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Fish protein hydrolysates (FPH) obtained by enzymatic hydrolysis allows for smart valorization of fish side streams. However, further treatments are normally needed to enhance bioactive and functional properties of the obtained FPH. At present, the commonly used methods to improve functional properties of FPH include chemical and enzymatic modification. Chemical treatments often cause environmental problems, while the enzymatic modification method requires the use of quite expensive enzymes. In recent years, emerging technologies such as ultrasound treatment (US-treatment) have shown great potential in protein modification with high efficiency and safety, low energy consumption, and low nutritional destructiveness. In this study, high-power ultrasound treatments were applied to fish protein hydrolysates (FPH) extracted from Atlantic mackerel (Scomber scombrus) side streams to improve their quality parameters. The effect of three different treatments of 300 W, 450 W and 600 W at the operating frequency of 20 kHz for 10 min on the physicochemical, structural, and functional characteristics of FPH, were examined. The results have shown that with an increase in ultrasound power, the protein solubility of FPH increased linearly, and the changes were significant for all US-treated samples compared to control (untreated) samples. US-treatment significantly increased the degree of hydrolysis of FPH samples treated with 450 W and 600 W compared to control samples. The carbonyl content of FPH increased (significantly for 450 W and 600 W), while thiol groups decreased (significantly for 300 W and 450 W). This indicated that some US-treatments induced oxidation of FPH, however the values of the protein oxidation were low. Amino acid composition of FPH revealed that US-treatment increased the proportion of essential amino acids in the sample treated with 300 W and 450 W, but the increase was not significant. After the US-treatment, all FPH samples became lighter and less yellowish and reddish, which suggest potentially higher attractiveness to consumers. In addition, the in vitro antioxidant activity was assessed using the DPPH, FRAP, and ABTS assays and the cell-free dipeptidyl peptidase IV (DPP-IV) inhibitory activity was also measured. Moreover, these biological activities were measured at cellular level utilizing human intestinal Caco-2 cells. Specifically, the FPH capacity to lower H2O2-induced reactive oxygen species (ROS) and lipid peroxidation levels was used to measure its antioxidant activity. The findings suggest that Scomber scombrus hydrolysates could find use as ingredients for promoting health.

The production of olive oil has important economic repercussions in Mediterranean countries but also a considerable impact on the environment. This production generates enormous quantities of waste and by-products, which can be exploited as new raw materials to obtain innovative ingredients and therefore make the olive production more sustainable. In a previous study, we decided to foster olive seeds by generating two protein hydrolysates using food-grade enzymes, alcalase (AH) and papain (PH). These hydrolysates have shown, both in vitro and at the cellular level, antioxidant and antidiabetic activities, being able to inhibit the activity of the DPP-IV enzyme and modulate the secretion of GLP-1. Given the multifunctional behavior of peptides, both hydrolysates displayed dual hypocholesterolemic activity, inhibiting the activity of HMGCoAR and impairing the PPI of PCSK9/LDLR, with an IC50 equal to 0.61 mg/mL and 0.31 mg/mL for AH and PH, respectively. Furthermore, both samples restored LDLR protein levels on the membrane of human hepatic HepG2 cells, increasing the uptake of LDL from the extracellular environment. Since intestinal bioavailability is a key component of bioactive peptides, the second objective of this work is to evaluate the capacity of AH and PH peptides to be transported by differentiated human intestinal Caco-2 cells. The peptides transported by intestinal cells have been analyzed using mass spectrometry analysis, identifying a mixture of stable peptides that may represent new ingredients with multifunctional qualities for the development of nutraceuticals and functional foods to delay the onset of metabolic syndrome, promoting the principles of environmental sustainability.

