Enzymes to unravel bioproducts architecture.

Enzymes are essential and ubiquitous biocatalysts involved in various metabolic pathways and used in many industrial processes. Here, we reframe enzymes not just as biocatalysts transforming bioproducts but also as sensitive probes for exploring the structure and composition of complex bioproducts, like meat tissue, dairy products and plant materials, in both food and non-food bioprocesses. This review details the global strategy and presents the most recent investigations to prepare and use enzymes as relevant probes, with a focus on glycoside-hydrolases involved in plant deconstruction and proteases and lipases involved in food digestion. First, to expand the enzyme repertoire to fit bioproduct complexity, novel enzymes are mined from biodiversity and can be artificially engineered. Enzymes are further characterized by exploring sequence/structure/dynamics/function relationships together with the environmental factors influencing enzyme interactions with their substrates. Then, the most advanced experimental and theoretical approaches developed for exploring bioproducts at various scales (from nanometer to millimeter) using active and inactive enzymes as probes are illustrated. Overall, combining multimodal and multiscale approaches brings a better understanding of native-form or transformed bioproduct architecture and composition, and paves the way to mainstream the use of enzymes as probes.

INFOGEST static in vitro simulation of gastrointestinal food digestion.

Developing a mechanistic understanding of the impact of food structure and composition on human health has increasingly involved simulating digestion in the upper gastrointestinal tract. These simulations have used a wide range of different conditions that often have very little physiological relevance, and this impedes the meaningful comparison of results. The standardized protocol presented here is based on an international consensus developed by the COST INFOGEST network. The method is designed to be used with standard laboratory equipment and requires limited experience to encourage a wide range of researchers to adopt it. It is a static digestion method that uses constant ratios of meal to digestive fluids and a constant pH for each step of digestion. This makes the method simple to use but not suitable for simulating digestion kinetics. Using this method, food samples are subjected to sequential oral, gastric and intestinal digestion while parameters such as electrolytes, enzymes, bile, dilution, pH and time of digestion are based on available physiological data. This amended and improved digestion method (INFOGEST 2.0) avoids challenges associated with the original method, such as the inclusion of the oral phase and the use of gastric lipase. The method can be used to assess the endpoints resulting from digestion of foods by analyzing the digestion products (e.g., peptides/amino acids, fatty acids, simple sugars) and evaluating the release of micronutrients from the food matrix. The whole protocol can be completed in ~7 d, including ~5 d required for the determination of enzyme activities.

ß-Casein(94-123)-derived peptides differently modulate production of mucins in intestinal goblet cells.

We recently reported the identification of a peptide from yoghurts with promising potential for intestinal health: the sequence (94-123) of bovine ß-casein. This peptide, composed of 30 amino acid residues, maintains intestinal homoeostasis through production of the secreted mucin MUC2 and of the transmembrane-associated mucin MUC4. Our study aimed to search for the minimal sequence responsible for the biological activity of ß-CN(94-123) by using several strategies based on (i) known bioactive peptides encrypted in ß-CN(94-123), (ii) in silico prediction of peptides reactivity and (iii) digestion of ß-CN(94-123) by enzymes of intestinal brush border membranes. The revealed sequences were tested in vitro on human intestinal mucus-producing HT29-MTX cells. We demonstrated that ß-CN(108-113) (an ACE-inhibitory peptide) and ß-CN(114-119) (an opioid peptide named neocasomorphin-6) up-regulated MUC4 expression whereas levels of the secreted mucins MUC2 and MUC5AC remained unchanged. The digestion of ß-CN(94-123) by intestinal enzymes showed that the peptides ß-CN(94-108) and ß-CN(117-123) were present throughout 1·5 to 3 h of digestion, respectively. These two peptides raised MUC5AC expression while ß-CN(117-123) also induced a decrease in the level of MUC2 mRNA and protein. In addition, this inhibitory effect was reproduced in airway epithelial cells. In conclusion, ß-CN(94-123) is a multifunctional molecule but only the sequence of 30 amino acids has a stimulating effect on the production of MUC2, a crucial factor of intestinal protection.

