Effect of In Vitro Enzyme Digestion and Bile Treatment on Milk Extracellular Vesicles Stability.

SCOPE: Milk extracellular vesicles (EVs) are nanosized particles with potential immune bioactivities. This study examines their fate during in vitro infant gastrointestinal digestion (GI). METHODS AND RESULTS: Bovine milk is digested using the in vitro INFOGEST method, adjusted for the infant. To unravel the contribution of digestive enzymes from bile, milk is treated with digestive enzymes, bile, or a combination of both. EVs are collected posttreatment using differential ultracentrifugation. EVs characterization includes electrophoresis, immunoblotting, nanoparticle tracking analysis, and atomic force microscopy. EVs protein markers programmed cell death 6-interacting protein (ALIX), tumor susceptibility gene 101 (TSG101), cluster of differentiation 9 (CD9), and xanthine dehydrogenase (XDH) are detected after gastric digestion (G60), but their signal intensity is significantly reduced by intestinal conditions (p < 0.05). Enzyme digestion, compared to bile treatment (I60 + bile), results in a significant reduction of signal intensities for TSG101 and CD9 (p < 0.05). Nanoparticle tracking analysis shows a significant reduction (p < 0.05) of EV numbers at the end of the intestinal phase. EVs are detected by atomic force microscopy at the end of the intestinal phase, showing that intact EVs can survive upper gut digestion. CONCLUSION: Intact EVs can be found at the end of the intestinal phase. However, digestive enzymes and bile reduce the quantity and characteristics of EVs, with digestive enzymes playing a larger role.

Infant milk formula, produced by membrane filtration, promotes mucus production in the upper small intestine of young pigs.

Human breast milk promotes maturation of the infant gastrointestinal barrier, including the promotion of mucus production. In the quest to produce next generation infant milk formula (IMF), we have produced IMF by membrane filtration (MEM-IMF). With a higher quantity of native whey protein, MEM-IMF more closely mimics human breast milk than IMF produced using conventional heat treatment (HT-IMF). After a 4-week dietary intervention in young pigs, animals fed a MEM-IMF diet had a higher number of goblet cells, acidic mucus and mucin-2 in the jejunum compared to pigs fed HT-IMF (P < 0.05). In the duodenum, MEM-IMF fed pigs had increased trypsin activity in the gut lumen, increased mRNA transcript levels of claudin 1 in the mucosal scrapings and increased lactase activity in brush border membrane vesicles than those pigs fed HT-IMF (P < 0.05). In conclusion, MEM-IMF is superior to HT-IMF in the promotion of mucus production in the young gut.

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.

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.

Dairy and plant based protein beverages: In vitro digestion behaviour and effect on intestinal barrier biomarkers.

The consumer demand for protein-enriched food products continues to grow, in parallel with consumers' interest in plant based alternatives. The replacement of milk protein by plant protein is likely to be occur predominantly in prepared consumer foods such as nutritional beverages. This study aimed to compare and contrast powder beverages formulated with commercially available dairy versus plant ingredients in terms of protein digestion and gut barrier health. After simulated static in vitro gastrointestinal digestion, the release of free amino acids increased for all model beverages. In addition, the majority of peptides present in digested beverages were < 0.8 kDa in size. Gastrointestinal digestion did not increase the degree of protein hydrolysis in beverages formulated with prehydrolysed milk protein, whey or pea ingredients. A 2 h permeability assessment of digested beverages across the intestinal barrier, using Caco-2/HT-29/MTX co-cultures, revealed reduced transcription of tight junction protein 1, claudin-1 and mucus protein 2 albeit gut barrier impedance was unchanged. IL-8 mRNA levels in cell monolayers was significantly increased with digested fluids treatment but even more so with digesta from hydrolysed milk protein beverage. Overall, the response observed on intestinal biomarkers with digested plant beverages was similar to dairy based beverages supporting the replacement of dairy with plant proteins in powder beverage formulations.

Membrane filtration processing of infant milk formula alters protein digestion in young pigs.

