A Shared Perspective on in Vitro and in Vivo Models to Assay Intestinal Transepithelial Transport of Food Compounds.

Assessing nutrient bioavailability is complex, as the process involves multiple digestion steps, several cellular environments, and regulatory-metabolic mechanisms. Several in vitro models of different physiological relevance are used to study nutrient absorption, providing significant challenges in data evaluation. However, such in vitro models are needed for mechanistic studies as well as to screen for biological functionality of the food structures designed. This collaborative work aims to put into perspective the wide-range of models to assay the permeability of food compounds considering the particular nature of the different molecules, and, where possible, in vivo data are provided for comparison.

CCK and GLP-1 response on enteroendocrine cells of semi-dynamic digests of hydrolyzed and intact casein.

A semi-dynamic gastrointestinal device was employed to explore the link between protein structure and metabolic response upon digestion for two different substrates, a casein hydrolysate and the precursor micellar casein. As expected, casein formed a firm coagulum that remained until the end of the gastric phase while the hydrolysate did not develop any visible aggregate. Each gastric emptying point was subjected to a static intestinal phase where the peptide and amino acid composition changed drastically from that found during the gastric phase. Gastrointestinal digests from the hydrolysate were characterized by a high abundancy of resistant peptides and free amino acids. Although all gastric and intestinal digests from both substrates induced the secretion of cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1) in STC-1 cells, GLP-1 levels were maximum in response to gastrointestinal digests from the hydrolysate. The enrichment of protein ingredients with gastric-resistant peptides by enzymatic hydrolysis is proposed as strategy to deliver protein stimuli to the distal gastrointestinal tract to control food intake or type 2 diabetes.

Coupling in vitro food digestion with in vitro epithelial absorption; recommendations for biocompatibility.

As food transits the gastrointestinal tract, food structures are disrupted and nutrients are absorbed across the gut barrier. In the past decade, great efforts have focused on the creation of a consensus gastrointestinal digestion protocol (i.e., INFOGEST method) to mimic digestion in the upper gut. However, to better determine the fate of food components, it is also critical to mimic food absorption in vitro. This is usually performed by treating polarized epithelial cells (i.e., differentiated Caco-2 monolayers) with food digesta. This food digesta contains digestive enzymes and bile salts, and if following the INFOGEST protocol, at concentrations that although physiologically relevant are harmful to cells. The lack of a harmonized protocol on how to prepare the food digesta samples for downstream Caco-2 studies creates challenges in comparing inter laboratory results. This article aims to critically review the current detoxification practices, highlight potential routes and their limitations, and recommend common approaches to ensure food digesta is biocompatible with Caco-2 monolayers. Our ultimate aim is to agree a harmonized consensus protocol or framework for in vitro studies focused on the absorption of food components across the intestinal barrier.

A multi-centre peptidomics investigation of food digesta: current state of the art in mass spectrometry analysis and data visualisation.

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods - skim milk powder (SMP), cooked chicken breast and tofu - were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow. The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.

Peptidomic Characterization and Amino Acid Availability after Intake of Casein vs. a Casein Hydrolysate in a Pig Model.

It is known that casein hydrolysis accelerates gastrointestinal transit in comparison to intact casein, although the effect of the protein hydrolysis on the composition of the digests is not fully understood. The aim of this work is to characterize, at the peptidome level, duodenal digests from pigs, as a model of human digestion, fed with micellar casein and a previously described casein hydrolysate. In addition, in parallel experiments, plasma amino acid levels were quantified. A slower transit of nitrogen to the duodenum was found when the animals received micellar casein. Duodenal digests from casein contained a wider range of peptide sizes and a higher number of peptides above five amino acids long in comparison with the digests from the hydrolysate. The peptide profile was markedly different, and although ß-casomorphin-7 precursors were also found in hydrolysate samples, other opioid sequences were more abundant in the casein digests. Within the same substrate, the evolution of the peptide pattern at different time points showed minimal changes, suggesting that the protein degradation rate relies more on the gastrointestinal location than on digestion time. Higher plasma concentrations of methionine, valine, lysine and amino acid metabolites were found in animals fed with the hydrolysate at short times (<200 min). The duodenal peptide profiles were evaluated with discriminant analysis tools specific for peptidomics to identify sequence differences between both substrates that can be used for future human physiological and metabolic studies.

