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.

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.

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.

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.

The effect of konjac glucomannan on enzyme kinetics and fluorescence spectrometry of digestive enzymes: An in vitro research from the perspective of macromolecule crowding.

Konjac glucomannan (KGM) can significantly prolong gastrointestinal digestion. However, it is still worth investigating whether the macromolecular crowding (MMC) induced by KGM is correlated with digestion. In this paper, the MMC effect was quantified by fluorescence resonance energy transfer and microrheology, and the digests of starch, protein, and oil were determined. The digestive enzymes were analyzed by enzyme reaction kinetic and fluorescence quenching. The results showed that higher molecular weight (604.85 âˆ¼ 1002.21 kDa) KGM created a larger MMC (>0.8), and influenced the digestion of macronutrients; the digests of starch, protein, and oil all decreased significantly. MMC induced by KGM decreased the Michaelis-Menten constants (Km and Vmax) of pancreatic α-amylase (PPA), pepsin (PEP), and pancreatic lipase (PPL). The larger MMC (>0.8) induced by KGM resulted in the decrease of fluorescence quenching constants (Ksv) in PPA and PPL, and the increase of Ksv in PEP. Therefore, varying degrees of MMC induced by KGM could play a role in regulating digestion and the inhibitory effect on digestion was more significant in a relatively more crowded environment induced by KGM. This study provides theoretical support for the strategies of nutrient digestion regulation from the perspective of MMC caused by dietary fiber.

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.

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.

Microfluidic-oriented green synthesis of pepsin-doped gold nanoparticles for colorimetric and photothermal dual-readout detection of food hazards.

Gold nanoparticles (AuNPs)-based immunochromatographic assay has gained popularity as a rapid detection method for food hazards. Synthesizing highly stable AuNPs in a rapid, simple and environmentally friendly manner is a key focus in this field. Here, we present a green microfluidic strategy for the rapid, automated, and size-controllable synthesis of pepsin-doped AuNPs (AuNPs@Pep) by employing glucose-pepsin as a versatile reducing agent and stabilizer. Through combining the colorimetric and photothermal (PoT) properties of AuNPs@Pep, both "signal-off" and "signal-on" formats of microfluidic paper analytical devices (PADs) were developed for detection of a small molecule antibiotic, florfenicol, and an egg allergen, ovalbumin. Compared to the colorimetric mode, a 4-fold and 3-fold improvement in limit of detection was observed in the "signal-off" detection of florfenicol and the "signal-on" detection of ovalbumin, respectively. The results demonstrated the practicality of AuNPs@Pep as a colorimetric/PoT dual-readout probe for immunochromatographic detection of food hazards at different molecular scales.

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.

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.

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.

Development of Highly Sensitive Fluorescent Sensors for Separation-Free Detection and Quantitation Systems of Pepsin Enzyme Applying a Structure-Guided Approach.

Two fluorescent molecularly imprinted polymers (MIPs) were developed for pepsin enzyme utilising fluorescein and rhodamine b. The main difference between both dyes is the presence of two (diethylamino) groups in the structure of rhodamine b. Consequently, we wanted to investigate the effect of these functional groups on the selectivity and sensitivity of the resulting MIPs. Therefore, two silica-based MIPs for pepsin enzyme were developed using 3-aminopropyltriethoxysilane as a functional monomer and tetraethyl orthosilicate as a crosslinker to achieve a one-pot synthesis. Results of our study revealed that rhodamine b dyed MIPs (RMIPs) showed stronger binding, indicated by a higher binding capacity value of 256 mg g-1 compared to 217 mg g-1 for fluorescein dyed MIPs (FMIPs). Moreover, RMIPs showed superior sensitivity in the detection and quantitation of pepsin with a linear range from 0.28 to 42.85 µmol L-1 and a limit of detection (LOD) as low as 0.11 µmol L-1. In contrast, FMIPs covered a narrower range from 0.71 to 35.71 µmol L-1, and the LOD value reached 0.34 µmol L-1, which is three times less sensitive than RMIPs. Finally, the developed FMIPs and RMIPs were applied to a separation-free quantification system for pepsin in saliva samples without interference from any cross-reactors.

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.

Advances in Understanding the Antioxidant and Antigenic Properties of Egg-Derived Peptides.

Pepsin, trypsin and proteinase K were used in the present study to hydrolyse the proteins from whole eggs, yolks or whites, and the resulting hydrolysates were characterised in terms of antioxidant and IgE-binding properties, using a combination of in vitro and in silico methods. Based on the degree of hydrolysis (DH) results, the egg yolk proteins are better substrates for all the tested enzymes (DH of 6.2-20.1%) compared to those from egg whites (DH of 2.0-4.4%). The SDS-PAGE analysis indicated that pepsin and proteinase K were more efficient compared to trypsin in breaking the intramolecular peptide bonds of the high molecular weight egg proteins. For all the tested substrates, enzyme-assisted hydrolysis resulted in a significant increase in antioxidant activity, suggesting that many bioactive peptides are encrypted in inactive forms in the parent proteins. The hydrolysates obtained with proteinase K exhibited the highest DPPH radical scavenging activity (124-311 µM Trolox/g protein) and the lowest residual IgE-binding capacity. The bioinformatics tools revealed that proteinase K is able to break the integrity of the main linear IgE-binding epitopes from ovalbumin and ovomucoid. It can be concluded that proteinase K is a promising tool for modulating the intrinsic properties of egg proteins.

