Peptide bonds cleaved by pepsin are affected by the morphology of heat-induced ovalbumin aggregates.

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.

In vitro gastrointestinal digestion of cow's and sheep's dairy products: Impact of species and structure.

Sheep's milk (SM) is known to differ from cow's milk (CM) in nutritional composition and physicochemical properties, which may lead to different digestion behaviours. This work aimed to investigate the impact of the species (cow vs sheep) and the structure (milk vs yogurt) on the digestion of dairy products. Using an in vitro static gastrointestinal digestion model, CM, SM, cow's milk yogurt (CY) and sheep's milk yogurt (SY) were compared on particle size evolution, microscopic observations, degree of lipolysis, degree of proteolysis, specific protein degradation and calcium bioaccessibility. Species and structure affected particle size evolution during the gastric phase resulting in smaller particles for yogurts compared to milks as well as for CM products compared to SM products. Species impacted lipid composition and lipolysis, with SM products presenting higher short/medium-chain fatty acids content and higher intestinal degree of lipolysis. Proteolysis was influenced by structure, with milks showing higher intestinal degree of proteolysis compared to yogurts. Caseins were digested faster in CM, ⍺-lactalbumin was digested faster in SM despite its higher concentration, and during gastric digestion ß-lactoglobulin was more degraded in CM products compared to SM products and more in yogurts compared to milks. Lastly, SM products released more bioaccessible calcium than CM products. In conclusion, species (cow vs sheep) impacted more the digestion compared to the structure (milk vs yogurt). In fact, SM was different from CM mainly due to a denser protein network that might slow down the accessibility of the enzyme to its substrate which induce a delay of gastric disaggregation and thus lead to slower the digestion of the nutrients.

Feasibility of using a realistic food bolus for semi-dynamic in vitro gastric digestion of hard cheese with pH-stat monitoring of protein hydrolysis.

Oral processing of solid foods leads to boluses made of a human saliva and particles distributed in the size range âˆ¼ 0 to 5 mm. However, studies on the release of nutrients from realistic solid food boluses during digestion are scarce because such mechanisms are difficult to investigate in vivo, and in vitro experiments generally recommend to extensively mince solid foods during the oral stage. Similarly, it has previously been shown that the peptic hydrolysis of protein solutions during in vitro gastric digestion can be monitored by acid titration in both static and dynamic pH conditions, but such approach has never been evaluated in the presence of particles of several millimetres in size. The first objective of the study was therefore to test the feasibility of using a realistic food bolus for gastric digestion studies with a pH-stat monitoring of proteolysis, using Emmental cheese as a solid food and with consideration of gastric acidifying kinetics. Degree of hydrolysis (DH) of proteins was monitored from two series of experiments performed in the presence and absence of pepsin. Other DH measurements, estimated from an independent approach based on the amount of free NH2 groups (OPA method) contained by peptides released in the supernatant (UV absorbance) validated the pH-stat results. A second objective of this work was to test the possible influence of human saliva on gastric proteolysis (in comparison with a water-based bolus). Results showed that saliva slightly delayed initiation of proteolysis, which could be explained by the slightly higher initial pH of the saliva-based bolus, but had no statistical effects on pepsin activity. We conclude that acid titration with a pH-stat system can be a valuable approach to monitor the gastric in vitro proteolysis of realistic solid food boluses in dynamic pH conditions.

Quantitative magnetic resonance imaging of in vitro gastrointestinal digestion of a bread and cheese meal.

