Thermolysin (TLN) is a microbial highly-priced thermostable metallo-endoprotease with complementary substrate specificity to those of proteases widely used in science and industry for protein digestion and milk-clotting. This study is the first to immobilize TLN on aminated superparamagnetic nanoparticles (Fe3O4@silica-NH2) aiming for higher stability, recoverability, reusability, and applicability in proteolysis and as a microbial rennet-like milk-clotting enzyme. The nanobiocatalyst developed (Fe3O4@silica-TLN) displays hydrolytic activity on a synthetic TLN substrate and, apparently, was fully recovered from reaction media by magnetic decantation. More importantly, Fe3O4@silica-TLN retains TLN catalytic properties in the presence of calcium ions even after exposure to 60 °C for 48 h, storage at 4 °C for 80 days and room temperature for 42 days, use in proteolyses, and in milk-clotting for up to 11 cycles. Its proteolytic activity on bovine milk casein in 24 h furnished 84 peptides, of which 29 are potentially bioactive. Also, Fe3O4@silica-TLN catalyzed the digestion of bovine serum albumin. In conclusion, Fe3O4@silica-TLN showed to be a new, less autolytic, thermostable, non-toxic, magnetically-separable, and reusable nanobiocatalyst with highly attractive properties for both science (peptide/protein chemistry and structure, proteomic studies, and the search for new bioactive peptides) and food industry (cheese manufacture).

The microreactor with two types of immobilized enzymes, exhibiting excellent orthogonal performance, represents an effective approach to counteract the reduced digestion efficiency resulting from the absence of a single enzyme cleavage site, thereby impacting protein identification. In this study, we developed a hydrophilic dual-enzyme microreactor characterized by rapid mass transfer and superior enzymatic activity. Initially, we selected KIT-6 molecular sieve as the carrier for the dual-IMER due to its three-dimensional network pore structure. Modification involved co-deposition of polyethyleneimine (PEI) and acrylamide (AM) as amine donors, along with dopamine to enhance material hydrophilicity. Remaining amino and double bond functional groups facilitated stepwise immobilization of trypsin and Glu-C. Digestion times for bovine serum albumin (BSA) and bovine hemoglobin (BHb) on the dual-IMER were significantly reduced compared to solution-based digestion (1 min vs. 36 h), resulting in improved sequence coverage (91.30% vs. 82.7% for BSA; 90.24% vs. 89.20% for BHb). Additionally, the dual-IMER demonstrated excellent durability, retaining 96.08% relative activity after 29 reuse cycles. Enhanced protein digestion efficiency can be attributed to several factors: (1) KIT-6\'s large specific surface area, enabling higher enzyme loading capacity; (2) Its three-dimensional network pore structure, facilitating faster mass transfer and substance diffusion; (3) Orthogonality of trypsin and Glu-C enzyme cleavage sites; (4) The spatial effect introduced by the chain structure of PEI and glutaraldehyde\'s spacing arm, reducing spatial hindrance and enhancing enzyme-substrate interactions; (5) Mild and stable enzyme immobilization. The KIT-6-based dual-IMER offers a promising technical tool for protein digestion, while the PDA/PEI/AM-KIT-6 platform holds potential for immobilizing other proteins or active substances.

This research assessed how three preprocessing techniques [soaking (S), soaking and reconstitution (SR), and soaking and dehulling (SD)] impact the protein digestibility and bioactivity of faba bean flours when combined with thermoplastic extrusion. Samples were compared against a control (C) of extruded faba bean flour without preprocessing. Applying preprocessing techniques followed by extrusion diminished antinutrient levels while enhancing protein hydrolysis and in vitro bioactivity in higher extent compared to C. Specifically, SD combined with extrusion was the most effective, achieving an 80% rate of protein hydrolysis and uniquely promoting the release of gastric digestion-resistant proteins (50-70 kDa). It also resulted in the highest release of small peptides (<3kDa, 22.51%) and free amino acids (15.50%) during intestinal digestion. Moreover, while all preprocessing techniques increased antioxidant (ABTS radical-scavenging), antidiabetic, and anti-hypertensive activities, SD extruded flour displayed the highest levels of dipeptidyl peptidase inhibition (DPP-IVi, IC50=13.20 µg/mL), pancreatic α-amylase inhibition (IC50=8.59 mg/mL), and angiotensin I-converting enzyme inhibition (ACEi, IC50=1.71 mg protein/mL). As a result, it was selected for further peptide and in silico bioactive analysis. A total of 24 bioactive peptides were identified in intestinal digests from SD extruded flour, all with potential DPP-IVi and ACEi activities, and six were also predicted as antioxidant peptides. VIPAGYPVAIK and GLTETWNPNHPEL were highlighted as resistant bioactive peptides with the highest antidiabetic and antioxidant potential. Our findings demonstrated that combining preprocessing (particularly SD) and thermoplastic extrusion enhances protein digestibility in faba beans and promotes the release of beneficial bioactive peptides in the intestine.

Chitosan (CTS) and chitosan oligosaccharides (COS) have been widely applied in food industry due to their bioactivities and functions. However, CTS and COS with positive charges could interact with proteins, such as whey protein isolate (WPI), influencing their digestion. Interaction among CTS/COS, FUC, and WPI/enzymes was studied by spectroscopy, chromatography, and chemical methods in order to reveal the role of FUC in relieving the inhibition of protein digestibility by CTS/COS and demonstrate the action mechanisms. As shown by the results, the addition of FUC increased degree of hydrolysis (DH) and free protein in the mixture of CTS and WPI to 3.1-fold and 1.8-fold, respectively, while raise DH value and free protein in the mixture of COS and WPI to 6.7-fold and 1.2-fold, respectively. The interaction between amino, carboxyl, sulfate, and hydroxyl groups from carbohydrates and protein could be observed, and notably, FUC could interact with CTS/COS preferentially to prevent CTS/COS from combining with WPI. In addition, the addition of FUC could also relieve the combination of CTS to trypsin, increasing the fluorescence intensity and concentration of trypsin by 83.3 % and 4.8 %, respectively. Thus, the present study demonstrated that FUC could alleviate the inhibitory effect of CTS/COS on protein digestion.

