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Summary: Ageing affects food choices, food-related life and the nutritional status of older people. The latter may be addressed through the targeted development of specific food products. For example, the development of protein-fortified foods could help older adults increase their protein consumption, thus contributing to the prevention of age-related diseases. This exploratory qualitative study focused on exploring the factors affecting the choice of protein-fortified foods amongst community-dwelling Irish adults ≥ 55 years of age. Sixteen community-dwelling adults (between 55 and 89 years) residing in Ireland were interviewed online in relation to their preferences and perceptions of these foods. Thematic analysis of the transcribed interviews revealed that the willingness to consume protein-fortified foods was low and that the interviewees generally preferred to consume foods naturally high in protein. However, if they had to consume protein-fortified foods, their choice and acceptance would depend on the product characteristics such as its sensory properties, functionality, composition, nutritional value and digestibility. Moreover, most of the interviewees would prefer to consume savoury (e.g. bread, soup) rather than sweet (e.g. pancake, ice cream) products. The study also revealed individual-related factors that affected acceptance of protein-fortified foods including sociodemographic characteristics (living situation), physiological factors (health issues), food-related life (diet and food habits) and food-related awareness (lack of information about protein). This preliminary qualitative study provides valuable information to help guide the development of protein-fortified foods acceptable by the target population.

Two blue whiting protein hydrolysates (BWPHs) with potent in situ and in vitro antidiabetic and antioxidant activities were fractionated. Potent fractions were analysed using LC-MS/MS. Peptides VPVE and IPQD exhibited potent in situ DPP-IV inhibitory activity (Caco-2 cells) with IC50 values of 88.79 ± 5.40 and 91.12 ± 19.73 µM, respectively. Significant decreases in ROS production observed (HepG2 cells) with MPKKE, GPAG and MPAH. MPAH and LPDDL significantly (p

This review presents a comparative analysis of gelation properties in plant-based versus animal-based proteins, emphasizing key factors such as pH, ionic environment, temperature, and anti-nutritional factors. Gelation, a crucial process in food texture formation, is influenced by these factors in varying ways for plant and animal proteins. Animal proteins, like casein, whey, meat, and egg, generally show stable gelation properties, responding predictably to pH, temperature, and ionic changes. In contrast, plant proteins such as soy, pea, wheat, and oilseed show more variable gelation, often requiring specific conditions, like the presence of NaCl or optimal pH, to form effective gels. Animal proteins tend to gel more reliably, while plant proteins require precise environmental adjustments for similar results. Understanding these factors is crucial for selecting and processing proteins to achieve desired textures and functionalities in food products. This review highlights how changing these key factors can optimize gel properties in both plant- and animal-based proteins.

A thermal diluted acid pretreatment using brewers spent grain (BSG) was optimised to improve enzymatic hydrolysis while minimising energy and chemical inputs. First, the use of hydrochloric or sulfuric acid for pretreatment was compared, using hydrochloric acid for the next steps. Three different dilute acid thermal pretreatment combinations were optimised in terms of acid concentration, temperature and time using a response surface methodology. Optimization was based on i) highest remaining protein content in the solid fraction (C1: 0.49% HCl; 87.7°C; 92 min), ii) highest liquid recovery (C2: 0.80% HCl; 121.0°C; 142 min), iii) lowest acid concentration applied to achieve largest protein and lowest remaining solid levels (C3: 0.10% HCl; 104.0°C; 70 min); and iv) a final condition based on the lowest water retention capacity when using HCl (C4: 0.20% HCl; 121.0°C; 20 min). The efficiency of enzymatic hydrolysis was evaluated, in the absence and presence of a large concentration of reducing carbohydrates, by centrifuging the slurry after acid pretreatment, recovering the solid fraction and resuspending it in fresh water. In C2, the enzyme (Depol 40L) was added directly to the entire slurry after pretreatment. For C1, C3, and C4 direct addition of enzyme to the whole slurry resulted in a higher release of carbohydrates during hydrolysis. Only in the case of C2 did the use of the resuspended solid result in a higher carbohydrate release. The overall carbohydrate recovery efficiency in the liquid fractions for C1, C2, C3 and C4 corresponded to 399.1 (±26.1), 535.8 (±28.7), 257.0 (±11.5), and 446.3 (±81.1) mg carbohydrate per Gram of BSG (dry weight), respectively. C1 and C4 were considered the optimal pretreatments as these combined a low acid concentration and energy input prior to enzymatic hydrolysis.