The monitoring of food degradation during gastrointestinal digestion is essential in understanding food structure impacts on the bioaccessibility and bioavailability of nutrients. Magnetic Resonance Imaging (MRI) has the unique ability to access information on changes in multi-scale structural features of foods in a spatially resolved and non-destructive way. Our objective was to exploit various opportunities offered by MRI for monitoring starch, lipid and protein hydrolysis, as well as food particle breakdown during the semi-dynamic in vitro gastrointestinal digestion of complex foods combined in a meal. The meal consisted of French bread, hard cheese and water (drink), with a realistic distribution of bolus particle sizes. The MRI approach was reinforced by parallel chemical analysis of all macronutrients in the supernatant. By combining different imaging protocols, quantitative MRI provided insights into a number of phenomena at the level of the cheese and bread particles and within the liquid phase that are hard to access through conventional approaches. MRI thus revealed the progressive ingress of fluids into the bread crust and the release of the gas trapped in the crumb, the erosion of cheese particles, the creaming of fat, the disappearance of small food particles and changes in liquid phase composition. Excellent agreement was obtained between the quantitative parameters extracted from the MRI images and the results of the chemical analysis, demonstrating the strong potential of MRI for the monitoring of in vitro gastrointestinal digestion. The present study proposes further improvements to fully exploit the capabilities of MRI and constitutes an important step towards the extension of quantitative MRI to in vivo studies.

Spatial-temporal mapping of the intra-gastric pepsin concentration and proteolysis in pigs fed egg white gels.

While there is a consensus that food structure affects food digestion, the underlying mechanisms remain poorly understood. A previous experiment in pigs fed egg white gels of same composition but different structures evidenced such effect on food gastric disintegration. In this study, we detailed the consequences on intra-gastric pH, pepsin concentration and proteolysis by sampling throughout the stomach over 6 h digestion. Subsequent amino acid absorption was investigated as well by blood sampling. While acidification was almost homogeneous after 6 h digestion regardless of the gel, pepsin distribution never became uniform. Pepsin started to accumulate in the pylorus/antrum region before concentrating in the body stomach beyond 4 h, time from which proteolysis really started. Interestingly, the more acidic and soft gel resulted in a soon (60 min) increase in proteolysis, an earlier and more intense peak of plasmatic amino acids, and a final pepsin concentration three times higher than with the other gels.

The contribution of gastric digestion of starch to the glycaemic index of breads with different composition or structure.

Breads of higher density exhibit lower glycaemic index (GI) both in vivo and in vitro, a phenomenon generally attributed to a slower intestinal starch digestion. The aim of this work was to gain a better understanding of the relationship between bread density, oral and gastric digestion, and GI. Three breads were studied: industrial-style and traditional-style French baguettes (similar composition, different densities), and whole-wheat baguette. In vitro GI predictions confirmed that, for an identical composition, higher bread density was associated with a lower GI. Subsequent oro-gastric digestions, using the dynamic system DIDGI®, showed extensive starch digestion at the gastric stage by salivary α-amylase, in line with recently published data. They further showed that higher bread density led to a lower hydrolysis rate. The concurrence of these results with those of in vivo studies, suggests a mediating role for gastric digestion in the relationship between bread density and GI, possibly via the repercussions on the starch proportion that remains to be hydrolysed in the small intestine. This study therefore adds to the scientific knowledge of the importance of salivary α-amylase to starch digestion, and draws special attention to the possible role of the gastric phase in determining the GI.

Glycemic response, satiety, gastric secretions and emptying after bread consumption with water, tea or lemon juice: a randomized crossover intervention using MRI.

PURPOSE: Numerous studies, including our previous work with lemon juice, have reported that low-pH meals reduce the glycemic response to starchy foods. However, the underlying mechanism is not yet understood. Tea, for its polyphenol content, has also been investigated. The main objective of this research was to concurrently study gastric emptying, appetite perceptions and glycemic responses to bread consumed with water, tea, or lemon juice. METHODS: In this randomized, crossover intervention, ten participants consumed equal portions of bread (100 g) with 250 mL of water, water-diluted lemon juice, or black tea at breakfast. Gastric volumes, blood glucose concentrations and appetite perceptions were alternately assessed over 180 min using magnetic resonance imaging, the finger-prick method and visual analogue scales, respectively. RESULTS: Compared to water, lemon juice led to a 1.5 fold increase of the volume of gastric contents, 30 min after the meal (454.0 ± 18.6 vs. 298.4 ± 19.5 mL, [Formula: see text] ± SEM P < 0.00001). Gastric emptying was also 1.5 times faster (P < 0.01). Conversely, lemon juice elicited a lower glycemic response than water (blood glucose concentrations at t = 55 min were 35% lower, P = 0.039). Tea had no effect. Changes in appetite perceptions and gastric volumes correlated well, but with no significant differences between the meals. CONCLUSIONS: Lemon juice lowered the glycemic response and increased both gastric secretions and emptying rate. The results are compatible with the hypothesis that the reduction of the glycemic response is mainly due to the interruption of starch hydrolysis via the acid-inhibition of salivary α-amylase. TRIAL REGISTRATION NUMBER: NCT03265392, August 29, 2017.