Due to the continuous growth of the world population, there is an urgent need to find sustainable sources of high-quality protein. Fish side streams rich in essential nutrients and accounting for 60–70% of the whole fish, represent a sustainable source for recovery of valuable protein compounds. The present study aimed at extensive characterization of physicochemical, antioxidant and techno-functional properties of fish protein hydrolysate (FPH) obtained from farmed rainbow trout (Oncorhynchus mykiss). The FPH was produced from a minced rainbow trout raw material by enzymatic hydrolysis performed at 50 °C with addition of 0.05% w/w papain and 0.05% w/w bromelain. After inactivation of the proteases at 90 °C for 10 min, the content of the bioreactor was centrifuged, and the soluble protein fraction (FPH) was collected and freeze-dried. The total protein content of the FPH with 17.24% degree of hydrolysis was high (88.9%) and mainly represented by water-soluble proteins, while the lipid content was below 1%. In addition to the high protein content, trout hydrolysate had low protein oxidation values characterized by a relatively low total carbonyl content together with high amount of thiol groups (3.64 ± 0.31 and 20.7 ± 0.6 nmol/mg protein, respectively). No glass transition was detected in the differential scanning calorimetry (DSC) heat flow curves, suggesting lack of unfreezable solution formation in the FPH at freezing temperatures. The viscosity of FPH showed typical Newtonian behaviour. A peptidomic investigation (using HPLC-MS/MS technique) displayed chemical composition of the trout hydrolysate and identified peptide sequences which are present in the hydrolysate mixture, as well as proteins to which each peptide belongs to. In conclusion, it was suggested to use the obtained trout hydrolysate as a functional ingredient in the food and nutraceutical industry.

The objective of the study was the evaluation of the potential pleiotropic effect of a commercial casein hydrolysate (CH). After an analysis of the composition, the BIOPEP-UWM database suggested that these peptides contained numerous sequences with potential inhibitory activities on angiotensin converting enzyme (ACE) and dipeptidyl-peptidase IV (DPP-IV). The anti-diabetic and anti-hypertensive effects of these peptides were thus assessed using either cell-free or cell-based assays. In the cell-free system, CH displayed inhibitory properties against DPP-IV (IC50 value equal to 0.38 ± 0.01 mg/mL) and ACE (IC50 value equal to 0.39 ± 0.01 mg/mL). Further, CH reduced the DPP-IV and ACE activities expressed by human intestinal Caco-2 cells by 61.10 ± 1.70% and 76.90 ± 4.47%, respectively, versus untreated cells, after 6 h of treatment at the concentration of 5 mg/mL. This first demonstration of the multifunctional behavior of this material suggests that it may become an anti-diabetic and/or anti-hypertensive ingredient to be included in the formulation of different functional food or nutraceutics.

MOMAST® is a patented natural phenolic complex, rich in tyrosol (9.0 g/kg, Tyr), hydroxityrosol (43,5 g/kg, OH-Tyr), and verbascoside (5.0 g/Kg), which is obtained from the OVW by-product of the Coratina cultivar with potent direct antioxidant activity (measured by DPPH and FRAP assays, respectively). Indeed, MOMAST® represents an innovative sustainable bioactive ingredient which has been obtained with ethical and empowering behavior by applying the principles of a circular economy. In the framework of research aimed at fostering its health-promoting activity, in this study it was clearly demonstrated that MOMAST® treatment reduced the oxidative stress and levels of total cholesterol (TC) and low-density lipoprotein (LDL) cholesterol, and increased the HDL levels, without changes in the triglyceride (TG) levels in Western diet (WD)-fed mice. The modulation of the plasmatic lipid profile is similar to red yeast rice (RYR) containing Monacolin K (3%). In addition, at the molecular level in liver homogenates, similarly to RYR, MOMAST® exerts cholesterol-lowering activity through the activation of LDL receptor, whereas, unlike RYR, MOMAST® reduces proprotein convertase subtilisin/kexin type 9 (PCSK9) protein levels via hepatic nuclear factor 1 (HNF1)-α activation. Hence, this study provides the proof of concept regarding the hypocholesterolemic activity of MOMAST, which could be successfully exploited as an active ingredient for the development of innovative and sustainable dietary supplements and functional foods.