IntroductionSprat (Sprattus sprattus) is an underutilized fish species that may act as an economic and sustainable alternative source of protein due to its good amino acid (AA) profile along with its potential to act as a source of multiple bioactive peptide sequences.Method and resultsThis study characterized the physicochemical, technofunctional, and in vitro antioxidant properties along with the AA profile and score of a sprat protein enzymatic hydrolysate (SPH). Furthermore, the impact of the SPH on the growth, proliferation, and muscle protein synthesis (MPS) in skeletal muscle (C2C12) myotubes was examined. The SPH displayed good solubility and emulsion stabilization properties containing all essential and non-essential AAs. Limited additional hydrolysis was observed following in vitro-simulated gastrointestinal digestion (SGID) of the SPH. The SGID-treated SPH (SPH-SGID) displayed in vitro oxygen radical antioxidant capacity (ORAC) activity (549.42 μmol TE/g sample) and the ability to reduce (68%) reactive oxygen species (ROS) production in C2C12 myotubes. Muscle growth and myotube thickness were analyzed using an xCELLigence™ platform in C2C12 myotubes treated with 1 mg protein equivalent.mL−1 of SPH-SGID for 4 h. Anabolic signaling (phosphorylation of mTOR, rpS6, and 4E-BP1) and MPS (measured by puromycin incorporation) were assessed using immunoblotting. SPH-SGID significantly increased myotube thickness (p < 0.0001) compared to the negative control (cells grown in AA and serum-free medium). MPS was also significantly higher after incubation with SPH-SGID compared with the negative control (p < 0.05).ConclusionsThese preliminary in situ results indicate that SPH may have the ability to promote muscle enhancement. In vivo human studies are required to verify these findings.

The impact of whey protein (WP) denaturation on the in vitro digestibility and biological activity of milk protein concentrate-85 (MPC85) was investigated. MPC85S1 and MPC85S2 having undenatured WP levels equal to 16.6 and 6.0 g/100 g overall protein, respectively, had similar in vitro protein digestibility corrected amino acid scores equal to 1.14. The samples were subjected to in vitro simulated gastrointestinal digestion while sampling was performed every 30 min during gastric (GD) followed by intestinal (GID) digestion. Liquid chromatography–mass spectroscopy showed that MPC85S1-GD, MPC85S2-GD, MPC85S1-GID and MPC85S2-GID had 50, 38, 47 and 66 unique peptides, respectively. The degree of hydrolysis, molecular mass distribution, dipeptidyl peptidase-IV inhibition, oxygen radical absorbance capacity and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity of the digests were compared. Overall, the results showed higher digestibility and bioactivities for low-denatured MPC85 compared to high-denatured MPC85 upon GD, however, following GID, both samples were digested to a similar extent.

The impact of different light conditions during culture on the nitrogen, protein, colour, total phenolic content (TPC) and amino acid profile of Palmaria palmata biomass was investigated. P. palmata was cultured using different light regimes, i.e., white (1 and 2), red, blue and green over 12 days. A significant decrease (p < 0.05) in total nitrogen (TN), non-protein nitrogen (NPN) and protein nitrogen (PN) was observed on day 6 while an increase was observed on day 12 in P. palmata samples cultured under blue light. The protein content (nitrogen conversion factor of 4.7) of the initial sample on day 0 was 15.0% (w/w) dw whereas a maximum protein content of 16.7% (w/w) was obtained during exposure to blue light following 12 days culture, corresponding to an 11.2% increase in protein content. Electrophoretic along with amino acid profile and score analyses showed light-related changes in protein composition. The lighting regime used during culture also influenced the colour parameters (lightness L*, redness a*, yellowness b* and colour difference ΔE) of milled algal biomass along with the TPC. Judicious selection of lighting regime during culture may allow the targeted production of sustainable high-quality proteins from P. palmata.

Silkworm (Bombyx mori) pupae is a by-product from the silk industry which is rich in protein. Hydrolysates from silkworm pupae generated using Alcalase®, Prolyve®, Flavourzyme® and Brewers Clarex® proteolytic preparations were characterised. The antioxidant activity of the hydrolysates was investigated using in vitro antioxidant assays and an in situ assay for reactive oxygen species (ROS) reduction using hepatic HepG2. Overall, Alcalase and Prolyve hydrolysates had highest scavenging activities, however, Flavourzyme and Brewers Clarex hydrolysates had enhanced ferric reducing antioxidant power (FRAP) activity compared to the other samples. Furthermore, the Flavourzyme hydrolysate significantly reduced ROS by 40% compared to untreated control HepG2 cells. Peptides identified by LC-MS/MS were synthetised and then tested for their in vitro and in situ antioxidant activity. Peptides SWFVTPF and NDVLFF showed highest antioxidant activity (ROS reduction, superoxide dismutase (SOD) expression and glutathione (GSH) production activity) in HepG2 cells, and therefore may have potential as natural antioxidants.