Pepsin activity as a function of pH and digestion time on caseins and egg white proteins under static in vitro conditions.

The activity of pepsin, the gastric protease, is generally considered to be negligible for pH ≥ 4, based on the results obtained with a few purified globular proteins. The present study aimed at studying the activity of porcine pepsin on egg white proteins (EWP) and casein micelle micro-aggregates (CA) over a broad range of pH (from 1 to 7) for short (3 min) and long (2 h) digestion times. For a short time, the results confirmed a tendency for a higher rate of hydrolysis with decreasing pH, but with different pH activity profiles for both the substrates. More remarkably, the degree of hydrolysis of CA after 2 h of digestion was constant from pH 1 to pH 5, and was only reduced by half at pH 6. This finding demonstrates that pepsin can hydrolyse caseins from the very beginning of gastric digestion. Interestingly, the trend of the reaction kinetics over 2 h appeared to be rather characteristic of the type of the substrate and was largely independent in terms of pH. Most hydrolysis profiles could be accurately fitted by a power law, an empirical model that was then successfully applied to the static in vitro gastric proteolysis of 6 other food matrices. Overall, our results support the idea that pepsin activity under weakly acidic conditions (pH ≥ 4) should not always be neglected, in particular, for milk caseins, and that pepsin reaction kinetics during static in vitro gastric digestion seems to evolve proportionally to the power of the digestion time.

Statistical modeling of in vitro pepsin specificity.

The specificity of pepsin, the major protease of gastric digestion, has been previously investigated, but only regarding the primary sequence of the protein substrates. The present study aimed to consider in addition physicochemical and structural characteristics, at the molecular and sub-molecular scales. For six different proteins submitted to in vitro gastric digestion, the peptide bonds cleaved were determined from the peptides released and identified by LC-MS/MS. An original statistical approach, based on propensity scores calculated for each amino acid residue on both sides of the peptide bonds, concluded that preferential cleavage occurred after Leu and Phe, and before Ile. Moreover, reliable statistical models developed for predicting peptide bond cleavage, highlighted the predominant role of the amino acid residues at the N-terminal side of the peptide bonds, up to the seventh position (P7 and P7'). The significant influence of hydrophobicity, charge and structural constraints around the peptide bonds was also evidenced.

Physiologically Based Modeling of Food Digestion and Intestinal Microbiota: State of the Art and Future Challenges. An INFOGEST Review.

This review focuses on modeling methodologies of the gastrointestinal tract during digestion that have adopted a systems-view approach and, more particularly, on physiologically based compartmental models of food digestion and host-diet-microbiota interactions. This type of modeling appears very promising for integrating the complex stream of mechanisms that must be considered and retrieving a full picture of the digestion process from mouth to colon. We may expect these approaches to become more and more accurate in the future and to serve as a useful means of understanding the physicochemical processes occurring in the gastrointestinaltract, interpreting postprandial in vivo data, making relevant predictions, and designing healthier foods. This review intends to provide a scientific and historical background of this field of research, before discussing the future challenges and potential benefits of the establishment of such a model to study and predict food digestion and absorption in humans.

Lemon juice, but not tea, reduces the glycemic response to bread in healthy volunteers: a randomized crossover trial.

