Current Evidence on the Bioavailability of Food Bioactive Peptides.

Food protein-derived bioactive peptides are recognized as valuable ingredients of functional foods and/or nutraceuticals to promote health and reduce the risk of chronic diseases. 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. This fact is mainly due to the lack of correlation of in vitro findings with in vivo functions of peptides because of their low bioavailability. Once ingested, peptides need to resist the action of digestive enzymes during their transit through the gastrointestinal tract and cross the intestinal epithelial barrier to reach the target organs in an intact and active form to exert their health-promoting properties. 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. This review summarizes the most current evidence on those factors affecting the digestive and absorptive processes of food bioactive peptides, the recently designed models mimicking the gastrointestinal environment, as well as the novel strategies developed and currently applied to enhance the absorption and bioavailability of peptides.

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.

Dynamic gastric digestion of a commercial whey protein concentrate†.

BACKGROUND: A dynamic gastrointestinal simulator, simgi® , has been applied to assess the gastric digestion of a whey protein concentrate. Samples collected from the outlet of the stomach have been compared to those resulting from the static digestion protocol INFOGEST developed on the basis of physiologically inferred conditions. RESULTS: Progress of digestion was followed by SDS-PAGE and LC-MS/MS. By SDS-PAGE, serum albumin and α-lactalbumin were no longer detectable at 30 and 60 min, respectively. On the contrary, ß-lactoglobulin was visible up to 120 min, although in decreasing concentrations in the dynamic model due to the gastric emptying and the addition of gastric fluids. Moreover, ß-lactoglobulin was partly hydrolysed by pepsin probably due to the presence of heat-denatured forms and the peptides released using both digestion models were similar. Under dynamic conditions, a stepwise increase in number of peptides over time was observed, while the static protocol generated a high number of peptides from the beginning of digestion. CONCLUSION: Whey protein digestion products using a dynamic stomach are consistent with those generated with the static protocol but the kinetic behaviour of the peptide profile emphasises the effect of the sequential pepsin addition, peristaltic shaking, and gastric emptying on protein digestibility. © 2017 Society of Chemical Industry.

Short communication: Identification of iron-binding peptides from whey protein hydrolysates using iron (III)-immobilized metal ion affinity chromatography and reversed phase-HPLC-tandem mass spectrometry.

Peptides with iron-binding capacity obtained by hydrolysis of whey protein with Alcalase (Novozymes, Araucaria, PR, Brazil), pancreatin, and Flavourzyme (Novozymes) were identified. Hydrolysates were subjected to iron (III)-immobilized metal ion affinity chromatography, and the bound peptides were sequenced by mass spectrometry. Regardless of the enzyme used, the domains f(42-59) and f(125-137) from ß-lactoglobulin enclosed most of identified peptides. This trend was less pronounced in the case of peptides derived from α-lactalbumin, with sequences deriving from diverse regions. Iron-bound peptides exhibited common structural characteristics, such as an abundance of Asp, Glu, and Pro, as revealed by mass spectrometry and AA analysis. In conclusion, this characterization of iron-binding peptides helps clarify the relationship between peptide structure and iron-chelating activity and supports the promising role of whey protein hydrolysates as functional ingredients in iron supplementation treatments.

Assessment on proximate composition, dietary fiber, phytic acid and protein hydrolysis of germinated Ecuatorian brown rice.

Germinated brown rice (GBR) is considered healthier than brown rice (BR) but its nutritive value has been hardly studied. Since nutritive quality of GBR depends on genetic diversity and germination conditions, six Ecuadorian BR varieties were germinated at 28 and 34 ºC for 48 and 96 h in darkness and proximate composition, dietary fiber fractions, phytic acid content as well as degree of protein hydrolysis and peptide content were studied. Protein, lipids, ash and available carbohydrate ranged 7.3-10.4%, 2.0-4.0%, 0.8-1.5% and 71.6 to 84.0%, respectively, in GBR seedlings. Total dietary fiber increased during germination (6.1-13.6%), with a large proportion of insoluble fraction, while phytic acid was reduced noticeably. In general, protein hydrolysis occurred during germination was more accused at 28 ºC for 48 h. These results suggest that GBR can be consumed directly as nutritive staple food for a large population worldwide contributing to their nutritional requirements.

Monitoring the large-scale production of the antihypertensive peptides RYLGY and AYFYPEL by HPLC-MS.