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.

Chagas disease is transmitted to humans by obligatory hematophagous insects of Triatominae subfamily, which feeds on various hosts to acquire their nutritional sustenance derived from blood proteins. Hemoglobin (Hb) digestion is a pivotal metabolic feature of triatomines, representing a key juncture in their competence toward Trypanosoma cruzi; however, it remains poorly understood. To explore the Hb digestion pathway in Rhodnius prolixus, a major Chagas disease vector, we employed an array of approaches for activity profiling of various midgut-associated peptidases using specific substrates and inhibitors. Dissecting the individual contribution of each peptidase family in Hb digestion has unveiled a predominant role played by aspartic proteases and cathepsin B-like peptidases. Determination of peptidase-specific cleavage sites of these key hemoglobinases, in conjunction with mass spectrometry-based identification of in vivo Hb-derived fragments, has revealed the intricate network of peptidases involved in the Hb digestion pathway. This network is initiated by aspartic proteases and subsequently sustained by cysteine proteases belonging to the C1 family. The process is continued simultaneously by amino and carboxypeptidases. The comprehensive profiling of midgut-associated aspartic proteases by quantitative proteomics has enabled the accurate revision of gene annotations within the A1 family of the R. prolixus genome. Significantly, this study also serves to illuminate a potentially important role of the anterior midgut in blood digestion. The expanded repertoire of midgut-associated proteases presented in this study holds promise for the identification of novel targets aimed at controlling the transmission of Chagas disease.

In the last two decades, biological mass spectrometry has become the gold standard for the identification of proteins in biological samples. The technological advancement of mass spectrometers and the development of methods for ionization, gas phase transfer, peptide fragmentation as well as for acquisition of high-resolution mass spectrometric data marked the success of the technique. This chapter introduces peptide-based mass spectrometry as a tool for the investigation of protein complexes. It provides an overview of the main steps for sample preparation starting from protein fractionation, reduction, alkylation and focus on the final step of protein digestion. The basic concepts of biological mass spectrometry as well as details about instrumental analysis and data acquisition are described. Finally, the most common methods for data analysis and sequence determination are summarized with an emphasis on its application to protein-protein complexes.

An infant in vitro digestion model was utilized to investigate protein digestion characteristics in human and diverse mammalian milk (i.e., cow, goat, sheep, mare, and camel milk) using electrophoresis and chromatography. Digestive differences among milks were mainly manifested in the infant gastric phase, as evidenced by varying degrees of protein digestion. Notably, proteins (i.e., lactoferrin, serum albumin, and immunoglobulin G-heavy chain) remained partially intact in human milk, whereas these proteins in animal milk were exclusively degraded after gastrointestinal digestion. The peptide spectra of human, mare, and camel milk were highly similar, with a predominant formation of low-intensity small peptides, whereas the other three milk showed the opposite phenomenon. Heatmap cluster analysis indicated that camel milk was the most comparable to human milk before digestion, yet sheep milk was the most similar to human milk regarding protein digestion behaviors following infant gastric digestion.

As plant-based diets become more popular, there is an interest in developing innovations to improve the bioaccessibility of plant protein. In this study, seven probiotic strains (Bifidobacterium animalis subsp. lactis B420, B. lactis Bl-04, Lactobacillus acidophilus NCFM, Lacticaseibacillus rhamnosus HN001, Lacticaseibacillus paracasei subsp. paracasei Lpc-37, Lactiplantibacillus plantarum Lp-115, and Lactococcus lactis subsp. lactis Ll-23) were evaluated for their capacity to hydrolyze soy and pea protein ingredients in an in vitro digestion model of the upper gastrointestinal tract (UGIT). Compared to the control digestion of protein without a probiotic, all the studied strains were able to increase the digestion of soy or pea protein, as evidenced by an increase in free α-amino nitrogen (FAN) and/or free amino acid concentration. The increase in FAN varied between 13 and 33% depending on the protein substrate and probiotic strain. The survival of probiotic bacteria after exposure to digestive fluids was strain-dependent and may have affected the strain\'s capacity to function and aid in protein digestion in the gastrointestinal environment. Overall, our results from the standardized in vitro digestion model provide an approach to explore probiotics for improved plant protein digestion and bioaccessibility of amino acids; however, human clinical research is needed to evaluate the efficacy of probiotics on amino acid absorption and bioavailability in vivo.

The leavening of wheat-based steamed bread is carried out either with a pure yeast culture or with traditional starter cultures containing both lactic acid bacteria and yeast/mold. The use of variable starter cultures significantly affects steamed bread\'s quality attributes, including nutritional profile. In this paper, differences in physicochemical properties, the type of digested starch, the production of free amino acids, and the specific volume of steamed bread under three fermentation methods (blank, yeast, and LP-GM4-yeast) were compared. The digestion characteristics (protein and starch hydrolysis) of steamed bread produced by using either yeast alone or a combination of Lactiplantibacillus plantrum and yeast (LP-GM4-yeast) were analyzed by an in vitro simulated digestion technique. It was found that the specific volume of steamed bread fermented by LP-GM4-yeast co-culture was increased by about 32%, the proportion of resistant starch was significantly increased (more than double), and soluble protein with molecular weight of 30-40 kDa was significantly increased. The results of this study showed that steamed bread produced by LP-GM4-yeast co-culture is more beneficial to human health than that by single culture.