Food peptides as inducers of CCK and GLP-1 secretion and GPCRs involved in enteroendocrine cell signalling.

The strong effect of protein digestion products on gastrointestinal-released hormones is recognised. However, little is known about the specific peptide sequences able to induce gastrointestinal hormone secretion and the receptors involved. Our objective was to identify peptides able to induce the secretion of cholecystokinin (CCK) and glucagon like peptide-1 (GLP-1) in the enteroendocrine cell line STC-1, and to evaluate the involvement of the calcium-sensing receptor and G-protein coupled receptor-93 in this cell signalling. The key role of the amino acidic sequence on CCK and GLP-1 secretion is demonstrated. Removing Ser from the N-terminus of κ-casein 33SRYPS37, or the N-terminal Trp-Ile in lysozyme 123WIRGCRL129 decreased the secretion of both hormones. However, substituting Tyr by Ala in peptide αs1-CN 90RYLG93 enhanced the CCK secretion levels but not the GLP-1 ones. In addition, the involvement of CaSR and GPR93 was evidenced, but our results pointed to the contribution of additional receptors or transporters.

In vitro digestion of milk proteins including intestinal brush border membrane peptidases. Transepithelial transport of resistant casein domains.

The use of enzymes from the brush border membrane (BBM) in simulated gastrointestinal digestion of milk proteins has been evaluated. With this purpose, the resistant sequences from casein and milk whey proteins after INFOGEST in vitro digestion with and without BBM have been analyzed by tandem mass spectrometry. The use of BBM revealed additional cleavages to those found with pancreatic enzymes, although the number of total identified peptides decreased due to the reduction of the peptide size. These new cleavages were mainly attributed to the activity of amino- and carboxy-peptidases, which was also reflected in the higher concentration of free amino acids found in the gastrointestinal digests with BBM. The peptidome of the simulated gastrointestinal digests was compared with that previously obtained in digests aspirated from human jejunum after oral administration of the same substrates. The addition of BBM did not change significantly the peptide profile, although it allowed the identification of peptides found in human digests. However, none of the models was able to reproduce the large variety of peptides found in vivo. In addition, in vitro transepithelial transport of six ß-casein derived peptides resistant to gastrointestinal digestion, including the opioid ß-casomorphin-7, was also evaluated. The results point to the importance of the nature of the N- and C-terminal end for the transport rate through the Caco-2 cell monolayer. Therefore, the use of BBM as a supplementary step after simulated pancreatic digestion can be considered in bioavailability studies since the final sequence can determine the absorption of peptides.

Gastrointestinal co-digestion of wine polyphenols with glucose/whey proteins affects their bioaccessibility and impact on colonic microbiota.

Interactions between food components during their gastrointestinal digestion are constant and could affect compounds digestibility and bioaccessibility. These interactions could have a key role in the bioactivity of dietary polyphenols. This study aimed to investigate the food matrix effects during the co-digestion of red wine with glucose and whey proteins using the gastrointestinal dynamic simulator simgi®. Bioaccessibility of wine polyphenols and nutrients and the effect of co-digestion on colonic microbiota composition and metabolism were evaluated. Co-digestion with red wine led to a reduction of over 50% of glucose bioaccessibility and lowered α-lactalbumin gastric degradation. Still, co-digestion with the food matrices modified polyphenols profiles, including their bioaccessible and non-bioaccessible fractions. For instance, the (-)-epicatechin bioaccessible fraction increased 70% when the wine was co-digested with glucose. Hence, the combined feeding of wine and each food matrix affected microbiota composition and functionality at colonic level. Glucose and whey proteins reduced bacterial diversity, but homogenization of beta-diversity by wine was observed. Moreover, wine presence favoured intestinal health-related taxa as Akkermansia or Bifidobacterium, and the co-digestion of wine and food matrices significantly increased total short- and medium-chain fatty acids production, especially butyric acid. Overall, this study provides evidence of the convenience of the simgi® system to evaluate the effects of co-digestion and highlights the importance of food matrix effects on our understanding of polyphenol bioactivity.

