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The aim of the present work was to evaluate and compare in vitro the antioxidant activity of raw, cooked and cooked-digested pork, beef and chicken burgers. The cooking process influenced the antioxidant capacity of the meat by decreasing the values of ABTS, FRAP and the content of free thiols. Conversely, a positive effect was observed after in vitro gastrointestinal digestion which increased the biological activity of the meat, characterised by greater antioxidant activity. The type of meat influenced the chemical composition and biological capacity of the burgers. In fact, both before and after the cooking process, beef burgers showed higher thiol content and, consequently, a higher oxidative stability of proteins than chicken and pork burgers. In vitro gastrointestinal digestion also improved the nutraceutical quality of beef burgers, which showed higher ABTS values and thiol content than pork burgers, which showed higher FRAP values. This work aims to support the potential of meat constituents as a natural antioxidant component that is essential to counteract the oxidative stress responsible for imbalances in the human organism and several cardiovascular diseases.

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Three Streptococcus thermophilus strains, namely RBC6, RBC20, and RBN16, were proven to release bioactive peptides during whey protein concentrate (WPC) fermentation, resulting in WPC hydrolysates with biological activities. However, these bioactive peptides can break down during gastro-intestinal digestion (GID), hindering the health-promoting effect of fermented WPC hydrolysates in vivo. In this work, the effect of simulated GID on three WPC hydrolysates fermented with S. thermophilus strains, as well as on unfermented WPC was studied in terms of protein hydrolysis, biological activities, and peptidomics profiles, respectively. In general, WPC fermentation enhanced protein hydrolysis compared to unfermented WPC. After in vitro GID, WPC fermented with S. thermophilus RBC20 showed the highest antioxidant activity, whereas WPC fermented with strain RBC06 displayed the highest angiotensin-converting enzyme (ACE)- and dipeptidyl peptidase IV (DPP-IV)-inhibitory activities. Peptidomics analysis revealed that all digested WPC samples were highly similar to each other in peptide profiles, and 85% of the 46 identified bioactive peptides were shared among fermented and unfermented samples. However, semi-quantitative analysis linked the observed differences in biological activities among the samples to differences in the amount of bioactive peptides. The anti-hypertensive peptides VPP and IPP, as well as the DPP-IV-inhibitory peptide APFPE, were quantified. In conclusion, WPC fermentation with S. thermophilus positively impacted protein hydrolysis and bioactive peptide release during GID.

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Opuntia cactus fruit (prickly pear flesh and agricultural residues such as peels and stalks) is an important source of bioactive compounds, including betalains and phenolic compounds. In this work, two double emulsion W1/O/W2 formulations (A and B) were designed to encapsulate green extracts rich in betalains and phenolic compounds obtained from Opuntia stricta var. dillenii (OPD) fruits with the aim of improving their stability and protecting them during the in vitro gastrointestinal digestion process. In addition, the characterization of the double emulsions was studied by microscopy and the evaluation of their physical and physico-chemical parameters. Formulation A, based on Tween 20, showed smaller droplets (1.75 µm) and a higher physical stability than Formulation B, which was achieved with sodium caseinate (29.03 µm). Regarding the encapsulation efficiency of the individual bioactives, betalains showed the highest values (73.7 ± 6.7 to 96.9 ± 3.3%), followed by flavonoids (68.2 ± 5.9 to 95.9 ± 7.7%) and piscidic acid (71 ± 1.3 to 70.2 ± 5.7%) depending on the formulation and the bioactive compound. In vitro digestive stability and bioaccessibility of the individual bioactives increased when extracts were encapsulated for both formulations (67.1 to 253.1%) in comparison with the non-encapsulated ones (30.1 to 64.3%), except for neobetanin. Both formulations could be considered as appropriate microcarrier systems for green OPD extracts, especially formulation A. Further studies need to be conducted about the incorporation of these formulations to develop healthier foods.

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In this study, a novel peptide, AEYLCEAC with high angiotensin-I-converting enzyme inhibitory (ACEI) activity was screened from oyster (Crassostrea gigas) hydrolysates, which was obtained from simulated gastro-intestinal digestion. Candidate peptides were confirmed to have a higher binding to angiotensin-I-converting enzyme (ACE) than the positive drug phosphoinic tripeptide calculated by Discovery Studio, and AEYLCEAC showed the highest ACE inhibition rate in vitro with a IC 50 of 4.287 mM. Lineweaver-Burk plots confirmed that the peptidic inhibitory type of ACE is competitive. The molecular docking showed that ACEI activity of the AEYLCEAC was mainly due to the hydrogen bonding interactions with the active pockets (S1 and S2) of ACE. In vivo, AEYLCEAC effectively reduced diastolic blood pressure (DBP) and Systolic blood pressure (SBP) in hypertensive rats. These results indicate that AEYLCEAC might act as a helpful ingredient in functional foods or pharmaceuticals for the prevention and treatment of hypertension.

