Bioactive peptides have been recognized as an important category of functional food ingredients, which exert a potential impact on human health beyond their nutritional value. However, although peptides have been demonstrated to exert multiple benefits by biochemical assays, cell culture, and animal models, the ability to translate the new findings into practical or commercial uses remains delayed because of several challenges that require to be addressed. Researchers have found that the in vitro bioactivity of peptides do not always correlate with in vivo effects. This is due to the fact that physiological effects of peptides depend largely on their ability to remain intact after digestive process. The peptides that resist the digestion and arrive intact at the intestinal absorption site can have a local function or may be able to cross the epithelium, enter the bloodstream, reach the target organ, and have a systemic effect. Therefore, as much as it is in pharmacology research, investigating the stability and bioavailability of bioactive peptides has become a new trend in the current functional food field.
In the last few years, food-derived bioactive peptides have attracted the interest of scientists because of their safety, low cost, and benefits on health beyond the nutritive role. Bioactive peptides have been demonstrated to positively affect the major body systems, notably, the cardiovascular, digestive, endocrine, immune, and nervous systems, while minimizing the risks of chronic disease development . Thus, they have become promising ingredients for functional foods and nutraceuticals . A lot of in vitro biochemical assays, cell models, and animal models have been optimized and applied for testing the bioactivity of these food bioactive peptides. However, although the research on the development of peptides-enriched products has notably increased, the ability to translate the new findings into practical or commercial uses remains delayed. Among the major reasons behind this delay, one of the most important is the lack of correlation of the in vitro bioactivities of peptides with in vivo functions due to their low bioavailability following oral administration . Peptides need to resist the action of digestive enzymes during their transit through the gastrointestinal tract and cross the intestinal epithelial barrier to reach intact the target organs where peptides can exert their health-promoting effects . Thus, when studying the effects of bioactive peptides in our organism, it is important to assess their under-digestive conditions, and if the peptide is absorbed, it is necessary to evaluate its distribution, metabolism, and excretion behavior . In this review, the most current evidence on the in vitro and in vivo models designed to evaluate the digestibility and bioavailability of food bioactive peptides is summarized, focusing on those limiting factors affecting both peptides resistance to digestive conditions and absorption capacity.
Human digestion is a complex process that involves the concerted action of digestive enzymes on dietary ingredients. In the case of digestion of food proteins, several factors influence this process such as the type of proteins, gastric and intestinal pH, activity of digestive enzymes, endogenous secretions, and motility . Digestion is considered a vital process for life because nutrients released from ingested foods are used by the body as an energy source for cell maintenance and growth . During the digestion of food proteins, peptides and amino acids are liberated, acting as signals of gastric or intestinal motility and pancreatic secretion, and/or exerting local and systemic physiological functions.
Digestion starts with a short food chewing step in the mouth, which is relevant for the complete digestive process, particularly for the gastric emptying rate . The food bolus resulting from mechanical and enzymatic degradations in the mouth is transported through the esophagus to the stomach by peristaltic movements. Once the bolus reaches the proximal part of the stomach, it is mixed with the gastric juice, which is mostly composed of hydrochloric acid (HCl), pepsin and lipases responsible for protein and lipid digestion, respectively, and mucus that protects the mucosal surface. In the distal part, peristaltic movements allow breaking large food particles into smaller ones by grinding and mixing gastric contents. The stomach ends at the pylorus that pumps small particles (chyme) to the duodenum, while the largest particles are maintained in the stomach for further digestion. Once the chyme enters the duodenum, its acidic pH is neutralized by sodium carbonate (NaHCO3) until reaching a pH appropriate for the activity of pancreatic (proteases, amylases, and lipases) and intestinal enzymes, which are responsible for the subsequent digestion of molecules contained in the chyme. Bile produced by the liver contributes to lipid digestion by emulsifying dietary fats into small droplets that favor the activity of lipase. Once digested, released nutrients are available for their absorption by villus enterocytes through different transport mechanisms, and non-absorbed material travels down to the large intestine. In the colon, water and electrolytes are absorbed, bile salts are reabsorbed, and non-digested polysaccharides and proteins are fermented by colonic microbiota, releasing new degradation products. Finally, at the end of the large intestine, the formation, storage, and elimination of feces occurs .