Brewers’ spent grain (BSG) is the major co-product of the brewing industry having a high content of protein and fibre. Enzymatic modification improves the properties of different ingredients when they are incorporated into confectionary products. This study characterised the rheological, textural and sensory properties of unmodified BSG (BSGA) and enzymatically modified BSG (BSGB, i.e., proteinase and carbohydrase modified BSG) when substituted at different levels (5, 10 and 15%) in a muffin ingredient mix. Rheological assessment showed a lower viscosity for the BSGB compared to the BSGA batters. Baked BSGB containing muffins had lower hardness values compared to BSGA and control without added BSG. Sensory assessment showed no significant differences in liking of the different attributes tested (overall appearance, texture, smell, colour and taste) between the 5% BSGA and BSGB supplemented muffins. Incorporation of BSGB at 10% had no negative impact on the sensory attributes in comparison to BSGA 10%. Overall, low level substitution of enzymatic modified BSG beneficially reduced batter viscosity and muffin hardness and gave comparable sensory attributes to those of unmodified BSG supplemented muffins. Therefore, prior enzymatic modification can enhance the ingredient functionality of BSG in confectionary products.

The proteins from two conventionally (CC1 and CC2) and one organically cultivated (OC) hempseed samples were extracted (by alkaline solubilization followed by isoelectric precipitation) and compared in terms of their physicochemical, digestibility and in vitro bioactivity properties. The OC hempseed had higher total protein and lower nonprotein nitrogen content. Protein extracts showed bimodal particle size distributions, with OC showing the smallest and CC1 the largest mean particle diameter (d(0.5)), i.e., 89.0 and 120.0 µm, respectively. Chromatographic analysis showed similar protein profiles for all three protein extracts. The protein extracts were subjected to in vitro simulated gastrointestinal digestion (SGID). Degree of hydrolysis (DH) measurement showed that the highest extent of digestion upon SGID was associated with CC1 (11.0 ± 1.5%), which also had the lowest in vitro antioxidant activity. Only the OC and OC digested samples had lipase inhibitory activity. The results indicate that the cultivation method impacted the composition, physicochemical, digestibility, and biofunctional properties of hempseed proteins.

Coastal countries have traditionally relied on the existing marine resources (e.g., fishing, food, transport, recreation, and tourism) as well as tried to support new economic endeavors (ocean energy, desalination for water supply, and seabed mining). Modern societies and lifestyle resulted in an increased demand for dietary diversity, better health and well-being, new biomedicines, natural cosmeceuticals, environmental conservation, and sustainable energy sources. These societal needs stimulated the interest of researchers on the diverse and underexplored marine environments as promising and sustainable sources of biomolecules and biomass, and they are addressed by the emerging field of marine (blue) biotechnology. Blue biotechnology provides opportunities for a wide range of initiatives of commercial interest for the pharmaceutical, biomedical, cosmetic, nutraceutical, food, feed, agricultural, and related industries. This article synthesizes the essence, opportunities, responsibilities, and challenges encountered in marine biotechnology and outlines the attainment and valorization of directly derived or bio-inspired products from marine organisms. First, the concept of bioeconomy is introduced. Then, the diversity of marine bioresources including an overview of the most prominent marine organisms and their potential for biotechnological uses are described. This is followed by introducing methodologies for exploration of these resources and the main use case scenarios in energy, food and feed, agronomy, bioremediation and climate change, cosmeceuticals, bio-inspired materials, healthcare, and well-being sectors. The key aspects in the fields of legislation and funding are provided, with the emphasis on the importance of communication and stakeholder engagement at all levels of biotechnology development. Finally, vital overarching concepts, such as the quadruple helix and Responsible Research and Innovation principle are highlighted as important to follow within the marine biotechnology field. The authors of this review are collaborating under the European Commission-funded Cooperation in Science and Technology (COST) Action Ocean4Biotech – European transdisciplinary networking platform for marine biotechnology and focus the study on the European state of affairs.

