time information about the digestion and efficacy of digestive drugs. In addition, the signals can be collected via a convenient wireless communication manner.
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Poliestirenos , Humanos , Poliestirenos/química , Digestión , Polímeros/química , Pepsina A/metabolismo , Pepsina A/química , Concentración de Iones de Hidrógeno , Temperatura , TiofenosRESUMEN
The 11S globulin legumin typically accounts for approximately 3% of the total protein in common beans (Phaseolus vulgaris). It was previously reported that a legumin peptide of approximately 20 kDa is resistant to pepsin digestion. Sequence prediction suggested that the pepsin-resistant peptide is located at the C-terminal end of the α-subunit, within a glutamic acid-rich domain, overlapping with a chymotrypsin-resistant peptide. Using purified legumin, the peptide of approximately 20 kDa was found to be resistant to pepsin digestion in a pH-dependent manner, and its location was determined by two-dimensional gel electrophoresis and LC-MS-MS. The location of the chymotrypsin-resistant peptide was confirmed by immunoblotting with peptide-specific polyclonal antibodies. The presence of a consensus site for proline hydroxylation and arabinosylation, the detection of hydroxyproline residues, purification by lectin affinity chromatography, and a difference in electrophoretic migration between the chymotrypsin- and pepsin-resistant peptides suggest the presence of a large O-glycan within these peptides.
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Secuencia de Aminoácidos , Quimotripsina , Pepsina A , Péptidos , Phaseolus , Phaseolus/química , Pepsina A/química , Pepsina A/metabolismo , Quimotripsina/química , Quimotripsina/metabolismo , Péptidos/química , Péptidos/aislamiento & purificación , Leguminas/química , Espectrometría de Masas en Tándem , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismoRESUMEN
The present work explores the specificity of supramolecular assemblies comprising dialkylaminostyrylhetarene dye molecules incorporated into phosphatidylcholine (PC) or phosphatidylserine (PS) aggregates. In PS-based assemblies, the dyes demonstrate a concentration-dependent fluorescent response, distinguishing anionic proteins such as bovine serum albumin (BSA) and pepsin from lysozyme (LYZ) in aqueous solutions. Conversely, no significant response is observed when the dyes are incorporated into the well-organized bilayers of neutral PC. The fluorescent response arises from the binding of dyes to proteins, leading to the detachment of dye molecules from the assemblies, rather than from the binding of proteins to the assemblies, although the latter process is facilitated by electrostatic attraction. Thus, both the poor ordering of PS molecules and the interfacial arrangement of the dyes are prerequisites for the fluorescent response of dye-PS aggregates. The structure of the dyes significantly impacts the spectral features of dye-PS and dye-protein assemblies. An optimal dye structure has been identified for the recognition of BSA, with a limit of detection (LOD) of 10.8â¯nM.
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Colorantes Fluorescentes , Fosfolípidos , Albúmina Sérica Bovina , Albúmina Sérica Bovina/química , Colorantes Fluorescentes/química , Fosfolípidos/química , Animales , Muramidasa/química , Muramidasa/metabolismo , Bovinos , Materiales Biomiméticos/química , Espectrometría de Fluorescencia , Pepsina A/química , Pepsina A/metabolismo , Fosfatidilcolinas/química , BiomiméticaRESUMEN
Pickering emulsions provide a promising platform for the efficient delivery of bioactive. However, co-delivery of fragile bioactives with different physicochemical properties for comprehensive effects still faces practical challenges due to the limited protection for bioactives and the lack of stimuli-responsive property for on-demand release. Herein, a stimuli-responsive co-delivery system is developed based on biomineralized particles stabilized Pickering emulsions. In this tailor co-delivery system, hydrophilic bioactive (pepsin) with the fragile structure is encapsulated and immobilized by biomineralization, the obtained biomineralized particles (PPS@CaCO3) are further utilized as emulsifiers to form O/W Pickering emulsions, in which the hydrophobic oxidizable bioactive (curcumin) is stably trapped into the dispersed phase. The results show that two bioactives are successfully co-encapsulated in Pickering emulsions, and benefiting from the protection capacities of biomineralization and Pickering emulsions, the activity of pepsin and curcumin shows a 7.33-fold and 144.83-fold enhancement compared to the free state, respectively. Moreover, In vitro study demonstrates that Pickering emulsions enable to co-release of two bioactives with high activity retention by the acid-induced hydrolyzation of biomineralized particles. This work provides a powerful stimuli-responsive platform for the co-delivery of multiple bioactive compounds, enabling high activity of bioactives for the comprehensive health effects.
Asunto(s)
Curcumina , Emulsionantes , Emulsiones , Tamaño de la Partícula , Emulsiones/química , Emulsionantes/química , Curcumina/química , Curcumina/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Carbonato de Calcio/química , Pepsina A/química , Pepsina A/metabolismo , Humanos , Propiedades de Superficie , Liberación de Fármacos , Biomineralización/efectos de los fármacosRESUMEN
In a hydrogen exchange-mass spectrometry (HX-MS) experiment, the enzymatic proteolysis of the deuterated protein is an essential step. Often the differences in the performance between different digestion protocols or between immobilized protease columns can be challenging to evaluate. To compare differences in the performance of immobilized protease columns, a new digestion efficiency metric known as digestible peptide scoring (DPS) was developed and is presented in this work. The measured response fraction of substance P peptide is used to assign a value between 0% and 100% based on the fraction of substance P digested by the enzyme, using angiotensin II as an undigested internal standard. In this work, the DPS approach was tested using multiple immobilized pepsin batches prepared using different protocols. The results demonstrate the repeatability of DPS values for batches prepared using the same conditions and the ability of the DPS evaluations to provide unique values when the immobilization conditions were altered. Protein digestions obtained with a higher scoring column were better than digestions obtained using a lower scoring column. The DPS evaluation is simple and quickly provides an unambiguous assessment which can be used to evaluate an immobilized enzyme column's suitability prior to performing an experiment, to track performance over a column's lifetime, to optimize protease immobilization protocols specifically for the quench conditions of a particular experiment, and to optimize the digestion conditions.
Asunto(s)
Pepsina A , Proteolisis , Pepsina A/metabolismo , Pepsina A/química , Péptidos/química , Péptidos/análisis , Péptidos/metabolismo , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio/métodos , Sustancia P/química , Sustancia P/metabolismo , Sustancia P/análisis , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismoRESUMEN
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.