Hempseed (Cannabis sativa) is one of the most promising sources of plant proteins. It contains approximately 24% (w/w) protein, and edestin accounts for approximately 60–80% (w/w) of its total proteins. In a framework of research aimed at fostering the proteins recovered from the press cake by-products generated after the extraction of hempseed oil, two hempseed protein hydrolysates (HH1 and HH2) were produced at an industrial level using a mixture of different enzymes from Aspergillus niger, Aspergillus oryzae, and Bacillus licheniformis for different times (5 h and 18 h). Using a combination of different direct antioxidant tests (DPPH, TEAC, FRAP, and ORAC assays, respectively), it has been demonstrated that HHs exert potent, direct antioxidant activity. A crucial feature of bioactive peptides is their intestinal bioavailability; for this reason, in order to solve this peculiar issue, the ability of HH peptides to be transported by differentiated human intestinal Caco-2 cells has been evaluated. Notably, by using mass spectrometry analysis (HPLC Chip ESI-MS/MS), the stable peptides transported by intestinal cells have been identified, and dedicated experiments confirmed that the trans-epithelial transported HH peptide mixtures retain their antioxidant activity, suggesting that these hempseed hydrolysates may be considered sustainable antioxidant ingredients to be exploited for further application, i.e., nutraceutical and/or food industries.

The present study aimed at characterizing the possible biological activities of the multifunctional low molecular weight fractions (Oncorhynchus mykiss) obtained by enzymatic hydrolysis. The fish protein hydrolysate (FPH) was tested for its antioxidant property along with its angiotensin converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities. In particular, the 2,2-diphenyl-1-picrylhydrazyl (DPPH), the ferric reducing antioxidant power (FRAP), the oxygen radical absorbance capacity (ORAC) assay and the 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays were carried out for the evaluation of the in vitro antioxidant activity. The cell-free ACE and DPP-IV inhibitory activity assays were also estimated, showing a dose-dependent inhibition. These biological properties were additionally quantified at the cellular level using human intestinal Caco-2 cells. Namely, the antioxidant activity was determined by evaluating the capability of the hydrolysate to reduce the H2O2-induced reactive oxygen species (ROS) and lipid peroxidation levels, and the DPP-IV activity assays show a reduction of enzyme activity of up to 27.57 ± 3.7% at 5 mg/mL. The results indicate that Oncorhynchus mykiss-derived peptides may have potential employment as health-promoting ingredients.

For improving the management of the production chain of PGI Mantua pears (which comprises many varieties, including Abate Fetel), applying the cardinal principles of circular economy and sustainability, the fruits with diseases or defects were recovered for producing dried rounds of pears from the Abate Fetel cultivar, a new product with high nutritional value that extends the remaining life. This process led to the production of secondary and residual by-products, which are mainly composed of the highest and lowest part of the fruits, comprising seeds, pulps, peels and petioles. Hence, this study was focused on the valorization of these secondary by-products of Abate Fetel pears through the production of pear extracts using traditional and “green” extraction methods that involve the use of supercritical CO2 fluid extraction. The produced extracts, together with a reference solvent-derived extract, were analyzed by HPLC-ESI-MS, and in parallel, their direct and cellular antioxidant activity were assessed. Evidence has indicated that all the tested extracts reduced the H2O2-induced reactive oxygen species (ROS), lipid peroxidation and nitric oxide (NO) levels, respectively, in human intestinal Caco-2 cells. Hence, this study clearly suggests that extracts obtained from Mantuan PGI pear by-products may be used as valuable sources of bioactive upcycled phytocomplex for the development of dietary supplements and/or functional foods.