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Arthrospira platensis (Spirulina) has been credited with multiple beneficial effects, many of which are attributed to bioactive peptides produced during the gastrointestinal digestion of this micro-alga. Many Spirulina-based nutraceuticals have been produced, and numerous functional foods enriched with Spirulina are available on the market. These are subjected to checks aimed at verifying the amount of algae actually present, but few studies relating to the bioavailability of the bioactive compounds in these products have been carried out. However, such investigations could be very important to elucidate the possible critical effects exerted by food matrices on protein digestion and bioactive peptide production. Here, in order to assess the suitability of Spirulina-enriched foods as a source of potentially bioactive peptides, a simulated digestion protocol was used in combination with mass spectrometry quantitative analysis to analyze functionalized pasta and sorbets. In the case of the pasta enriched with Spirulina, the production of peptides was quite similar to that of the Spirulina powder. On the other hand, the type of fruit present in the food matrix influenced the digestion of Spirulina inside the sorbets. In particular, the high concentration of protease inhibitors in kiwifruit drastically reduced the production of peptides from Spirulina in kiwi sorbet.

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Food proteins and peptides are able to exert a variety of well-known bioactivities, some of which are related to well-being and disease prevention in humans and animals. Currently, an active trend in research focuses on chronic inflammation and oxidative stress, delineating their major pathogenetic role in age-related diseases and in some forms of cancer. The present study aims to investigate the potential effects of pseudocereal proteins and their derived peptides on chronic inflammation and oxidative stress. After purification and attribution to protein classes according to classic Osborne's classification, the immune-modulating, antioxidant, and trypsin inhibitor activities of proteins from quinoa (Chenopodium quinoa Willd.), amaranth (Amaranthus retroflexus L.), and buckwheat (Fagopyrum esculentum Moench) seeds have been assessed in vitro. The peptides generated by simulated gastro-intestinal digestion of each fraction have been also investigated for the selected bioactivities. None of the proteins or peptides elicited inflammation in Caco-2 cells; furthermore, all protein fractions showed different degrees of protection of cells from IL-1ß-induced inflammation. Immune-modulating and antioxidant activities were, in general, higher for the albumin fraction. Overall, seed proteins can express these bioactivities mainly after hydrolysis. On the contrary, higher trypsin inhibitor activity was expressed by globulins in their intact form. These findings lay the foundations for the exploitation of these pseudocereal seeds as source of anti-inflammatory molecules.

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Okara oil is a by-product remaining from defatting okara, the solid residue generated after extracting the aqueous fraction of grounded soybeans in the elaboration of soy beverages. The goal of this work was to encapsulate the probiotic Lactobacillus plantarum CIDCA 83114 into W/O emulsions composed of a block-copolymer constituted of pluronic® and acrylic acid (PPP12) and okara oil, prepared in microfluidic devices. For comparative purposes, alginate was also included as a second dispersed phase. Lactobacillus plantarum CIDCA 83114 was suspended in PPP12 or alginate giving rise to dispersed phases with different compositions, named I, II, III and IV. Controls were prepared by suspending microorganisms in water as dispersed phase. 6-carboxyfluorescein was added as bacterial marker in all the emulsions. The presence of green dyed bacteria in the dispersed phases, inside the droplets of the emulsions and the absence of fluorescence outside them, confirmed the complete encapsulation of bacteria in the dispersed phases. After being prepared, emulsions were freeze-dried. The exposure to gastric conditions did not lead to significant differences among the emulsions containing polymers. However, in all cases bacterial counts were significantly lower than those of the control. After exposing emulsions to the simulated intestinal environment, bacterial counts in assays I, II and III (emulsions composed of only one dispersed phase or of two dispersed phases with bacteria resuspended in the PPP12 one) were significantly greater than those of the control (p < 0.05) and no detectable microorganisms were observed for assay IV (emulsions composed of two dispersed phases with bacteria resuspended in the alginate one). In particular, bacterial cultivability in emulsions corresponding to assay I (only PPP12 as dispersed phase) exposed to the intestinal environment was 8.22 ± 0.02 log CFU/mL (2 log CFU higher than the values obtained after gastric digestion). These results support the role of PPP12 as an adequate co-polymer to protect probiotics from the gastric environment, enabling their release in the gut, with great potential for food or nutraceutical applications.