Sequential release of milk protein-derived bioactive peptides in the jejunum in healthy humans.

BACKGROUND: The digestive hydrolysis of dietary proteins leads to the release of peptides in the intestinal tract, where they may exert a variety of functions, but their characterization and quantification are difficult. OBJECTIVES: We aimed to characterize and determine kinetics of the formation of peptides present in the jejunum of humans who ingested casein or whey proteins by using mass spectrometry and to look for and quantify bioactive peptides. DESIGN: Subjects were equipped with a double-lumen nasogastric tube that migrated to the proximal jejunum. A sample collection was performed for 6 h after the ingestion of 30 g (15)N-labeled casein (n = 7) or whey proteins (WPs; n = 6). Nitrogen flow rates were measured, and peptides were identified by using mass spectrometry. RESULTS: After casein ingestion, medium-size peptides (750-1050 kDa) were released during 6 h, whereas larger peptides (1050-1800 kDa) were released from WPs in the first 3 h. A total of 356 and 146 peptides were detected and sequenced in the jejunum after casein and WP ingestion, respectively. ß-casein was the most important precursor of peptides, including bioactive peptides with various activities. The amounts of ß-casomorphins (ß-casein 57-, 58-, 59-, and 60-66) and ß-casein 108-113 released on the postprandial window were sufficient to elicit the biological action of these peptides (ie, opioid and antihypertensive, respectively). CONCLUSIONS: Clear evidence is shown of the presence of bioactive peptides in the jejunum of healthy humans who ingested casein. Our findings raise the question about the physiologic conditions under which these peptides can express their bioactivity in humans. This trial was registered at clinicaltrials.gov as NCT00862329.

A novel bioactive peptide from yoghurts modulates expression of the gel-forming MUC2 mucin as well as population of goblet cells and Paneth cells along the small intestine.

Several studies demonstrated that fermented milks may provide a large number of bioactive peptides into the gastrointestinal tract. We previously showed that beta-casomorphin-7, an opioid-like peptide produced from bovine ß-casein, strongly stimulates intestinal mucin production in ex vivo and in vitro models, suggesting the potential benefit of milk bioactive peptides on intestinal protection. In the present study, we tested the hypothesis that the total peptide pool (TPP) from a fermented milk (yoghurt) may act on human intestinal mucus-producing cells (HT29-MTX) to induce mucin expression. Our aim was then to identify the peptide(s) carrying the biological activity and to study its impact in vivo on factors involved in gut protection after oral administration to rat pups (once a day, 9 consecutive days). TPP stimulated MUC2 and MUC4 gene expression as well as mucin secretion in HT29-MTX cells. Among the four peptide fractions that were separated by preparative reversed-phase high-performance liquid chromatography, only the C2 fraction was able to mimic the in vitro effect of TPP. Interestingly, the sequence [94-123] of ß-casein, present only in C2 fraction, also regulated mucin production in HT29-MTX cells. Oral administration of this peptide to rat pups enhanced the number of goblet cells and Paneth cells along the small intestine. These effects were associated with a higher expression of intestinal mucins (Muc2 and Muc4) and of antibacterial factors (lysozyme, rdefa5). We conclude that the peptide ß-CN(94-123) present in yoghurts may maintain or restore intestinal homeostasis and could play an important role in protection against damaging agents of the intestinal lumen.

Phosphorylation and coordination bond of mineral inhibit the hydrolysis of the beta-casein (1-25) peptide by intestinal brush-border membrane enzymes.