This work reports the quantitative analysis of two novel antihypertensive peptides alpha(s1)-CN f(90-94), with sequence RYLGY, and alpha(s1)-CN f(143-149), with sequence AYFYPEL, by high-performance liquid chromatography-mass spectrometry in food-grade hydrolysates of milk proteins. The method was validated and showed sufficient specificity, reproducibility, linearity and recovery. Linear calibrations of the molecular ions m/z 671.2 and 902.3 were selected for the determination of the peptides RYLGY and AYFYPEL, respectively, and showed good statistical results (R(2) > or = 0.995 and with no significant lack-of-fit). The simplicity of RP-HPLC-MS method allowed the automated quantification of both antihypertensive peptides without any sample pretreatment. The application of this method permitted the evaluation of some hydrolysis variables, i.e., substrate, temperature, hydrolysis time or enzyme/substrate ratio, on the formation of antihypertensive peptides. The quantitative analysis of RYLGY and AYFYPEL showed that ultrafiltration was not effective to improve the content in active peptides, containing the hydrolysates and their respective permeates similar peptide amounts.

Introduction to the Special Issue: New Advances in the Research of Antioxidant Food Peptides.

During cell metabolism, oxygen is partially reduced to reactive oxygen species (ROS) that play a physiological role in cellular processes, including proliferation, cell cycle and death, and signal transduction [...].

In Silico and In Vitro Analysis of Multifunctionality of Animal Food-Derived Peptides.

Currently, the associations between oxidative stress, inflammation, hypertension, and metabolic disturbances and non-communicable diseases are very well known. Since these risk factors show a preventable character, the searching of food peptides acting against them has become a promising strategy for the design and development of new multifunctional foods or nutraceuticals. In the present study, an integrated approach combining an in silico study and in vitro assays was used to confirm the multifunctionality of milk and meat protein-derived peptides that were similar to or shared amino acids with previously described opioid peptides. By the in silico analysis, 15 of the 27 assayed peptides were found to exert two or more activities, with Angiotensin-converting enzyme (ACE) inhibitory, antioxidant, and opioid being the most commonly found. The in vitro study confirmed ACE-inhibitory and antioxidant activities in 15 and 26 of the 27 synthetic peptides, respectively. Four fragments, RYLGYLE, YLGYLE, YFYPEL, and YPWT, also demonstrated the ability to protect Caco-2 and macrophages RAW264.7 cells from the oxidative damage caused by chemicals. The multifunctionality of these peptides makes them promising agents against oxidative stress-associated diseases.

Induction of CCK and GLP-1 release in enteroendocrine cells by egg white peptides generated during gastrointestinal digestion.

The effect of dietary protein on the induction of intestinal hormones is recognised. However, little is known about the nature of the digestion products involved in this intestinal signalling. Our aim was to characterise egg white protein digestion products and study their ability to induce CCK and GLP-1 release in enteroendocrine STC-1 cells. Intestinal digests triggered GLP-1 release at a higher rate than gastric digests. Peptides, but not free amino acids, showed a potent GLP-1 secretagogue effect, while proteins only had a modest effect. CCK was released in response to peptides and free amino acids but not proteins. Two hydrophobic negatively charged peptides triggered CCK release, while the highest GLP-1 response was found with a hydrophobic positively charged peptide, pointing to the involvement of different receptors or active sites. Identifying peptide sequences and receptors involved in hormonal secretion could open up new ways to control food intake and glucose metabolism.

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.

Identification of the initial binding sites of alphas2-casein f(183-207) and effect on bacterial membranes and cell morphology.

The aim of this work was to identify the initial binding sites to the bacterial membranes of the antimicrobial peptide alphas2-casein f(183-207) and also to acquire further insight into membrane permeabilization of this peptide. Furthermore, cell morphology was studied by transmission electron microscopy. In all the experiments, bovine LFcin was employed as a comparison. Results showed that initial binding sites of alphas2-casein f(183-207) peptide were lipoteichoic acid in Gram-positive bacteria and lipopolysaccharide in Gram-negative. The peptide was able to permeabilize the outer and inner membranes. Moreover, the alphas2-casein peptide f(183-207) generated pores in the outer membrane of Gram-negative bacteria and in the cell wall of Gram-positive bacteria. In the Gram-negative bacteria, f(183-207) originated cytoplasm condensation, and in the Gram-positive bacteria the cytoplasmic content leaked into the extracellular medium. Furthermore, the experiments of inner and outer membrane permeabilization performed with LFcin-B showed that this peptide also has the ability to permeabilize both the inner and outer membranes.