Absorption of most of the digestion products occurrs in the jejunum, where chyme enters from the stomach, and it is further broken down into nutrients (including peptides, fatty acids, mono- and oligosaccharides, vitamins, and minerals) that cross the intestinal wall, reaching the systemic circulation. Traditionally, it was thought that once ingested, all peptides and proteins were hydrolyzed by digestive enzymes to their constituent amino acids that were absorbed across the intestinal epithelial barrier. It was also believed that proteins and peptides were only absorbed under pathological conditions. However, in the last few years, it has been found that many peptides are absorbed by intestinal cells under normal conditions, being detected in both newborn and adults’ bloodstream and/or target organs where they exert their biological activities .
To date, four different routes of peptides absorption have been described: paracellular diffusion, transcellular passive diffusion, transcytosis, and carrier-mediated transport. Following, the main characteristics of these absorption pathways and examples of peptides using them are summarized.
Gut endogenous proteins represent a larger and more constant supply of protein in the gastrointestinal tract in comparison with dietary protein . They are constituted by gastrointestinal tract epithelial turnover, gut microflora proteins, and soluble secreted proteins such as mucins, digestive enzymes, hormones, serum albumin, immunoglobulins, and lysozymes, among others . Although these endogenous proteins have been exhaustively studied to estimate the dietary amino acid requirements and digestibility, the data on their potential as a source of bioactive peptides are still scarce. However, given the high amount of endogenous proteins present in the gastrointestinal tract, a wide array of potentially bioactive peptides are expected to be liberated during the digestion process. In a preliminary in silico gastrointestinal digestion prediction model, 26 gut endogenous proteins were evaluated as a source of bioactive peptides . The total number of bioactive peptides predicted to be released ranged from 1 (secretin) to 39 (mucin-5AC), of which ACE-inhibitory peptides were the most frequently observed. These results were confirmed by an in vitro digestion assay of endogenous proteins, resulting in the release of a high number of antioxidant, ACE, and DPP-IV inhibitory peptides . Similarly to dietary bioactive peptides that have been demonstrated to bind to specific receptors in the gut modulating gut motility, satiety, and the secretion of gastrointestinal endogenous proteins, peptides released from these endogenous proteins might also have effects on gut physiology and functions . Therefore, little modifications of both dietary and endogenous sources of bioactive peptides offers a great opportunity to modulate gut processes.
Once the mechanisms involved in the transport of bioactive peptides and the factors influencing their absorption are known and understood, it is possible to design valuable strategies that improve the bioavailability of peptides and maintain their potent in vivo bioactivities. These strategies aim at achieving the following objectives: (i) reduction of the detrimental effects of food processing on peptides bioactivities; (ii) promotion of the desiderable interactions between peptides and other food matrix components, reducing the undesiderable ones; (iii) protection of bioactive peptides from gastrointestinal conditions and digestive enzyme activity; (iv) control of the sustained peptides’ release directed at their target organs; and (v) improvement of the transport of bioactive peptides across the intestinal epithelium and target cells .
It has been recognized worldwide that food-derived bioactive peptides are valuable ingredients of functional foods and/or nutraceuticals to promote health and reduce the risk of chronic diseases. However, although oral administration is the preferred route for bioactive peptides, the translation of in vitro activity to in vivo effects when peptides are orally ingested is not always realistic. These discrepancies are due to the molecular characteristics of peptides as well as to both dietary and non-dietary factors. Molecular mass, amino acid sequence, and additional structure modifications are determinant properties for the resistance of peptides to digestive enzymes and the preferred transport route to cross the intestinal barrier. Among dietary factors, the interactions between peptides and other compounds of the food matrix are considered relevant, since these components may reversibly or irreversibly react with bioactive peptides, modulating their digestibility and/or altering the routes of absorption of peptides, influencing their bioavailability. However, to date, the existing evidence on the effects of the food matrix is still limited. In addition, the behavior of peptides during their transit through the gastrointestinal tract depends on health and pathological conditions that can alter the digestive and absorptive gut environment. Thus, for a better understanding of the in vivo physiological effects of food bioactive peptides, extensive research studies on their gastrointestinal stability and transport are needed. Combined in vitro studies simulating gastrointestinal digestion conditions and cell culture mimicking the intestinal absorptive environment are being optimized, becoming an interesting and valuable approach to confirm the beneficial role of peptides on health at doses that are physiologically relevant. Due to the low bioavailability of most food peptides, efforts are being focused on the design of new strategies that increase their resistance to the action of digestive enzymes during their transit through the gastrointestinal tract and allow the controlled release of intact and active peptides in the target organs where they exert their biological activity. Among these strategies, delivery systems with natural, safe, and biocompatible materials are becoming the most promising; thus, further research should be needed to optimize the encapsulation conditions enhancing the digestibility and bioavailability of food bioactive peptides.