The manufacture of vegetal beverages has the drawback of producing large amounts of press cakes that are generally used as feed components. This work had the objective of valorizing the press cakes deriving from almond and coconut drinks production by using ultrasound-assisted extraction (UAE) to obtain protein ingredients for human use. Starting from coconut and almond press cakes, whose initial protein contents were 19.7% and 18.6%, respectively, the UAE treatment allowed liquid fractions to be obtained that were then freeze-dried: the extraction yields were 24.4 g dry extract/100 g press cake in case of coconut and 49.3 g dry extract/100 g press cake in case of almond. The protein contents of these dried materials were 30.10% and 22.88%, respectively. The quality of the extracted protein ingredients was assessed in term of phytic acid content, protein profile, techno-functional features, and antioxidant properties. The sonication had also a favorable effect on digestibility.

Agri-food industry wastes and by-products include highly valuable components that can upgraded, providing low-cost bioactives or used as an alternative protein source. In this context, by-products from olive production and olive oil extraction process, i.e., seeds, can be fostered. In particular, this work was aimed at extracting and characterizing proteins for Olea europaea L. seeds and at producing two protein hydrolysates using alcalase and papain, respectively. Peptidomic analysis were performed, allowing to determine both medium- and short-sized peptides and to identify their potential biological activities. Moreover, an extensive characterization of the antioxidant properties of Olea europaea L. seed hydrolysates was carried out both in vitro by 2,2-diphenyl-1-picrylhydrazyl (DPPH), by ferric reducing antioxidant power (FRAP), and by 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays, respectively, and at cellular level by measuring the ability of these hydrolysates to significant reduce the H2O2-induced reactive oxygen species (ROS) and lipid peroxidation levels in human intestinal Caco-2 cells. The results of the both hydrolysates showed significant antioxidant properties by reducing the free radical scavenging activities up to 65.0 ± 0.1% for the sample hydrolyzed with alcalase and up to 75.7 ± 0.4% for the papain hydrolysates tested at 5 mg/mL, respectively. Moreover, similar values were obtained by the ABTS assays, whereas the FRAP increased up to 13,025.0 ± 241.5% for the alcalase hydrolysates and up to 12,462.5 ± 311.9% for the papain hydrolysates, both tested at 1 mg/mL. According to the in vitro results, both papain and alcalase hydrolysates restore the cellular ROS levels up 130.4 ± 4.24% and 128.5 ± 3.60%, respectively, at 0.1 mg/mL and reduce the lipid peroxidation levels up to 109.2 ± 7.95% and 73.0 ± 7.64%, respectively, at 1.0 mg/mL. In addition, results underlined that the same hydrolysates reduced the activity of dipeptidyl peptidase-IV (DPP-IV) in vitro and at cellular levels up to 42.9 ± 6.5% and 38.7 ± 7.2% at 5.0 mg/mL for alcalase and papain hydrolysates, respectively. Interestingly, they stimulate the release and stability of glucagon-like peptide 1 (GLP-1) hormone through an increase of its levels up to 660.7 ± 21.9 pM and 613.4 ± 39.1 pM for alcalase and papain hydrolysates, respectively. Based on these results, olive seed hydrolysates may represent new ingredients with antioxidant and anti-diabetic properties for the development of nutraceuticals and functional foods for the prevention of metabolic syndrome onset.