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Lipid oxidation products generated during meat digestion may contribute to the apparent epidemiological link between red meat intake and the risk of cardiovascular diseases and colorectal cancer. The aim of this work was to assess the lipid oxidation inhibitory activity of black, green, and pink pepper during cooking and in vitro digestion of meat. Peppers were characterized for their phenolic profiles by LC-ESI-MS and the antioxidant properties. Pink pepper showed the highest phenolic content and antioxidant activities. Then, the peppers were added to meat either before or after cooking, and the meat was subjected to in vitro digestion. Pink pepper added before cooking was the most effective, with an inhibition of 80% and 72% in lipid hydroperoxides and TBA-RS formation after digestion, respectively. These findings suggest that peppers, particularly pink pepper, can be used to minimize lipid oxidation in the gastro-intestinal tract and for the design of healthy dietary patterns.

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The establishment of the INFOGEST in vitro static digestion method, a standardized international consensus, was an important milestone in the field of food digestion. We evaluated the contribution of iron and zinc in reagents used in the INFOGEST method in relation to sample iron and zinc and the potential interference of reagent-derived iron and zinc with bioaccessibility measurements. In most cases, reagent-derived iron and zinc contributed more than 50% of the total iron or zinc in the digesta containing selected cereals and legumes. Moreover, the chemical behaviour of reagent-derived iron and zinc was matrix dependent such that the application of a blanket blank correction was not appropriate. We therefore propose an improved approach involving isotopic labelling of reagent iron and zinc in order to discriminate between reagent-derived and sample-derived iron and zinc in each matrix. This stable isotope approach could improve the accuracy and reliability of iron and zinc bioaccessibility studies.

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Would an engineered nanomaterial (ENM) still have the same identity once it reaches a secondary target tissue after a journey through several physiological compartments? Probably not. Does it matter? ENM pre-treatments may enhance the physiological relevance of in vitro testing via controlled transformation of the ENM identity. The implications of material transformation upon reactivity, cytotoxicity, inflammatory, and genotoxic potential of Ag and SiO2 ENM on advanced gastro-intestinal tract cell cultures and 3D liver spheroids are demonstrated. Pre-treatments are recommended for certain ENM only.

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The present study was conducted to evaluate effect of ethephon and acetylene treatments on phenolics, flavonoids and antioxidant activity of banana flesh and their bioaccessibility. Total phenolics, flavonoids and antioxidant activity (DPPH, ABTS, FRAP) were measured at different phases of simulated gastrointestinal digestion of banana treated with 1000 ppm ethephon and 1000 ppm acetylene against natural ripening. The results revealed that inducing ripening lowers the content of phenolics, flavonoids and antioxidant activity considerably in the fresh fruit. Bioavailability of phenolics, flavonoids and FRAP activity were increased significantly (p < 0.05) after gastric digestion regardless of the treatment. The release of polyphenols and flavonoids during gastric digestion in treated banana was more significant than in naturally ripened banana. Recovery of polyphenols after dialysis was significantly high in naturally ripened banana. Dialyzable flavonoids, DPPH and ABTS activities of dialyzed fractions were not significantly affected by ethephon or acetylene treatments.

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Eggplant is an important component of the Mediterranean Diet, which becomes edible after cooking. This study determined the fate of dark purple eggplant phenolic compounds after baking, boiling, frying, grilling and digestion. Thirty-seven phenolic compounds were identified and quantified in raw eggplant. Frying determined a 74% increase in total hydroxycinnamic acids whereas a decrease was observed after boiling (27%), grilling (51%), and baking (60%). After digestion, 45%, 33% and 22% of total phenolic compounds resulted bioaccessible in baked, grilled and fried dark purple eggplant. Fried eggplant displayed the highest amount of phenolic compounds (751.46 mg/100 g) after digestion. The cooking methods differently affected the release of individual phenolic compounds. Baking and grilling resulted in higher amount of bioaccessible caffeoylquinic acids whereas frying in di-caffeoylquinic acids and hydroxycinnamic acid-amides. A careful design of the cooking method may be pivotal to modulate the release of specific phenolic compounds.