This study investigated the antihyperglycaemic effectiveness of an oral Palmaria palmata protein hydrolysate (PPPH), versus metformin, upon metabolic control in streptozotocin (STZ)-induced diabetic mice. Mice were administered PPPH (50 mg/kg bodyweight) or metformin (200 mg/kg bodyweight) by oral gavage twice-daily for 18 days. Blood glucose and plasma insulin were measured every third day. PPPH caused a significant reduction in blood glucose (p

Independent of glucose, ingestion of protein evokes an insulinotropic response due to enterogastric incretin hormones and hyperaminoacidemia. The aim of this study was to characterise insulin and incretin secretion in healthy young women (age 22.0 ± 1.2 y, n = 8) following acute ingestion of a milk based protein matrix (MPM) containing casein, whey protein and whey protein hydrolysates with in vitro dipeptidyl peptidase IV (DPP-IV) inhibitory activity. Following ingestion of 0.33 g protein/kg body mass, plasma insulin, glucose, glucagon, total glucose-dependent insulinotropic polypeptide (GIP), active glucagon-like peptide-1 (GLP-1), DPP-IV activity and amino acids were measured over a 3 h period. These biomarkers were also determined following ingestion of an isocaloric maltodextrin drink (MALT) in order to estimate the equivalent glucose-dependent response. Ingestion of MPM evoked ~50% of the insulin response obtained with MALT without hyperglycaemia. Time-dependent changes in plasma GIP, GLP-1 and amino acid levels indicated these factors were probably regulators of insulin secretion on MPM ingestion.

Marine organisms produce a vast diversity of metabolites with biological activities useful for humans, e.g., cytotoxic, antioxidant, anti-microbial, insecticidal, herbicidal, anticancer, pro-osteogenic and pro-regenerative, analgesic, anti-inflammatory, anti-coagulant, cholesterol-lowering, nutritional, photoprotective, horticultural or other beneficial properties. These metabolites could help satisfy the increasing demand for alternative sources of nutraceuticals, pharmaceuticals, cosmeceuticals, food, feed, and novel bio-based products. In addition, marine biomass itself can serve as the source material for the production of various bulk commodities (e.g., biofuels, bioplastics, biomaterials). The sustainable exploitation of marine bio-resources and the development of biomolecules and polymers are also known as the growing field of marine biotechnology. Up to now, over 35,000 natural products have been characterized from marine organisms, but many more are yet to be uncovered, as the vast diversity of biota in the marine systems remains largely unexplored. Since marine biotechnology is still in its infancy, there is a need to create effective, operational, inclusive, sustainable, transnational and transdisciplinary networks with a serious and ambitious commitment for knowledge transfer, training provision, dissemination of best practices and identification of the emerging technological trends through science communication activities. A collaborative (net)work is today compelling to provide innovative solutions and products that can be commercialized to contribute to the circular bioeconomy. This perspective article highlights the importance of establishing such collaborative frameworks using the example of Ocean4Biotech, an Action within the European Cooperation in Science and Technology (COST) that connects all and any stakeholders with an interest in marine biotechnology in Europe and beyond.

During the generation of functional food ingredients by enzymatic hydrolysis, parameters such as choice of enzyme, reaction pH and the drying process employed may contribute to the physicochemical and bio-functional properties of the resultant protein hydrolysate ingredients. This study characterised the properties of spray- (SD) and freeze-dried (FD) whey protein hydrolysates (WPHs) generated using Alcalase® and Prolyve® under pH-stat and free-fall pH conditions. The enzyme preparation used affected the physicochemical and antioxidative properties but had no impact on powder composition, morphology or colour. SD resulted in spherical particles with higher moisture content (~6%) compared to the FD powders (~1%), which had a glass shard-like structure. The SD-WPHs exhibited higher antioxidative properties compared to the FD-WPHs, which may be linked to a higher proportion of peptides ® under free-fall pH conditions, followed by SD, which had oxygen radical absorbance capacity and Trolox equivalent (TE) antioxidant capacity values of 1132 and 686 µmol TE/g, respectively. These results demonstrate that both the hydrolysis and the drying process impact the biofunctional (antioxidant) activity of WPHs.