Nowadays, notwithstanding their nutritional and technological properties, food bioactive peptides from plant sources garner increasing attention for their ability to impart more than one beneficial effect on human health. Legumes, which stand out thanks to their high protein content, represent valuable sources of bioactive peptides. In this context, this study focused on the characterization of the potential pleotropic activity of two commercially available soybean (SH) and pea (PH) protein hydrolysates, respectively. Since the biological activity of a specific protein hydrolysate is strictly correlated with its chemical composition, the first aim of the study was to identify the compositions of the SH and PH peptides. Peptidomic analysis revealed that most of the identified peptides within both mixtures belong to storage proteins. Interestingly, according to the BIOPEP-UWM database, all the peptides contain more than one active motive with known inhibitory angiotensin converting enzyme (ACE) and dipeptidyl-dipeptidases (DPP)-IV sequences. Indeed, the results indicated that both SH and PH inhibit DPP-IV and ACE activity with a dose-response trend and IC50 values equal to 1.15 ± 0.004 and 1.33 ± 0.004 mg/mL, and 0.33 ± 0.01 and 0.61 ± 0.05 mg/mL, respectively. In addition, both hydrolysates reduced the activity of DPP-IV and ACE enzymes which are expressed on the surface of human intestinal Caco-2 cells. These findings clearly support that notion that SH and PH may represent new ingredients with anti-diabetic and hypotensive effects for the development of innovative multifunctional foods and/or nutraceuticals for the prevention of metabolic syndrome.

Hempseed (Cannabis sativa) protein is an important source of bioactive peptides. H3 (IGFLIIWV), a transepithelial transported intestinal peptide obtained from the hydrolysis of hempseed protein with pepsin, carries out antioxidant and anti-inflammatory activities in HepG2 cells. In this study, the main aim was to assess its hypocholesterolemic effects at a cellular level and the mechanisms behind this health-promoting activity. The results showed that peptide H3 inhibited the 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGCoAR) activity in vitro in a dose-dependent manner with an IC50 value of 59 μM. Furthermore, the activation of the sterol regulatory element binding proteins (SREBP)-2 transcription factor, followed by the increase of low-density lipoprotein (LDL) receptor (LDLR) protein levels, was observed in human hepatic HepG2 cells treated with peptide H3 at 25 µM. Meanwhile, peptide H3 regulated the intracellular HMGCoAR activity through the increase of its phosphorylation by the activation of AMP-activated protein kinase (AMPK)-pathways. Consequently, the augmentation of the LDLR localized on the cellular membranes led to the improved ability of HepG2 cells to uptake extracellular LDL with a positive effect on cholesterol levels. Unlike the complete hempseed hydrolysate (HP), peptide H3 can reduce the proprotein convertase subtilisin/kexin 9 (PCSK9) protein levels and its secretion in the extracellular environment via the decrease of hepatic nuclear factor 1-α (HNF1-α). Considering all these evidences, H3 may represent a new bioactive peptide to be used for the development of dietary supplements and/or peptidomimetics for cardiovascular disease (CVD) prevention.

(1) Background: Proprotein convertase subtilisin/kexin 9 (PCSK9) is responsible for the degradation of the hepatic low-density lipoprotein receptor (LDLR), which regulates the circulating cholesterol level. In this field, we discovered natural peptides derived from lupin that showed PCSK9 inhibitory activity. Among these, the most active peptide, known as P5 (LILPHKSDAD), reduced the protein-protein interaction between PCSK9 and LDLR with an IC50 equals to 1.6 µM and showed a dual hypocholesterolemic activity, since it shows complementary inhibition of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR). (2) Methods: In this study, by a computational approach, the P5 primary structure was optimized to obtain new analogs with improved affinity to PCSK9. Then, biological assays were carried out for fully characterizing the dual cholesterol-lowering activity of the P5 analogs by using both biochemical and cellular techniques. (3) Results: A new peptide, P5-Best (LYLPKHSDRD) displayed improved PCSK9 (IC50 0.7 µM) and HMG-CoAR (IC50 88.9 µM) inhibitory activities. Moreover, in vitro biological assays on cells demonstrated that, not only P5-Best, but all tested peptides maintained the dual PCSK9/HMG-CoAR inhibitory activity and remarkably P5-Best exerted the strongest hypocholesterolemic effect. In fact, in the presence of this peptide, the ability of HepG2 cells to absorb extracellular LDL was improved by up to 254%. (4) Conclusions: the atomistic details of the P5-Best/PCSK9 and P5-Best/HMG-CoAR interactions represent a reliable starting point for the design of new promising molecular entities endowed with hypocholesterolemic activity.