Reducing heat treatment (HT) during processing of infant milk formula (IMF) is desirable to produce a product that more closely resembles breast milk. By employing membrane filtration (MEM), we produced an IMF (60:40 whey to casein ratio) at pilot scale (250 kg). MEM-IMF had a significantly higher content of native whey (59.9 %) compared to HT-IMF (4.5 %) (p < 0.001). Pigs, at 28 days old, were blocked by sex, weight and litter origin and assigned to one of two treatments (n = 14/treatment): (1) starter diet containing 35 % of HT-IMF powder or (2) starter diet containing 35 % of MEM-IMF powder for 28 days. Body weight and feed intake were recorded weekly. Pigs at day 28 post weaning were sacrificed 180 min after their final feeding, for the collection of gastric, duodenal, jejunum and ileal contents (n = 10/treatment). MEM-IMF diet resulted in more water-soluble proteins and higher levels of protein hydrolysis in the digesta at various gut locations compared to HT-IMF (p < 0.05). In the jejunal digesta, a higher concentration of free amino acids were present post MEM-IMF consumption (247 ± 15 µmol g-1 of protein in digesta) compared to HT-IMF (205 ± 21 µmol g-1 of protein). Overall, average daily weight gain, average dairy feed intake and feed conversion efficiency were similar for pigs fed either MEM-IMF or HT-IMF diets, but differences and trends to difference of these indicators were determined in particular intervention periods. In conclusion, reducing heat treatment during processing of IMF influenced protein digestion and revealed minor effects on growth parameters providing in vivo evidence that babies who are fed with IMF processed by MEM are likely to have different protein digestion kinetics but minimal effect on overall growth trajectories as babies fed IMF processed by traditional thermal processing.

Critical features of an in vitro intestinal absorption model to study the first key aspects underlying food allergen sensitization.

New types of protein sources will enter our diet in a near future, reinforcing the need for a straightforward in vitro (cell-based) screening model to test and predict the safety of these novel proteins, in particular their potential risk for de novo allergic sensitization. The Adverse Outcome Pathway (AOP) for allergen sensitization describes the current knowledge of key events underlying the complex cellular interactions that proceed allergic food sensitization. Currently, there is no consensus on the in vitro model to study the intestinal translocation of proteins as well as the epithelial activation, which comprise the first molecular initiation events (ME1-3) and the first key event of the AOP, respectively. As members of INFOGEST, we have highlighted several critical features that should be considered for any proposed in vitro model to study epithelial protein transport in the context of allergic sensitization. In addition, we defined which intestinal cell types are indispensable in a consensus model of the first steps of the AOP, and which cell types are optional or desired when there is the possibility to create a more complex cell model. A model of these first key aspects of the AOP can be used to study the gut epithelial translocation behavior of known hypo- and hyperallergens, juxtaposed to the transport behavior of novel proteins as a first screen for risk management of dietary proteins. Indeed, this disquisition forms a basis for the development of a future consensus model of the allergic sensitization cascade, comprising also the other key events (KE2-5).

An investigation of extracellular vesicles in bovine colostrum, first milk and milk over the lactation curve.

Extracellular vesicles (EVs) in milk have claimed benefits ranging from conveying immunological privilege to infants to being suitable as natural delivery vehicles for therapeutic drugs. However, a longitudinal study of bovine EVs quantities and characteristics in colostrum (COL), first milk (FM) and throughout the lactation curve of mature milk (MM) had never been performed and so was our aim. COL, FM and 9 months of MM samples were collected. Caseins -overlapping size with EVs- were removed. EVs were collected by density gradient ultracentrifugation and characterised by SDS-PAGE, Bradford assay, nanoparticle tracking analysis, immunoblotting, imaging flow cytometry analysis, and transmission electron microscopy. COL and FM had substantially more EVs than MM, with COL enriched in small EVs. No significant differences were observed between months 1-9 of MM. Altogether, although COL and FM are particularly rich sources of EVs, mature milk throughout the lactation curve is also an abundant source of intact EVs.

Comparison of conventional heat-treated and membrane filtered infant formulas using an in vitro semi-dynamic digestion method.

Introducing membrane filtration steps into infant milk formula (IMF) manufacture can partly preserve native whey proteins in the final products. In this study, the IMF produced by membrane filtration (MEM-IMF) and conventional heat treatment (HT-IMF) were compared by using a novel semi-dynamic infant in vitro digestion method. MEM-IMF exhibited a fragmented curd during gastric digestion, and confocal laser light microscopy showed that protein aggregates had disassociated from the fat droplets within 93 min in the MEM-IMF digesta. In contrast, the digesta of HT-IMF showed a more extensive curd formation and denser protein aggregates, which remained intact until the end of gastric digestion. Molecular weight profiles and the primary amine assay suggested that protein degradation and peptide release were faster in the MEM-IMF. In conclusion, the presence of native whey protein in the IMF altered the gastric digestion kinetics by changing coagulation and formation of aggregates, potentially accelerating the rate of gastric emptying in vivo.