In vitro simulated semi-dynamic gastrointestinal digestion: evaluation of the effects of processing on whey proteins digestibility and allergenicity.

The effect of thermal processing on digestibility of milk proteins should be better understood as this can greatly affect their immunoreactivity. The aim of this study was to evaluate the effects of thermal processing and lactosylation on digestibility and allergenicity, by comparing non heat-treated with industrially processed whey proteins. A semi-dynamic model was used to mimic the kinetics of digestion, and ELISA inhibition tests against human specific serum IgE were performed on the mass-spectrometry characterized products. A quicker gastric digestion of the industrially treated sample produced a lower immunogenic response in comparison with the raw sample, where intact conformational epitopes remained. In later digests, greater IgE reactivity was shown in the heat treated product, probably due to the release of linear epitopes, while at intestinal level the immunogenic response was negligible. Moreover, transepithelial transport of a reported ß-lactoglobulin-derived allergen, KIDALNENVLVL, produced during digestion was assayed. It was found that the epitope-belonging peptide could be transported through the cell monolayer, both in the native and mono-lactosylated forms, with a favored passage of the native peptide.

Composition of Brazil Nut (Bertholletia excels HBK), Its Beverage and By-Products: A Healthy Food and Potential Source of Ingredients.

The consumption of plant-based beverages is a growing trend and, consequently, the search for alternative plant sources, the improvement of beverage quality and the use of their by-products, acquire great interest. Thus, the purpose of this work was to characterize the composition (nutrients, phytochemicals and antioxidant activity) of the Brazil nut (BN), its whole beverage (WBM), water-soluble beverage (BM-S), and its by-products of the beverage production: cake, sediment fraction (BM-D), and fat fraction (BM-F). In this study, advanced methodologies for the analysis of the components were employed to assess HPLC-ESI-QTOF (phenolic compounds), GC (fatty acids), and MALDI-TOF/TOF (proteins and peptides). The production of WBM was based on a hot water extraction process, and the production of BM-S includes an additional centrifugation step. The BN showed an interesting nutritional quality and outstanding content of unsaturated fatty acids. The investigation found the following in the composition of the BN: phenolic compounds (mainly flavan-3-ols as Catechin (and glycosides or derivatives), Epicatechin (and glycosides or derivatives), Quercetin and Myricetin-3-O-rhamnoside, hydroxybenzoic acids as Gallic acid (and derivatives), 4-hydroxybenzoic acid, ellagic acid, Vanillic acid, p-Coumaric acid and Ferulic acid, bioactive minor lipid components (ß-Sitosterol, γ-Tocopherol, α-Tocopherol and squalene), and a high level of selenium. In beverages, WBM had a higher lipid content than BM-S, a factor that influenced the energy characteristics and the content of bioactive minor lipid components. The level of phenolic compounds and selenium were outstanding in both beverages. Hydrothermal processing can promote some lipolysis, with an increase in free fatty acids and monoglycerides content. In by-products, the BM-F stood out due to its bioactive minor lipid components, the BM-D showed a highlight in protein and mineral contents, and the cake retained important nutrients and phytochemicals from the BN. In general, the BN and its beverages are healthy foods, and its by-products could be used to obtain healthy ingredients with appreciable biological activities (such as antioxidant activity).

Protein Profile and Simulated Digestive Behavior of Breast Milk from Overweight and Normal Weight Mothers.