MOMAST® is a patented phenolic complex derived from the olive oil vegetation water, a by-product of the olive oil supply chain, in which hydroxytyrosol (OH-Tyr) and tyrosol (Tyr) and verbascoside are the main compounds. This study was aimed at investigating its hypocholesterolemic effect by assessing the ability to modulate the low-density lipoprotein (LDL) receptor (LDLR)/sterol regulatory element-binding protein 2 (SREBP-2), and proprotein convertase subtilisin/kexin type 9 (PCSK9) pathways. MOMAST® inhibits the in vitro activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCOAR) with a dose-response trend. After the treatment of HepG2 cells, MOMAST® increases the SREBP-2, LDLR, and HMGCoAR protein levels leading, from a functional point of view to an improved ability of hepatic cells to up-take LDL from the extracellular environment with a final cholesterol-lowering effect. Furthermore, MOMAST® decreased the PCSK9 protein levels and its secretion in the extracellular environment, presumably via the reduction of the hepatic nuclear factor 1-α (HNF1-α). The experiments were performed in parallel, using pravastatin as a reference compound. Results demonstrated that MOMAST® may be exploited as a new ingredient for the development of functional foods and/or nutraceuticals for cardiovascular disease prevention.

Food bioactive peptides are increasingly used for formulating food products, nutraceuticals, and functional food, since they are generally considered safe for human consumption and metabolic syndrome prevention. They are also becoming popular as sustainable sources of novel functional biomaterials such as hydrogels, edible nanonutraceuticals, delivery systems, and packing materials. However, such food peptides are mostly unstable, and degrade during food processing, or in a gastrointestinal environment, thus resulting in low bioavailability precluding their practical applications. Here, we decided to functionalize the well-known and characterized self-assembling peptide RADA16 with two synthetic analogues of food bioactive peptides deriving from the hydrolysis of soybean glycinin and lupin β-conglutin (namely IAVPTGVA and LTFPGSAED) for control of and improvement in their gel-forming nanostructures, biomechanics, and biological features. Extensive characterization was performed via Circular Dichroism (CD) spectroscopy, Fourier Transform Infrared spectroscopy (FT-IR), Thioflavin T (ThT) binding assay, rheological measurements, and Atomic Force Microscopy (AFM) analysis. Lastly, since self-assembling peptides (SAPs) can be co-assembled with diluent SAPs (without a bioactive epitope) as an approach to control the density of biological signals and therefore attain enhanced bioactivity, we investigated the effect of the co-assembly of RADA16 and functionalized food bioactive SAPs (dubbed cAP-Soy1 and cAP-Lup1) for the growth of Caco-2 human intestinal cells and contextually we characterized their biological activities as DPP-IV and ACE inhibitors, in order to demonstrate their potential use for the prevention of metabolic syndrome.

Two extra virgin olive oil (EVOO) phenolic extracts (BUO and OMN) modulate DPP-IV activity. The in vitro DPP-IV activity assay was performed at the concentrations of 1, 10, 100, 500, and 1000 μg/mL, showing a dose-dependent inhibition by 6.8 ± 1.9, 17.4 ± 6.1, 37.9 ± 2.4, 57.8 ± 2.9, and 81 ± 1.4% for BUO and by 5.4 ± 1.7, 8.9 ± 0.4, 28.4 ± 7.2, 52 ± 1.3, and 77.5 ± 3.5% for OMN. Moreover, both BUO and OMN reduced the DPP-IV activity expressed by Caco-2 cells by 2.9 ± 0.7, 44.4 ± 0.7, 61.2 ± 1.8, and 85 ± 4.2% and by 3 ± 1.9, 35 ± 9.4, 60 ± 7.2, and 82 ± 2.8%, respectively, at the same doses. The concentration of the most abundant and representative secoiridoids within both extracts was analyzed by nuclear magnetic resonance (1H-NMR). Oleuropein, oleacein, oleocanthal, hydroxytyrosol, and tyrosol, tested alone, reduced the DPP-IV activity, with IC50 of 472.3 ± 21.7, 187 ± 11.4, 354.5 ± 12.7, 741.6 ± 35.7, and 1112 ± 55.6 µM, respectively. Finally, in silico molecular docking simulations permitted the study of the binding mode of these compounds.