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Chronic neuroinflammation associated with neurodegenerative disorders has been reported to be prevented by dietary components. Particularly, dietary (poly)phenols have been identified as having anti-inflammatory and neuroprotective actions, and their ingestion is considered a major preventive factor for such disorders. To assess the relation between (poly)phenol classes and their bioactivity, we used five different raspberry genotypes, which were markedly different in their (poly)phenol profiles within a similar matrix. In addition, gastro-intestinal bio-accessible fractions were produced, which simulate the (poly)phenol metabolites that may be absorbed after digestion, and evaluated for anti-inflammatory potential using LPS-stimulated microglia. Interestingly, the fraction from genotype 2J19 enriched in ellagitannins, their degradation products and ellagic acid, attenuated pro-inflammatory markers and mediators CD40, NO, TNF-α, and intracellular superoxide via NF-κB, MAPK and NFAT pathways. Importantly, it also increased the release of the anti-inflammatory cytokine IL-10. These effects contrasted with fractions richer in anthocyanins, suggesting that ellagitannins and its derivatives are major anti-inflammatory (poly)phenols and promising compounds to alleviate neuroinflammation.

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Drug release from a lipid-based drug delivery system (LbDDS) is typically studied in vitro using a one-step intestinal digestion model. However, lately the importance of incorporating gastric digestion has been stressed. The aim of the present study was to compare a two-step gastro-intestinal (GI) in vitro digestion model to the commonly used one-step intestinal digestion model. The models were evaluated by studying release of the model drug A1260 from two LbDDSs (F-I and F-II), for which in vivo pharmacokinetic data from oral administration to beagle dogs were available. The amount of A1260 recovered in the aqueous phases during and after the GI digestion of F-I and F-II was related to the Cmax and AUC0-48h of the plasma concentration-time profiles of each formulation and produced a rank order in vitro-in vivo (IVIV) relation. In comparison, a similar IVIV rank ordering was obtained when relating the amount of A1260 recovered in the aqueous phase prior (t = 0 min), and following 15 min of intestinal digestion, to the plasma concentration-time profiles. However, after 60 min of intestinal digestion, the LbDDSs performed equally in the one-step in vitro digestion model, contrary to what was observed in the two-step digestion model, and in vivo. As the GI digestion model produced a clearer distinction in terms of LbDDS rank ordering of the two LbDDSs, compared to the intestinal digestion model, it was found to be a promising in vitro model to study and estimate the LbDDS behavior in vivo.

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The effects of microwave (90 W and 180 W), hot air (60 and 70 °C) and vacuum (60 and 70 °C with 200 and 300 mbar) drying methods on drying characteristics, total phenolic content, antioxidant capacity, color and in vitro gastrointestinal digestion of medlar pestil were investigated. Medlar showed a good potential for pestil production while being the most applicable in microwave treatments. For drying kinetics, five thin-layer drying models were applied and the Page and Modified Page were the best fitted models. L*, b*, chroma and hue angle decreased while a* generally increased in dried pestils. Dried samples showed a general decrement in phenolics and antioxidant capacity. According to in vitro gastrointestinal digestion, intestinal phase of all the samples resulted with an increment in phenolics, FRAP and DPPH compared to undigested extracts. In conclusion, different drying methods may affect the release of phenolics and antioxidant capacity, while leading to increased bioaccessibility during intestinal digestion.

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A two-stage simulated gastro-intestinal (GI) digestion model (2 h pepsin treatment and subsequent 2 h pancreatin treatment at 37 °C) was used to explore the antioxidant activity of the digested products of Cyprinus carpio haematopterus scale gelatin with different molecular weights (MW). From the gastric phase to the intestinal phase, the hydrolysis degree of the products increased from 2.6 ± 0.4% to 16.9 ± 0.7%. The fraction of 0-1 kDa (JCP3) exhibited the best antioxidant activities in hydroxyl radical scavenging, reducing power, and metal chelating activity. The fraction of 1-3 kDa (JCP2) exhibited the best 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. However, the fractions of 0-1 kDa (JCP3) and 1-3 kDa (JCP2) showed similar inhibitory activity of lipid peroxidation. The results indicated that Cyprinus carpio haematopterus scale gelatin can be digested in the gastrointestinal tract. Furthermore, the digested products had antioxidant activity.