This study determined the physicochemical properties (apparent viscosity (ηapp), turbidity (A550nm), particle size and molecular mass distribution) of hydrolysates generated from whey protein concentrate (WPC), milk protein concentrate (MPC) and sodium caseinate (NaCN), following incubation with Debitrase HYW20™ and Prolyve™ at 50 °C, pH 7.0 for 1 and 4 h, before and after heat inactivation (80 °C for 10 min). The degree of hydrolysis (DH) increased with incubation time, giving values of 6.56%, 8.17% and 9.48%, following 1 h hydrolysis of WPC, MPC and NaCN with Debitrase HYW20™, and 12.04%, 15.74% and 17.78%, respectively, following 4 h incubation. These DHs were significantly higher compared to those obtained following 4 h incubation with Prolyve™. Hydrolysis with Debitrase HYW20™ gave >40% of peptides with molecular masses < 1 kDa for all substrates, which was higher than the value obtained following hydrolysis with Prolyve™. The effect of hydrolysis on the physicochemical properties was substrate dependent, since ηapp decreased in WPC and NaCN hydrolysates, particle size decreased for all the substrates, with aggregate formation for MPC, and turbidity decreased in WPC and MPC hydrolysates, while it increased in NaCN hydrolysates. The physical properties of the hydrolysates were influenced by the enzyme thermal inactivation step in a DH-dependent manner, with no significant effect on turbidity and viscosity for hydrolysates at higher DHs.

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

Whey protein concentrate (WPC) hydrolysates generated using Neutrase®, Alcalase® and Flavourzyme® and their associated ultrafiltration fractions inhibited angiotensin-1-converting enzyme activity (71.14 ± 1.05, 73.22 ± 0.99 and 51.52 ± 5.80% inhibition when assayed at 14.3 µg/mL). Incubation of human umbilical vein endothelial cells with 5 kDa permeates of Neutrase®- and Alcalase®-hydrolysed WPC for 48 h resulted in the beneficial differential expression of genes relevant to blood pressure control, as measured by microarray. Furthermore, real-time reverse transcriptase polymerase chain reaction demonstrated an upregulation of endothelial nitric oxide synthase (+2.14 ± 0.45 and 2.36 ± 0.27-fold) and down-regulation of endothelin-1 (−0.58 ± 0.09 and −0.82 ± 0.11-fold) following incubation with the 5 kDa permeates of Alcalase®- and Neutrase®-hydrolysates, respectively. Peptide sequences within the 5 kDa permeate of the Alcalase®-hydrolysed WPC were identified, many of which have previously been demonstrated as having ACE-inhibitory and/or other bioactivities. These WPC hydrolysates potentially represent sources of bioactive peptides for the beneficial regulation of endothelial cell function.

A blue whiting (Micromesistius poutassou) protein hydrolysate generated using Alcalase 2.4L and Flavourzyme 500L and its simulated gastrointestinal digestion (SGID) sample was assessed for antidiabetic potential in vitro and in vivo. In addition to inhibiting dipeptidyl peptidase-IV (DPP–IV), the hydrolysates mediated insulin and glucagon-like peptide-1 (GLP-1) release from BRIN-BD11 and GLUTag cells, respectively. No significant difference was observed in insulinotropic and DPP-IV inhibitory activity following SGID, while GLP-1 secretion increased significantly (p

This study investigated the antioxidant and immunomodulatory potential of six blue whiting soluble protein hydrolysates (BWSPHs, BW-SPH-A to -F) and their simulated gastrointestinal digests (SGID, BW-SPH-A-GI to -F-GI) in murine RAW264.7 macrophages. Hydrolysate BW-SPH-A, both pre- and post-SGID, increased endogenous antioxidant glutathione (GSH) in tert-butylhydroperoxide (tBOOH)-treated cells and reduced reactive oxygen species (ROS) in H2O2-challenged RAW264.7 cells compared with treated controls in the absence of BWSPHs (p < 0.05). BW-SPH-A-GI also exhibited higher ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) activities than the other BWSPHs tested (p < 0.05). All BWSPHs and SGID BWSPH samples induced immunostimulating effects in lipopolysaccharide (LPS)-activated RAW264.7 macrophages through the upregulation of NO production. BW-SPH-F-GI increased IL-6 and TNF-α levels compared with the LPS controls indicating the liberation of immunomodulatory peptide/amino acids during the SGID process. Therefore, BW-SPH-A and BW-SPH-F may have potential use against oxidative stress and immunosuppression-related diseases, respectively.