Chlorella pyrenoidosa (C. pyrenoidosa) is a microalgae species with a remarkably high protein content that may potentially become a source of hypotensive and hypoglycemic peptides. In this study, C. pyrenoidosa proteins were extracted and hydrolyzed overnight with pepsin and trypsin with final degrees of hydrolysis of 18.7% and 35.5%, respectively. By LC-MS/MS, 47 valid peptides were identified in the peptic hydrolysate (CP) and 66 in the tryptic one (CT). At the concentration of 1.0 mg/mL, CP and CT hydrolysates inhibit in vitro the angiotensin-converting enzyme (ACE) activity by 84.2 ± 0.37% and 78.6 ± 1.7%, respectively, whereas, tested at cellular level at the concentration of 5.0 mg/mL, they reduce the ACE activity by 61.5 ± 7.7% and 69.9 ± 0.8%, respectively. At the concentration of 5.0 mg/mL, they decrease in vitro the DPP-IV activity by 63.7% and 69.6% and in Caco-2 cells by 38.4% and 42.5%, respectively. Short peptides (≤10 amino acids) were selected for investigating the potential interaction with ACE and DPP-IV by using molecular modeling approaches and four peptides were predicted to block both enzymes. Finally, the stability of these peptides was investigated against gastrointestinal digestion.

P5 (LILPKHSDAD) is a hypocholesterolemic peptide from lupin protein with a multi-target activity, since it inhibits both 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) and proprotein convertase subtilisin/kexin type-9 (PCSK9). This work shows that, during epithelial transport experiments, the metabolic transformation mediated by intestinal peptidases produces two main detected peptides, ILPKHSDAD (P5-frag) and LPKHSDAD (P5-met), and that both P5 and P5-met are linearly absorbed by differentiated human intestinal Caco-2 cells. Extensive comparative structural, biochemical, and cellular characterizations of P5-met and the parent peptide P5 demonstrate that both peptides have unique characteristics and share the same mechanisms of action. In fact, they exert an intrinsically multi-target behavior being able to regulate cholesterol metabolism by modulating different pathways. The results of this study also highlight the dynamic nature of bioactive peptides that may be modulated by the biological systems they get in contact with.

This study was aimed at the valorization of the okara byproduct deriving form soy food manufacturing, by using ultrasound at different temperatures for extracting the residual proteins. The physicochemical and conformational changes of the extracted proteins were investigated in order to optimize the procedure. Increasing the temperature from 20 up to 80 °C greatly enhanced the yields and the protein solubility without affecting the viscosity. The protein secondary and tertiary structures were also gradually modified in a significant way. After the ultrasonication at the highest temperature, a significant morphological transition from well-defined single round structures to highly aggregated ones was observed, which was confirmed by measuring the water contact angles and wettability. After the ultrasound process, the improvement of peptides generation and the different amino acid exposition within the protein led to an increase of the antioxidant properties. The integrated strategy applied in this study allows to foster the okara protein obtained after ultrasound extraction as valuable materials for new applications.