Solid lipid nanoparticles to improve bioaccessibility and permeability of orally administered maslinic acid.

Maslinic acid (MA) is a plant-derived, low water-soluble compound with antitumor activity. We have formulated MA in the form of solid lipid nanoparticles (SLNs) with three different shell compositions: Poloxamer 407 (PMA), dicarboxylic acid-Poloxamer 407 (PCMA), and HA-coated PCMA (PCMA-HA). These SLNs improved the solubility of MA up to 7.5 mg/mL, are stable in a wide range of pH, and increase the bioaccessibility of MA after in vitro gastrointestinal (GI) digestion. Gastrointestinal digested SLNs afforded MA delivery across in vitro gut barrier models (21 days old Caco-2 and mucus-producing Caco-2/HT29-MTX co-cultures). The cellular fraction of Caco-2/HT29-MTX co-cultures retained more MA from GI digested PCMA-HA than the Caco-2 monolayers. The concentration of MA reached in the basolateral chamber inhibited growth of pancreatic cancer cells, BxPC3. Finally, confocal microscopy images provided evidence that Nile Red incorporated in MA SLNs was capable of crossing Caco-2 monolayers to be taken up by basolaterally located BxPC3 cells. We have demonstrated that SLNs can be used as nanocarriers of hydrophobic antitumor compounds and that these SLNs are suitable for oral consumption and delivery of the bioactive across the gut barrier.

Blue Whiting (Micromesistius poutassou) Protein Hydrolysates Increase GLP-1 Secretion and Proglucagon Production in STC-1 Cells Whilst Maintaining Caco-2/HT29-MTX Co-Culture Integrity.

Inducing the feeling of fullness via the regulation of satiety hormones presents an effective method for reducing excess energy intake and, in turn, preventing the development of obesity. In this study, the ability of blue whiting soluble protein hydrolysates (BWSPHs) and simulated gastrointestinal digested (SGID) BWSPHs, to modulate the secretion and/or production of satiety hormones, such as glucagon-like peptide-1 (GLP-1), cholecystokinin (CCK) and peptide YY (PYY), was assessed in murine enteroendocrine STC-1 cells. All BWSPHs (BW-SPH-A to BW-SPH-F) (1.0% w/v dw) increased active GLP-1 secretion and proglucagon production in STC-1 cells compared to the basal control (Krebs-Ringer buffer) (p < 0.05). The signaling pathway activated for GLP-1 secretion was also assessed. A significant increase in intracellular calcium levels was observed after incubation with all BWSPHs (p < 0.05) compared with the control, although none of the BWSPHs altered intracellular cyclic adenosine monophosphate (cAMP) concentrations. The secretagogue effect of the leading hydrolysate was diminished after SGID. Neither pre- nor post-SGID hydrolysates affected epithelial barrier integrity or stimulated interleukin (IL)-6 secretion in differentiated Caco-2/HT-29MTX co-cultured cells. These results suggest a role for BWSPH-derived peptides in satiety activity; however, these peptides may need to be protected by some means to avoid loss of activity during gastrointestinal transit.

Sodium butyrate converts Caco-2 monolayers into a leaky but healthy intestinal barrier resembling that of a newborn infant.

A simple and reliable in vitro model of the infant intestinal barrier is needed to study nutrient absorption and drug permeability specifically for this life stage. This study investigated the treatment of 20 day old differentiated Caco-2 monolayers with sodium butyrate at various concentrations (0-250 mM). Monolayer integrity, cytotoxicity, permeability and inflammatory response were tracked. An intestinal barrier model, with infant gut characteristics, was developed based on the treatment of mature monolayers with 125 mM sodium butyrate for 24 h. Such treatment was not cytotoxic but caused a stable transepithelial electrical resistance value of 408 ± 52 Ω cm2. The ratio of lactulose to mannitol transport across the intestinal barrier increased 1.79-fold. Redistribution of the tight junction proteins, occludin and ZO-1, in response to sodium butyrate treatment was visualized with immunofluorescence. Levels of the cytokines, TNF-α and IL-6, although modestly increased did not indicate an inflammatory response by Caco-2 to sodium butyrate. This intestinal barrier demonstrated physiologically relevant transport rates for dairy protein of 0.01-0.06%, suggesting it may be used to track permeability of proteins in infant nutritional products.

Protein quality and quantity influence the effect of dietary fat on weight gain and tissue partitioning via host-microbiota changes.