A targeted enzymatic approach was employed for the generation of angiotensin converting enzyme (ACE) inhibitory/anti-hypertensive peptides. Porcine skin gelatin was hydrolysed with Aspergillus niger prolyl endoproteinase (An-PEP) to produce an hydrolysate with potent ACE inhibitory activity (mean IC50: 220.2 µg mL−1) after 4-h hydrolysis. Peptide identification was achieved by UPLC-ESI-MS/MS. The ACE-inhibitory activity of a selection of the identified peptides was determined. The most potent peptide was Met-Gly-Pro with an ACE IC50 of 51.11 ± 1.14 µM. Furthermore, oral administration (50 mg/kg body weight) of the hydrolysate to spontaneously hypertensive rats resulted in decreases in systolic and diastolic blood pressure of −28.89 ± 5.11 and −22.86 ± 5.65 mm Hg, respectively. Mean arterial pressure and heart rate were also reduced (−25.99 ± 5.66 mm Hg and −53.63 ± 17.67 bpm, respectively). The beneficial in vivo effects may be related to the potent C-terminal containing proline ACE-inhibitory peptides in the hydrolysate.

Dipeptidyl peptidase IV (DPP-IV) inhibitory peptides were identified in silico within camel milk proteins. Camel milk was hydrolysed with trypsin using a design of experiments (DOE, temperature (40–60 °C), enzyme to substrate (E:S) ratio (0.50–2.00% (w/w)) and time (60–240 min)). Fifteen hydrolysates (H1–H15) having DPP-IV half maximal inhibitory concentration (IC50) values between 0.52 ± 0.06 (H9) and 1.26 ± 0.13 (H1) mg mL−1 were produced. Camel and bovine milk proteins hydrolysed at 40 °C, 1.8% E:S and 218 min had DPP-IV IC50 values of 0.68 ± 0.08 and 0.85 ± 0.10 mg mL−1 (p

Milk protein derived peptides have numerous well-documented bioactive properties. The conventional approach for the generation, identification and validation of bioactive peptides (BAPs) has involved (i) protein hydrolysis, (ii) bioactivity screening and (iii) validation in vivo. The low potency (in comparison to conventional drugs), susceptibility to breakdown during gastrointestinal transit and low intestinal permeability are key challenges in the development of highly bioactive food protein hydrolysates/peptides. However, the generation of potent and effective health enhancing hydrolysates/peptides can benefit from a range of in silico techniques including the application of structure bioactivity relationship modelling (e.g., quantitative structure activity relationship (QSAR) modelling), molecular docking and design of experiments (DOE) approaches to optimise BAP production and identification. Some examples of how these approaches have been employed in BAP discovery and generation will be outlined.

Brewer's spent grain (BSG) is a co-product of the brewing industry and is a potentially rich source of bioactive peptides. A BSG protein hydrolysate, manufactured at a semi-pilot scale, mediated significant hypotensive effects 6 h after ingestion in spontaneously hypertensive rats. This hydrolysate was fractionated and further analysed by UPLC-ESI-MS/MS for peptide identification. The peptides, IPY and LPY demonstrated the highest antioxidant activity (0.37 ± 0.02 and 0.34 ± 0.02 µmol Trolox equivalents µM peptide−1, respectively), IPLQP and LPLQP had the highest ACE inhibitory activity (IC50: 3.10 ± 0.60 and 3.17 ± 0.60 µM, respectively) while IPVP had the highest DPP-IV inhibitory activity (IC50: 38.67 ± 5.94 µM). Overall, peptides containing I had higher in vitro bioactivities when compared to peptides having L. Novel BSG peptides were identified with potential for application as naturally derived ingredients for the management of cardiovascular disease and type 2 diabetes.

The conchocelis life cycle stage of P. dioica represents an unexplored source of bioactive compounds. The aim of this study was to generate and characterise, for the first time, hydrolysates of conchocelis using a specific combination of proteases (Prolyve® and Flavourzyme®). Hydrolysate molecular mass distribution and free amino acid contents were assessed, and the antioxidant activity was determined using a range of in vitro assays. The protein content and the total amino acid profiles of conchocelis were also studied. Conchocelis contained ~25% of protein (dry weight basis) and had a complete profile of essential amino acids. Direct sequential enzymatic treatment modified the profile of the generated compounds, increasing the amount of low molecular weight peptides (