PURPOSE: The inhibition of enzymes that hydrolyze starch during digestion could constitute an opportunity to slow down the release, and ultimately the uptake, of starch-derived glucose. Simple dietary approaches consisting in pairing starch-rich foods with beverages that have the capacity to inhibit such enzymes could be an effective and easily implementable strategy. The objective of this work was to test the impact of black tea and lemon juice on the glycemic response to bread and subsequent energy intake in healthy adults. METHODS: A randomized crossover study was conducted with equal portions of bread (100 g) and 250 ml of water, black tea or lemon juice. Capillary blood glucose concentrations were monitored during 180 min using the finger-prick method. Ad libitum energy intake was assessed 3 h later. RESULTS: Tea had no effect on the glycemic response. Lemon juice significantly lowered the mean blood glucose concentration peak by 30% (p < 0.01) and delayed it more than 35 min (78 vs. 41 min with water, p < 0.0001). None of the tested beverages had an effect on ad libitum energy intake. CONCLUSION: These results are in agreement with previous in vitro studies showing that lowering the pH of a meal can slow down starch digestion through premature inhibition of salivary α-amylase. Furthermore, the effect of lemon juice was similar to what has been repeatedly observed with vinegar and other acidic foods. Including acidic beverages or foods in starchy meals thus appears to be a simple and effective strategy to reduce their glycemic impact.

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.

A standardised semi-dynamic in vitro digestion method suitable for food - an international consensus.

The link between food and human health is increasingly a topic of interest. One avenue of study has been to assess food disintegration and interactions within the gastrointestinal tract. In vitro digestion models have been widely used to overcome the constrictions associated with in vivo methodology. The COST Action INFOGEST developed an international, harmonised protocol for static simulation of digestion in the upper gastrointestinal tract of adults. This protocol is widely used; however, it is restricted to providing end-point assessment without considering the possible structural changes. On the other hand, there are dynamic models that provide more physiologically relevant data but are expensive and difficult to access. There is a gap between these models. The method outlined in this article provides an intermediate model; it builds upon the harmonised static model and now includes crucial kinetic aspects associated with the gastric phase of digestion, including gradual acidification, fluid and enzyme secretion and emptying. This paper provides guidance and standardised recommendations of a physiologically relevant semi-dynamic in vitro simulation of upper gastrointestinal tract digestion, with particular focus on the gastric phase. Adaptations of this model have already been used to provide kinetic data on nutrient digestion and structural changes during the gastric phase that impact on nutrient absorption. Moreover, it provides a simple tool that can be used in a wide range of laboratories.

Inhibitory effect of black tea, lemon juice, and other beverages on salivary and pancreatic amylases: What impact on bread starch digestion? A dynamic in vitro study.

Certain food properties (ex.: pH, polyphenolic composition) can inhibitdigestive amylases and thereby slow down starch digestion. Our aim was twofold. (1) To determine the impact of21 beverages and condiments (coffees, teas, wines, vinegars and lemon juice)on salivary and pancreatic amylolysis: inhibition ranged from 10% to 100%in our experimental conditions. (2) To investigate the effect of one black tea and lemon juice (selected for their strong inhibitory capacity) on starch hydrolysis during dynamicoro-gastro-intestinal digestion of bread. Compared to water (control), the effect of black tea was limited to a ≈20% reduction of released oligosaccharides during the intestinal phase. Lemon juice had a remarkable effect, completely interrupting gastric amylolysis by salivary amylase via a preliminary acidification of gastric contents. These results provide a strong biochemical rationale for the development of dietary strategies to improve the glycaemic impact of starch-rich meals which could be tested in vivo.

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.

Oro-gastro-intestinal digestion of starch in white bread, wheat-based and gluten-free pasta: Unveiling the contribution of human salivary α-amylase.

Starch is a major determinant of the glycemic responses elicited by our diets, but the exact contribution of the two main amylolytic enzymes (salivary and pancreatic α-amylases) remains a matter of debate. Our aim was to investigate the contribution of the oral, gastric and intestinal phases to the hydrolysis of starch in bread and pasta during dynamic in vitro digestions using DiDGI®. Before its inactivation by the low gastric pH, salivary α-amylase released about 80% of the starch in bread and 30% of that in pasta, hydrolysing over half of it into oligosaccharides. Accordingly, the contribution of pancreatic α-amylase during the intestinal phase was lower for bread than pasta. Our results are well correlated with in vivo data, and demonstrate the importance of salivary α-amylase during oro-gastric processing of starchy foods. This finding is discussed in relation with observations regarding salivary α-amylase from other fields of knowledge.