We investigated how protein quantity (10%-30%) and quality (casein and whey) interact with dietary fat (20%-55%) to affect metabolic health in adult mice. Although dietary fat was the main driver of body weight gain and individual tissue weight, high (30%) casein intake accentuated and high whey intake reduced the negative metabolic aspects of high fat. Jejunum and liver transcriptomics revealed increased intestinal permeability, low-grade inflammation, altered lipid metabolism, and liver dysfunction in casein-fed but not whey-fed animals. These differential effects were accompanied by altered gut size and microbial functions related to amino acid degradation and lipid metabolism. Fecal microbiota transfer confirmed that the casein microbiota increases and the whey microbiota impedes weight gain. These data show that the effects of dietary fat on weight gain and tissue partitioning are further influenced by the quantity and quality of the associated protein, primarily via effects on the microbiota.

Assessment of the biological activity of fish muscle protein hydrolysates using in vitro model systems.

The generation of biologically active fish protein hydrolysates (FPH) is a useful technique to produce value-added products with potential application in the functional food and nutraceutical industries. Fish muscle is an attractive substrate for the production of protein hydrolysates due to its rich protein content, containing 15-25% of total fish protein. This paper reviews the production of protein hydrolysates from fish muscle, most commonly via enzymatic hydrolysis, and their subsequent bioactivities including anti-obesity, immunomodulatory, antioxidant, angiotensin I-converting enzyme (ACE)-inhibitory, anti-microbial, and anti-cancer activities as measured by in vitro testing methods. Disease prevention with FPH potentially offers a safe and natural alternative to synthetic drugs. Small molecular weight (MW) FPHs generally exhibit favourable bioactivity than large MW fractions via enhanced absorption through the gastrointestinal tract. This review also discusses the relationship between amino acid (AA) composition and AA sequence of FPH and peptides and their exhibited in vitro bioactivity.

Thermal or membrane processing for Infant Milk Formula: Effects on protein digestion and integrity of the intestinal barrier.

Infant Milk Formula (IMF) is designed as a breastmilk substitute to satisfy the nutritional requirements during the first months of life. This study investigates the effects of two IMF processing technologies on cow milk protein digestion using an infant static in vitro gastrointestinal model. The degree of protein hydrolysis at the end of the gastric phase was 3.7-fold higher for IMF produced by high temperature (IMF-HT), compared to IMF produced by cascade membrane filtration (IMF-CMF), as assessed by free N-terminal group analysis. The processing type also influenced the panel of bioavailable peptides detected in basolateral compartments of Caco-2 monolayers exposed to gastrointestinal digested IMFs. In addition, IMF-CMF significantly increased tight junction protein, claudin 1, whilst IMF-HT significantly reduced tight junction integrity. In conclusion, producing IMF by CMF may preserve intestinal barrier integrity and can deliver its own unique inventory of bioavailable peptides with potential bioactivity.

Whey for Sarcopenia; Can Whey Peptides, Hydrolysates or Proteins Play a Beneficial Role?

As the human body ages, skeletal muscle loses its mass and strength. It is estimated that in 10% of individuals over the age of 60, this muscle frailty has progressed to sarcopenia. Biomarkers of sarcopenia include increases in inflammatory markers and oxidative stress markers and decreases in muscle anabolic markers. Whey is a high-quality, easily digested dairy protein which is widely used in the sports industry. This review explores the evidence that whey protein, hydrolysates or peptides may have beneficial effects on sarcopenic biomarkers in myoblast cell lines, in aged rodents and in human dietary intervention trials with the older consumer. A daily dietary supplementation of 35 g of whey is likely to improve sarcopenic biomarkers in frail or sarcopenia individuals. Whey supplementation, consumed by an older, healthy adult certainly improves muscle mTOR signaling, but exercise appears to have the greatest benefit to older muscle. In vitro cellular assays are central for bioactive and bioavailable peptide identification and to determine their mechanism of action on ageing muscle.

Whey proteins: targets of oxidation, or mediators of redox protection.