Multifunctionality of lunasin and peptides released during its simulated gastrointestinal digestion.

Oxidative stress, inflammation, and hypertension are recognized risk factors for non-communicable diseases. Because of the preventable character of these factors, the searching of dietary compounds with counteracting effects against them would provide a new framework for the development of novel multifunctional foods or nutraceuticals. Lunasin is a naturally occurring soybean peptide with chemopreventive and anti-inflammatory properties. Upon oral intake, lunasin is susceptible to the action of digestive enzymes during its transit through gastrointestinal tract. In spite of its cleavage into smaller peptides, these fragments have been suggested to contribute on the health beneficial effects attributed to lunasin. To confirm this hypothesis, the multifunctionality of lunasin derived-fragments was investigated. In vitro, peptides corresponding to the N-terminal and central regions of lunasin were demonstrated to inhibit angiotensin converting enzyme and to scavenge peroxyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radicals. Moreover, lunasin and fragments released during its gastrointestinal digestion exerted potent protective effects on cell viability and oxidative status in macrophages RAW264.7 challenged with chemicals tert-butylhydroperoxide and hydrogen peroxide. These peptides were also able to reduce the nitric oxide production in pro-inflammatory lipopolysaccharide-induced macrophages. These results confirm the promising role of lunasin and its derived-fragments as protective agents against oxidative damage and inflammation-associated diseases.

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.

ACE-inhibitory and radical-scavenging activity of peptides derived from beta-lactoglobulin f(19-25). Interactions with ascorbic acid.

In this work, the angiotensin-converting enzyme (ACE)-inhibitory and radical-scavenging activities of the beta-lactoglobulin (beta-Lg)-derived peptides WY f(19-20), WYS f(19-21), WYSL f(19-22), WYSLA f(19-23), WYSLAM f(19-24), and WYSLAMA f(19-25) have been determined. The ACE-inhibitory activity (IC50) varied from 38.3 to 90.4 microM, with the exception of WYS (>500 microM). All beta-Lg-derived peptides also exhibited radical-scavenging activity (oxygen radical absorbance capacity (ORAC) values ranged from 4.45 to 7.67 micromol Trolox equivalents/micromol of peptide). The presence and position of amino acids Trp, Tyr, and Met were proposed to be responsible for the antioxidant activity. The equimolar amino acid mixtures of all the peptides showed ORAC values lower than those of the corresponding peptides, indicating that the peptidic bond or the structural conformation had a positive influence on this activity. Finally, positive antioxidant effects of WYS, WYSL, and WYLA with ascorbic acid were observed, whereas WY and WYSLAM showed negative effects, both cases for different molar ratio mixtures. These results should be taken into account in the development of new food ingredients on the basis of peptides from beta-Lg.

Influence of Protein-Phenolic Complex on the Antioxidant Capacity of Flaxseed (Linum usitatissimum L.) Products.

The impact of the naturally present phenolic compounds and/or proteins on the antioxidant capacity of flaxseed products (phenolic fraction, protein concentrates, and hydrolysates) before and after simulated gastrointestinal digestion was studied. For that, whole and phenolic reduced products were assessed. Four glycosylated phenolic compounds (secoisolariciresinol and ferulic, p-coumaric, and caffeic acids) were identified in flaxseed products. Phenolic fraction exerts the highest antioxidant capacity that increased by alkaline hydrolysis and by simulated gastrointestinal digestion. The action of Alcalase and digestive enzymes resulted in an increase of the antioxidant capacity of whole and phenolic reduced products. Principal component analysis showed that proteinaceous samples act as antioxidant is by H+ transfer, while those samples containing phenolic compounds exert their effects by both electron donation and H+ transfer mechanisms. Protein/peptide-phenolic complexation, confirmed by fluorescence spectra, exerted a positive effect on the antioxidant capacity, mainly in protein concentrates.

Effect of beta-lactoglobulin hydrolysis with thermolysin under denaturing temperatures on the release of bioactive peptides.