Enzymatic hydrolysis has been employed to modify protein functional properties and discover new sources of antioxidants. In this study, the effect of different enzymatic treatments on antioxidant activity of Porphyra dioica (blades and protein isolate (PI)) was investigated. Protein nitrogen content of P. dioica blades and PI were 23 and 50% (dry weight), respectively. Blades and PI were hydrolyzed with Prolyve® and Prolyve® plus Flavourzyme®. Peptide profiles and molecular mass distribution of the hydrolysates were investigated. The hydrolysis promoted generation of peptides and low molecular mass components P. dioica blades and PI enhanced the in vitro antioxidant activity. Direct hydrolysis of blades improved ORAC values up to 5-fold (from 610 to 3054 μmol Trolox eq./g freeze dried sample (FDS). The simultaneous release of phenolic compounds suggested a potential synergistic activity (ORAC and ABTS·+ assays). Such hydrolysates may be of value as functional food ingredients.

Bovine whey protein concentrate (WPC) was hydrolysed under pH-stat (ST) and non pH-controlled (free-fall, FF) conditions using Debitrase (DBT) and FlavorPro Whey (FPW). The resultant whey protein hydrolysates (WPHs) were assessed for the impact of hydrolysis conditions on the physicochemical and the in vitro antioxidant and intracellular reactive oxygen species (ROS) generation in oxidatively stressed HepG2 cells. Enzyme and hydrolysis condition dependent differences in the physicochemical properties of the hydrolysates were observed, however, the extent of hydrolysis was similar under ST and FF conditions. Significantly higher (p < 0.05) in vitro and cellular antioxidant activities were observed for the DBT compared to the FPW–WPHs. The WPHs generated under ST conditions displayed significantly higher (p < 0.05) oxygen radical absorbance capacity (ORAC) and Trolox equivalent antioxidant capacity (TEAC) values compared to the FF-WPHs. The impact of hydrolysis conditions was more pronounced in the in vitro compared to the cellular antioxidant assay. WPH peptide profiles (LC-MS/MS) were also enzyme and hydrolysis conditions dependent as illustrated in the case of β-lactoglobulin. Therefore, variation in the profiles of the peptides released may explain the observed differences in the antioxidant activity. Targeted generation of antioxidant hydrolysates needs to consider the hydrolysis conditions and the antioxidant assessment method employed.

Inhibition of dipeptidyl peptidase IV (DPP-IV) may be exploited to maintain the incretin effect during the postprandial phase. As a result, glycemic regulation and energy homeostasis may be improved. Food protein-derived peptides have been identified as natural agents capable of inhibiting DPP-IV. Ile-Pro-Ile is the most potent DPP-IV inhibitory peptide identified to date. A minimum analog peptide set approach was used to study peptide analogs of Ile-Pro-Ile. The DPP-IV half maximal inhibitory concentration (IC50) values of the 25 peptides evaluated ranged from 3.9 ± 1.0 µM (Ile-Pro-Ile) to 247.0 ± 32.7 µM (Phe-Pro-Phe). The presence of Pro at position 2 of tripeptides was required to achieve high DPP-IV inhibition. Most peptides behaved as competitive inhibitors of DPP-IV with the exception of peptides with a N-terminal Trp, which were mixed-type inhibitors. While possessing the structure of preferred DPP-IV substrates, most peptides studied were particularly stable during 30 min incubation with DPP-IV. Molecular docking revealed that Ile-Pro-Ile and its peptide analogs interacted in a very similar manner with the active site of DPP-IV. In addition, no correlation was found between the Hydropathic INTeraction score and the DPP-IV IC50 values of the peptides studied. This outcome suggests that free energy may not be directly responsible for enzyme inhibition by the peptides. Finally, novel DPP-IV inhibitory peptides were identified using the strategy employed herein. These results may be relevant for the development of food protein-derived peptides with serum glucose lowering and food intake regulatory properties in humans.

Milk proteins are a good source of bioactive peptides (BAPs). BAPs can positively affect various health biomarkers in vitro. The role of milk protein-derived BAPs in humans was reviewed herein. To date, a limited number of BAPs have been identified in the gastrointestinal tract of humans and in the circulation at concentrations which may be sufficient to mediate in vivo bioactivity. While the outcomes of several human intervention studies have shown promising results, some studies have failed to demonstrate additional health benefits. This may be related to a low potency and bioavailability of the BAPs evaluated as well as confounding factors (i.e. genotype, health status and other sources of inter-individual variability). Universal guidelines for the evaluation of BAPs in humans such as adequately powered double-blind randomised clinical trials are needed.