Bovine whey proteins are highly valued dairy ingredients. This is primarily due to their amino acid content, digestibility, bioactivities and their processing characteristics. One of the reported bioactivities of whey proteins is antioxidant activity. Numerous dietary intervention trials with humans and animals indicate that consumption of whey products can modulate redox biomarkers to reduce oxidative stress. This bioactivity has in part been assigned to whey peptides using a range of biochemical or cellular assays in vitro. Superimposing whey peptide sequences from gastrointestinal samples, with whey peptides proven to be antioxidant in vitro, allows us to propose peptides from whey likely to exhibit antioxidant activity in the diet. However, whey proteins themselves are targets of oxidation during processing particularly when exposed to high thermal loads and/or extensive processing (e.g. infant formula manufacture). Oxidative damage of whey proteins can be selective with regard to the residues that are modified and are associated with the degree of protein unfolding, with α-Lactalbumin more susceptible than ß-Lactoglobulin. Such oxidative damage may have adverse effects on human health. This review summarises how whey proteins can modulate cellular redox pathways and conversely how whey proteins can be oxidised during processing. Given the extensive processing steps that whey proteins are often subjected to, we conclude that oxidation during processing is likely to compromise the positive health attributes associated with whey proteins.

Effects of Protein-Derived Amino Acid Modification Products Present in Infant Formula on Metabolic Function, Oxidative Stress, and Intestinal Permeability in Cell Models.

Proteins present in infant formulas are modified by oxidation and glycation during processing. Modified amino acid residues released from proteins may be absorbed in the gastrointestinal tract, and pose a health risk to infants. In this study, the markers of glycation furosine (1.7-3.5 µg per milligram of protein) and Nε-(carboxymethyl)lysine (28-81 ng per milligram of protein) were quantitated in infant formulas. The effects of these species, and other amino acid modifications, at the levels detected in infant formulas, on 3T3-L1 (murine preadipocyte) and Caco-2 (human intestinal epithelial) cells were assessed. Incubation of 3T3-L1 cells for 48 h with amino acid side chain oxidation and glycation products (1 and 10 µM) resulted in a loss (up to 40%, p < 0.05) of cell thiols and decreased metabolic activity compared with those of the controls. In contrast, Caco-2 cells showed a stimulation (10-50%, p < 0.05) of cellular metabolism on exposure to these products for 24 or 48 h. A 28% ( p < 0.05) increase in protein carbonyls was detected upon incubation with 200 µM modified amino acids for 48 h, although no alteration in transepithelial electrical resistance was detected. Oxidation products were detected in the basolateral compartments of Caco-2 monolayers when modified amino acids were applied to the apical side, consistent with limited permeability (up to 3.4%) across the monolayer. These data indicate that modified amino acids present in infant formulas can induce effects on different cell types, with evidence of bioavailability and induction of cellular stress. This may lead to potential health risks for infants consistently exposed to high levels of infant formulas.

Bovine whey peptides transit the intestinal barrier to reduce oxidative stress in muscle cells.

Health benefits are routinely attributed to whey proteins, their hydrolysates and peptides based on in vitro chemical and cellular assays. The objective of this study was to track the fate of whey proteins through the upper gastrointestinal tract, their uptake across the intestinal barrier and then assess the physiological impact to downstream target cells. Simulated gastrointestinal digestion (SGID) released a selection of whey peptides some of which were transported across a Caco-2/HT-29 intestinal barrier, inhibited free radical formation in muscle and liver cells. In addition, SGID of ß-lactoglobulin resulted in the highest concentration of free amino acids (176 nM) arriving on the basolateral side of the co-culture with notable levels of branched chain and sulphur-containing amino acids. In vitro results indicate that consumption of whey proteins will deliver bioactive peptides to target cells.

Personalized nutrition in ageing society: redox control of major-age related diseases through the NutRedOx Network (COST Action CA16112).

A healthy ageing process is important when it is considered that one-third of the population of Europe is already over 50 years old, although there are regional variations. This proportion is likely to increase in the future, and maintenance of vitality at an older age is not only an important measure of the quality of life but also key to participation and productivity. So, the binomial "nutrition and ageing" has different aspects and poses considerable challenges, providing a fertile ground for research and networks. The NutRedOx network will focus on the impact of redox-active compounds in food on healthy ageing, chemoprevention, and redox control in the context of major age-related diseases. The main aim of the NutRedOx network is to gather experts from Europe, and neighbouring countries, and from different disciplines that are involved in the study of biological redox active food components and are relevant to the ageing organism, its health, function, and vulnerability to disease. Together, these experts will form a major and sustainable EU-wide cluster in form of the NutRedOx Centre of Excellence able to address the topic from different perspectives, with the long-term aim to provide a scientific basis for improved nutritional and lifestyle habits, to train the next generation of multidisciplinary researchers in this field, to raise awareness of such habits among the wider population, and also to engage with industry to develop age-adequate foods and medicines.