Water-Soluble Coffee Melanoidins Inhibit Digestive Proteases.

Coffee is one of the most popular beverages around the world and its consumption contributes to the daily intake of dietary melanoidins. Despite the emerging physiological role of food melanoidins, their effect on digestive processes has not been studied so far. In this study, the activity of the gastrointestinal enzymes pepsin and trypsin was investigated in the presence of water-soluble coffee melanoidins. The gastric enzyme pepsin is only slightly affected, whereas the intestinal enzyme trypsin is severely inhibited by coffee melanoidins. The intestinal digestibility of casein was significantly inhibited by coffee melanoidins at a concentration achievable by regular coffee consumption. The inhibition of proteolytic enzymes by coffee melanoidins might decrease the nutritional value of dietary proteins.

Rational design of soluble expressed human aldehyde dehydrogenase 2 with high stability and activity in pepsin and trypsin.

Acetaldehyde dehydrogenase 2 (ALDH2) is a crucial enzyme in alcohol metabolism, and oral administration of ALDH2 is a promising method for alcohol detoxification. However, recombinant ALDH2 is susceptible to hydrolysis by digestive enzymes in the gastrointestinal tract and is expressed as inactive inclusion bodies in E. coli. In this study, we performed three rounds of rational design to address these issues. Specifically, the surface digestive sites of pepsin and trypsin were replaced with other polar amino acids, while hydrophobic amino acids were incorporated to reshape the catalytic cavity of ALDH2. The resulting mutant DE2-852 exhibited a 45-fold increase in soluble expression levels, while its stability against trypsin and pepsin increased by eightfold and twofold, respectively. Its catalytic efficiency (kcat/Km) at pH 7.2 and 3.2 improved by more than four and five times, respectively, with increased Vmax and decreased Km values. The enhanced properties of DE2-852 were attributed to the D457Y mutation, which created a more compact protein structure and facilitated a faster collision between the substrate and catalytic residues. These results laid the foundation for the oral administration and mass preparation of highly active ALDH2 and offered insights into the oral application of other proteins.

A millimeter water-in-oil droplet as an alternative back exchange prevention strategy for hydrogen/deuterium exchange mass spectrometry of peptides/proteins.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a versatile bioanalytical technique for protein analysis. Since the reliability of HDX-MS analysis considerably depends on the retention of deuterium labels in the post-labeling workflow, deuterium/hydrogen (D/H) back exchange prevention strategies, including decreasing the pH, temperature, and exposure time to protic sources of the deuterated samples, are widely adopted in the conventional HDX-MS protocol. Herein, an alternative and effective back exchange prevention strategy based on the encapsulation of a millimeter droplet of a labeled peptide solution in a water-immiscible organic solvent (cyclohexane) is proposed. Cyclohexane was used to prevent the undesirable uptake of water by the droplet from the atmospheric vapor through the air-water interface. Using the pepsin digest of deuterated myoglobin, our results show that back exchange kinetics of deuterated peptides is retarded in a millimeter droplet as compared to that in the bulk solution. Performing pepsin digestion directly in a water-in-oil droplet at room temperature (18-21 °C) was found to preserve more deuterium labels than that in the bulk digestion with an ice-water bath. Based on the present findings, it is proposed that keeping deuterated peptides in the form of water-in-oil droplets during the post-labelling workflow will facilitate the preservation of deuterium labels on the peptide backbone and thereby enhance the reliability of the H/D exchange data.

Biochemical characterization of acid proteases from the stomach of palometa (Pygocentrus nattereri, Kner 1858) with potential industrial application.

Pepsin is one of the major enzymes with significant importance in the food industry, biomedicines, and pharmaceutical formulations. In this work, the main objective was to biochemically characterize a pepsin-like enzymatic extract obtained from Pygocentrus nattereri, a predatory freshwater fish, focusing on their potential industrial application. The obtained extract exhibited optimal activity at 45 °C and pH 1.0-2.0. These proteases remained stable after 2 h of incubation at temperatures ranging from 0° to 45 °C and within pH range of 1.0 to 7.0. Their activity was significantly affected in presence of pepstatin A and SDS, 10 µM and 3.46 mM respectively, while EDTA and PMSF showed partial inhibitory effects. Divalent cations (Ca2+ and Mg2+) did not inhibit the proteolytic activity of the extract; in fact, it improved at a 5 mM CaCl2 concentration. As the NaCl concentration increased, the enzyme activity decreased. However, after desalination, 90 % of the activity was recovered within the tested exposure time. Besides, this extract demonstrated exceptional versatility across diverse industrial applications, including collagen extraction augmentation, IgG hydrolysis facilitation, and silver and polyester recovery from X-ray films. Our results suggest that the obtained enzymatic extract has a wide range of potential applications.

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.