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In the present study, the antioxidant hydrolysates obtained from watermelon seed protein (WSP) after divergent ultrasound and ultrafiltration treatment were studied. The results showed that the slit divergent ultrasound (SDU, 20/28 KHz) pretreatment had considerable influence on the structure and enzymatic efficiency of WSP. Besides, compared with hydrolysates without ultrasonic and ultrafiltration treatment, watermelon protein hydrolysates with molecular weight <1 kDa (WSPHs-I) showed the highest antioxidant activities and could protect RAW 264.7 cells from H2O2-induced oxidative stress damage via activating the Nrf2/HO-1 pathway. Interestingly, WSPHs-I had good stability against oxidation at temperature under 100 °C or in the acidic or neutral condition and still exhibited strong antioxidant activity after simulated gastrointestinal digestion. Taken together, SDU pretreatment could significantly increase the antioxidant activities and stability of WSPHs by improving the structure and facilitating enzymolysis of the WSP.

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During the past two decades, a range of in vitro models simulating the digestion processes occurring in the stomach and small intestine have been developed to characterize lipid based drug delivery systems (LbDDSs). This review describes the presently existing range of in vitro digestion models and their use in the field of oral drug delivery. The models are evaluated in terms of their suitability to assess LbDDSs, and their ability to produce in vitro - in vivo correlations (IVIVCs). While the pH-stat lipolysis model is by far the most commonly utilized in vitro digestion model in relation to characterizing LbDDSs, a series of recent studies have shown a lack of IVIVCs limiting its future use. Presently, no single in vitro digestion model exists which is able to predict the in vivo performance of various LbDDSs. However, recent research has shown the potential of combined digestion-permeation models as well as species specific digestion models.

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The consumption of supplements based on dairy or plant proteins may be associated with bioactive potential, including angiotensin-1-converting enzyme inhibitory (ACE-1i) activity, which is linked with blood pressure reduction in vivo. To gain insight into this proposed mechanism, the ACE-1i potential of protein-based supplements, including a selection of dairy (n = 10) and plant (n = 5) proteins were in vitro digested. The total digest was filtered and permeate and retentate were obtained. ACE-1i activity was measured as the ability of proteins (pre-digestion, 'gastric', permeate, and retentate) to decrease the hydrolysis of furanacroloyl-Phe-Glu-Glu (FAPGG) substrate for the ACE-1 enzyme. Permeate and retentate of dairy proteins exerted a significantly higher ACE-1i activity (mean of 10 proteins: 27.05 ± 0.2% and 20.7 ± 0.2%, respectively) compared with pre-digestion dairy proteins (16.7 ± 0.3%). Plant protein exhibited high ACE-1i in 'gastric' and retentate fractions (mean of five proteins: 54.9 ± 0.6% and 35.7 ± 0.6%, respectively). The comparison of the in vitro ACE-1i activity of dairy and plant proteins could provide valuable knowledge regarding their specific bioactivities, which could inform their use in the formulation of specific functional supplements that would require testing for blood pressure control in human randomly-controlled studies.

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Proteins and peptides able to resist gastrointestinal digestion and reach the intestinal mucosa have the potential to influence human health. Chickpea (Cicer arietinum L.) seed proteins are able to resist cooking (86.9% total protein) and/or in vitro simulated human digestion (15.9% total protein resists soaking, cooking and digestion with pepsin and pancreatin). To identify and characterize proteins resisting digestion we made use of different MS methodologies. The efficiency of several proteases (trypsin, AspN, chymotrypsin and LysC) was tested, and two technologies were employed (MALDI-MS/MS and LC-nESI-MS/MS). Digestion with trypsin and AspN were most successful for the identification of seed proteins. When analyzed by MALDI- MS/MS, trypsin allowed the identification of at least one protein in 60% of the polypeptide bands, while AspN allows the identification in 48%. The use of LC-nESI-MS/MS, allowed the identification of much more proteins/polypeptides from digested seeds (232 vs 17 using trypsin). The majority of the proteins found to be able to resist simulated digestion were members of the 7S vicilin and 11S legumin seed storage protein classes, which are reported to contain bio-active functions. In addition, we have found proteins that had not yet been described as potentially able to cause an impact on human health. SIGNIFICANCE: This is the first proteomic study to analyze the effect of processing and simulated human gastrointestinal digestion on the proteome of chickpea seed. Chickpea is reported to have anti-nutritional effects as well as nutraceutical properties, so the identification and characterization of the proteins able to resist digestion is crucial to understand the targets underlying such properties.

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