Acid induced reduction of the glycaemic response to starch-rich foods: the salivary α-amylase inhibition hypothesis.

Numerous studies have reported that the glycaemic response to starch-rich meals can be reduced by 20-50% with acidic drinks or foods. A number of candidate explanations have been put forward, but this phenomenon still remains vaguely understood. This study intends to demonstrate the remarkable effect of acid inhibition of salivary α-amylase during oro-gastric hydrolysis of starch, shedding light on this often overlooked mechanism. Oro-gastric digestions of bread, wheat and gluten-free pastas, combined with either water or lemon juice were performed using a dynamic in vitro system that reproduces gastric acidification kinetics observed in humans. In the presence of water, large proportions of starch (25-85%) and oligosaccharides (15-50%) were released from all foods within the first hour of gastric digestion (pH > 3.5). In the presence of lemon juice (pH < 3.5 at all time), starch release was about twice as low, and amylolysis into oligosaccharides was completely interrupted. Acid-inhibition of salivary α-amylase may explain, at least in part, the reduction of the blood glucose response through acidification of starch-rich foods/meals. This offers new perspectives for the development of strategies to improve the glycaemic response elicited by starch-rich diets.

Monitoring protein hydrolysis by pepsin using pH-stat: In vitro gastric digestions in static and dynamic pH conditions.

This study intends to demonstrate that acid titration at low pH is very well adapted to the monitoring of pepsin activity. After a description of the underlying principles, this approach was used during in vitro gastric digestions of a model of complex food containing 15wt% of whey proteins, according to both static (2h at pH = 3, Infogest protocol) and dynamic pH conditions (from pH 6.3 down to 2 in 1h). Pepsin activity was quantitatively assessed in all experiments through the calculation of degrees of hydrolysis (DH). Final values of 3.7 and 3.0% were obtained in static and dynamic pH conditions, respectively, and validated using an independent method. Results also show that about 92% of the peptides were detected at pH = 3, and 100% for pH≤2.5. Overall, the proposed approach proved to be very worthy to study protein hydrolysis during in vitro gastric digestions.

Exploring the breakdown of dairy protein gels during in vitro gastric digestion using time-lapse synchrotron deep-UV fluorescence microscopy.

A novel time-lapse synchrotron deep-UV microscopy methodology was developed that made use of the natural tryptophan fluorescence of proteins. It enabled the monitoring in situ of the microstructural changes of protein gels during simulated gastric digestion. Two dairy gels with an identical composition, but differing by the coagulation mode, were submitted to static in vitro gastric digestion. The kinetics of gel particle breakdown were quantified by image analysis and physico-chemical analyses of digesta. The results confirm the tendency of rennet gels, but not acid gels, to form compact protein aggregates under acidic conditions of the stomach. Consequently, the kinetics of proteolysis were much slower for the rennet gel, confirming the hypothesis of a reduced pepsin accessibility to its substrate. The particle shapes remained unchanged and the disintegration kinetics followed an exponential trend, suggesting that erosion was the predominant mechanism of the enzymatic breakdown of dairy gels in these experimental conditions.

The important role of salivary α-amylase in the gastric digestion of wheat bread starch.

The role of salivary α-amylase (HSA) in starch digestion is often overlooked in favour of that of pancreatic α-amylase due to the short duration of the oral phase. Although it is generally accepted that the amylase of salivary origin can continue to be active in the stomach, studies ascertaining its contribution are lacking. This study aimed to address this issue by coupling in vitro oral processing with an in vitro dynamic system that mimicked different postprandial gastric pH reduction kinetics observed in vivo following a snack- or lunch-type meal. The digestion of both starch and protein from wheat bread as well as the interplay between the two processes were studied. We have observed that the amylolytic activity of saliva plays a preponderant role hydrolysing up to 80% of bread starch in the first 30 min of gastric digestion. Amylolysis evolved exponentially and nearly superimposing curves were obtained regardless of the acidification profiles, revealing its high efficiency.