Human milk proteins have shown to vary in concentration and distribution through lactation. However, while some regulatory components, such as hormones, have shown associations with regard to the mothers' body mass index, there is limited information on the possible influence of this condition on the whole protein distribution. The objective of this study was to evaluate the protein profile of human milk from normal weight and overweight or obese mothers to identify differences in protein expression in colostrum, transitional and mature milk. The mass spectrometry analysis showed the ability to class with a high degree of confidence the lactation state and the milk profile according to the mother's condition. Individual milk samples were subjected to a digestion in vitro model that takes into account the specificities of the gastrointestinal conditions of full-term newborn infants. The digestion products were compared with available data from the digestive contents in newborns. The behavior of the most abundant proteins and the overall peptide generation and survival, showed good correspondence with in vivo data.

Study of the phenolic compound profile and antioxidant activity of human milk from Spanish women at different stages of lactation: A comparison with infant formulas.

Human milk (HM) has been proven to have important and essential antioxidant properties to counteract infant susceptibility to oxidative stress. Phenolic compounds are secondary metabolites which come from plants and are potent natural antioxidants. The ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method used in the present study allowed the quantification of 26 phenolic compounds (ten hydroxybenzoic acids, seven hydroxycinnamic acids, four flavonoids, three hydroxybenzaldehydes and two other polyphenols) in HM samples at different stages of lactation (colostrum, transitional milk and mature milk) and infant formulas (IF). Many of the phenolic compounds identified have been reported to be present in HM for the first time. The total phenolic compound content (TPC) was quantified using the Folin assay and the antioxidant activity (AC) was evaluated with the DPPH, ABTS and FRAP assays. Significant differences were evidenced between HM and IF. HM from mothers with an adherence to a Mediterranean diet contained twice as many individual phenolic compounds as infant formulas, with a higher proportion of hydroxybenzoic acids. Conversely, IF showed a higher proportion of hydroxycinnamic acids. Overall, the antioxidant activity of HM showed small variations during lactation.

Compared digestibility of plant protein isolates by using the INFOGEST digestion protocol.

The use of ingredients based on plant protein isolates is being promoted due to sustainability and health reasons. However, it is necessary to explore the behaviour of plant protein isolates during gastrointestinal digestion including the profile of released free amino acids and the characterization of resistant domains to gastrointestinal digestion. The aim of the present study was to monitor protein degradation of four legume protein isolates: garden pea, grass pea, soybean and lentil, using the harmonized Infogest in vitro digestion protocol. In vitro digests were characterized regarding protein, peptide and free amino acid content. Soybean was the protein isolate with the highest percentage of insoluble nitrogen at the end of the digestion (12%), being this fraction rich in hydrophobic amino acids. Free amino acids were mainly released during the intestinal digestion, comprising 21-24% of the total nitrogen content, while the percentage of nitrogen corresponding to peptides ranged from 66 to 76%. Legume globulins were resistant to gastric digestion whereas they were hydrolysed into peptides and amino acids during the intestinal phase. However, the molecular weight (MW) distribution demonstrated that all intestinal digests, except those from soybean, contained peptides with MW > 4 kDa at the end of gastrointestinal digestion. The profile of free amino acids released during digestion supports legume protein isolates as an excellent source of essential amino acids to be used in protein-rich food products. Peptides released during digestion matched with previously reported epitopes from the same plant species or others, explaining the ability to induce allergic reactions and cross-linked reactivity.

Peptidomic data of egg white gastrointestinal digests prepared using the Infogest Harmonized Protocol.