Caseinophosphopeptides (CPP) are food mineral-rich components that may resist intestinal enzyme hydrolysis. We wondered whether phosphorylation and/or mineral binding induces resistance of CPP to intestinal hydrolysis. We used intestinal brush-border membrane vesicles to digest different forms of the beta-casein (1-25) peptide: unphosphorylated and phosphorylated carrier of varied cations. The results showed that the activity of alkaline phosphatase seems not to be specific to either the phosphorylation degree or the phosphorylation sites whereas phosphorylations limited the action of peptidases. Studying the mechanism and the kinetics of hydrolysis of the different peptides allows understanding how some cations prevent more CPP from hydrolysis than others. The action of both exo- and endopeptidases was limited for the beta-CN (1-25) peptide bound to zinc or copper. Actually the peptide bound to copper was almost not hydrolyzed during the digestion, suggesting that coordination bond of copper to CPP inhibits the action of both phosphatase and peptidases.

The (193-209) 17-residues peptide of bovine ß-casein is transported through Caco-2 monolayer.

Although the bioavailability of large peptides with biological activity is of great interest, the intestinal transport has been described for peptides up to only nine residues. ß-casein (ß-CN, 193-209) is a long and hydrophobic peptide composed of 17 amino acid residues (molecular mass 1881 Da) with immunomodulatory activity. The present work examined the transport of the ß-CN (193-209) peptide across Caco-2 cell monolayer. In addition, we evaluated the possible routes of the ß-CN (193-209) peptide transport, using selective inhibitors of the different routes for peptide transfer through the intestinal barrier. The results showed that the ß-CN (193-209) peptide resisted the action of brush-border membrane peptidases, and that it was transported through the Caco-2 cell monolayer. The main route involved in transepithelial transport of the ß-CN (193-209) peptide was transcytosis via internalized vesicles, although the paracellular transport via tight-junctions could not be excluded. Our results demonstrated the transport of an intact long-chain bioactive peptide in an in vitro model of intestinal epithelium, as an important step to prove the evidence for bioavailability of this peptide.

Glycosylations of kappa-casein-derived caseinomacropeptide reduce its accessibility to endo- but not exointestinal brush border membrane peptidases.

Caseinomacropeptide (CMP) is a peptide obtained from kappa-casein hydrolysis by gastric proteinases and which exhibits various biological activities. The aim of this study was to analyze the intestinal processing of CMP at the brush border membrane (BBM) level. Intestinal BBM vesicles (BBMV) were used to digest glycosylated and unglycosylated CMP. Our results demonstrated that whatever was the glycosylated state of CMP, they were digested by BBMV intestinal enzymes, from macropeptides to free amino acids. The digestion of unglycosylated and glycosylated CMP throughout the action of exopeptidases was similar, but the activity of endopeptideases on glycosylated CMP was limited, certainly due to the attached O-glycosylations. Consequently, much more peptides were identified from the unglycosylated than from the glycosylated CMP. In addition, the glycosylation core as well as the number of the attached glycosylated chain modified the kinetic of digestion; the most heavily glycosylated forms being the slowest digested.

Structure development in a soft cheese curd model during manufacture in relation to its biochemical characteristics.

The structure development of a soft cheese curd model has been studied in relationship to its rheological properties and its biochemical characteristics (pH, amount and partition of minerals, casein proteolysis) at different technical steps including cutting, drawing, three turns and demoulding. Scanning electron microscopy was used to observe structural changes during the drainage of a fat-free soft cheese. The micrographs provided visual evidence of changes in the casein matrix from casein particles aggregated in clusters to uniform strands observed at the demoulding. The initial increase of loss tangent and of the exponent of the power law between G' and G" and frequency (that were maximal at the second turn) was related to the solubilization of micellar calcium phosphate, while intact caseins and large casein fragments accumulated in the curd. After the second turn, the strength, Youngs' and loss moduli of the curd increased greatly. The hydrolysis of alpha(s1)-casein into alpha(s1)-I-CN f(24-199) may facilitate the rearrangement of casein particles within the curd. The pH-induced solubilization of calcium phosphate continued throughout the manufacture process but was unexpectedly incomplete at the end of the drainage. Combination of electron microscopic observations with dynamic rheological measurements and chemical and biochemical assessments provided increased knowledge about the structure of soft cheese during drainage, an important but poorly understood cheese making stage.