Phlorotannins are a group of complex polymers of phloroglucinol (1,3,5-trihydroxybenzene) unique to macroalgae. These phenolic compounds are integral structural components of the cell wall in brown algae, but also play many secondary ecological roles such as protection from UV radiation and defense against grazing. This study employed Ultra Performance Liquid Chromatography (UPLC) with tandem mass spectrometry to investigate isomeric complexity and observed differences in phlorotannins derived from macroalgae harvested off the Irish coast (Fucus serratus, Fucus vesiculosus, Himanthalia elongata and Cystoseira nodicaulis). Antioxidant activity and total phenolic content assays were used as an index for producing phlorotannin fractions, enriched using molecular weight cut-off dialysis with subsequent flash chromatography to profile phlorotannin isomers in these macroalgae. These fractions were profiled using UPLC-MS with multiple reaction monitoring (MRM) and the level of isomerization for specific molecular weight phlorotannins between 3 and 16 monomers were determined. The majority of the low molecular weight (LMW) phlorotannins were found to have a molecular weight range equivalent to 4–12 monomers of phloroglucinol. The level of isomerization within the individual macroalgal species differed, resulting in substantially different numbers of phlorotannin isomers for particular molecular weights. F. vesiculosus had the highest number of isomers of 61 at one specific molecular mass, corresponding to 12 phloroglucinol units (PGUs). These results highlight the complex nature of these extracts and emphasize the challenges involved in structural elucidation of these compounds.

Tryptic hydrolysis of whey protein isolate under specific incubation conditions including a relatively high enzyme:substrate (E:S) ratio of 1:10 is known to preferentially hydrolyse β-lactoglobulin (β-LG), while retaining the other major whey protein fraction, i.e., α-lactalbumin (α-LA) mainly intact. An objective of the present work was to explore the effects of reducing E:S (1:10, 1:30, 1:50, 1:100) on the selective hydrolysis of β-LG by trypsin at pH 8.5 and 25 °C in a 5% (w/v) WPI solution during incubation periods ranging from 1 to 7 h. In addition, the use of a pilot-scale continuous high-temperature, short-time (HTST) heat exchanger with an extended holding time (EHT) of 5 min as a means of inactivating trypsin to terminate hydrolysis was compared with laboratory-based acidification to 90% β-LG hydrolysis after respective incubation periods of 4 and 6 h, with

Dipeptides with a C terminal Pro inhibit dipeptidyl peptidase IV (DPP-IV), a key enzyme in incretin hormone processing. It was hypothesised that tri- and tetrapeptides with a proline at the C-terminus may also be DPP-IV inhibitors. Therefore, an in silico hydrolysis approach was used to release short (4 ⩽ amino acids) C terminal Pro peptides from the individual caseins which constitute Pro rich substrates. This was achieved using theoretical digestion of caseins with a prolyl oligopeptidase activity. Fifteen peptides were subsequently selected for in vitro DPP-IV inhibitory analysis. Stability of these peptides to gastrointestinal enzymes was also evaluated in silico and the predicted breakdown peptides were assessed for their DPP-IV inhibitory and antioxidant potential. New DPP-IV inhibitors were identified, the most potent being Phe-Leu-Gln-Pro (IC50 65.3 ± 3.5 μM). A low in vitro antioxidant (2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging) activity was also associated with the peptides studied. The strategy presented highlights the utility of employing an in silico approach for the prediction of food-derived peptides with a potential role in glycaemic management for subsequent development of functional foods.

Marine organisms such as fish and shellfish are rich sources of structurally diverse bioactive nitrogenous components. Based on emerging evidence of potential health benefits, these components show significant promise as functional food ingredients. Activities including antihypertensive, antioxidant, anti-microbial, anti-coagulant, anti-diabetic, anti-cancer, immunostimulatory, calcium-binding, hypocholesteremic and appetite suppression have been reported. Fish and shellfish waste components contain significant levels of high quality protein (10–23% (w/w)) which represents a source for biofunctional peptide mining. This review summarises the protein-derived bioactive peptides identified in marine processing waste, molluscs and crustaceans. Moreover, it highlights the potential of proteins derived from these marine organisms as substrates for the generation of biofunctional peptides. It outlines current technologies used in the production, fractionation and purification of marine protein-derived peptides and lists some commercially available products containing marine derived bioactive protein hydrolysates and peptides. Finally, bioactive proteins, non-protein peptides and amino acids found in fish and shellfish are briefly discussed.