These data are related to the research article entitled "Induction of CCK and GLP-1 release in enteroendocrine cells by egg white peptides generated during gastrointestinal digestion". In this article, the peptide and free amino acid profile of egg white gastrointestinal in vitro digests is shown. Egg white proteins were digested following the INFOGEST gastrointestinal digestion protocol. Different time points of gastric and intestinal digestion were characterized regarding protein, peptide and amino acid content. Protein degradation was followed by SDS-PAGE where some electrophoretic bands were identified by MALDI-TOF/TOF after tryptic digestion. Moreover, the molecular weight distribution of egg white peptides found at different times of gastrointestinal digestion was performed using MALDI-TOF. Peptides identified from the most abundant egg white proteins by tandem mass spectrometry were represented using a peptide profile tool and raw data are given in table format. These results reveal the protein regions resistant to digestion and illustrate the free amino acid profile of egg white protein at the end of the digestion process. These data can be used for nutritional purposes and to identify allergen epitopes or bioactive sequences.

Implication of Opioid Receptors in the Antihypertensive Effect of a Bovine Casein Hydrolysate and αs1-Casein-Derived Peptides.

The antihypertensive activity of two αs1-casein-derived peptides and casein hydrolysate containing these sequences was evaluated in the presence of naloxone. The activity was abolished by this opioid antagonist at 2, 4, and 6 h post-administration. Similarly, the antihypertensive effect of the αs1-casein peptides 90RYLGY94 (-23.8 ± 2.5 mmHg) and 143AYFYPEL149 (-21.1 ± 3.2 mmHg) at 5 mg/kg of body weight was antagonized by the co-administration of naloxone. Because peptide 143AYFYPEL149 had recently shown opioid activity, a molecular dynamic simulation of this peptide with human µ-opioid receptor was performed to demonstrate its favorable structure and interaction energy, despite the presence of Ala at the N terminus. Altogether, these results revealed that the in vivo effect on systolic blood pressure of the studied αs1-casein peptides is mediated by interaction with opioid receptors and the antihypertensive activity of casein hydrolysate can be very likely ascribed to them with the possible contribution of other mechanisms.

Gastrointestinal digestion of food-use silver nanoparticles in the dynamic SIMulator of the GastroIntestinal tract (simgi®). Impact on human gut microbiota.

The increasing use of silver nanoparticles (AgNPs) in consumer products has led to concern about their impact on human health. This paper aims to provide new scientific evidence about the modifications and potential effects of AgNPs with food applications during their passage through the digestive tract. For that, two types of AgNPs [solid polyethylene glycol-stabilised silver nanoparticles (PEG-AgNPs 20) and liquid glutathione-stabilised silver nanoparticles (GSH-AgNPs)] were initially subjected to gut-microbial digestion simulation in an in vitro static model. Based on these experiments, digestion of GSH-AgNPs was carried out in a dynamic model (simgi®) that simulated the different regions of the digestive tract (stomach, small intestine and the ascending, transverse and descending colon) in physiological conditions. Dynamic transport of GSH-AgNPs in the simgi® was similar to that observed for the inert compound Cr-EDTA, which discarded any alterations in the intestinal fluid delivery due to the AgNPs. Also, feeding the simgi® with GSH-AgNPs seemed not to induce significant changes in the composition and metabolic activity (i.e., proteolytic activity) of the gut microbiota. Concerning monitoring of AgNps, it was observed that the GSH-AgNPs underwent several transformations in the gastrointestinal fluids and appeared to expose the intestine in ways that were structurally different from the original forms. In compliance with European guidelines, the simgi® model can be considered a useful in vitro tool to evaluate the effects of nanoparticles at the digestive level, prior to human studies, and, therefore, minimising animal testing.

Release of multifunctional peptides from kiwicha (Amaranthus caudatus) protein under in vitro gastrointestinal digestion.