In the framework of research aimed at promoting the nutraceutical properties of the phenolic extract (BUO) obtained from an extra virgin olive oil of the Frantoio cultivar cultivated in Tuscany (Italy), with a high total phenols content, this study provides a comprehensive characterization of its antioxidant properties, both in vitro by Trolox equivalent antioxidant capacity, oxygen radical absorbance capacity, ferric reducing antioxidant power, and 2,2-diphenyl-1-picrylhydrazyl assays, and at the cellular level in human hepatic HepG2 and human intestinal Caco-2 cells. Notably, in both cell systems, after H2O2 induced oxidative stress, the BUO extract reduced reactive oxygen species, lipid peroxidation, and NO overproduction via modulation of inducible nitric oxide synthase protein levels. In parallel, the intestinal transport of the different phenolic components of the BUO phytocomplex was assayed on differentiated Caco-2 cells, a well-established model of mature enterocytes. The novelty of our study lies in having investigated the antioxidant effects of a complex pool of phenolic compounds in an extra virgin olive oil (EVOO) extract, using either in vitro assays or liver and intestinal cell models, rather than the effects of single phenols, such as hydroxytyrosol or oleuropein. Finally, the selective trans-epithelial transport of some oleuropein derivatives was observed for the first time in differentiated Caco-2 cells.

This study was aimed at demonstrating the substantial equivalence of two extra virgin olive oil samples extracted from the same batch of Coratina olives with (OMU) or without (OMN) using ultrasound technology, by performing chemical, biochemical, and cellular investigations. The volatile organic compounds compositions and phenolic profiles were very similar, showing that, while increasing the extraction yields, the innovative process does not change these features. The antioxidant and hypocholesterolemic activities of the extra virgin olive oil (EVOO) phenol extracts were also preserved, since OMU and OMN had equivalent abilities to scavenge the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals in vitro and to protect HepG2 cells from oxidative stress induced by H2O2, reducing intracellular reactive oxygen species (ROS) and lipid peroxidation levels. In addition, by inhibiting 3-hydroxy-3-methylglutarylcoenzyme a reductase, both samples modulated the low-density lipoprotein receptor (LDLR) pathway leading to increased LDLR protein levels and activity.

This study was aimed at investigating the hypocholesterolemic effects of extra virgin olive oil (EVOO) phenols and the mechanisms behind the effect. Two phenolic extracts were prepared from EVOO of different cultivars and analyzed using the International Olive Council (IOC) official method for total phenols, a recently validated hydrolytic procedure for total hydroxytyrosol and tyrosol, and 1H-NMR analysis in order to assess their secoiridoid profiles. Both of the extracts inhibited in vitro the 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGCoAR) activity in a dose-dependent manner. After the treatment of human hepatic HepG2 cells (25 µg/mL), they increased the low-density lipoprotein (LDL) receptor protein levels through the activation of the sterol regulatory element binding proteins (SREBP)-2 transcription factor, leading to a better ability of HepG2 cells to uptake extracellular LDL molecules with a final hypocholesterolemic effect. Moreover, both of the extracts regulated the intracellular HMGCoAR activity through the increase of its phosphorylation by the activation of AMP-activated protein kinase (AMPK)-pathways. Unlike pravastatin, they did not produce any unfavorable effect on proprotein convertase subtilisin/kexin 9 (PCSK9) protein level. Finally, the fact that extracts with different secoiridoid profiles induce practically the same biological effects suggests that the hydroxytyrosol and tyrosol derivatives may have similar roles in hypocholesterolemic activity.

Arthrospira platensis (spirulina) is a cyanobacterium, which contains mainly two phycobiliproteins (PBP), i.e., C-phycocyanin (C-PC) and allophycocyanin (APC). In this study, PBP were hydrolyzed using trypsin, and the composition of the hydrolysate was characterized by HPLC-ESI-MS/MS. Furthermore, the potential anti-diabetic activity was assessed by using either biochemical or cellular techniques. Findings suggest that PBP peptides inhibit DPP-IV activity in vitro with a dose-response trend and an IC50 value falling in the range between 0.5 and 1.0 mg/mL. A lower inhibition of the DPP-IV activity expressed by Caco-2 cells was observed, which was explained by a secondary metabolic degradation exerted by the same cells.