In this study, bovine beta-lactoglobulin A (beta-Lg A) was hydrolysed with thermolysin under non-denaturing and heat-denaturing conditions. The peptides released during hydrolysis were identified by HPLC-MS/MS. A total of 25 peptides were identified in the hydrolysate obtained at 37 degrees C for 5 min. Some of these peptides survived to further proteolysis even at higher incubation temperatures. Furthermore, novel cleavage sites localised in the most buried zones of beta-Lg and available for thermolysin were recognised when the incubation temperature increased in the range between 60 and 80 degrees C. Three new peptides, LDA, LKPTPEGD, and LQKW, appeared after 30 min hydrolysis at these incubation temperatures, but they were not identified in the 30-min hydrolysates obtained at 37 and 50 degrees C. Of special interest was the peptide LQKW, corresponding to the fragment f(58-61) that had been previously described as a potent angiotensin-converting enzyme-inhibitor (IC50 value of 34.7 microM).

Identification of antibacterial peptides from bovine kappa-casein.

The objective of the present study was to identify antimicrobial peptides present in several digests of commercial caseins with gastric enzymes. The most active hydrolysate against Escherichia coli ATCC 25922 and Listeria innocua CECT 910T corresponded to a pepsin digest of bovine kappa-casein. The protein digest was first separated by semipreparative high-performance liquid chromatography (HPLC), and the most active fractions were again subjected to a second chromatographic step. Finally, identification of the active peptides was carried out by online and offline HPLC-electrospray ionization-tandem mass spectrometry. By means of this technique, 21 peptides were identified in the active HPLC fractions. Although most were derived from bovine kappa-casein, some of the identified fragments corresponded to beta-casein and alpha(s)-casein fragments, a result of the presence of small amounts of these proteins in the preparation of kappa-casein. Some of the peptides identified were chemically synthesized and showed antibacterial effects against several gram-positive and gram-negative bacteria. Among the synthesized peptides, kappa-casein f(18-24), f(30-32), and f(139-146) were most effective against all bacteria tested. The antibacterial effect of these peptides is discussed in relation to their amino acid sequences.

Preparation of antioxidant enzymatic hydrolysates from alpha-lactalbumin and beta-lactoglobulin. Identification of active peptides by HPLC-MS/MS.

We have investigated the antioxidant activity of hydrolysates from whey proteins bovine alpha-lactalbumin (alpha-La) and beta-lactoglobulin A (beta-Lg A) by commercial proteases (pepsin, trypsin, chymotrypsin, thermolysin, and Corolase PP). Corolase PP was the most appropriate enzyme to obtain antioxidant hydrolysates from alpha-La and beta-Lg A (ORAC-FL values of 2.315 and 2.151 micromol of Trolox equivalent/mg of protein, respectively). A total of 42 peptide fragments were identified by HPLC-MS/MS in the beta-Lg A hydrolysate by Corolase PP. One of the sequences (Trp-Tyr-Ser-Leu-Ala-Met-Ala-Ala-Ser-Asp-Ile) possessed radical scavenging (ORAC-FL value of 2.621 micromol of Trolox equivalent/micromol of peptide) higher than that of butylated hydroxyanisole (BHA). Our results suggest that whey protein hydrolysates could be suitable as natural ingredients in enhancing antioxidant properties of functional foods and in preventing oxidation reaction in food processing.

The protective role of the Bowman-Birk protease inhibitor in soybean lunasin digestion: the effect of released peptides on colon cancer growth.

Lunasin is a naturally-occurring peptide demonstrating chemopreventive, antioxidant and anti-inflammatory properties. To exhibit these activities, orally ingested lunasin needs to survive proteolytic attack of digestive enzymes to reach target tissues in active form/s. Preliminary studies suggested the protective role of protease inhibitors, such as the Bowman-Birk inhibitor and Kunitz-trypsin inhibitor, against lunasin's digestion by both pepsin and pancreatin. This work describes in depth the behaviour of lunasin under conditions simulating the transit through the gastrointestinal tract in the absence or presence of soybean Bowman-Birk isoinhibitor 1 (IBB1) in both active and inactive states. By liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS), the remaining lunasin at the end of gastric and gastro-duodenal phases was quantified. Protection against the action of pepsin was independent of the amount of IBB1 present in the analyzed samples, whereas an IBB1 dose-dependent protective effect against trypsin and chymotrypsin was observed. Peptides released from lunasin and inactive IBB1 were identified by MS/MS. The remaining lunasin and IBB1 as well as their derived peptides could be responsible for the anti-proliferative activity against colon cancer cells observed for the digests obtained at the end of simulated gastrointestinal digestion.