BACKGROUND: The multifactorial origin of many chronic diseases provides a new framework for the development of multifunctional foods. In this study, the effect of in vitro simulated gastrointestinal digestion of kiwicha (Amaranthus caudatus) proteins on the release of multifunctional peptides was evaluated. RESULTS: Gastric digest showed higher angiotensin I converting enzyme (ACE) inhibitory activity while 60 min gastroduodenal digest showed the highest antioxidant, dipeptidyl peptidase IV (DPP-IV), α-amylase and Caco-2 cell viability inhibitory activities. Peptides >5 kDa were more effective in inhibiting colon cancer cell viability, whereas peptides <5 kDa were mainly responsible for the antioxidant, ACE, DPP-IV and α-amylase inhibitory activities. Thirteen peptides from amaranth sequenced proteins were identified. Structure-activity relationship analysis of the identified sequences pointed to three amaranth fragments, namely FLISCLL, SVFDEELS and DFIILE, as potential peptides able to concurrently exert antioxidant capacity and ability to inhibit both ACE and α-amylase. CONCLUSIONS: Five of thirteen peptides identified in kiwicha protein digests show high potential to exert multifunctional properties. Thus kiwicha proteins might start to gain importance as ingredients for functional foods for the prevention and/or management of chronic diseases related to oxidative stress, hypertension and/or diabetes. © 2018 Society of Chemical Industry.

Critical Review and Perspectives on Food-Derived Antihypertensive Peptides.

Food-derived peptides with antihypertensive properties have received great interest during the past 30 years. There is solid evidence of the effect of various peptide sequences in clinical trials, but their use in preventive or therapeutic treatments is not extensive. There are certain issues, such as the bioavailability or the mechanism of action, that would need to be clarified to establish a direct cause/effect relationship between the administered molecule and the observed effect. This perspective emphasizes the advances in the study of antihypertensive peptides and proposes future research topics that might encourage industry and health policy to exploit these food constituents.

Intestinal Signaling of Proteins and Digestion-Derived Products Relevant to Satiety.

Luminal nutrients stimulate enteroendocrine cells through the activation of specific receptors to release hormones that inhibit appetite and promote glucose homeostasis. While food protein is the macronutrient with the highest effect on satiety, the signaling on the protein digestion products at the gut is poorly understood. This perspective aims to highlight the existing gaps in the study of protein digestion products as signaling molecules in gastrointestinal enteroendocrine cells. Because dietary protein digestion can be modulated by the technological processes applied to food, it is possible to target gut receptors to control food intake by formulating specific food ingredients or protein preloads.

Bioaccessible peptides released by in vitro gastrointestinal digestion of fermented goat milks.

In this study, ultrafiltered goat milks fermented with the classical starter bacteria Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarus subsp. thermophilus or with the classical starter plus the Lactobacillus plantarum C4 probiotic strain were analyzed using ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) and/or high performance liquid chromatography-ion trap (HPLC-IT-MS/MS). Partial overlapping of the identified sequences with regard to fermentation culture was observed. Evaluation of the cleavage specificity suggested a lower proteolytic activity of the probiotic strain. Some of the potentially identified peptides had been previously reported as angiotensin-converting enzyme (ACE) inhibitory, antioxidant, and antibacterial and might account for the in vitro activity previously reported for these fermented milks. Simulated digestion of the products was conducted in the presence of a dialysis membrane to retrieve the bioaccessible peptide fraction. Some sequences with reported physiological activity resisted digestion but were found in the non-dialyzable fraction. However, new forms released by digestion, such as the antioxidant αs1-casein 144YFYPQL149, the antihypertensive αs2-casein 90YQKFPQY96, and the antibacterial αs2-casein 165LKKISQ170, were found in the dialyzable fraction of both fermented milks. Moreover, in the fermented milk including the probiotic strain, the k-casein dipeptidyl peptidase IV inhibitor (DPP-IV) 51INNQFLPYPY60 as well as additional ACE inhibitory or antioxidant sequences could be identified. With the aim of anticipating further biological outcomes, quantitative structure activity relationship (QSAR) analysis was applied to the bioaccessible fragments and led to potential ACE inhibitory sequences being proposed. Graphical abstract Ultrafiltered goat milks were fermented with the classical starter bacteria (St) and with St plus the L. plantarum C4 probiotic strain. Samples were analyzed using HPLC-IT-MS/MS and UPLC-Q-TOF-MS/MS. After simulated digestion and dialysis, some of the active sequences remained and new peptides with reported beneficial activities were released.